Fungal Diseases

FUNGAL DISEASES
An Emerging Threat to Human,

Animal, and Plant Health
Workshop Summary
LeighAnne Olsen, Eileen R. Choffnes, David A. Relman,

and Leslie Pray, Rapporteurs
Forum on Microbial Threats

Board on Global Health
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Financial support for this project was provided by the U.S. Department of Health and Hu-
man Services: National Institutes of Health, National Institute of Allergy and Infectious
Diseases, Centers for Disease Control and Prevention, Food and Drug Administration,
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Army: Global Emerging Infections Surveillance and Response System, Medical Research
and Materiel Command, and the Defense Threat Reduction Agency; U.S. Department of
Veterans Affairs; U.S. Department of Homeland Security; U.S. Agency for International
Development; American Society for Microbiology; sanofi pasteur; Burroughs Wellcome
Fund; Pfizer, Inc.; GlaxoSmithKline; Infectious Diseases Society of America; and the
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view of the organizations or agencies that provided support for this project.
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Cover images: Front (upper): Little brown bats with white-nose syndrome, New York,
photo courtesy of New York Department of Environmental Conservation; Front (lower):
Yellow stripe rust on wheat, photo courtesy of Stephen A. Harrison, Louisiana State Uni-
versity Agricultural Center. Spine: The Panamanian golden frog (Atelopus zeteki), photo
courtesy of Wikimedia Commons, photo by Brian Gratwicke, Smithsonian Conservation
Biology Institute. Back: Geomyces destructans, shown in a false-color SEM image (fungus
hyphae are yellow, green, and orange; spores are blue), image reprinted from Chaturvedi
et al. (2010) Morphological and Molecular Characterizations of Psychrophillic Fungus
Geomyces destructans from New York Bats with White Nose Syndrome (WNS). PLoS
ONE 5(5): e10783. Doi: 10.1371/journal.pone.0010783.
Suggested citation: IOM (Institute of Medicine). 2011. Fungal Diseases: An Emerging
Threat to Human, Animal, and Plant Health. Washington, DC: The National Academies
Press.
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FORUM ON MICROBIAL THREATS1
DAVID A. RELMAN (Chair), Stanford University and Veterans Affairs Palo
Alto Health Care System, Palo Alto, California
JAMES M. HUGHES (Vice-Chair), Global Infectious Diseases Program,
Emory University, Atlanta, Georgia
LONNIE J. KING (Vice-Chair), Ohio State University, Columbus
KEVIN ANDERSON, Department of Homeland Security, Washington, DC
RUTH L. BERKELMAN, Center for Public Health Preparedness and
Research, Rollins School of Public Health, Emory University, Atlanta,
Georgia
DAVID BLAZES,2 Armed Forces Health Surveillance Center, Division of
Global Emerging Infectious Surveillance, Silver Spring, Maryland
ENRIQUETA C. BOND, Burroughs Wellcome Fund (Emeritus), Marshall,
Virginia
ROGER BREEZE, Lawrence Livermore National Laboratory, Livermore,
California
STEVEN J. BRICKNER,3 SJ Brickner Consulting, LLC, Ledyard,
Connecticut
PAULA R. BRYANT, Defense Threat Reduction Agency, Medical S&T
Division, Fort Belvoir, Virginia
JOHN E. BURRIS, Burroughs Wellcome Fund, Research Triangle Park, North
Carolina
ARTURO CASADEVALL,2 Albert Einstein College of Medicine, Bronx,
New York
PETER DASZAK, EcoHealth Alliance, New York, New York
JEFFREY S. DUCHIN, Public Health–Seattle and King County, Seattle,
Washington
JONATHAN EISEN, Genome Center, University of California, Davis
MARK B. FEINBERG, Merck Vaccine Division, Merck & Co., West Point,
Pennsylvania
JACQUELINE FLETCHER, Oklahoma State University, Stillwater
S. ELIZABETH GEORGE,3 Department of Homeland Security,
Washington, DC
JESSE L. GOODMAN, Food and Drug Administration, Rockville, Maryland
EDUARDO GOTUZZO, Instituto de Medicina Tropical–Alexander von
Humbolt, Universidad Peruana Cayetano Heredia, Lima, Peru
CAROLE A. HEILMAN, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, Maryland
1â•›â•›Institute of Medicine Forums and Roundtables do not issue, review, or approve individual docu-
ments. The responsibility for the published workshop summary rests with the workshop rapporteurs
and the institution.
2â•›â•›Forum member since September 1, 2011.
3â•›â•›Forum member until December 31, 2010.
v
DAVID L. HEYMANN, Health Protection Agency, London, United Kingdom
PHILIP HOSBACH, sanofi pasteur, Swiftwater, Pennsylvania
STEPHEN ALBERT JOHNSTON, Arizona BioDesign Institute, Arizona
State University, Tempe
KENT KESTER, Walter Reed Army Institute of Research, Silver Spring,
Maryland
GERALD T. KEUSCH, Boston University School of Medicine and Boston
University School of Public Health, Boston, Massachusetts
RIMA F. KHABBAZ, Centers for Disease Control and Prevention, Atlanta,
Georgia
STANLEY M. LEMON, School of Medicine, University of North Carolina,
Chapel Hill
EDWARD McSWEEGAN, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, Maryland
MARK A. MILLER, Fogarty International Center, Bethesda, Maryland
PAUL F. MILLER,4 Pfizer, Inc., Groton, Connecticut
STEPHEN S. MORSE,5 Center for Public Health Preparedness, Columbia
University, New York, New York
GEORGE POSTE, Complex Adaptive Systems Initiative, Arizona State
University, Tempe, Arizona
JOHN C. POTTAGE, JR., ViiV Healthcare, Collegeville, Pennsylvania
DAVID RIZZO,6 Department of Plant Pathology, University of California,
Davis
GARY A. ROSELLE, Veterans Health Administration, Department of Veterans
Affairs, Cincinnati, Ohio
ALAN S. RUDOLPH, Defense Threat Reduction Agency, Fort Belvoir,
Virginia
KEVIN RUSSELL, Armed Forces Health Surveillance Center, Department of
Defense, Silver Spring, Maryland
JANET SHOEMAKER, American Society for Microbiology, Washington, DC
P. FREDERICK SPARLING, University of North Carolina, Chapel Hill,
North Carolina
TERENCE TAYLOR, International Council for the Life Sciences, Arlington,
Virginia
MURRAY TROSTLE, U.S. Agency for International Development,
Washington, DC
MARY E. WILSON, Harvard School of Public Health, Harvard University,
Boston, Massachusetts
4 Forum member until July 31, 2011.

5 Forum member until December 31, 2010.
6 Forum member since September 1, 2011.
vi
Staff
EILEEN CHOFFNES, Director
LEIGHANNE OLSEN, Program Officer
KATHERINE McCLURE, Senior Program Associate
COLLIN WEINBERGER, Research Associate (until May 2011)
REBEKAH HUTTON, Research Associate (from June 2011)
ROBERT GASIOR, Senior Program Assistant (until March 2011)
PAMELA BERTELSON, Senior Program Assistant (since September 2011)
vii
BOARD ON GLOBAL HEALTH1
Richard Guerrant (Chair), Thomas H. Hunter Professor of International
Medicine and Director, Center for Global Health, University of Virginia
School of Medicine, Charlottesville
Jo Ivey Boufford (IOM Foreign Secretary), President, New York Academy of
Medicine, New York
Claire V. Broome, Adjunct Professor, Division of Global Health, Rollins
School of Public Health, Emory University, Atlanta, Georgia
Jacquelyn C. Campbell, Anna D. Wolf Chair, and Professor, Johns Hopkins
University School of Nursing, Baltimore, Maryland
Thomas J. Coates, Professor, David Geffen School of Medicine, University of
California, Los Angeles, Los Angeles, California
Gary Darmstadt, Director, Family Health Division, Global Health Program,
Bill & Melinda Gates Foundation, Seattle, Washington
Valentin Fuster, Director, Wiener Cardiovascular Institute Kravis
Cardiovascular Health Center Professor, Cardiology, Mount Sinai School
of Medicine, Mount Sinai Medical Center, New York, New York
James Hospedales, Coordinator, Chronic Disease Project, Health Surveillance
and Disease Management Area, Pan American Health Organization/World
Health Organization, Washington, DC
Peter J. Hotez, Professor and Chair, Department of Microbiology,
Immunology, and Tropical Medicine, The George Washington University,
Washington, DC
Clarion Johnson, Global Medical Director, Medicine and Occupational
Medicine Department, Exxon Mobil, Fairfax, Virginia
Fitzhugh Mullan, Professor, Department of Health Policy, George Washington
University, Washington, DC
Guy Palmer, Regents Professor of Pathology and Infectious Diseases, Director
of the School for Global Animal Health, Washington State University
Jennifer Prah-Ruger, Associate Professor, Division of Health Policy and
Administration, Yale University School of Public Health, New Haven,
Connecticut
Staff
Patrick Kelley, Director
Angela Mensah, Program Associate
1â•›â•›Institute of Medicine boards do not review or approve individual workshop summaries. The
responsibility for the content of the workshop summary rests with the authors and the institution.
viii
Reviewers
This report has been reviewed in draft form by individuals chosen for their
diverse perspectives and technical expertise, in accordance with procedures ap-
proved by the National Research Council’s Report Review Committee. The pur-
pose of this independent review is to provide candid and critical comments that
will assist the institution in making its published report as sound as possible and
to ensure that the report meets institutional standards for objectivity, evidence,
and responsiveness to the study charge. The review comments and draft manu-
script remain confidential to protect the integrity of the process. We wish to thank
the following individuals for their review of this report:
Beth Bell, Centers for Disease Control and Prevention

Michael Jeger, Imperial College London
Karen Lips, University of Maryland
Victoria McGovern, Burroughs Wellcome Fund
John W. Taylor, University of California at Berkeley
Brett Tyler, Virginia Bioinformatics Institute
Although the reviewers listed above have provided many constructive com-
ments and suggestions, they were not asked to endorse the final draft of the report
before its release. The review of this report was overseen by Dr. Melvin Worth.
Appointed by the Institute of Medicine, he was responsible for making certain
that an independent examination of this report was carried out in accordance with
institutional procedures and that all review comments were carefully considered.
Responsibility for the final content of this report rests entirely with the authoring
committee and the institution.
ix
Acknowledgments
The Forum on Emerging Infections was created by the Institute of Medicine

(IOM) in 1996 in response to a request from the Centers for Disease Control
and Prevention (CDC) and the National Institutes of Health (NIH). The purpose
of the Forum is to provide structured opportunities for leaders from govern-
ment, academia, and industry to regularly meet and examine issues of shared
concern regarding research, prevention, detection, and management of emerg-
ing, reemerging, and novel infectious diseases in humans, plants, and animals.
In pursuing this task, the Forum provides a venue to foster the exchange of
information and ideas, identify areas in need of greater attention, clarify policy
issues by enhancing knowledge and identifying points of agreement, and inform
decision makers about science and policy issues. The Forum seeks to illuminate
issues rather than resolve them. For this reason, it does not provide advice or
recommendations on any specific policy initiative pending before any agency or
organization. Its value derives instead from the diversity of its membership and
from the contributions that individual members make throughout the activities
of the Forum. In September 2003, the Forum changed its name to the Forum on
Microbial Threats.
The Forum on Microbial Threats and the IOM wish to express their warmest
appreciation to the individuals and organizations who gave their valuable time
to provide information and advice to the Forum through their participation in
the planning and execution of this workshop. A full list of presenters, and their
biographical information, may be found in Appendixes B and F, respectively.
The Forum gratefully acknowledges the contributions of the members of the
xi
xii ACKNOWLEDGMENTS
planning committee1: Gerald Keusch (Boston University), Rima Khabbaz (Cen-

ters for Disease Control and Prevention), Lonnie King (Ohio State University),
Victoria McGovern (Burroughs Wellcome Fund), Carol Meteyer (United States
Geological Service, National Wildlife Health Center), John Perfect (Duke Uni-
versity), Erica Rosenblum (University of Idaho), Kevin Russell (Department of
Defense), Fred Sparling (University of North Carolina), and James Stack (Kansas
State University).
The Forum is indebted to IOM staff who tirelessly contributed throughout
the planning and execution of the workshop and the production of this workshop
summary report. On behalf of the Forum, we gratefully acknowledge these efforts
led by Dr. Eileen Choffnes, director of the Forum; Dr. LeighAnne Olsen, program
officer; Katherine McClure, senior program associate; Collin Weinberger and
Rebekah Hutton, research associates; and Robert Gasior and Pamela Bertelson,
senior program assistants, for dedicating much effort and time to developing this
workshop’s agenda and for their thoughtful and insightful approach and skill in
planning for the workshop and in translating the workshop’s proceedings and
discussion into this workshop summary report. We would also like to thank
the following IOM staff and consultants for their valuable contributions to this
activity: Greta Gorman, Jill Grady, Laura Penny, Heather Phillips, Leslie Pray,
Elisabeth Reese, Vilija Teel, and Jordan Wyndelts.
Finally, the Forum wishes to recognize the sponsors that supported this ac-
tivity. Financial support for this project was provided by the U.S. Department of
Health and Human Services: NIH, National Institute of Allergy and Infectious
Diseases, CDC, Food and Drug Administration, and the Fogarty International
Center2; U.S. Department of Defense, Department of the Army: Global Emerg-
ing Infections Surveillance and Response System, Medical Research and Ma-
teriel Command, and the Defense Threat Reduction Agency; U.S. Department
of Veterans Affairs; U.S. Department of Homeland Security; U.S. Agency for
International Development; American Society for Microbiology; sanofi pasteur;
Burroughs Wellcome Fund; Pfizer, Inc.; GlaxoSmithKline; Infectious Diseases
Society of America; and the Merck Company Foundation. The views presented
in this workshop summary report are those of the workshop participants and
rapporteurs and are not necessarily those of the Forum on Microbial Threats or
its sponsors.
1â•›â•›Institute of Medicine (IOM) planning committees are solely responsible for organizing the work-
shop, identifying topics, and choosing speakers. The responsibility for the published workshop sum-
mary rests with the workshop rapporteurs and the institution.
2â•›â•›Sponsor as of October 1, 2010.
Contents
Workshop Overview 1
Workshop Overview References, 84
Appendixes
A Contributed Manuscripts 101
A1 The Emergence of Cryptococcus gattii in British Columbia and the

Pacific Northwest, 101
Karen H. Bartlett, Sarah E. Kidd, and James W. Kronstad
A2 The Good, the Bad, and the Ugly: Fungi Mold Your World, 116
Meredith Blackwell
A3 The Fungi: 1, 2, 3 … 5.1 Million Species?, 140
Meredith Blackwell
A4 Bat White-Nose Syndrome in North America, 167
David S. Blehert, Jeffrey M. Lorch, Anne E. Ballmann, Paul M.
Cryan, and Carol U. Meteyer
A5 Mammalian Endothermy Optimally Restricts Fungi and Metabolic
Costs, 177
Aviv Bergman and Arturo Casadevall
A6 Vertebrate Endothermy Restricts Most Fungi as Potential
Pathogens, 181
Vincent A. Robert and Arturo Casadevall
xiii
xiv CONTENTS
A7 Surveillance for Emerging Diseases in Wildlife, 188

Peter Daszak, Carlos Zambrana-Torrelio, and Tiffany Bogich
A8 Geography, Climate, Dust, and Disease: Epidemiology of Valley
Fever (Coccidioidomycosis) and Ways It Might Be Controlled, 196
John N. Galgiani
A9 Cryptococcus gattii: An Emerging Pathogen in the United
States, 207
Julie R. Harris
A10 Sexual Reproduction, Evolution, and Adaptation of Cryptococcus
gattii in the Pacific Northwest Outbreak, 226
Joseph Heitman, Edmond J. Byrnes III, and John R. Perfect
A11 Yeast Infections—Human Genetics on the Rise, 248
Steven M. Holland and Donald C. Vinh
A12 The Increased Risk of Global Wheat Rust Pandemics: Putting Yellow
Rust into Perspective, 252
Mogens Støvring Hovmøller
A13 Fungal Pathogenesis in Plants and Animals: Similarities and
Differences, 264
Barbara Howlett
A14 Climate, Globalization, and Trade: Impacts on Dispersal and
Invasion of Fungal Plant Pathogens, 273
Michael Jeger, Marco Pautasso, and James Stack
A15 Emerging Fungal Diseases of Wild Animal Species, 296
Luis R. Padilla
A16 The Emergence of Phytophthora ramorum in North America and
Europe, 321
David M. Rizzo, Ross K. Meentemeyer, and Matteo Garbelotto
A17 Climate Change, Extreme Weather Events, and Fungal Disease
Emergence and Spread, 324
Compton J. Tucker, Karina Yager, Assaf Anyamba, and Kenneth
J. Linthicum
A18 Host-Pathogen Dynamics of Amphibian Chytridiomycosis: The Role
of the Skin Microbiome in Health and Disease, 342
Vance T. Vredenburg, Cheryl J. Briggs, and Reid Harris
A19 The Effect of Trade-Mediated Spread of Amphibian Chytrid on
Amphibian Conservation, 355
Ché Weldon and Matthew C. Fisher
A20 White-Nose Syndrome Fungus (Geomyces destructans) in Bats,
Europe, 368
Gudrun Wibbelt, Andreas Kurth, David Hellmann, Manfred
Weishaar, Alex Barlow, Michael Veith, Julia Prüger, Tamás
Görföl, Lena Grosche, Fabio Bontadina, Ulrich Zöphel, Hans-
Peter Seidl, Paul M. Cryan, and David S. Blehert
CONTENTS xv
A21 Pan-European Distribution of White-Nose Syndrome Fungus

(Geomyces destructans) Not Associated with Mass Mortality, 380
Sébastien J. Puechmaille, Gudrun Wibbelt, Vanessa Korn,
Hubert Fuller, Frédéric Forget, Kristin Mühldorfer, Andreas
Kurth, Wieslaw Bogdanowicz, Christophe Borel, Thijs Bosch,
Thomas Cherezy, Mikhail Drebet, Tamás Görföl, Anne-Jifke
Haarsma, Frank Herhaus, Guénael Hallart, Matthias Hammer,
Christian Jungmann, Yann Le Bris, Lauri Lutsar, Matti Masing,
Bart Mulkens, Karsten Passior, Martin Starrach, Andrzej
Wojtaszewski, Ulrich Zöphel, and Emma C. Teeling
B Agenda 403
C Acronyms 409
D Glossary 413
E Forum Member Biographies 427
F Speaker Biographies 455
Tables, Figures, and Boxes
TABLES
WO-1 Number of Individual Animals Traded by the United States (2000–
2006), 21
WO-2 Disease Types and Associated Symptoms Caused by P. ramorum, 56
A2-1 Examples of Fungal Associations with Plants, 127

A2-2 Examples of Fungal Associations with Insects, 130
A6-1 Growth Tolerances for Fungi from Soils, Animals, and Plants at 2
Temperatures, 184
A9-1 Characteristics of C. gattii Patients in the United States,

2004–2010, 212
A9-2 Comparison Between Outbreak-Strain (VGIIa/b/c) and Other
Genotypes of Infection with C. gattii, United States, 2004–2010, 213
A9-3 Sources and Species of Isolates of Cryptococcus Submitted Following
a Request Through ClinMicroNet, United States, October 2010–
February 2011, 216
A13-1 General Similarities and Differences Between Fungal Pathogens of

Plants and Animals, 266
A13-2 Fungicides Used to Control Plant and Animal Diseases, 269
A14-1 Selected Papers Illustrating the Effects of Climate and Global Change
Factors on Specific Pathogen–Host Systems, 280
xvii
xviii TABLES, FIGURES, AND BOXES
A20-1 Bats Tested for Geomyces destructans by Using Microscopy, Fungal

Culture, or PCR Analysis, by Country, Europe, 373
A20-2 Fungal Culture and PCR Results for 23 Bats with Evidence of Fungal
Colonization Tested by Light or Electron Microscopy, Europe, 374
A21-1 Confirmed Records of Geomyces destructans on Hibernating Bats in

Europe and Details of the Culture and Genetic Analyses, 384
A21-2 Suspected Photographic Records of Geomyces destructans on
Hibernating Bats in Europe, 385
A21-3 Suspected Visual Records of Geomyces destructans on Hibernating
Bats in Europe, 386
FIGURES
WO-1 The fungal kingdom, 5
WO-1-1 Leafcutter ants tending their fungal garden, 10
WO-2 Diversity of fungal morphology, 6
WO-3 Depiction of starving Irish children in 1847 potato famine, 13
WO-4 The epidemiological triad, 16
WO-5 Global aviation network, 20
WO-6 Selected dispersal events of fungal pathogens, 22
WO-7 Environmental disturbances and dust storms contribute to the dispersal
of fungal spores, 24
WO-8 Change in precipitation between the 1971–2000 average and the
2091–2100 average in inches of liquid water/year, 27
WO-9 Incidence of systemic fungal disease has increased since the 1950s, 30
WO-10 Damage response framework, 31
WO-11 Microbial flora as a host defense, 33
WO-12 Map of the Pacific Northwest, comprising parts of British Columbia,
Canada, and the states of Washington and Oregon in the United States,
showing human and veterinary Cryptococcus gattii cases, 36
WO-13 Environmental sampling for Cryptococcus gattii in British Columbia
(2001–2009), 40
WO-14 Signs of bat white-nose syndrome (WNS), 42
WO-15 Spread of bat white-nose syndrome (WNS) in North America as of
April 21, 2011, 44
WO-16 Species affected by bat white-nose syndrome (WNS), 45
WO-17 Global distribution of Bd, 48
WO-18 A chytridiomycosis outbreak in southern mountain yellow-legged
frogs, 50
WO-19 Sudden oak death and ramorum blight, 55
WO-20 P. ramorum “migration” pathways, 57
WO-21 Wheat production regions worldwide, 59
TABLES, FIGURES, AND BOXES xix
WO-22 Yellow “stripe” rust on wheat, 61

WO-23 Presence of “trace” and “severe” levels of yellow rust in North
America since 2000, 62
WO-24 Roles and responsibilities for monitoring pathogens in humans,
animals, plants, food, and the environment in the United States, 65
WO-25 Risk for sudden oak death in the continental United States, based on
agreement among five spatially referenced models, 71
WO-26 Mechanisms of action of selected antifungals, 78
WO-27 Frogs in the Sierra Nevada region, being treated in baths containing
a fungicidal bacterium in hopes of eliminating infection by the
fungal pathogen (Bd) associated with the deadly disease: amphibian
chytridiomycosis, 79
WO-28 Panamanian golden frog (Atelopus zeteki), 82
A1-1 Map of the forecasted ecologic niche and region of emergence of C.

gattii in British Columbia (BC), 103
A2-1 Diagrammatic representation of relationships of fungal taxa, examples

(ex.), and approximate number of species in each group, 120
A2-2 Images of representative fungal groups, 121
A2-3 Saccharomyces cerevisiae (Y-2235), baker’s yeast and model
organism, 123
A2-4 Anaptychia ciliaris, 128
A2-5 Ectomycorrhizal root, 129
A2-6 Excavation of deeply entrenched nest of the ant Atta texana requires
heavy equipment or, alternatively, ground-penetrating radar to map
such nests, 132
A2-7 Hirsutella citriformis (Ophiostomataceae) on a delphacid
planthopper, 133
A3-1 Fungal phyla and approximate number of species in each group, 142
A3-2 Lemonniera sp., 144
A3-3 The aero-aquatic ascomycete Helicoon gigantisporum produces
distinctive tightly coiled conidia, 144
A3-4 The smut Testicularia sp. develops in the ovary of grasses and (as
shown here) sedges, 144
A3-5 Perithecia of Pyxidiophora sp. (Laboulbeniomycetes) developed
in moist chamber on moose dung from Meredith Station, New
Brunswick, Canada, 144
A3-6 The ca. 8 cm wide basidiomata of Pycnoporus sp., a wide-ranging,
brightly colored, wood-decaying polypore, photographed at Barro
Colorado Island, Panama, 144
xx TABLES, FIGURES, AND BOXES
A3-7 Peniphorella baculorubrensis, a bark-decaying basidiomycete common

on and restricted to living live oak (Quercus virginiana), decays the
bark and changes its water-holding capacity, 144
A3-8 Basidiomata of Perenniporia phloiophila on the bark of living Quercus
virginiana, 144
A3-9 A basidioma (8 cm diameter) of the wood-decaying fungus, Favolus
tenuiculus, a favorite food of several species of mushroom-feeding
beetles, 144
A3-10 The small (>10 mm long) brightly colored beetle, Mycotretus sp.
(Erotylidae), was collected at Barro Colorado Island, Panama, 144
A3-11 Numbers of known fungi from the Dictionary of the Fungi (editions
1–10, 1950–2008), 146
A4-1 Occurrence of white-nose syndrome and/or Geomyces destructans in

the United States (by county) and Canada (by county or district) from
winter 2005/2006 through April 2011, 169
A4-2 Micrograph of Geomyces destructans showing distinctive
asymmetrically curved conidia either free or borne singly at the tips
and sides of branched conidiophores, 170
A4-3A Three little brown bats (Myotis lucifugus) photographed by Alan
Hicks (New York State Department of Environmental Conservation) in
Graphite Mine, New York in November, 2008, 172
A4-3B Periodic acid-Schiff (PAS) stained microscopic section of wing
membrane from a little brown bat with white-nose syndrome collected
in Pennsylvania in February, 2009, 172
A4-4 Colony expansion rates of Geomyces destructans when grown on
cornmeal agar at 3, 7, 14, and 20°C, 173
A5-1 Organism fitness as a function of body temperature, 180
A6-1 Frequency histogram of thermal growth tolerance for 4802 fungal

strains, 184
A7-1 Proportion of emerging infectious diseases caused by different

taxonomic groups of pathogens, 190
A8-1 Annual cases of coccidioidomycosis, 199
A9-1 Human infections with C. gattii, United States, December 2004–

January 2011, 210
A9-2 U.S. human cases of C. gattii, by year of illness onset, 211
TABLES, FIGURES, AND BOXES xxi
A10-1 The C. gattii outbreak expanded into, and emerged within, the United
States, 229
A10-2 Cryptococcus pathogenic species complex, 230
A10-3 Cryptococcus neoformans can reproduce unisexually and
bisexually, 235
A10-4 Sexual reproduction and the origin of an outbreak, 240
A11-1 Mechanisms of fungal sensing and control, 250
A12-1 Typical macroscopic symptoms of rust infections on adult wheat

plants, 254
A12-2 Map indicating the distribution of global wheat production and regions
of recent yellow rust epidemics, 255
A14-1 The increase in goods (109 tons × km) moved in the United Kingdom
from the 1930s to the 1990s, 275
A14-2 The world in 1897, with British possessions marked in red, 282
A16-1 Current distribution of Phytophthora ramorum in California and

Oregon forests, 316
A17-1 Summary of observations that show the Earth is warming (red arrows)
while the Sun has been constant over the same period of time, 326
A17-2 A comparison of the existing four global surface temperature datasets
that are used in climate analyses, 327
A17-3 Sea-level rise based on radar altimeters from TOPEX and Jason, with
seasonal variations removed, 329
A17-4 A comparison between the total solar irradiance and the NASA/GISS
surface temperature data, both from 1979 to 2010, 330
A17-5 Representation of a general circulation model, 331
A17-6 Change in precipitation between the 1971–2000 average and the
2091–2100 average in inches of liquid water/year, 331
A17-7 Rift Valley fever major outbreak events plotted against time and
the Southern Oscillation Index, a measure of the phase of El Niño/
Southern Oscillation events, 333
A17-8 Summary Rift Valley fever (RVF) risk maps for (A) Eastern Africa:
September 2006–May 2007; (B) Sudan: May 2007–December
2007; (C) Southern Africa: September 2007–May 2008; and (D)
Madagascar: September 2007–May 2008, 335
A17-9 Stem rust symptoms on wheat, 336
A17-10 False-color Landsat satellite data (RGB 642) showing glaciers as the
blue colors. The green colors represent green vegetation and the red
colors represent areas of rock, sand, and soil, 337
xxii TABLES, FIGURES, AND BOXES
A18-1 Decline of (A) Sierra Nevada mountain yellow-legged frog, Rana

sierrae, and (B) southern mountain yellow-legged frog, Rana muscosa,
in California, USA, 345
A18-2 Maps of the three study metapopulations showing the spread of Bd and
frog population status (adults only) during a 4-year period following
the initial detection of Bd, 347
A18-3 Frog Bd dynamics in eight intensively sampled populations in
Milestone and Sixty Lake basins before and after detection of Bd, 349
A19-1 Maps indicating (A) the global prevalence of Batrachochytrium

dendrobatidis. (B) Regional U.S. prevalence of Batrachochytrium
dendrobatidis, 361
A20-1 (A) Greater mouse-eared bat (Myotis myotis) with white fungal growth
around its muzzle, ears, and wing membranes. (B) Scanning electron
micrograph of a bat hair colonized by Geomyces destructans, 371
A20-2 Locations in Europe of bats positive for Geomyces destructans by
PCR alone (circles) or by PCR and culture (solid stars) and bats
negative for G. destructans but positive for other fungi (square), 375
A21-1 Distribution of confirmed and suspected records of G. destructans on

hibernating bats in Europe, 387
A21-2 Photographic evidence showing bats with confirmed or suspected
growth of G. destructans, 388
A21-3 Seasonal changes of the number of live bats reported with white
fungal growth in Europe, 390
A21-4 Indirect evidence of bats grooming off G. destructans during
hibernation, 391
A21-S1 Monitoring of bats at an hibernaculum in Germany during (A) the
winter 2006/2007 (September 5, 2006 until April 19, 2007) and (B)
the winter 2007/2008 (August 28, 2007 until April 23,
2008), 399
BOXES
WO-1 The Fungal Gardens of Leafcutter Ants, 10
WO-2 Factors in the Emergence of Infectious Diseases, 17
Workshop Overview
FUNGAL DISEASES: AN EMERGING THREAT TO

HUMAN, ANIMAL, AND PLANT HEALTH
Will the blight end the chestnut?
The farmers rather guess not.
It keeps smouldering at the roots
And sending up new shoots
Till another parasite
Shall come and end the blight.
—Robert Frost (1936)
Fungi are the only group of organisms that have been

convincingly shown to cause extinction.
—Arturo Casadevall (2010)
At the beginning of the 20th century, the American chestnut population

counted nearly 4 billion trees. The American chestnut tree, once dominant in the
forests of the Eastern United States, was decimated by an accidentally introduced
and previously unknown fungal pathogen. Within a span of 40 years, this once
abundant, iconic forest tree was all but annihilated by this microscopic fungus. In
the middle of the 20th century, an epidemic of Dutch elm disease—a vector-borne
fungal disease, also unknown to science at the time—ravaged the elm trees of
North America, Europe, and England (Brasier and Buck, 2001). Together, these
diseases rapidly and radically transformed the landscape of America’s cities and
forests (Money, 2007).
1
2 FUNGAL DISEASES
Fungal diseases of plants, animals, and humans have altered tree population
diversity and forest ecosystem dynamics, devastated agricultural crops, triggered
global population declines and extinctions in wildlife, and contributed to death
and disability in humans. Cryptococcus gattii (C. gattii), a pathogenic fungus
that emerged in 1999 on Vancouver Island, British Columbia, Canada, is caus-
ing a growing epidemic of human and animal infections and deaths (Galanis
and MacDougall, 2010). Since its initial recognition, the pathogen has spread
from Vancouver Island to mainland British Columbia and south into the Pacific
Northwest of the United States. This fungal pathogen has been associated with
338 confirmed human infections and 40 deaths1 in these regions, which represents
the largest documented population of C. gattii infected people in the world (Datta
et al., 2009a; Galanis and MacDougall, 2010). Bat white-nose syndrome (WNS)
and amphibian chytridiomycosis2 have caused massive population declines and
threaten local extinctions of New World bat and amphibian species, respectively
(Frick et al., 2010; Skerratt et al., 2007). By 2009, the geographic range of two
virulent and highly aggressive strains3 of yellow “stripe” rust—first detected in
North America in 2000—expanded to include major wheat-producing areas on
five continents, threatening the global wheat supply (Hovmøller et al., 2010).
The recent observation that a fungus (Nosema spp.), in combination with a DNA
virus, might be associated with “colony collapse” disorder—a disease that has
destroyed 20–40 percent of the honeybee colonies in the United States since
2006—underscores the direct and indirect impacts and ecosystem dynamics of
fungal diseases in human, plant, and animal communities (Bromenshenk et al.,
2010).
Fungal organisms interact with humans, animals, and plants in beneficial as
well as pathogenic ways. A dozen fungal diseases are considered “life threaten-
ing” to humans. At the same time, human health has benefited immensely from
fungal-derived antibiotics, such as penicillin (Blackwell et al., 2009; Buckley,
2008; Casadevall, 2007). Indeed, fungi are indispensible to life on this planet
through their ability to break down complex organic matter and recycle essential
nutrients back into the environment (Wainwright, 1992).
The fungal kingdom is among the most diverse kingdoms in the Tree of Life
(Blackwell, 2011). Yet, fewer than 10 percent of fungal organisms have been
formally described (Hawksworth, 1991, 2001). For the purposes of this chapter,
the terms fungi, fungal, and fungus are used inclusively to describe all organisms
traditionally studied by mycologists—including species that are now excluded
from Kingdom Fungi (e.g., Phytophthora spp. which are members of Oomycota)
or whose relationship to the fungal kingdom have yet to be determined (e.g., the
1â•›â•›As
of December 2010.
2â•›â•›In
this chapter, we will refer to this disease as amphibian chytridiomycosis and to the associated
pathogen (Batrachochytrium dendrobatidis) as Bd.
3â•›â•›Puccinia striiformis Westend. f.sp. tritici Eriksson.
WORKSHOP OVERVIEW 3
microsporidia Nosema spp. and the newly discovered cryptomycota) (see Jones
et al., 2011; Stajich et al., 2009).
Despite the extensive influence of fungi on economic well-being, as well as
on human, animal, plant, and ecosystem health, the threats posed by emerging
fungal pathogens are often unappreciated and poorly understood. On December
14 and 15, 2010, the Institute of Medicine’s (IOM’s) Forum on Microbial Threats
hosted a public workshop on this topic in order to explore the scientific and policy
dimensions associated with the causes and consequences of emerging fungal
diseases. Through invited presentations and discussions, the workshop explored
the environmental, host (plant, animal, and human), and pathogen-related factors
influencing the emergence, establishment, and spread of fungal pathogens, as well
as the impacts of these diseases on human and animal health, agriculture, and
biodiversity. Workshop participants also considered and discussed opportunities
to improve surveillance, detection, and response strategies for identifying and
mitigating the impacts of these diseases in order to better prepare for future out-
breaks. Convened in response to the perceived threat posed by emerging fungal
diseases to human, animal, and plant health, this was the first workshop in the
Forum’s 15-year history that focused exclusively on fungal pathogens.
Organization of the Workshop Summary

This workshop summary was prepared by the rapporteurs for the Forum’s
members and includes a collection of individually authored papers and commen-
tary. Sections of the workshop summary not specifically attributed to an individ-
ual reflect the views of the rapporteurs and not those of the Forum on Microbial
Threats, its sponsors, or the IOM. The contents of the unattributed sections are
based on presentations and discussions at the workshop.
The summary is organized into sections as a topic-by-topic description of
the presentations and discussions that took place at the workshop. Its purpose is
to present lessons from relevant experience, to delineate a range of pivotal is-
sues and their respective challenges, and to offer potential responses as discussed
and described by the workshop participants. Manuscripts and reprinted articles
submitted by some, but not all, of the workshop’s participants may be found, in
alphabetical order, in Appendix A.
Although this workshop summary provides a description of the individual
presentations, it also reflects an important aspect of the Forum’s philosophy. The
workshop functions as a dialogue among representatives from different sectors
and allows them to present their beliefs about which areas merit further atten-
tion. This report only summarizes the statements of workshop participants. This
workshop summary report is not intended to be an exhaustive exploration of the
subject matter nor does it represent the findings, conclusions, or recommenda-
tions of a consensus committee process.
4 FUNGAL DISEASES
THE HIDDEN KINGDOM

Fungi are among the most evolutionarily and ecologically diverse organisms
on the planet, comprising a kingdom of organisms that provide valuable ecosys-
tem services through their decomposition of organic matter, symbiotic associa-
tions with numerous plant and animal species, and as food sources (Blackwell,
2011; Taylor et al., 2004). Initially thought by early taxonomists to be members of
the plant kingdom, fungi are actually more closely related to animals than plants
(Figure WO-1) (McLaughlin et al., 2009).
According to keynote speaker Arturo Casadevall, of the Albert Einstein
College of Medicine, fungal organisms—in terms of sheer numbers of spe-
cies—constitute the most successful kingdom in the tree of life. (Dr. Casadevall’s
contribution to the workshop summary report can be found in Appendix A, pages
177–188.) Yet fewer than 10 percent of the estimated 1.5 million species of fungi
have been formally identified and described4 (Blackwell, 2011; Hawksworth,
2001). Forum Chair David Relman, of Stanford University, observed that, “We
are blind to a lot of their biology and what it is that they spend most of their time
doing and why and for whom. I think many in this room would agree that fungi
are ignored and underappreciated.” This “blindspot,” he continued, “leaves us
with fairly poor situational awareness: a relatively poor understanding of fungal
biogeography—meaning their spatial distribution patterns—the factors that de-
termine their distribution in space and time, and the factors that underlie their
evolution, especially within short time-frames.”
Fungal Diversity
Existing as single-celled organisms, such as yeasts, or complex communities
of filamentous mycelial networks covering hundreds of acres, fungi are ubiqui-
tous in nature and display a dazzling array of sizes, shapes, and colors, including
many that are bioluminescent (Figure WO-2) (Blackwell, 2011; Desjardin et al.,
2010; Lutzoni et al., 2004).
The fungal life cycle is equally varied. Fungi can reproduce asexually or
sexually through life cycles that range from simple to complex—including “di-
morphic” switching between yeast and filamentous forms and the use of multiple
host species (Blackwell et al., 2009). Spores5 are produced during the fungal life
cycle and may be passively or actively dispersed through a variety of environ-
mental media including air, water, wind, animals, and materials (Blackwell et al.,
2009). Fungal growth, reproduction, spore production, and dispersal are also ex-
quisitely sensitive to environmental conditions including temperature, humidity,
4â•›â•›This number is considered by many to be an underestimate of the actual number of fungal species;
see contributed manuscripts by Blackwell in Appendix A (pages 116–167).

5â•›â•›Spores are well-protected structures that can survive in adverse environmental conditions, such as
freezing or drying (better than mycelia and yeast cells), for months and even years.
WORKSHOP OVERVIEW 5
Animals (outgroup)
(1 293 642 species)
? Microsporidia
(1300 species)
Chytridiomycota
“ Chytrids”
(706 species)
Neocallimastigomycota
(20 species)
Blastocladiomycota
(98 000 species)

(179 species)
Fungi
Zygomycota 1
“ Zygomycetes”
? (327 species)
Zygomycota 2
(744 species)
Entomophthorales
? (277 species)
Glomeromycota
(169 species)
Ascomycota
(64 163 species)
Basidiomycota
(31 515 species)
FIGURE WO-1â•‡ The fungal kingdom. The classification of species within kingdom Fungi
continues to evolve. The diagram above provides an overview of some of the primary
lineages of fungal organisms andFigure WO-1.eps
the estimated number of species for each lineage.
SOURCE: Blackwell (2010).
winds, and water (Bahn et al., 2007; Judelson and Blanco, 2005; Kauserud et al.,
2008; Kumamoto, 2008).
Fungi are highly adaptable to new environmental niches including what
might be considered “extreme” environments (Gostinčar et al., 2010; Le Calvez
et al., 2009). Some have suggested the ability of fungi to access multiple strate-
gies for reproduction contributes to why fungi are so “adept at adaptation.” Under
different environmental conditions, fungal reproduction can maintain character-
istics adapted to a particular environmental niche or generate genetically diverse
offspring that can quickly respond to changing host or environmental factors
(Heitman, 2006). (Dr. Blackwell’s contribution to the workshop summary report
can be found in Appendix A, pages 116–167.) Keynote speaker Meredith Black-
well, of Louisiana State University, noted that scientists continue to find new
species of fungi in a wide range of environments—from tropical and temperate
forests to the guts of insects (e.g., Arnold et al., 2003; Gostinčar et al., 2010;
Miller et al., 2001; Suh and Blackwell, 2006). These discoveries often reveal the
unique capabilities of these microorganisms. As observed by Casadevall, some
fungal species can survive and thrive in high radiation and other extreme envi-
ronments. Zhdanova et al. (2000) reported extensive fungal growth on the walls
6 FUNGAL DISEASES
A B C
D E F
G H
I
.
WO-2 new
WORKSHOP OVERVIEW 7
FIGURE WO-2â•‡ Diversity of fungal morphology. (A) Two flagellated fungal cells from
the recently discovered group of fungi known as cryptomycota. This ancient group of
organisms is thought to be distinct from other fungi because of the absence of a cell wall
made of chitin; (B) asexual, spore-producing culture of Cryphonectria parasitica (chestnut
blight fungus); (C–F) multicellular, spore-producing structures (fruiting bodies) are pro-
duced during the sexual phase of the fungal life cycle. Many fruiting bodies are familiar
as mushrooms—including species that are consumed by humans as food: (C) Morchella
conica (morel) and (D) Crucibulum laeve (bird’s nest fungus). Mushrooms of some species
are known to be toxic or poisonous to humans: (E) Amanita muscaria. Fungal fruiting bod-
ies can exhibit a wide range of shapes and sizes, including (F) the bioluminescent “shelf”
fungus, Panellus stipticus; (G) Micrograph of Phytophthora ramorum chlamydospores;
(H) SEM photomicrograph prepared from G. destructans culture isolated from bat tissue
samples collected from Williams Hotel Mine; note curved conidia borne in whorls on
septate hyphae; bar is 2 µm. All images are pseudo-colored in Adobe Photoshop 9.0; (I)
“fairy rings” in which mushrooms sprout along the outer edge of a sprawling, underground
mycelial network. These networks (mycelia) have been known to cover several hundred
acres. One of largest known mycelia has been estimated to encircle 900 hectares (3.4
square miles).
SOURCE: (A) Micrograph kindly provided by Meredith Jones, Exeter University; (B)
photo by Kent Loeffler, provided by Alice C.L. Churchill, Cornell University; (C–F) Wi-
kimedia Commons; (G) photo provided courtesy of Paul Reeser, Oregon State University;
(H) Chaturvedi et al. (2010); (I) Wikimedia Commons.
and other areas of the shelter installed around the damaged unit of the Chernobyl
nuclear power plant, including 37 species among 19 genera 6; fungi are also
known to inhabit high-radiation space environments and have even colonized the
International Space Station (Dadachova and Casadevall, 2008).
The fungal pathogen responsible for sudden oak death and ramorum blight,
Phytophthora ramorum, was only identified as a new species in 2000. Since
then, according to speaker David Rizzo of the University of California at Davis,
researchers have identified an additional 50 Phytophthora7 species. (Dr. Rizzo’s
312–324.) As Rizzo observed, these new discoveries do not reflect recent fungal
evolution, but are a reflection of the fact that “we just haven’t really been looking
6â•›â•›Many of the species inhabiting the most heavily contaminated sites of the Chernobyl nuclear
power plant were rich in melanin (a high molecular weight pigment). Dadachova et al. (2007) reported
that radiation enhances the growth of melanized Wangiella dermatitidis, Cryptococcus neoformans,
and Cladosporium sphaerospermum cells.
7â•›â•›Phytophthora (“plant destroyer”) is a genus of approximately 100 species that includes several
notorious plant pathogens, including Phytophthora infestans, which caused the Irish Potato Famine.
Phytophthora species are oomycetes, which are fungus-like organisms in the kingdom Stramenopila.
8 FUNGAL DISEASES
for them.” Several other forest fungi that have caused major damage in the past,
including the fungi responsible for chestnut blight and Dutch elm disease, were
unknown to science until they started causing noticeable damage and die-off of
forest and urban trees (Brasier and Webber, 2010).
Ecosystem Services8 and Interactions

The ability of fungi to process complex organic matter into essential nutrients
(e.g., nitrogen, phosphorus) makes them indispensible members of virtually all
ecosystems and “invisible” shapers of the world around us (Wainwright, 1992).
The vast majority of described fungal species are saprophytic,9 surviving on
dead plant matter and animal tissue (Blackwell et al., 2000). Fungi can be “free
living”10 or form mutualistic, commensalistic, or parasitic relationships with
plants, animals, and microbes—deriving benefits from and contributing to their
living hosts (Blackwell et al., 2009).
Humans have used fungi as a direct source of food (e.g., truffles, mush-
rooms), as a leavening agent for bread, and in the fermentation of various food
products, including, but not limited to, beer, wine, and soy products (Buckley,
2008). Some fungi contain psychotropic compounds that may be consumed
recreationally or in traditional spiritual ceremonies, and they have been used
for millennia for medicinal purposes (Capasso, 1998). The fruiting structures of
a few species are highly valued in China for their purported medicinal benefits
including as a “libido booster”11 (Roach, 2011). Blackwell stated that since the
early 1940s, fungi have been exploited for their life-saving antibiotics.12 More
recently, various enzymes and pigments produced by fungi have been used indus-
trially and in the manufacture of a wide variety of products, including furniture,
musical instruments, and clothing (Blanchette et al., 1992; Buckley, 2008; Keller
et al., 2005). These organisms have been used extensively as biological pesticides
to control weeds, plant diseases, and insect pests (Buckley, 2008). Blackwell ob-
served that biomedical researchers have used certain species of fungi extensively
as model organisms for genetic and other scientific research for decades.
Many fungi maintain close associations with their insect hosts. Blackwell
discussed the symbiotic fungi that inhabit insect guts and are essential to the
8â•›â•›Services
provided by ecosystems that benefit humans and are necessary for a healthy planet like
oxygen production, water purification, pollination, soil formation, and nutrient recycling. See www.
conservation.org/resources/glossary/Pages/e.aspx (accessed on June 13, 2011).
9â•›â•›Deriving nutrients from dead organic matter.
10â•›â•›Not dependent on a host for survival.
11â•›â•›For example: A parasitic fungus, Ophiocordyceps sinensis, grows in the Tibetan Plateau in China
and is highly valued for its “purported medicinal benefits,” including uses as “a treatment for cancer
and aging and as a libido booster.” The nutty-tasting fungus is considered “fungal gold” because it
can be sold for high prices in Chinese markets (see Roach, 2011).
12â•›â•›Other medicines such as the immunosuppressant cyclosporine A and statin drugs also are derived
from fungi.
WORKSHOP OVERVIEW 9
nutrition of many insects (e.g., Nardi et al., 2006; Suh et al., 2003, 2005). Fungi
also are cultivated by fungus-farming termites and ants (Aanen et al., 2002;
Currie et al., 2003; Dentinger et al., 2009; Munkacsi et al., 2004) (Box WO-1).
Not all fungal–insect associations are mutualistic. Blackwell described the
parasitic but not usually pathogenic fungi in the order Laboulbeniales. She noted
the reports of extreme host specificity exhibited by different species in this
order—sometimes inhabiting only certain parts of the host insect (Weir and
Beakes, 1995). Most laboulbenialean species are associated with beetles (Cole-
optera), and flies (Diptera), but they are also associated with a diverse array of
host species in other insect orders, mites and millipedes (Weir and Beakes, 1995).
Blackwell discussed a number of fungal–plant symbioses. She estimated
that:
• Half of all ascomycetes (Phylum Ascomycota) are lichens [symbiotic

associations between fungi and photosynthetic partners (algae)] (Lutzoni
et al., 2001; Schoch et al., 2009);
• 90 percent of all photosynthetic plants have mycorrhizal associates
(Ruehle and Marx, 1979); and
• 95 percent of all plants have fungal endophytes (Arnold, 2007; Rodriguez
et al., 2009).
Endophytes—fungi that live inside the plant tissue but without causing
any obvious negative effects—are less well known than other plant–fungal as-
sociations, but mycologists find them wherever they look (Arnold et al., 2003;
Rodriguez et al., 2009). Numerous endophytic fungal infections have been ob-
served in cocoa trees (Theobroma cacao) and they may play an important role
in host defense by decreasing the damage associated with Phytophthora spp.
infections (Arnold et al., 2003). To illustrate the complexity of these relationships,
Blackwell noted interactions among the fungus Curvularia protuberata, the grass
Dichanthelium lanuginosum,13 and a fungal virus. The grass infected with the
fungus infected with “Curvularia thermal tolerance virus” provides thermal re-
sistance benefits for the host plant. This tripartite relationship allows the grass to
grow in the high-temperature soils of Yellowstone National Park (Márquez et al.,
2007). Blackwell pointed to the red-cockaded woodpecker (Picoides borealis)
as just one example of the many ways that fungi confer benefits to the health of
ecosystems. These woodpeckers usually nest in trees infected with red heart rot
(Phellinus pini) (Hooper et al., 1991).
13â•›â•›Commonly referred to as Panic Grass.

10 FUNGAL DISEASES
BOX WO-1
The Fungal Gardens of Leafcutter Ants
Over the past 50 million years, a unique symbiosis has developed between
attine (fungal growing) “leafcutter” ants and fungi in the Lepiotacea family. In what
biologists consider the earliest form of agriculture, leafcutter ant colonies grow
and meticulously maintain a specific fungal cultivar for food (Schultz and Brady,
2008; Wade, 1999).
Inhabiting forest ecosystems throughout Mexico and Central and South Amer-
ica, these ant colonies can number more than 8 million individuals. Foraging ants
bring cut pieces of leaves back to the colony where they are broken down and fed
to the fungus by worker ants (see Figure WO-1-1).
A second symbiotic relationship protects these fungal gardens. Pseudono-
cardia bacteria, which grow on the bodies of the worker ants, produce antibiotic
compounds that prevent the growth of parasitic molds (Currie et al., 1999).
FIGURE WO-1-1â•‡ Leafcutter ants tending their fungal garden.

Figure WO- figure for Box WO-1.eps
SOURCE: © Alex Wild.
For more information on leafcutterbitmap

ants, visit the PBS video
segment: “Ancient Farmers of the Amazon,” © WGBH Educa-
tional Foundation and Clear Blue Sky Productions, Inc., 2001,
available at: http://www.youtube.com/watch_popup?v=RH3KY
BMpxOU&vq=medium#t=11.
Or, use your smart phone to link directly to the video using the
QR code at right:
Figure WO-QRtode.eps
bitmap
WORKSHOP OVERVIEW 11
Fungi as Pathogens
The longstanding utility of fungi to all life on earth has often been matched
by their ability to directly or indirectly cause devastating disease in human,
animal, and plant hosts. Fungi are the predominant pathogen species in plants,
remarked Casadevall, and fungi can also cause disease in healthy humans and
animals. Described by several workshop participants as “formidable pathogens,”
many fungi can also endure adverse environmental conditions and thrive outside
of their host (Casadevall, 2007).
Fungal pathogens in general execute a series of sequential steps in order to
cause disease, remarked speaker Barbara Howlett of the University of Melbourne.
(Dr. Howlett’s contribution to the workshop summary report can be found in Ap-
pendix A, pages 264–273.) These pathogens must:
• Recognize and attach to the host;

• Germinate, colonize, and derive nutrition from the host;
• Subvert host defense responses;
• Reproduce, exit, and disperse; and
• Find another host14 (Sexton and Howlett, 2006).
Very few fungal pathogens are able to cause disease in hosts from the plant
and animal kingdoms; those that do are referred to as trans-kingdom pathogens
(De Lucca, 2007).15 Fungi can also form different associations with different host
types. For example, the fungus Cryptococcus gattii is pathogenic in animals in-
cluding humans, but forms non-pathogenic associations with plants –which play
an essential role in the maintenance of C. gattii spores in certain environmental
niches (Bartlett et al., 2007; Xue et al., 2007).Once outside of a host, fungal
pathogens of animals and plants often have different requirements for survival.
Animal pathogens, noted Howlett, are often soil saprophytes that are free-living
rather than obligate.16 In contrast, some plant pathogens can only survive on the
tissue of a specific plant host(s).
14â•›â•›For more information, see contributed manuscript by Barbara Howlett in Appendix A (pages
264–273).
15â•›â•›Howlett noted two trans-kingdom pathogens during her remarks: Fusarium oxysporum f. sp.
lycopersici, which causes vascular wilt in plants and is an emerging human pathogen (Ortoneda et al.,
2004); and Aspergillus flavus, which infects corn and is an emerging pathogen in immunocompro-
mised humans (Krishnan et al., 2009).
16â•›â•›Capable of existing only in a particular environment; an obligate parasite cannot survive inde-
pendently of its host (Science dictionary).

12 FUNGAL DISEASES
Fungal Pathogens of Plants

In addition to contributing heavily to annual losses in global crop producÂ�
tion,17 fungal plant pathogens are associated with many notable episodes of hu-
man suffering and economic and ecological loss, including:
• Irish Potato Famine: The mid-19th-century epidemic of potato late

blight in Ireland led to the Irish Potato Famine, which caused or con-
tributed to the starvation and death of well over 1 million people and the
emigration of another 1 million (Money, 2007; Vurro et al., 2010). At
the time of the Potato Famine, one-third of Ireland’s population of eight
million was dependent upon the potato as a primary food source (Money,
2007) (Figure WO-3). See also Large (1965) and Woodham-Smith (1962).
• Southern corn leaf blight: The 1970 southern corn leaf blight epidemic
led to the loss of 710 million bushels of corn—valued at more than $1
billion at the time, or about $5.6 billion in 2009 dollars (Tatum, 1971).
• Dutch elm disease: The impact of Dutch elm disease extends well be-
yond the death of 100 million mature elm trees in the midle of the 20th
century. It not only transformed the landscape of cities and forests, but it
has continued to alter associated ecosystem dynamics to this day through
reduced food sources and nesting sites for wildlife, altered tree composi-
tion, and density (Loo, 2009; Money, 2007).
Fungal plant diseases have far-reaching health impacts that extend beyond
the infected plant species—including, but not limited to, negative impacts on
associated flora and fauna (Giraud et al., 2010; Loo, 2009). As the Irish Potato
Famine illustrated, crop losses can have devastating impacts on populations that
are heavily, or solely, dependent on a single food source for their caloric needs.
Speaker Jim Stack of Kansas State University observed that with 59 percent of
calories consumed by humans being derived from just four plant species (rice,
wheat, maize, and potatoes), fungal diseases in these staple crops may cata-
strophically threaten local and global food security (Strange and Scott, 2005;
Vurro et al., 2010). (Dr. Stack’s contribution to the workshop summary report
can be found in Appendix A, pages 273–296.)
Fungal Pathogens of Humans and Animals

Given the ubiquity and diversity of fungi, it is perhaps surprising that, of the
nearly 1,400 recognized human pathogens, a little more than 20 percent (~ 325)
are fungal, and fewer than a dozen are associated with “life-threatening” disease
(Casadevall, 2007; Woolhouse and Gaunt, 2007). Historically, fungal diseases
17â•›â•›Crop losses due to all pathogens (1988–1990) totaled $33 billion for rice, $14 billion for wheat,
$7.8 billion for maize, and $9.8 billion for potatoes (Oerke et al., 1995; Rosenzweig et al., 2001).
FIGURE WO-3â•‡ Depiction of starving Irish children in 1847 potato famine; by Cork artist
James Mahony (1810–1879). Figure WO-3.eps
SOURCE: Wikimedia Commons.
bitmap
14 FUNGAL DISEASES
of humans have had a lower disease burden than bacterial, viral, or parasitic
infections, although this disease burden may be changing. It has been noted that
fungal diseases are increasing in incidence in the growing populations of immu-
nocompromised human hosts (Romani, 2004). Once established, fungal diseases
are often difficult to treat (Casadevall, 2007; Romani, 2004).
Disease in humans most often results from opportunistic18 infections (Shoham
and Levitz, 2005). Only a few fungal diseases (e.g., coccidioidomycosis, histo-
plasmosis) are caused by “primary” fungal pathogens19 that induce symptomatic
disease in otherwise healthy people (Casadevall, 2007; Cutler et al., 2007). The
“apparent” resistance of humans to fungal disease may be a reflection of the host
immune response, coupled with the high basal temperature of mammals, which
often exceeds the thermotolerance20 range for many fungi (Casadevall, 2005;
Garcia-Solache and Casadevall, 2010; Robert and Casadevall, 2009).
Primary fungal pathogens of humans can also infect other mammals, such as
domesticated livestock and companion animals. These diseases are generally not
considered contagious and are acquired via inhalation of aerosolized infectious
propagules21 from environmental reservoirs, such as soil or trees (Casadevall and
Pirofski, 2007). According to speaker Luis Padilla of the Smithsonian Conserva-
tion Biology Institute, wildlife are also affected by opportunistic and primary
fungal pathogens, but the epidemiology of these diseases in wildlife is not well
understood. (Dr. Padilla’s contribution to the workshop summary report can be
found in Appendix A, pages 296–312.) Two fungal diseases of wildlife, am-
phibian chytridiomycosis and bat white-nose syndrome, emerged rapidly and
unexpectedly over the past several decades. These diseases are associated with
unprecedented local and global population declines of amphibian and bat species,
and pose serious threats to biodiversity and ecosystem stability (Frick et al., 2010;
Wake and Vredenburg, 2008).
Fungal Pathogens as “Invasive Species”

“Fungi are the only group of organisms that have been convincingly shown
to cause extinction,” Casadevall remarked, referring to the extinction of the land
snail Partula turgida by a parasitic microsporidian fungus (Cunningham and
Daszak, 1998). As Casadevall observed, this capacity for destruction may be
18â•›â•›Resulting from pathogen entry via wounds or weakened state of the host, or as a disturbance of
a normally benign host–fungus relationship.

19â•›â•›Medically important fungi can be categorized as opportunists or primary pathogens. The op-
portunists rarely cause disease in an immunocompetent host whereas the primary pathogens do. For
more information see: Cutler et al. (2007).
20â•›â•›Garcia-Solache and Casadevall (2010) define thermotolerance as the ability to grow at mam-
malian (37°C) and higher temperatures. Most fungi thrive in the range of 12°C to 30°C, but there are
wide temperature tolerances among species, with some growing at temperatures as low as –10°C or
as high as 65°C. See contributed manuscript by Casadevall in Appendix A (pages 181–188).
21â•›â•›Spores or encapsulated yeast cells.
due, in part, to the fact that “when [fungal pathogens] get into an ecosystem—a
vertebrate host, for example—they simply don’t care. They have no need for that
host in order to go forward. They will take down every last member of the spe-
cies.” In contrast, most newly introduced viral and bacterial pathogens in a naïve
host eventually attenuate their virulence such that infection does not kill the host.
Such adaptations are beneficial to both the host and pathogen in that the host sur-
vives and the pathogen avoids an “evolutionary dead end” (IOM, 2009). As noted
by Casadevall and Pirofski (2007), the host independence of “environmental”
microbes,22 including many fungi, may confer advantages that promote survival
and virulence in other niches, including new ecosystems and novel host species.
The term “invasive species” is used to describe “non-native”23 plants and
animals that, when introduced to new environments, reproduce or spread so ag-
gressively that they harm their adopted ecosystems (Carlton, 2004; Dybas, 2004).
They compete with native organisms for food and habitat, act as predators or
parasites of native species, and cause or carry diseases, often with devastating
ecological and economic consequences (Pimentel et al., 2005). As observed by
Morse (2004), infectious diseases represent another form of biological invasion—
often arising “out of nowhere” with devastating effects.
Discussions during the workshop illuminated the capacity of many fungal
pathogens to persist in environmental reservoirs and to readily adapt to new envi-
ronmental niches and host species. Like invasive species, these fungal pathogens
have been able to thrive in new environments and are changing the ecosystem in
ways that are difficult to anticipate and even more daunting to prevent (Desprez-
Loustau et al., 2007; Giraud et al., 2010; Rizzo, 2005). Given both the links and
similarities between invasive species and many pathogenic fungi, it may be useful
to view the origins of disease emergence, and the strategies deployed to prevent
or mitigate the threats associated with fungal pathogens, through the larger lens
of biological invasiveness.
FACTORS OF EMERGENCE
Diseases are categorized as “emerging” if their incidence24 or virulence25 has
recently increased or if they begin to infect a novel host or population (WHO,
2010). As illustrated in Figure WO-4, disease26 results from a complex interplay
of interactions among the pathogen, host, and environment.
22â•›â•›Microbes acquired from the environment (in contrast to acquisition from other living hosts)
(Casadevall and Pirofski, 2007).
23â•›â•›Also called “exotic,” “alien,” and “nonindigenous” species.
24â•›â•›As used in epidemiology, the number of new cases of a disease that occur in a defined population
within a specified time period; the rate of occurrence (IOM, 1992).

25â•›â•›The degree of pathogenicity of an organism as evidenced by the severity of resulting disease and
the organism’s ability to invade the host tissues (IOM, 1992).

26â•›â•›A situation in which infection has elicited signs and symptoms in the infected individual; the
infection has become clinically apparent (IOM, 1992). Some exposures to infectious disease-causing
agents can also produce asymptomatic illnesses that can be spread to others.
16 FUNGAL DISEASES
FIGURE WO-4â•‡ The epidemiological triad. The familiar “epidemiological triad” concept
(host–pathogen–environment), as illustrated in the famous diagram of Snieszko (1974),
FigureofWO-4.eps
neatly illustrates the complex interplay factors that result in disease at the individual
and population levels. The presence of abitmap
pathogen is a necessary, but not sufficient, cause
of a particular disease (IOM, 2008b).
SOURCE: Snieszko (1974, Figure 1).
The range of factors identified as influencing the interactions between these

elements (see Box WO-2) underscores the fact that exposure to a potential
pathogenic agent is a necessary but insufficient condition for infectious disease
emergence (IOM, 2003).
Significant factors for infectious disease emergence include the introduction
of a pathogen into a new ecosystem or the disruption of an established ecosys-
tem (IOM, 1992, 2003, 2010; Woolhouse and Gaunt, 2007). Such changes often
expose immunologically naïve hosts to potential disease-causing organisms that
have been released from the constraints imposed on them in their native environ-
ments (Woolhouse and Gaunt, 2007). Important catalysts for such disruptions
and subsequent disease emergence include human activity, weather, and climate
(Anderson et al., 2004; Daszak et al., 2000; Harvell et al., 2002; IOM, 2008a,
2010).
Anthropogenic and environmental factors play integral roles in the introduc-
tion and spread of many emerging fungal diseases. In recent years, the emergence
of new plant diseases has been attributed to the evolution of hybrid patho-
gen species (Brasier, 2000). These hybrid species are thought to result from
trade-mediated geographic redistribution of plants infected with the parental
BOX WO-2
Factors in the Emergence of Infectious Diseases
Thirteen factors of emergence of infectious diseases were elucidated in a 2003

Institute of Medicine report, Microbial Threats to Health: Emergence, Detection,
and Response:
• Microbial adaptation and change
• Human susceptibility to infection
• Climate and weather
• Changing ecosystems
• Human demographics and behavior
• Economic development and land use
• International travel and commerce
• Technology and industry
• Breakdown of public health measures
• Poverty and social inequality
• War and famine
• Lack of political will
• Intent to harm
SOURCE: IOM (2003).
pathogens27 (Brasier, 2000). As Rizzo noted, “for some of these tree pathogens,
I don’t think there is anything extraordinary about the pathogens themselves.”
Rather, “it is the movement of pathogens from one environment to another that
seems to be driving much of the destruction.”
As described below, discussion at the workshop considered the influence of
human activity and behavior, winds and weather, host susceptibility, and patho-
gen adaptation and change on fungal disease emergence.
Human Activity and Behavior

During the past century, human activities have dramatically influenced local
environments and ecosystems, breaking down natural habitats and exposing new
27â•›â•›Stack
explained that interspecific hybridization is another unexpected outcome of pathogen glo-
balization. Two different (and previously isolated) fungal species mate and produce novel “hybrid”
offspring. Fungal pathogens of plants produced by interspecific hybridization are more aggressive
than either parental phenotype and may occupy a new host range (see Brasier, 2000). Stack noted
that even in a single nursery, the normal process of taking care of plants, which includes watering,
can result in a water splash that brings two different fungal species together in a single pot, where
interspecific hybridization can occur.
18 FUNGAL DISEASES
hosts to infectious disease agents (Anderson et al., 2004; Brasier, 2008; Daszak
et al., 2000). Travel, trade, migration, agricultural practices, and land use patterns
have all contributed to increased opportunities for contact between introduced
pathogens and naïve and susceptible host populations (IOM, 2010, and refer-
ences within).
Speaker Matthew Fisher of the Imperial College London, remarked that
migrating humans have been globalizing pathogens for thousands of years. (Dr.
Fisher’s contribution to the workshop summary report can be found in Appendix
A, pages 355–367.) He pointed to the spread of Coccidioides posadasii that ac-
companied human migration between 5,000 and 10,000 years ago through North,
Central, and South America as an example (Fisher et al., 2001). Stack agreed,
noting that “global trade is not new; we have had 3,000 years of global trade.” He
went on to state that, “what is new is the magnitude of trade in plants and plant
products and the speed at which they move around the world” [emphasis added].
Trade, Travel, and Tourism

The increase in international transportation, travel, and trade associated with
globalization in the 20th century has amplified the frequency of interactions
between people, plants, animals, and microbes—providing novel opportunities
for the rapid introduction, emergence, and spread of infectious diseases (IOM,
2010). The explosive growth of globalization—with dramatic increases in both
the quantity and diversity of goods—has been enabled by a simultaneous decrease
in travel time (IOM, 2010). Goods can be transported between most places in the
world in less time than the incubation period for most infectious diseases (Cliff
and Haggett, 2004; IOM, 2010). A study of factors associated with the emergence
of diseases in crop plants demonstrated that the majority were spread via trade
and travel (Anderson et al., 2004). Stack said this should not be surprising: In
2007 alone, the United States imported more than 48 million tons of agricultural
products, only 1–2 percent of which were inspected for possible pathogens and
other pests (Becker, 2009; Stack, 2010).28
Local and global transportation of ornamental plants, trees, and timber also
contribute to the introduction and spread of fungal diseases. The pathogens re-
sponsible for Dutch elm disease and chestnut blight were transported to America
in shipments of beetle-infested timber imported from Asia and live chestnut trees
imported from Japan, respectively (Money, 2007). Molecular epidemiological
analyses of many P. ramorum isolates support the hypothesis that nursery plants
infected with Phytophthora ramorum were the initial “source” for the epidemic
of sudden oak death that began in California in 1995 (Mascheretti et al., 2008). P.
ramorum has since emerged in the United Kingdom and Europe and now infects
28â•›â•›According to Stack (2010), each year, 12,000–14,000 potential pathogen and pest problems are
intercepted during these inspections.

more than 100 plant species (Grünwald et al., 2008). Stack noted that potential
pathogens can be transported in plants, plant associated material (e.g., soil),
seeds, and objects manufactured using plant products, such as wooden instru-
ments and packing materials.
Human spatial mobility has increased at least 1,000-fold in the past 200
years, with more people traveling faster, farther, and less expensively than ever
(Figure WO-5) (Cliff and Haggett, 2004; Hufnagel et al., 2004).
Travelers are now able to easily explore once-remote areas that serve as
both sources and sinks for emerging infectious diseases (Choffnes, 2008; IOM,
2010). Adventure travelers intrude on once-remote environments and often make
contact with exotic wildlife, encountering microbes that have never before been
recognized as human pathogens in the “developed” world (IOM, 2010). These
ecotourists become unwitting vectors of disease when they bring these exotic in-
fectious diseases back with them—on their person/clothing/luggage, etc.—when
they return to their home countries. If the conditions are favorable, an introduced
pathogen may persist and spread (Wilson, 2003). White-nose syndrome, which
is currently decimating New World bat populations in the United States, may
have been accidentally introduced by recreational cavers from Europe29 (Wibbelt
et al., 2010).
Infectious disease pandemics have also been associated with the legal and
illegal trade in and transportation of animals (IOM, 2010; Karesh et al., 2005;
Smith et al., 2009). Between 2000 and 2006, the United States traded approxi-
mately 1.5 billion animals, according to speaker and Forum member Peter Daszak
of EcoHealth Alliance. (Dr. Daszak’s contribution to the workshop summary
report can be found in Appendix A, pages 188-196.) These animals come from
a wide range of species, and most animal imports into the United States come
from emerging infectious disease “hot spots” (see Table WO-1) (Jones et al.,
2008; Smith et al., 2009).
The international amphibian trade is thought to have contributed to the emer-
gence and global spread of amphibian chytridiomycosis (Catenazzi et al., 2010;
Daszak et al., 2003; Fisher and Garner, 2007; Schloegel et al., 2010; Weldon
et al., 2004). Since the 1990s, this fungal disease has been implicated in the wide-
spread population declines—including some local extinction events—of more
than 200 species of frogs, toads, and salamanders (Fisher et al., 2009; Kilpatrick
et al., 2009; Lips et al., 2006; Schloegel et al., 2006; Skerratt et al., 2007).
Speaker Ché Weldon of North-West University of South Africa noted the
many points in the global amphibian trade pathway where traded species from
different origins come into contact including collector-supplier facilities, breed-
ing facilities, end-user facilities, etc. (Dr. Weldon’s contribution to the workshop
summary report can be found in Appendix A, pages 355–367.) Weldon discussed
two widely traded amphibian species—the African clawed frog, Xenopus laevis,
29â•›â•›Other possible explanations include the importation of horticultural soils from Europe.
20
FIGURE WO-5â•‡ Global aviation network. A geographical representation of the civil aviation traffic among the 500 largest international air-
ports in 100 countries is shown. Each line represents a direct connection between airports. The color reflects the number of passengers per day
traveling between two airports, with the most intense traffic (25,000) noted in yellow.
SOURCE: Hufnagel et al. (2004).
TABLE WO-1â•‡ Number of Individual Animals Traded by the United States

(2000–2006)
Class â•…Import â•…Export
Amphibia 27,631,172 1,594,961
Annelida 485,011 76,737
Arachnida 1,175,483 208,553
Aves 1,195,014 48,117
Chilopoda 4,358 274
Cnidaria 3,265,622 59,699
Crustacea 80,275,054 2,752,200
Diplopoda 13,926 1,218
Echinodermata 53,351 634
Insecta 469,606 88,686
Mammalia 184,682 32,879
Merostomata 60 0
Miscellaneous 5,430,083 154,195
Mollusca 3,187,671 555,829
Null 4,017,720 244,026
Pisces 1,316,977,591 138,404,653
Polychaeta 437 0
Reptilia 10,211,806 35984,895
TOTAL 1,458,805,947 180,207,556
SOURCE: Daszak (2010).
and the American bullfrog, Rana catesbeiana—that are “asymptomatic” carri-

ers of the causative agent of amphibian chytridiomycosis Bd. X. laevis has been
traded internationally since the 1930s (Weldon et al., 2007). Between 1998 and
2004 alone, more than 10,000 specimens of X. laevis were exported from South
Africa to over 100 institutions in more than 30 countries worldwide (Weldon
et al., 2007). Rana catesbeiana is one of more than 200 amphibian species in the
international food trade, which altogether moves tens of millions of individual
amphibians around the globe every year (Schloegel et al., 2010). He added that
both of these species have now established feral populations in import countries,
placing “native” species at risk of exposure to Bd (Weldon et al., 2007).
Winds and Weather

Along with anthropogenic introductions, wind and weather—including ex-
treme weather events 30—are associated with the introduction, establishment, and
spread of fungal diseases (Anderson et al., 2004). Because many fungal patho-
30â•›â•›Includes
weather phenomena that are at the extremes of the historical distribution, especially
severe or unseasonable weather (e.g., extreme heat or cold, tropical cyclones, tornadoes). http://
en.wikipedia.org/wiki/Extreme_weather.
22 FUNGAL DISEASES
FIGURE WO-6â•‡Selected dispersal events of fungal pathogens. Red and blue arrows
Figure
indicate invasions of new territories WO-6.eps
(first year recorded in brackets). Red arrows indicate
dispersal that probably occurred by direct movements of airborne spores (I, II, III, and
bitmap
IV). Blue arrows indicate pathogens that were probably transported to the new territory
in infected plant material or by people and spread thereafter as airborne spores (V, VI,
VII, and VIII). Orange circles indicate the worldwide spread of black Sigatoka disease of
banana; the first outbreak on each continent is marked (IX). Green arrows indicate periodic
migrations of airborne spores in extinction-recolonization cycles (X, XI, XII, XIII, XIV).
SOURCE: From J. K. M. Brown and M. S. Hovmøller. 2002. Aerial dispersal of pathogens
on the global and continental scales and its impact on plant disease. Science 29(5581):537–
541, reprinted with permission from AAAS. Background map provided courtesy of
Christopher Lukinbeal, University of Arizona.
gens are soil-associated, wind and other factors associated with soil disturbances
can disperse spore-associated dusts into the air. Once airborne, spores may pas-
sively travel on the wind over great distances—often hundreds or thousands of
miles—to new geographic areas and new host environments (Figure WO-6 [red])
(Brown and Hovmøller, 2002).
Aerial Dispersal—Winds and Extreme Weather Events

Limited dispersal of fungal spores carried by the wind is common and is
considered a key factor in the local spread of some fungal diseases. Sporadic
outbreaks of valley fever have occurred when spores of Coccidioides spp. are
swept up from the soil and carried by winds to be inhaled by susceptible hosts.
Long recognized as a threat to the health of military personnel stationed in arid
regions of California, Valley Fever outbreaks have also been associated with
land use changes and occupational or recreational exposures to dust (Chiller
et al., 2003; Crum-Canflone, 2007; Warnock, 2006). In 1994, the magnitude 6.7
Northridge earthquake led to an outbreak of valley fever in southern California
(Figure WO-7A) (Schneider et al., 1997). More recently, the massive dust storm
that swept through Arizona on July 5, 2011 is predicted to cause a similar increase
in cases of valley fever (Chan, 2011) (Figure WO-7B). Speaker John Galgiani,31
of the University of Arizona, explained that even small winds or soil disturbances
can easily loft spore-laden dusts into the air. (Dr. Galgiani’s contribution to the
workshop summary report can be found in Appendix A, pages 196–207.) Galgiani
further observed that inhalation of a single spore “at the right time” can cause
disease. Approximately 40 percent of infected persons develop symptoms, which
initially manifest as pneumonia (i.e., cough, chest pain, fever, and weight loss);
fatigue; bone and joint pains (“desert rheumatism”); or skin rashes (Hector and
Laniado-Laborin, 2005; Tsang et al., 2010). While dust storms and environmental
disturbances are clearly an important driver of the spread of Coccidioides spp.,
Galgiani said that simply living in an endemic region,32 without any direct contact
with the soil, puts one at risk of exposure. Yet the fungus is only sparsely distrib-
uted. Galgiani noted that, “you can do a lot of desert digging and disrupting and
not even be close to the fungus.”33
Airborne spore dispersal may also synergize with intercontinental trade and
travel to rapidly spread diseases between and within continents (Figure WO-6
[blue]). Yellow rust (Puccinia striiformis f. sp. tritici) is believed to have been
introduced into Western Australia from southern Europe, in 1979, as an adherent
spore on an air traveler’s clothing (Wellings, 2007). Once introduced into Aus-
tralia, the pathogen spread across Australia’s wheat belt and into New Zealand
via wind dispersal (Brown and Hovmøller, 2002). Indeed, winds allow many ag-
riculturally important fungal plant diseases to gradually expand their geographic
range (Brown and Hovmøller, 2002).
Pandemics caused by intercontinental aerial dispersal of spores can and do
occur—often facilitated by hurricanes and other extreme weather events. Ex-
amples include:
• Sugarcane rust (Puccinia melanocphala) is believed to have been intro-

duced from West Africa into America by cyclonic winds (Figure WO-6
[red]) (Brown and Hovmøller, 2002).
31â•›â•›Dr. Galgiani is also Chief Medical Officer at Valley Fever Solutions, Inc. which has licensed the
development of nikkomycin Z as a treatment for valley fever from the University of Arizona.
32â•›â•›Although widely perceived as endemic to the southwestern United States, Galgiani observed that
the endemicity of the disease extends through Mexico into Central and some parts of South America
(Tsang et al., 2010).
33â•›â•›It has been suggested that this spotty distribution is a result of the abundant fungal sporulation
that may accompany fungal decomposition of infected animal remains (whether or not fungal infec-
tion was responsible for an animal’s death) (Sharpton et al., 2009).
24 FUNGAL DISEASES
Figure WO-7.eps
B bitmap
FIGURE WO-7 Environmental disturbances and dust storms contribute to the dispersal
of fungal spores. (A) Dust from landslides caused by a 5.6 magnitude aftershock of the
1994 Northridge earthquake blows out of the Santa Susana Mountains into the Simi Valley.
An outbreak of valley fever occurred in the Simi Valley following the January 17, 1994,
6.7 magnitude Northridge earthquake. (B) The leading edge of a violent dust and sand
storm (known as a haboob). The wall of dust and sand that swept through Arizona on July
5, 2011, was estimated to be more than 50 miles wide. The storm travelled over 150 miles
and reached peak heights of 8,000 to 10,000 feet.
SOURCE: Photos courtesy of Tom Freeman; National Oceanic and Atmospheric
Administration.
• Coffee leaf rust (Hemileia vastatrix) may have been transported via trans-
atlantic winds between Angola to Bahia in Brazil in 1970 (Figure WO-6
[red]) (Brown and Hovmøller, 2002).
• Asian soybean rust was brought into the United States from South Amer-
ica by Hurricane Ivan in 2004 (Schneider et al., 2005).
Some scientists are concerned that the frequency and duration of such ex-
treme weather events could increase with global climate change, which in turn
could influence the incidence and intensity of fungal disease outbreaks (Garrett
et al., 2006; Greer et al., 2008).
Temperature, Humidity, and Climate Change

Like most microorganisms, fungi are highly sensitive to changes in weather
and climate34—particularly temperature, humidity, and wind—that can directly
influence their growth, spread, and survival (Harvell et al., 2002). One of the
most tragic outcomes of a weather-induced fungal disease outbreak was the Irish
Potato Famine, in which a sustained pattern of cool, rainy weather enabled the
emergence and spread of the “fungus-like” oomycete,35 Phytophthora infestans,
the causative agent of potato late blight (Fry and Goodwin, 1997; Large, 1965;
Woodham-Smith, 1962). In 1845 and 1846, late blight led to yield reductions of
40 and 90 percent, respectively, in the potato—at that time Ireland’s staple food
crop (Money, 2007). As previously noted, the resulting “Great Famine” led to the
death of more than 1 million and the emigration of over 1 million more Irish peo-
ple, primarily to the United States (Strange and Scott, 2005; Vurro et al., 2010).
When combined with reduced genetic diversity in the host plant, weather
can contribute to a “perfect storm” for a devastating agricultural disease epidemic
(Rosenzweig et al., 2001; Vurro et al., 2010). Unusually warm, moist weather,
coupled with a wholly susceptible host, provided the ideal conditions for the
emergence and spread of Helminthosporium maydis (also known as Cochliobolus
heterostrophu and Bipolaris maydis), the causative agent of Southern corn leaf
blight (SCLB) (Rosenzweig et al., 2001). Over the course of the 1970–1971
growing season, the SCLB epidemic spread from the tip of Florida up to Alberta,
Canada, destroying a significant proportion of the corn crop in its path (Ullstrup,
1972). Yield reductions were most severe in the southern states, with many farms
34â•›â•›As explained on the National Aeronautics and Space Administration website (www.nasa.gov), the
difference between weather and climate is a measure of time. Weather is the state of the atmosphere
over a short period of time; climate is how the atmosphere “behaves” over relatively long periods
of time.
35â•›â•›As noted by speaker David Rizzo, Phytophthora spp. is not a “true fungus”; it is an oomycete
or “water mold” that belongs to the Kingdom Stramenopila (a major eukaryotic group that includes
diatoms and brown algae, and is distinct from plants, fungi, and animals). Like fungi, oomycetes
“exhibit filamentous growth, produce sexual and asexual spores, and can feed on decaying matter or
be obligate parasites of plants” (Kliejunas, 2010).
26 FUNGAL DISEASES
experiencing total crop loss. Average yield loss in the Corn Belt states 36 was
20–30 percent, with some parts of Illinois and Indiana reporting yield losses of
50–100 percent (Ullstrup, 1972). In the 1970 season alone, the SCLB epidemic
led to the loss of 710 million bushels of corn—valued at more than $1 billion at
the time (or about $5.6 billion in 2009 dollars) (Tatum, 1971).
Compton Tucker, of the National Aeronautics and Space Administration
(NASA) Goddard Space Flight Center, presented data from a variety of satellite
and ground sources37 documenting increases in global temperatures worldwide,
as well as changes in the atmospheric concentration of carbon dioxide. (Dr.
Tucker’s contribution to the workshop summary report can be found in Appendix
A, pages 324-342.) He also explained how general circulation models, which
simulate the atmosphere, accounting for wind, humidity, clouds, temperature,
composition of the atmosphere (e.g., presence of trace gases), and other weather-
related variables, can be used to predict where on the surface of the earth (both
land and water) temperature and precipitation levels are likely to change.38 Ac-
cording to Tucker, these models predict that over the next century, average surface
temperatures will increase by 2–5°C, and regions of the world will get wetter or
drier (Figure WO-8).
Fungal diseases are influenced by weather fluctuations and display
“seasonality”—suggesting the possible influence of long-term climate changes
(IOM, 2003, 2008a; Rosenzweig et al., 2001). Stack noted that the onset of potato
late blight has been occurring earlier and earlier over the past 20 years in some
regions of the world and has resulted in more severe losses and greater mitigation
challenges (Hannukkala et al., 2007). In part, this is due to changing temperatures
and increased frequency of precipitation (Hannukkala et al., 2007).
Stack observed that modeling studies predict many negative impacts on plant
health in response to climate change, including shifts in the range, timing, and
severity of fungal diseases of plants39 (Jeger and Pautasso, 2008; Pautasso et al.,
2010). A 3°C increase in temperature, for example, is anticipated to alter the
phenology40 and conditions of the host species enough to result in expansion of
the geographic range of Phytophthora cinnamomi, which has already decimated
forests across southeastern Australia (Lonsdale and Gibbs, 1996). An enormous
effect is predicted for the severity of phoma stem canker (Leptosphaeria macu-
36â•›â•›The area in the Midwestern United States, roughly covering western Indiana, Illinois, Iowa, Mis-
souri, eastern Nebraska, and Eastern Kansas, in which corn (maize) and soybeans are the predominant
field crops (Encyclopedia Britannica: eb.com).
37â•›â•›These data include NASA satellite data on solar irradiance (i.e., energy output of the sun); Na-
tional Oceanic and Atmospheric Administration, NASA, and other surface data on land and ocean
temperatures worldwide; U.S. military and other satellite and ground data on sea ice; sea-level data;
NASA gravity data; and data on the atmospheric concentration of carbon dioxide and other compo-
nents of the atmosphere.
38â•›â•›See also contributed manuscript by Tucker in Appendix A (pages 324–342).
39â•›â•›See contributed manuscript by Jeger in Appendix A (page 273–296).
40â•›â•›The scientific study of cyclical biological events, such as flowering, breeding, and migration.
FIGURE WO-8â•‡ Change in precipitation between the 1971–2000 average and the 2091–
Figure WO-8.eps
2100 average in inches of liquid water/year.
SOURCE: Geophysical Fluid Dynamicsbitmap Laboratory, National Oceanic and Atmospheric
Administration.
lans) on oilseed rape, with many regions of the United Kingdom expected to
experience a 40–50 percent yield loss by 2050 (Butterworth et al., 2010).
Modeling studies predict that it is not just the plant pathogens themselves
that are likely to be impacted by continued climate change, Stack observed, but
host species as well (Loustau, 2006; Pautasso et al., 2010). Stack remarked that
while modeling studies forecast climate change effects on the distribution or
severity of many fungal plant pathogens, for most crop plants the future is un-
certain—both with regard to plant disease occurrence and the associated impacts
on food security.
Host and Pathogen Characteristics

Whether caused by anthropogenic or natural forces, the mere introduction
of a fungal organism is a necessary but insufficient condition for infectious
disease emergence. Or, as viewed through the lens of biological invasion: Not
all introduced species become “invasive.” Indeed, when introduced into new
environments, invasive species become quickly established and spread in a new
environment, while many other introduced organisms do not. As discussed at the
workshop and summarized below, host and pathogen characteristics are important
determinants for whether fungal pathogens will “thrive” in a new host or environ-
ment. For many emerging fungal pathogens, environmental factors have the great-
est influence on the interactions between a naïve host and an introduced pathogen.
28 FUNGAL DISEASES
Host Defenses in Plants and Animals

As discussed previously, fungal diseases of plant or animal hosts involve
several common steps (see “Fungi as Pathogens”). When it comes to host de-
fenses, remarked Howlett, animals and plants have several important similarities
and differences. Basal innate immunity41 is an important defense system shared
by organisms that infect plants, animals, and insects, with immunity activated
by recognition of pathogen-associated molecular patterns (PAMPs) (Nürnberger
et al., 2004). Other plant defense systems include a complex physical barrier (a
thick and impervious cuticle and cell wall), a repertoire of pathogen-specific re-
sistance genes, and systemic acquired resistance (i.e., if one leaf is infected and
the plant does not die, the plant mounts a strong immune defense in the event that
another leaf is infected later). By contrast, animals have a less complex physical
barrier (i.e., the skin and respiratory surface) and distinct innate immune system
components (with the complement system and phagocytes and other circulat-
ing cells) as well as a battery of adaptive,42 antibody-mediated, immune system
defenses (i.e., T and B cells) (Sexton and Howlett, 2006). “Most serious human
fungal diseases occur in immunocompromised hosts,” noted Howlett, suggesting
that “the mammalian immune defense system is very effective.”
Fungal Disease and the Mammalian Immune System

Fungal disease in humans usually reflects some underlying immune dysfunc-
tion (Holland and Vinh, 2009). Speaker Steven Holland of the National Institute
of Allergy and Infectious Diseases noted several examples of fungal diseases in
otherwise healthy individuals that were ultimately associated with previously un-
known primary immune disorders.43 (Dr. Holland’s contribution to the workshop
summary report can be found in Appendix A, pages 248–252.) Holland described
a healthy and young individual with no previously recognized immunodeficiency
who presented to an emergency department with acute shortness of breath that
rapidly progressed to severe respiratory distress. The woman was eventually
diagnosed with chronic granulomatous disease (CGD).44 Infection by the ubiq-
uitous fungus, Aspergillus fumigatus, probably occurred when she was handling
soil and plant debris and led to the onset of symptoms (Siddiqui et al., 2007).
Holland also reviewed the discovery of rare genetic immune deficiencies that
41â•›â•›Immune response (of both vertebrates and invertebrates) to a pathogen that involves the preex-
isting defenses of the body (e.g., barriers formed by skin and mucosa, antimicrobial molecules and
phagocytes). Such a response is not specific for the pathogen (Alberts et al., 2002).
42â•›â•›Response of the vertebrate immune system to a specific antigen that typically generates immu-
nological memory (Alberts et al., 2002).

43â•›â•›See contributed manuscript by Holland in Appendix A (pages 248–252).
44â•›â•›CGD is characterized by recurrent bacterial and fungal infections and inflammatory complica-
tions (Holland, 2010; Holland and Vinh, 2009).

underlie two serious diseases associated with fungal infection: Job’s syndrome 45
and severe coccidioidiomycosis (Buckley et al., 1972; Davis et al., 1996; Holland
et al., 2007; Vinh et al., 2009).
The incidence of opportunistic fungal infections46 has increased recently
and is associated with the growing populations of vulnerable, immunocompro-
mised individuals (e.g., people living with HIV/AIDS, recent organ transplant
recipients) (Romani, 2004). In Casadevall’s opinion, the period since the 1950s
should be viewed as a transition decade in which “fungi become more important
to human health” (Figure WO-9).
In the 1950s, only about 100 reported cases of disease were caused by Cryp-
tococcus neoformans, Casadevall observed; today, there are about 1 million cases
worldwide, mostly among persons with HIV/AIDS (Park et al., 2009). The yeast
infection caused by Candida spp. was also uncommon until the 1950s. Many
have associated the increase in Candida infections to the increased number of
immunocompromised individuals (Dixon et al., 1996). Casadevall speculated
that this may also be linked to the introduction of antibiotics, which altered the
microbial flora in the human host.
Forum member Fred Sparling of the University of North Carolina, Chapel
Hill, remarked “that there was significant cryptococcal disease47 in the pre-HIV
era,” and that he continued to observe the disease in apparently healthy individu-
als. Holland agreed and noted that he expected that “we may find new mecha-
nisms for susceptibility that might not be ‘Mendelian,’48 because it is not familial,
but something that comes on, typically, in adulthood.”
Casadevall considers host immune status in humans so important in the
development of fungal disease that, in his opinion, fungal virulence can only be
properly defined as a function of it. Casadevall went on to explain that pathoge-
nicity is not an invariant, absolute quality in an infectious disease agent, but that
the pathogenicity of a microorganism varies depending on the host and over time
(Casadevall, 2007). He reviewed the “damage response framework”—illustrated
in Figure WO-10—that was developed by Pirofski and Casadevall as a way to
illustrate these concepts (Casadevall and Pirofski, 2003; Pirofski and Casadevall,
2008).
Host damage can derive from either the pathogen (e.g., among immuno-
45â•›â•›A rare, inherited disease associated with abnormalities of the skin, sinuses, lungs, bones, and
teeth. People with this condition have chronic and severe skin infections (also known as hyper
immunoglobulin E [IgE] syndrome). MedlinePlus: http://www.nlm.nih.gov/medlineplus/ency/
article/001311.htm.
46â•›â•›These opportunistic fungal diseases include invasive aspergillosis and aspergilloma (Aspergillus
spp.), invasive fusariosis (Fusarium spp.), Pneumocystis pneumonia (Pneumocystis jirovecii), and
invasive candidiasis (Candida sp.) (Nucci and Marr, 2005; Pfaller and Diekema, 2010).
47â•›â•›Disease caused by Cryptococcus neoformans or gattii infection.
48â•›â•›A single gene disorder caused by a defect in one particular gene, and characterized by how they
are passed down in families. MedlinePlus: http://www.nlm.nih.gov/medlineplus/ency/article/002048.

htm.
30 FUNGAL DISEASES
HIV pandemic
Fungal
diseases
VULNERABLE HOSTS
Anti-neoplastic therapy
Anti-inflammatory therapy
Organ transplantation Antibiotic
resistance
Case report level
1900 1925 1950 1975 2000-2010
Antibiotics
Germ Intensive care units

theory Dialysis units
FIGURE WO-9â•‡ Incidence of systemic fungal disease has increased since the 1950s. Over
the same period, the use of medical technology and the HIV/AIDS pandemic have led to an
Figure WO-9.eps
increased number of immunocompromised individuals. The emergence of systemic fungal
disease in humans is considered by many to be a 20th-century phenomenon.
SOURCE: Adapted from Casadevall (2010).
compromised individuals with weak immune systems) or the host (e.g., among
healthy host individuals whose microbial flora has been disturbed by antibiotic
use, triggering a disproportionately strong immune response) (Casadevall and
Pirofski, 2003). This model predicts that not just immunocompromised individu-
als are at risk of disease from fungal infection(s), but also healthy hosts who
mount a disproportionately strong immune response (Casadevall and Pirofski,
2003).
Casadevall also suggested that there may be additional “subtle” effects of
fungal infection that we are just beginning to observe on a population level.
c. 1999 2010
Cryptococcosis HIV-associated cryptococcosis
Transplant-associated cryptococcosis
Sporadic (?)
Host damage
Host damage
Asthma
IRIS-associated cryptococcosis
Disease Disease
WEAK STRONG WEAK STRONG

Immune response Immune response
Candidiasis Disseminated candidiasis

Neonatal candidiasis
Candidemia
Host damage
Host damage
Vaginitis
Oral thrush
Mucocutaneous disease
Disease Disease
WEAK STRONG WEAK STRONG

Immune response Immune response
FIGURE WO-10â•‡ Damage response Figure WO-10.eps

framework. When the framework was proposed in
the late 1990s, conventional thinking was that the stronger the immune system response,
the less damage to the host, as depicted in the two lefthand graphs (Cryptococcus spp. on
the top, Candida spp. on the bottom). Since then, it has become clear that this is not the
case and that people with very strong immune systems can also become sick, as depicted in
the two righthand graphs. On the top righthand graph, notice that HIV-positive individuals
with weak immune systems are at risk of HIV-associated cryptococcosis, but HIV-positive
individuals with reconstituted immune systems (i.e., via antiretroviral therapy) are at risk
of immune reconstitution inflammatory syndrome (IRIS)–associated cryptococcosis. On
the bottom righthand graph, notice that candida vaginitis is believed to be associated with
an overreactive immune system.
SOURCE: Casadevall (2010).
He noted that “we are dealing with things now that we never saw 30–40 years
ago. The elimination of many viral and bacterial exposures, especially early in
life, without the concomitant elimination of fungal diseases could be a factor in
asthma and other atopic diseases.” Relman added, “We are not good at measuring
subtle damage. If there are fundamentally important but less obvious forms of
damage going on in the environment due to the emergence of fungi, we are not
going to be very swift at detecting them, or insightful about understanding their
implications.” Infectious propagules49 of C. neoformans spp. are everywhere,
Casadevall stated, and “we are all exposed to them.” Despite this high level of
49â•›â•›Spores or desiccated, encapsulated yeast cells.

32 FUNGAL DISEASES
exposure, Casadevall observed, C. neoformans infection rarely causes illness

in non-immunocompromised individuals. Casadevall went on to suggest that
asthma in children may be linked to an immune system that has been thrown out
of immunological balance by chronic exposure to C. neoformans. To support this
hypothesis, Casedevall pointed to two studies: Goldman et al. (2001) reported
that C. neoformans infects the majority of immunocompetent children ages 2
and older,50 and animal studies that demonstrated that even when asymptomatic,
chronic cryptococcal infection predisposes an individual to asthma (Goldman
et al., 2006).
Microbial Flora
Host immune defenses against fungal disease extend to their microbial flora.
As speaker Vance Vredenburg of San Francisco State University explained, am-
phibians “wear their defenses on their skin” (e.g., glands produce defensive
toxins). (Dr. Vredenburg’s contribution to the workshop summary report can be
found in Appendix A, pages 342–355.) Indeed, Brucker et al. (2008) isolated a
strain of bacteria (Janthinobacterium lividum) from the skin of the red-backed
salamander (Plethodon cinereus) and demonstrated that the bacteria produced
antifungal metabolites at concentrations lethal to the causative agent of amphibian
chytridiomycosis (Bd) (Figure WO-11).
Host Behavior and Thermal Tolerance

Other host characteristics including individual or group behavior can con-
tribute in unexpected ways to disease establishment and spread. Several partici-
pants noted that behaviors such as clustering for warmth (amphibians) or during
hibernation (bats) may increase opportunities for pathogen transmission between
animals. According to Casadevall, having a body temperature that exceeds the
thermal tolerance of fungi51 may also be a significant host defense (Robert and
Casadevall, 2009). Casadevall explained that most fungi thrive in the temperature
range of 12°C to 30°C. The mammalian body temperature of 37°C, he speculated,
may represent a balance between warding off fungal infection (not too cold, or
too close to ambient) and keeping metabolic costs down (not too hot) (Bergman
and Casadevall, 2010).
50â•›â•›Based on sera obtained from individuals who were being evaluated in an urban emergency
department.
51â•›â•›Robert and Casadevall (2009) found that of the 4,802 fungal strains examined (from 144 genera),
most could not grow at mammalian temperatures, and that every “1°C increase in the 30°C–40°C
range excluded an additional 6 percent of fungal isolates,” implying that fever could significantly
increase the thermal exclusion zone. This led them to conclude that, “Mammalian endothermy and
homeothermy are potent nonspecific defenses against most fungi that could have provided a strong
evolutionary survival advantage against fungal diseases.” See contributed manuscripts by Casadevall
in Appendix A (pages 177–188).
FIGURE WO-11â•‡ Microbial flora Figure WO-11.eps

as a host defense. Components of a host’s microbial
flora can be beneficial to the host by competing for resources or by secreting compounds
bitmap
that affect the survival of other, potentially harmful microbial components of the flora.
SOURCE: With kind permission from Springer Science+Business Media: Journal of
Chemical Ecology, Amphibian Chemical Defense: Antifungal Metabolites of the Micro-
symbiont Janthinobacterium lividum on the Salamander Plethodon cinereus, 34, 2008,
1422–1429, R.M. Brucker, Figure 1.
Pathogen Adaptation
Many fungi do not need a living host to survive. Fungi are well adapted to
exploit winds and water as a means for their dispersal. Moreover, a variety of
“environmental” cues trigger fungal growth, sexual and asexual reproduction,
sporulation, and continued existence during adverse environmental conditions
(Bahn et al., 2007; Judelson and Blanco, 2005; Kauserud et al., 2008; Kumamoto,
2008). In response to environmental stimuli, such as heat or drought, fungal
organisms can become “dormant”—an inactive state during which growth and
development cease but from which the organisms can be revived—or transform
into forms that are resilient to heat, drought, and winds. As discussed at the meet-
ing, the environment and environmental stimuli may also serve as a reservoir and
34 FUNGAL DISEASES
trigger for fungal pathogen adaptation and evolution (Casadevall, 2007; Lin and
Heitman, 2006; Stukenbrock and McDonald, 2008).
Sexual reproduction in fungi typically requires the presence of two differ-
ent mating types (Heitman, 2006). Two signals that regulate the sexual cycle of
C. gattii are interactions with plants and extreme desiccation (Lin and Heitman,
2006; Xue et al., 2007). According to Heitman, evidence suggests that when only
one mating type is present in an environment, C. gattii will adopt a “same-sex”
mating strategy for reproduction (Fraser et al., 2005; Lin et al., 2005; Saul et
al., 2008). This adaptability may be a widespread phenomenon, one that enables
recombination, the generation of genetic diversity, and the geographic expansion
of fungi (Heitman, 2006, 2009).52
Same-sex mating may have contributed to the expansion of C. gattii’s geo-
graphical range to Vancouver Island and the U.S. Pacific Northwest (Fraser et al.,
2005). Heitman also discussed how recombination between C. gattii lineages of
the same “sex” may have resulted in a “hypervirulent” recombinant genotype as-
sociated with the outbreak. Two of the three pathogen genotypes associated with
the C. gattii outbreak (VGIIa and VGIIc) are considered highly virulent (Byrnes
et al., 2010; Fraser et al., 2005). Moreover, VGIIa is considerably more virulent
than VGIIa isolates from other parts of the world (Fraser et al., 2005). Although
the reason why the VGIIa and VGIIc genotypes are so virulent is unclear, there
may be a link between the capacity for mating and production of spores and viru-
lence (Byrnes et al., 2010; Fraser et al., 2005; Lin and Heitman, 2006).
Howlett explained that plant breeders consider fungal pathogens to have a
“high evolutionary potential’’ if organisms undergo prolific sexual reproduction
and produce large numbers of genetically diverse spores that then act as inocu-
lum. This capacity leads to frequent breakdowns in a host plant’s resistance to
infection by particular strains of a fungal pathogen. Howlett also explained how
the agricultural environment plays a role in the breakdown of resistance.
Agricultural crops are large swaths of genetically identical plants that “exert
high levels of selection pressure” on populations of fungal strains produced dur-
ing sexual reproduction. Of the billions of offspring produced, the few fungal
strains that can infect these “resistant” plant strains will be amplified with each
subsequent disease cycle. As the frequency of virulent pathogens increases, host
resistance to disease eventually breaks down. Howlett noted that this is exactly
what happened with Leptosphaeria maculans, the causative agent of blackleg in
canola. In 2000, a new cultivar53 of L. maculans with a major resistance gene was
released on the Eyre Peninsula, Australia. Within 3 years, the fungal pathogen had
developed the capacity to overcome the host species’ genetic resistance resulting
in yield losses of more than 90 percent (Sprague et al., 2006).
The environment can also serve as a reservoir for pathogen adaptation and
52â•›â•›See
contributed manuscript by Heitman in Appendix A (pages 226–248).
53â•›â•›A
variety of a plant that has been created or selected intentionally and maintained through
cultivation.
evolution. Fungal pathogens that are free living in the environment may acquire
what Casadevall called “accidental virulence.” He noted that the soil can be
an extreme environment and that soil-dwelling microbes must adapt to rapidly
changing, often harsh, conditions (Casadevall and Pirofski, 2007). Traits acquired
in this environment, which allow fungal species to survive predation from amoeba
and other protozoan organisms, may also contribute to virulence capabilities in
hosts never before encountered by fungal pathogens. Casadevall suggested that
the concept of “accidental virulence” might best describe how environmentally
acquired fungi can be so virulent in “new” mammalian and other host organisms
(Casadevall, 2007; Casadevall and Pirofski, 2007).
EMERGING FUNGAL DISEASES OF

HUMANS, ANIMALS, AND PLANTS
Several case studies of emerging fungal disease were discussed at the work-
shop. These case studies illustrate the many factors that influence disease emer-
gence, the myriad direct and indirect impacts of fungal diseases on human and
ecosystem health, and the challenges of detecting and responding to these infec-
tious diseases.
Cryptococcus gattii
C. gattii already had the ability to survive in a wide range of environmental

variations, but the Western North America outbreak teaches us that it may
exploit hitherto unrecognized but clement environments and provide a wider
exposure, and thereby, risk of infection to the human and animal populations.
—Datta et al. (2009b, p. 5)
Cryptococcus gattii (C. gattii) is a pathogenic, environmental fungus that

emerged in humans and domestic animals on Vancouver Island, British Columbia,
Canada, in 1999, causing a growing epidemic of human and animal infections
and deaths. The fungus, which causes deadly infections of the lung and brain,
had been previously restricted to the tropical or subtropical regions of Australia,
the South Pacific, Southeast Asia, and Africa (Datta et al., 2009a,b). Since its
initial recognition in 1999 as an emerging disease, the outbreak has spread from
Vancouver Island to the British Columbia mainland and south into the Pacific
Northwest of the United States (Datta et al., 2009b) (Figure WO-12).
According to speakers Julie Harris, of the Centers for Disease Control and
Prevention, and Karen Bartlett, from the University of British Columbia, as of
December 2010, this fungal pathogen has been associated with approximately
338 confirmed human infections and at least 40 deaths. (Dr. Harris’ contribution
to the workshop summary report can be found in Appendix A, pages 207–225;
Dr. Bartlett’s contribution to the workshop summary report can be found in
36 FUNGAL DISEASES
FIGURE WO-12â•‡ Map of the Pacific Northwest, comprising parts of British Columbia,
Figure
Canada, and the states of Washington WO-12.eps
and Oregon in the United States, showing human and
veterinary Cryptococcus gattii cases (including
bitmap mammals) by place of residence or
marine
detection, and locations of environmental isolation of C. gattii during 1999–2008 (strain
NIH444 [Seattle] or CBS7750 [San Francisco] not included). Data were collected from
various state health departments and published reports referenced in the text. The map and
icons have been used at a scale that shows gross geographic areas, effectively masking any
personally identifiable patient locality information. Use of the map is courtesy of exclusive
permission from Google Maps: ©2008 Google, map data ©2008 NAVTEQ.
SOURCE: Datta et al. (2009a).
Appendix A, pages 101–116.) Investigators still do not know the origins of the
current epidemic, how C. gattii was introduced into the Pacific Northwest, or how
this invasive fungal pathogen is spreading (Datta et al., 2009a).
Phenomenology
C. gattii is a basidiomycetous yeast that colonizes tree bark, decaying wood,
and nearby soil and is a cause of cryptococcosis, a potentially fatal infection in
humans and animals (Galanis and MacDougall, 2010; Levitz, 1991; Lin and
Heitman, 2006; MacDougall et al., 2007). Before the current outbreak in British
Columbia, Canada and the Pacific Northwest, the environmental source with
which C. gattii had been most often associated was the wood, bark, and detritus
of eucalyptus trees (Levitz, 1991). More recent and widespread global surveil-
lance has established that the fungus also colonizes other tree species (Lin and
Heitman, 2006).54
Individuals become exposed to C. gattii by inhaling the organism or its
spores from soils or trees that have been colonized by the fungus (Lin and
Heitman, 2006; Sorrell, 2001). Once inhaled, C. gattii can cause severe infection
of the lungs and brain, including pneumonia, meningoencephalitis, and cryp-
tococcomas. Unlike C. neoformans, which has become a major cause of death
in HIV-infected individuals around the world, C. gattii also infects apparently
healthy, immunocompetent individuals (Galanis and MacDougall, 2010). The
disease affects a wide variety of humans and animals, but no case of transmission
between animals and/or humans has ever been documented (CDC, 2010; Datta
et al., 2009a).
Timely diagnosis of a C. gattii infection can be difficult. Patients infected
with this fungal pathogen often remain asymptomatic for 6 months or more.
When symptoms do present, fungal agents are not commonly considered by
physicians when evaluating pulmonary disease in an otherwise healthy patient
(Knox, 2010). Treating infected individuals can also be challenging because the
disease tends to require prolonged antifungal therapy, sometimes with multiple
drug courses (Iqbal et al., 2010; Sorrell, 2001). Some have suggested that when
compared with C. neoformans, C. gattii infections tend to require more prolonged
and invasive treatment (Sorrell, 2001). Harris remarked that “existing data sug-
gest that not all cryptococcal infections are alike” and “it is not clear which fac-
tors are the most influential on the patient’s presentation—the species, subtype,
host immune status, or host genetics, or some combination of factors.” 55
54â•›â•›See
contributed manuscript by Bartlett in Appendix A (pages 101–116).
55â•›â•›For
more information, see contributed manuscripts by Harris and Heitman in Appendix A (pages
207–225 and 226–248).
38 FUNGAL DISEASES
Discovery and Spread

Veterinarians and clinicians first observed cases of C. gattii infections in ani-
mals and humans on Vancouver Island in 1999 (Datta et al., 2009a). Until 2004,
all known human cases of C. gattii infection in the region occurred in individuals
who either resided on or visited the island, specifically its eastern coast (Bartlett
et al., 2007). In 2004, cases of C. gattii infections emerged on the British Co-
lumbia mainland in humans and animals that had not visited Vancouver Island,
suggesting an expansion of the endemic zone of the fungus (Datta et al., 2009a;
MacDougall et al., 2007). Also in 2004, cases of humans infected with C. gattii
who had not traveled to British Columbia emerged in Washington and Oregon,
marking the southern expansion of the fungus into the United States (CDC, 2010;
Datta et al., 2009a; MacDougall et al., 2007). The emergence and recognition
of a new, more virulent strain of the fungus accompanied C. gattii’s expansion
into Oregon (Byrnes et al., 2009, 2010). Recently, investigators found evidence
that the outbreak may have also expanded into other states, as researchers have
collected isolates from humans and animals in California and Idaho (Iqbal et al.,
2010).
Animal sentinels were instrumental to the study of C. gattii emergence and
spread in British Columbia (Bartlett et al., 2007). Bartlett noted that “human
cases, in all cases, were preceded by veterinary cases.” There are at least three
to four times as many pet cases as human cases, she continued, and “it was a
veterinarian that tipped us off that we had an outbreak.” Harris remarked that C.
gattii “is not a picky pathogen,” infecting a wide variety of animals, including,
but not limited to dogs, cats, dolphins, porpoises, elks, llamas, Bactrian camels,
alpacas, horses, and sheep.
The exact means by which C. gattii spread from Vancouver Island to the
British Columbia mainland, and south to the United States, remains unknown.
Researchers suspect that a number of factors may be responsible, including
human-mediated dispersal. A 2007 sampling study in British Columbia found C.
gattii in areas of its endemic zone that were subject to high foot and vehicle traf-
fic. These observations, combined with the finding of positive fungal samples on
peoples’ shoes and in the wheel wells of vehicles that had been driven in fungal
endemic regions, support the contention that dispersal may be partially anthropo-
genic (Kidd et al., 2007). Forestry activities could also facilitate C. gattii dispersal
by aerosolizing fungus particles during tree cutting and/or mechanically “seed-
ing” the fungus during the transfer of cut tree products, such as mulch, to new
areas (Kidd et al., 2007). In addition to these human-mediated dispersal methods,
Kidd and colleagues (2007) suggested that birds and animals might also play a
role by passively transporting the fungus during migration.
As reviewed by Bartlett, environmental sampling of endemic areas has
helped to describe how C. gattii is distributed in the environment and how easily
it might spread. Sampling revealed high levels of C. gattii in the soil as well as
its presence in the air, freshwater, saltwater, trees, and even dead wood, such as
fence posts, Bartlett remarked. Sampling results have also illustrated the effects
of forestry activities on the abundance of C. gattii in the air (measured in colony-
forming units,56 or CFUs). According to Bartlett, air samples taken in endemic
areas, where trees were being removed, revealed a baseline concentration on the
order of 100 CFU/m3, compared to 10,000 CFU/m3 during tree chainsawing and
wood chipping in the same area (Kidd et al., 2007). Once it is in the air, Bartlett
observed “the organism can travel 10 kilometers, easily, probably further than
that.” The organism is also resilient: Bartlett noted that she can still isolate viable
propagules from sawdust samples taken in 2001.
Origins of the Outbreak in the Pacific Northwest

The origins of the Pacific Northwest outbreak of C. gattii remain a mystery
(Datta et al., 2009a). Some investigators have suggested that the fungus was in-
troduced through the importation of contaminated trees, shoes, wooden pallets, or
shipping crates (Kidd et al., 2007). Supporting this hypothesis is the finding that
the VGIIb minor subtype found in British Columbia and the Pacific Northwest
is similar and may be related to VGIIb strains found in Australia (Byrnes et al.,
2010). Another origin hypothesis suggests that the VGIIa subtype has existed
in the Pacific Northwest for some time. The latter hypothesis is supported by a
case reported in 1971 of a patient in Seattle, Washington, who was infected with
a VGIIa strain of C. gattii similar to the strain in the current outbreak (Byrnes
et al., 2010; Datta et al., 2009a). However C. gattii was introduced, it is clear that
the fungus is now established in the region and appears to be evolving into new,
more virulent strains, as evidenced by the newly discovered and highly virulent
VGIIc strain (Byrnes et al., 2010; Knox, 2010).
Perhaps more interesting than the question of how C. gattii was introduced
to British Columbia and the Pacific Northwest is why it has now colonized the
region. Bartlett emphasized that the fungus previously was endemic only in areas
with tropical or subtropical climates—never in a temperate rainforest. Research-
ers have speculated that global warming may play a role, with the temperature
in the region having increased enough for the fungus to become established
(Bartlett et al., 2007; Kidd et al., 2004). Indeed, between 1998 and 2004, British
Columbia experienced six consecutive seasons of above-average temperatures,
with increases of more than 3°C in some seasons (Kidd et al., 2004).
Another possible explanation for C. gattii’s establishment in British Colum-
bia and the U.S. Pacific Northwest is that the organism itself has adapted such
that it can now successfully colonize a “novel” environment. Investigators have
found environmental isolates of C. gattii in trees that had never previously been
found to harbor the fungus, such as the Douglas fir and Western hemlock (Datta
56â•›â•›CFUs
are a standard unit of measurement for environmental sampling. Colonies reflect the num-
ber of “viable” organisms (i.e., organism capable of forming colonies when provided with nutritional
elements necessary for growth).
40 FUNGAL DISEASES
et al., 2009a). Bartlett remarked that the environmental sampling data may help to
define C. gattii’s ecological niche in British Columbia. The distribution of C. gat-
tii in the environment, thus far, appears heterogeneous, with colonization levels
differing significantly in regions such as the west and east coasts of Vancouver
Island, which, observed Bartlett, are “dramatically different in terms of rainfall,
soil type and vegetation” (Figure WO-13).
Further research into the reasons why C. gattii emerged in the temperate rain-
forest of the Pacific Northwest is needed because it could help researchers predict
the fungus’s future spread and further the scientific community’s understanding of
how environmental pathogens establish themselves in new environmental niches.
Molecular Epidemiology, Virulence, and Drug Resistance

Heitman explained that C. gattii spans four genetically isolated species
groups: VGI, VGII, VGIII, and VGIV. Examination of the molecular genotypes
of fungal isolates from infected patients reveals that nearly all of the observed
infections in British Columbia and the U.S. Pacific Northwest have been caused
FIGURE WO-13â•‡ Environmental sampling for Cryptococcus gattii in British Columbia

(2001–2009). Colonization levels of C. gattii reflect a heterogeneous distribution of C.
Figure WO-13.eps
gattii in the environment. Biogeoclimatic zones are also indicated.
SOURCE: Bartlett (2010). bitmap
by one molecular subtype of C. gattii—the VGII type (Byrnes et al., 2009). In

other regions of the world where C. gattii is endemic, two other molecular sub-
types predominate—VGI and VGIII (Byrnes et al., 2009; Kidd et al., 2004). The
VGII genotype is further subdivided into three subtypes: the majority genotype
VGIIa, which is unique to the Pacific Northwest region and not found in other
endemic regions; the less common VGIIb genotype; and the VGIIc subtype,
which has appeared in Oregon within the past several years (Byrnes et al., 2010;
Kidd et al., 2004).
Some researchers have suggested that the predominant VGIIa and the newly
discovered VGIIc C. gattii subtypes are more virulent than strains found in other
endemic countries such as Australia (Byrnes et al., 2010). This is supported by
the high rate of C. gattii infections in the current outbreak, which, at 25.1 cases/
million people on Vancouver Island, is among the highest in the world (Galanis
and MacDougall, 2010). Bartlett cautioned, however, that the high rate of C. gat-
tii infection could be a result of increased surveillance or exposure, not increased
virulence.
Recently, researchers have compared the drug susceptibility of the three
VGII subtypes found in British Columbia and the Pacific Northwest to the
more common VGI and VGIII genotypes. The VGIIc strain was found to be
significantly more drug resistant to nearly all of the tested antifungal compounds
(voriconazole, fluconazole, flucytosine, and amphotericin B) than the VGI or
VGIII genotypes. The VGIIa and VGIIb strains were also observed to be more
resistant to some antifungal drugs (fluconazole, flucytosine, and amphotericin B
for VGIIa; fluconazole for VGIIb) than the VGI and VGIII strains, though their
levels of resistance were lower than those of the VGIIc strain (Iqbal et al., 2010).
White-Nose Syndrome in Bats
Last year we estimate that we found between 10,000 and 20,000 dead bats on
the cave floorÂ€.Â€.Â€.Â€and to be honest the mortality is so disturbing.Â€.Â€.Â€. We just
can’t crawl through so many piles of dead bats.
—Scott Darling, Vermont Fish and Wildlife Department
(Buchen, 2010, p. 144)
Since the winter of 2006, a mysterious and previously unknown disease—

bat white-nose syndrome (WNS)—has decimated hibernating bat populations in
the eastern and central United States. Named after the visually distinctive white
fungus—Geomyces destructans—that grows on the muzzles, ears, and wings of
affected bats, the disease has spread rapidly across the United States and Canada.
Since it was first discovered, WNS has killed more than 1 million bats in the
United States, with some hibernation sites (hibernacula) losing 90–100 percent
of their bat populations (Figure WO-14) (FWS, 2011).
The population declines have been so rapid and dramatic that bat biologists
42 FUNGAL DISEASES
FIGURE WO-14â•‡ Signs of bat white-nose syndrome (WNS). (A) Mortality caused by
Figure WO-14.eps
WNS. (B) Geomyces destructans fungus, forming the visually distinctive white growth
2 bitmaps
on the muzzle, ears, and wings of an infected bat.
SOURCE: Photos provided courtesy of Alan C. Hicks, New York State Department of
Environmental Conservation.
at the U.S. Fish and Wildlife Service fear the extinction of entire New World57
bat populations in the United States and Canada (FWS, 2011).
Such extinctions could have devastating ecological and economic conse-
quences. Bats play a critical role in plant pollination, seed dissemination, and the
control of flying insects, including mosquitoes, moths, beetles, and other night-
flying insect populations (Blehert et al., 2009; Boyles et al., 2011). Large-scale
declines or complete disappearances of bat populations could result in reduced
plant pollination (which is already under siege by colony collapse disorder in
honeybees), significant increases in “nuisance” insect populations, and increased
insect damage to agricultural and forestry resources (Blehert et al., 2009; Boyles
et al., 2011; FWS, 2011). Because bats have very low reproductive rates—pro-
ducing one pup a year, sometimes two, in a single litter—WNS is predicted to
have long-lasting effects (Barclay et al., 2004; FWS, 2011). The value of bats to
the agricultural industry has been estimated to be roughly $22.9 billion/year, with
a range per year of $3.7 billion to $53 billion (Boyles et al., 2011).
57â•›â•›Refers
to the Western Hemisphere; in a biological context, New World species are those from
the Nearctic Neotropic ecological zones, versus Old World species from the Palearctic and Afrotropic
ecological zones.
Phenomenology
Geomyces destructans hyphae58 and conidia59 invade the hair follicles and
sebaceous and sweat glands of bats hibernating in caves and mines with seasonal
temperature ranges between 2°C and 14°C (Blehert et al., 2009). The skin of af-
fected bats does not typically show signs of inflammation or an immune response
at the site of fungal invasion (Meteyer et al., 2009). Hibernating bats infected with
WNS often have severely depleted fat reserves, which are critical for successful
hibernation (Blehert et al., 2009).60
Researchers have not yet confirmed whether Geomyces destructans is the
primary pathogen that causes WNS and the eventual death of affected bats, or if
it is an opportunistic infection that invades animals already immunocompromised
by some other, yet to be defined pathogen (Puechmaille et al., 2010). How WNS
kills bats is also unclear. Infection by the pathogen may irritate the animals, rous-
ing them from hibernation, and causing them to deplete their fat reserves to such
an extent that they are unable to survive through the winter (Blehert et al., 2009;
Cryan et al., 2010; FWS, 2011). Speaker David Blehert of the National Wildlife
Health Center at the U.S. Geological Survey presented recent research suggest-
ing that the fungus causes damage to the wing epidermis and skin structures that
help protect against water loss, causing bats to lose too much water to survive
their winter hibernation (Cryan et al., 2010). (Dr. Blehert’s contribution to the
workshop summary report can be found in Appendix A, pages 167–176.)

WNS was first documented in February 2006 in a single cave near Albany,
New York (FWS, 2011). Since then, WNS has spread rapidly across the United
States and Canada, killing more than a million bats of six different species, mak-
ing it the “worst wildlife health crisis in memory” (FWS, 2011). WNS is thought
to spread primarily through bat-to-bat contact. However, human cavers and
tourists may also be contributing to the spread of this pathogen by inadvertently
transporting spores of Geomyces destructans from cave to cave on their clothing
and equipment (Frick et al., 2010; FWS, 2011).
During routine hibernacula surveys in 2006–2007, biologists with the New
York Department of Environmental Conservation discovered bats exhibiting signs
of WNS in five caves, all within a 15-km radius of what is now recognized as
the likely “index” site near Albany, New York, recounted Blehert. The following
winter (2007–2008), researchers reported the discovery of WNS at 33 sites across
58â•›â•›Slender tubes that develop from germinated spores and form the structural parts of the body of
a fungus. A large mass of hyphae is known as a mycelium, which is the growing form of most fungi.
59â•›â•›Asexually produced fungal spore. Most conidia are dispersed by the wind and can endure
extremes of cold, heat, and dryness. When conditions are favorable, they germinate and grow into
hyphae.
60â•›â•›See contributed manuscript by Blehert in Appendix A (pages 167–176).
44 FUNGAL DISEASES
Connecticut, Massachusetts, New York, and Vermont—all within a 210 km radius

of the index site (Blehert et al., 2009). By the end of the 2010–2011 hibernation
season, the disease had been confirmed in bats in 18 U.S. states (FWS, 2011). The
disease has also spread north into Ontario, Quebec, New Brunswick, and Nova
Scotia in Canada (FWS, 2011) (Figure WO-15).
Blehert remarked that the magnitude of the mortality events being observed
is not only unprecedented among U.S. bat species but also among the 1,100-plus
bat species worldwide. Indeed, a recent modeling study predicted that for one
of the most common bat species in North America—the little brown bat (Myotis
lucifugus)—there is a 99 percent chance of regional extinction within the next
16 years as a result of mortality from white-nose syndrome (Frick et al., 2010).
Affected New World bat species include the big brown bat (Eptesicus fus-
cus), Eastern small-footed bat (Myotis leibii), little brown bat (M. lucifugus),
Northern long-eared bat (M. septentrionalis), endangered Indiana bat (M. soda-
lis), and tricolored bat (Perimyotis subflavus) (Figure WO-16). Although disease
pathology has not been confirmed, DNA from G. destructans has been detected
FIGURE WO-15â•‡ Spread of bat white-nose syndrome (WNS) in North America as of

June 6, 2011.
SOURCE: Figure courtesy of Bat Conservation International, www.batcon.org.
WO-15 new
Little brown bat Big brown bat Northern long-eared bat

(Myotis lucifugus) (Eptesicus fuscus) (Myotis septentrionalis)
Eastern small-footed bat Tri-colored bat Indiana bat

(Myotis leibii) (Perimyotis subflavus) (Myotis sodalis)
Figure
FIGURE WO-16â•‡ Species affected by batWO-16.eps
white-nose syndrome (WNS).
SOURCE: Merlin D. Tuttle, Bat Conservation International, www.batcon.org.
6 bitmaps w vector type
on skin samples from the endangered grey bat (M. grisescens), the southeastern
bat (M. austroriparius) and the cave bat (M. veilfer) (FWS, 2011). Investigators
are concerned that the endangered Virginia big-eared bat (Corynorhinus townsen-
dii virginianus) may also be “at risk.” Although the fungus has been detected on
other bat species that share their hibernacula, there has yet to be confirmed case
of an infected animal (FWS, 2011).
Investigators are concerned that WNS could eventually spread to infect all 25
of the hibernating bat species native to the United States, threatening more than
50 percent of the native U.S. bat populations (Bat Conservation International,
2010). In late May 2010, FWS officials reported that a live bat from Oklahoma
was PCR-positive for G. destructans DNA. This finding alarmed many, because
the infected species, the cave bat M. velifer, frequently shares hibernacula with
other bat species with migratory ranges that extend across the western United
States into Mexico, increasing the potential for further spread of the disease to the
west and south (Bat Conservation International, 2010; Oklahoma Department of
Wildlife Conservation and FWS, 2011). This finding, however, was not confirmed
by fungal culture or histopathology; and, to date, WNS has not been confirmed
in states west of the Mississippi River.
Bat researchers and human cavers may also be contributing to the rapid
spread of this pathogen within and across states. Blehert noted that the index site
for WNS, Howes Cave, is connected to Howe Caverns, a commercial tourist cave
that entertains up to a quarter-million visitors per year. Although there are no data
supporting (or refuting) the hypothesis that humans are serving as transmission
46 FUNGAL DISEASES
“vectors,” Blehert noted that the U.S. Fish and Wildlife Service recommends that
all people who enter caves (e.g., researchers and speleologists) employ decon-
tamination protocols for potentially contaminated clothes and equipment.
Origins
The origins of WNS and its relationship to G. destructans remain unknown
(Qaummen, 2010). However, recent evidence from Europe and characterization
of the newly described pathogen may provide clues. In early 2010, researchers
published several reports confirming that a number of bats (all Myotis spp.) in
France, Hungary, Germany, and Switzerland, while infected with G. destructans,
remained healthy (Puechmaille et al., 2010; Wibbelt et al., 2010). Speaker Gudrun
Wibbelt, of the Leibniz Institute for Zoo and Wildlife Research, reported that not
all of the European hibernacula surveyed have infected bats, and of those that do,
often only a small number of bats within the colony are infected. (Dr. Wibbelt’s
368–403.) Researchers have now confirmed the presence of G. destructans on 8
species of Old World bats in 12 countries in Europe (Puechmaille et al., 2011). 61
In addition, photographic evidence suggests that the fungus was present on bats
in Europe at least as early as the 1980s (Wibbelt et al., 2010). These findings,
which have important implications for future WNS research, could help explain
the origins of the disease and may also provide clues for understanding the
mechanisms of the infection.
One possible interpretation of these data is that G. destructans may have
originated in Europe and that Old World bats and this pathogen may have co-
evolved (Puechmaille et al., 2011; Wibbelt et al., 2010). This hypothesis is sup-
ported anecdotally by reports from routine winter bat surveys in Europe from
the past 30 years. The surveys occasionally noted the presence of a white fungus
similar in appearance to G. destructans on otherwise healthy bats, Wibbelt noted.
If European bats have coevolved with the fungus, they might be able to muster
a sufficient immune response to control and survive infection by G. destructans
(Puechmaille et al., 2010). Some have proposed the possibility that the microbial
flora of bat skin or other abiotic surfaces in European hibernacula “may have
also coevolved to incorporate G. destructans as a non pathogenic component of
the microbial community” (Wibbelt et al., 2010). Wibbelt also reported the pos-
sibility that Old World bats may only be colonized in a superficial fashion on the
outer epidermis without any invasion into deeper tissues.
A second possible interpretation of the discovery of healthy European bats
infected with G. destructans is that disease transmission in Old World bat popu-
lations may be affected for some biological or behavioral reason (Puechmaille
et al., 2010, 2011; Wibbelt et al., 2010). European bats tend to hibernate in
61â•›â•›See contributed manuscripts by Wibbelt in Appendix A (pages 368–403).

relatively small groups (rarely more than 100 individuals per cluster), Wibbelt
explained. This might make it more difficult for the disease to spread. In the
United States, bats hibernate in groups that can reach into the hundreds of thou-
sands (Wibbelt et al., 2010).
Additional explanations for why European bats infected with G. destructans
do not succumb to WNS include the possibility that the fungal strain in the United
States is more virulent than the strain in Europe or that G. destructans is not the
primary cause of death in WNS. However, Blehert did note that his team’s and
others’ diagnostic investigations of infected New World bats had ruled out tox-
ins, parasites, and known viral and bacterial pathogens as associated with WNS
(Blehert et al., 2009; Chaturvedi et al., 2010; Gargas et al., 2009). Wibbelt ob-
served that G. destructans isolates from North America and Europe appear identi-
cal in morphology and in the sequence of two genes (ITS and SSU) commonly
used as a marker to distinguish between different species (Puechmaille et al.,
2011). Further research will be necessary to determine the true cause of differ-
ences between North American and European bats infected with G. destructans.
Amphibian Chytridiomycosis
The effect of chytridiomycosis on amphibians has been described as the greatest

loss of vertebrate biodiversity attributable to disease in recorded history.
—Vredenburg et al. (2010, p. 9689)
Amphibians are currently the most threatened class of vertebrates on the

planet (Stuart et al., 2004). Researchers classify approximately one third of
the more than 6,500 known amphibian species as threatened and more than 40
percent of species have experienced population declines in recent decades (Lips
et al., 2006; Lötters et al., 2010; Stuart et al., 2004). The proximate cause of many
of these declines is a recently described disease associated with the fungus, Ba-
trachochytrium dendrobatidis (Bd), which infects more than 350 species of frogs,
toads, and salamanders on every continent except Antarctica (Fisher et al., 2009)
(Figure WO-17). In susceptible animals, Bd infection causes the deadly disease
chytridiomycosis, which has been implicated as the catalyst for the global decline
of more than 200 amphibian species, including local extinctions of several species
in the wild (Fisher et al., 2009; Kilpatrick et al., 2009; Lips et al., 2006; Lötters
et al., 2010; Schloegel et al., 2006; Skerratt et al., 2007).
Until effective control measures are in place, investigators expect Bd to con-
tinue to threaten more species with extinction, making the fungus a candidate for
the most destructive emergent, infectious epizootic disease ever recorded (Fisher,
2008). Despite its widespread impact, little is known about Bd’s origins, how it
has spread across the globe, the specific mechanism by which it causes death,
and why it is so devastating to some amphibian species while others are appar-
48
FIGURE WO-17â•‡ Global distribution of Bd.

SOURCE: The Global Bd-Mapping Project, http://www.bd-maps.net/.
Figure WO-17.eps
landscape, bitmap
ently able to control infections without significant morbidity or to resist infection

entirely (Rosenblum et al., 2009).
Phenomenology
B. dendrobatidis is an aquatic chytrid fungus—an early diverging class of
fungi—that infects keratinized epidermal cells of amphibians, causing rapidly
progressing and deadly chytridiomycosis in susceptible species (Fisher et al.,
2009). First identified in 1998 and characterized in 1999, Bd is unique among
other chytrids (Berger et al., 1998; James et al., 2006; Longcore et al., 1999).
It is one of only two known chytrid fungi to parasitize vertebrates and the only
known species to infect the keratinized skin of living amphibians (Berger et al.,
1998; Fisher et al., 2009). Bd’s asexual spore is a “free living” and motile zoo-
spore, possessing a single flagellum that allows the spore to travel small distances
(usually less than 2 cm) and thrive in aquatic habitats such as streams and ponds
(Fisher et al., 2009; Kilpatrick et al., 2009; Kriger and Hero, 2007; Rosenblum
et al., 2010).
According to speaker Vance Vredenburg, from San Francisco State Univer-
sity, investigators believe that amphibians become infected with Bd through both
casual contacts with zoospores in the water as well as through direct animal-
to-animal transmission. Once infected, the susceptible animals carry Bd for
24 to 220 days before they succumb to chytridiomycosis (Lips et al., 2006).
Vredenburg remarked that the susceptibility to Bd colonization and subsequent
development of chytridiomycosis varies widely across species, populations, and
individuals. Indeed, laboratory experiments have found mortality rates from 0 to
100 percent, depending on temperature, species, and age of the infected animals
(Berger et al., 2005; Daszak et al., 2004; Kilpatrick et al., 2009; Lamirande and
Nichols, 2002; Woodhams et al., 2003).
Many of the most notable and rapid declines in amphibia have occurred
among those populations living at high altitudes in mountainous regions, leading
some to associate outbreaks of fatal chytridiomycosis with cooler climates and
high altitudes (see Fisher et al., 2009). Several amphibian species that live near
sea level also have experienced notable population declines from this fungal dis-
ease, however, suggesting that this association may be an “oversimplification of
a complex host–pathogen relationship” (Fisher et al., 2009). The preponderance
of the evidence supports the observation that Bd does prefer cooler temperatures,
growing and reproducing between 4°C and 25°C (Berger et al., 2004; Drew
et al., 2006; Kilpatrick et al., 2009; Kriger and Hero, 2007). Indeed, the available
evidence suggests that the virulence of Bd is inversely related to temperature,
perhaps in a species-dependent manner (Fisher et al., 2009; Walker et al., 2010).
The specific mechanisms by which some species suffer rapid declines when
Bd is introduced while others are able to tolerate varying levels of infection
without the development of disease—or even resist infection entirely—remain
50 FUNGAL DISEASES
FIGURE WO-18â•‡ A chytridiomycosis outbreak in southern mountain yellow-legged frogs

Figure
(Rana muscosa), Sixty Lake Basin, KingsWO-18.eps
Canyon National Park, CA, USA.
SOURCE: Photo kindly provided by V. bitmap
Vredenburg (August 15, 2006).
unknown. Nevertheless, chytridiomycosis apparently results from the complex

interplay of pathogen, host, and environmental factors (Briggs et al., 2010;
Rosenblum et al., 2010; Vredenburg et al., 2010; Walker et al., 2010). Recount-
ing his investigations of disease dynamics in multiple populations of Bd-infected
amphibians (Rana muscosa and Rana sierrae) in Sequoia and Kings Canyon
National Parks in the Californian Sierra Nevada, Vredenburg noted that even
populations of the same species can have “very different outcomes of infection,
depending on where you are in the Sierra Nevada.”62
Although numerous studies have established that Bd is the proximate cause
of the observed declines in amphibian populations around the world, the specific
ways by which chytridiomycosis causes death remains a mystery (Figure WO-18).
In susceptible animals, chytridiomycosis causes a thickening of the skin
(hyperkeratosis), abnormal proliferation of epidermal cells (hyperplasia), and
sometimes increased skin shedding (Berger et al., 1998, 2005). Only rarely do
infected animals have visible skin lesions or other pathologies generally associ-
62â•›â•›See contributed manuscript by Vredenburg in Appendix A (pages 342–355).

ated with lethal infections. Bd may disrupt the normal regulatory functions of am-
phibian skin, causing osmotic imbalances, electrolyte depletion, and ultimately
death (Rosenblum et al., 2010). Although some investigators have suggested that
Bd might release lethal toxins, no specific toxin has been identified (Fisher et al.,
2009; Rosenblum et al., 2010). As discussed by Vredenburg, investigators are
also exploring the possibility that other microbial components of the amphibian
skin microbiome can contribute to disease mitigation (Harris et al., 2009).
Is Bd a Newly Emergent or Previously Endemic Pathogen?

One of the many unresolved debates among scientists who study this disease
is whether Bd is a newly emerging pathogen that recently spread across the globe
(known as the novel pathogen hypothesis) or if it has existed for some time as a
commensal or symbiont of amphibians, and only recently became more virulent
as a result of environmental changes that altered its relationship with its hosts
(the endemic pathogen hypothesis) (Fisher and Garner, 2007; Fisher et al., 2009;
Rachowicz et al., 2005; Rosenblum et al., 2009, 2010).
What is known is that Bd has existed at a low prevalence in some popula-
tions of amphibians since at least the 1930s, but massive species declines were
not reported until recently (Weldon et al., 2004). A number of studies established
significant associations between Bd, declines in amphibian populations, and
global warming (Fisher et al., 2009; Pounds et al., 2006). Fisher remarked that
more research is needed to illuminate the specific mechanisms through which
climate change or other environmental factors might influence the host–pathogen
dynamic between Bd and certain species of amphibians (Fisher et al., 2009). The
preponderance of the evidence generated to date supports the novel pathogen
hypothesis—Bd appears to be a newly emergent disease (Fisher et al., 2009;
James et al., 2009; Rosenblum et al., 2009, 2010).
The most compelling evidence in support of the novel pathogen hypothesis
comes from recent studies mapping the genome of Bd isolates from around the
world (Rosenblum et al., 2009). All global diversity of Bd can be explained by a
single ancestral diploid strain that subsequently spread across the world, diver-
sifying through mitotic and meiotic recombination (James et al., 2009; Morgan
et al., 2007). According to Fisher, investigators still do not know the origin of
this single diploid strain; however, all known Bd strains most likely came from
a small, genetically “bottlenecked” ancestral population (James et al., 2009).
These data provide strong, inferential evidence in support of the theory that Bd
is a recently emergent fungal pathogen that rapidly expanded its geographic
range across the globe in the first half of the 20th century (Fisher et al., 2009;
Rosenblum et al., 2009).
52 FUNGAL DISEASES
Origins and Spread

Despite its widespread “footprint,” little is known about Bd’s origins and
how it has spread across the planet (Fisher et al., 2009; Kilpatrick et al., 2009;
Rosenblum et al., 2009, 2010). Bd clearly is widely distributed, but its distribution
is not homogeneous (Fisher et al., 2009). Indeed, a number of areas around the
world—most notably Madagascar—have a rich diversity of amphibian species,
but Bd has not spread there yet (Fisher et al., 2009; Weldon et al., 2008).
Skin samples from the African clawed frog (Xenopus laevis) collected in
1938 on the Western Cape of South Africa provide the earliest evidence of Bd-
associated amphibian skin infections (Weldon et al., 2004). As noted by Weldon,
X. laevis is able to asymptomatically carry the fungus without developing chytrid-
iomycosis and has not experienced any population declines as a result of Bd in-
fections. In the early 20th century (between the 1930s and 1960s), the frogs were
globally marketed and used in human pregnancy assays (Weldon et al., 2004). X.
laevis is also widely used as a model organism in developmental biology because
its metamorphosis from zygote to tadpole can be easily observed in the laboratory
(Weldon et al., 2004). The global trade in African clawed frogs has led investiga-
tors to suggest that Bd originated in Africa and that X. laevis served as the natural
reservoir host for this fungal pathogen (Weldon et al., 2007). The fungus then
spread to new hosts and environmental niches as a result of the human-mediated
movement of amphibians, globally and locally (Weldon et al., 2004).
Some studies have cast doubt on the hypothesis that the fungus originated
in Africa (Fisher et al., 2009; Rosenblum et al., 2009, 2010). As noted by Fisher,
studies of the genetic diversity of Bd in different species found that fungal isolates
from North American bullfrogs (Rana catesbeiana), another species that is widely
traded and is able to carry Bd without any associated morbidity, are significantly
more genetically diverse than isolates from African clawed frogs (James et al.,
2009). This is contrary to what researchers would expect if African clawed frogs
were the original reservoir for Bd and suggests that the fungus origins may be
outside of Africa (Fisher et al., 2009; Rosenblum et al., 2010). Fisher observed
that genomic sequencing of global Bd isolates reveals that “what we have been
calling Bd actually consists of at least three highly divergent lineages.”
A number of theories have been put forward to explain Bd’s spread around
the globe (Fisher and Garner, 2007). As Weldon observed, many believe the
spread was at least partially human-mediated, the result of international trading
and transportation of amphibians that were infected with the fungus, but did not
show signs of obvious illness. Anthropogenic spread through the amphibian trade,
however, cannot explain how the fungus was introduced into and spread across
environments where there has been minimal human activity.
Phytophthora ramorum in Europe and North America
In the USA, the economic impact of losses due to P. ramorum is estimated to be

in the tens of millions of dollars due to the direct loss of nursery and ornamen-
tal crops, the decrease of property values due to dead/dying trees, the cost of
monitoring, tracking, and eradicating the disease, the societal impact through
loss of recreational value and cultural value, and the ecological impact through
loss of food resources for native fauna.
—Grünwald et al. (2008, p. 2)
The first reports of sudden oak death occurred in California forests in 1994–
1995 (Rizzo and Garbelotto, 2003). While the origins of the associated pathogen,
Phytophthora ramorum (P. ramorum), remain unknown, investigators believe the
“source” of the subsequent epidemic in California was an infected ornamental
Rhododendron plant(s) (Kliejunas, 2010; Mascheretti et al., 2009). Now known
to cause disease (commonly known as ramorum blight) in more than 100 plant
species, P. ramorum has also emerged as a novel plant pathogen in the United
Kingdom and Europe (Grünwald et al., 2008). Collectively, these diseases have
led to the rapid decline of oak forests on the west coast of the United States and
have led to widespread disease in trees and woody ornamental plants throughout
the United Kingdom and Europe (Grünwald et al., 2008).
P. ramorum is just one of the many invasive tree diseases that were intro-
duced into the forests of North America in the 20th century. As in the case of
chestnut blight and Dutch elm disease, the transportation and trade of plants and
plant materials contributed to the movement of this pathogen across oceans and
continents, as well as between suburban and forest ecosystems (Brasier and Web-
ber, 2010; Goss et al., 2009; IOM, 2010). As reviewed by speaker Rizzo, once P.
ramorum is established in the landscape, treatment options are extremely limited.
The discovery in 2009 that P. ramorum was reproducing in Japanese larch trees
in the United Kingdom led to the immediate clear cutting of 4 million larch
trees—more than 10,000 acres of forest—in a heroic effort to slow the spread of
the disease (Hardman, 2011).
Phenomenology
P. ramorum is a fungus-like oomycete63 or “water mold” that thrives in
the cool, wet climate of California coastal forests (Kliejunas, 2010). In con-
trast to most species of Phytophthora, P. ramorum exhibits a remarkably broad
63â•›â•›As
noted by speaker Rizzo, Phytophthora spp. is not a “true fungus.” It is an oomycete or “water
mold” that belongs to the Kingdom Stramenopila (a major eukaryotic group that includes diatoms
and brown algae, and is distinct from plants, fungi, and animals). Like fungi, oomycetes “exhibit fila-
mentous growth, produce sexual and asexual spores, and can feed on decaying matter or be obligate
parasites of plants” (Kliejunas, 2010).
54 FUNGAL DISEASES
host range, infecting a variety of tree and non-tree species—ranging from hard-
wood and conifer trees, to shrubs and leafy plants64 (Brasier and Webber, 2010;
Grünwald et al., 2008). P. ramorum colonizes the leaves of many plants and the
inner bark and sapwood of trees. The fungus can also survive in a dormant state
in decaying matter, such as leaf litter on the forest floor, and in the soil (Parke and
Lucas, 2008). Rizzo remarked that within forest ecosystems in California, “once
we started looking, we started finding it on just about every plant we looked at,
ranging from ferns to redwood trees.”
Infection occurs when spores and zoospores—dispersed by winds or wa-
ter—come into contact with susceptible plants. Moisture is not only essential
for the production of infectious propagules, but free water on plants—from fog,
dew, or rainfall—enhances infection and dispersal (Kliejunas, 2010; Rizzo and
Garbelotto, 2003). Indeed, monitoring streams that are “baited” with Rhododen-
dron leaves, Rizzo said, has been an effective method for early detection of this
pathogen in forest ecosystems. Disease manifests differently depending on the
plant species and the part of the host plant that is infected (Grünwald et al., 2008)
(Figure WO-19 and Table WO-2). Bleeding lesions and stem cankers develop
in forest trees, followed by rapid declines and “sudden” death. Ramorum blight
causes shoot-tip dieback and leaf spots in woody shrubs and ornamental trees.
Unlike sudden oak death, ramorum blight rarely kills its host (Grünwald et al.,
2008).

Observers first spotted symptoms of sudden oak death and ramorum blight
in the mid-1990s in Marin County, California, and in European nurseries
(Mascheretti et al., 2009). In 2000, researchers identified the common causative
fungal pathogen for these diseases, P. ramorum (Garbelotto and Rizzo, 2005;
Kliejunas, 2010). Trade and transportation of ornamental plants enabled the intro-
duction of this novel pathogen into the United States and Europe. P. ramorum has
since spread north to southern Oregon and south to Big Sur in California, where
tree mortality rates are among the highest (Kliejunas, 2010). In 2009, symptoms
of the disease were first detected in Japanese larch trees in the English counties of
Devon, Cornwall, and Somerset (Forestry Commission, 2010). By August 2010,
the disease had spread to Japanese larch trees in the counties of Waterford and
Tipperary in Ireland (Brasier and Webber, 2010; Forestry Commission, 2010).
Many now consider this disease to be a serious threat to Japanese larch and pos-
sibly other tree species in Europe (Brasier and Weber, 2010).
The ornamental trade and other anthropogenic factors continue to serve as
a source for global and local spread of P. ramorum. In 2004, “pre-symptomatic”
Rhododendron plants infected with the fungal pathogen were discovered in a
64â•›â•›See contributed manuscript by Rizzo in Appendix A (pages 312–324).

A B
FIGURE WO-19â•‡ Sudden oak death and ramorum blight. (A) Aerial view of a forest in
Humbolt County with patches of trees dying of sudden oak death; (B) canker on tanoak;
(C) signs of ramorum blight on a variety of host plant species.
SOURCE: Rizzo (2010). WO-19 New
56 FUNGAL DISEASES
TABLE WO-2â•‡ Disease Types and Associated Symptoms Caused by

P. ramorum
Geography and
Disease Symptoms Host Categories Typical Hosts* Environment
Sudden Stem cankers; Forest trees; Coast live oak, tanoak, North American
oak death bleeding cankers garden trees European beech forests; European
gardens
Ramorum Foliar and twig Ornamental Viburnum, European nurseries
blight blight; tip and trees and woody rhododendron, pieris, and gardens; North
shoot dieback; shrubs; forest lilac; coast redwood, American nurseries
leaf blight understory plants Douglas fir; tanoak, and forest
California bay laurel
* Only a small selection of typical hosts is presented. For a complete list of hosts refer to
Grünwald et al., 2008, and references within.
SOURCE: Adapted from Grünwald et al. (2008).
Southern California plant nursery, but not before the nursery had shipped poten-
tially infected plants to more than 40 states (Figure WO-20) (Goss et al., 2009).
As Rizzo noted, many of the fungicides used in the nursery trade do not kill
the pathogen, but rather suppress symptoms; this is probably how P. ramorum
spreads over very long distances via the plant trade. Leaves, flowers, and stems
of infected plants carry the pathogen, which is also spread by the transporta-
tion of plant or associated plant materials, including soil (Kliejunas, 2010).
Waterways are an effective means of spreading P. ramorum. Investigators have
detected the pathogen in streams contaminated with run-off from infected nurser-
ies (Kliejunas, 2010). P. ramorum may also provide an interesting case study of
humans acting as vectors for fungal disease, as sudden oak death “has potentially
been spread to new areas by hikers, mountain bikers, and equestrians” (IOM,
2010). Rizzo also pointed to the movement of “green waste,” that is, compost,
firewood, mulch, and other plant matter, as an another possible means of spread-
ing the fungus within and across ecosystems.
As of 2009, the U.S. Department of Agriculture (USDA) Animal and Plant
Health Inspection Service reported detection of P. ramorum in 11 states (Ala-
bama, California, Georgia, Maryland, Mississippi, New Jersey, North Carolina,
Oregon, Pennsylvania, South Carolina, and Washington) at 30 sites (24 nurseries
and 6 in the landscape) (Kliejunas, 2010). The pathogen’s eastward spread has
placed Eastern native forests at risk and has led many experts to worry that P.
ramorum could have ecological consequences comparable to those of chestnut
blight and Dutch elm disease (Goss et al., 2009).
The distribution of sudden oak death in California forests is heterogeneous,
Rizzo noted. Weather—including winds, temperature, humidity—contribute to
the pathogen’s establishment and spread within a new environment (Kliejunas,
FIGURE WO-20â•‡ P. ramorum “migration” pathways. The ornamental plant trade contin-
ues to serve as a major pathwayFigure WO-20.eps
for the spread of P. ramorum. Arrows indicate confirmed
P. ramorum-positive nursery trace forwards. Blue arrows are 2004 trace forwards and red
bitmap
arrows are 2006 trace forwards. There were no confirmed trace forwards in 2005 or 2007.
Pie charts show the distribution of the two groups of isolates (of NA 1 lineage) among
sampled states.
SOURCE: Goss et al. (2009).
2010). Infected tanoaks do not contribute significantly to disease spread, but,

as Rizzo observed, many other plants in forested areas (e.g., understory and
canopy trees) are also host species for P. ramorum. The pathogen’s very broad
host range, therefore, is also an important transmission factor for disease. In
California forests, the California bay laurel (Umbellularia californica) drives
transmission of the pathogen (Kliejunas, 2010; Rizzo and Garbelotto, 2003). Leaf
tips of these trees serve as a prolific source of inocula. Dissemination of spores
can occur via wind and rainsplash (Rizzo and Garbelotto, 2003). In the United
Kingdom, Japanese larch trees not only developed canker and died but the leaves
also served as foliar hosts and the source of massive amounts of inocula (Brasier
and Weber, 2010).
Origins
The lack of reports of P. ramorum in the United States before the mid-
1990s, combined with its aggressiveness and limited geographic range relative
to its hosts’ distribution, suggests that P. ramorum was only recently introduced
58 FUNGAL DISEASES
into the United States (Grünwald et al., 2008). Genetic evidence supports the
hypothesis that the U.S. and European strains are distinct and that both strains
likely originated from a third, as yet unknown, source (Grünwald et al., 2008).
Molecular studies have identified three main lineages: (1) EU1—found in both
North American and European nurseries and some European woodlands; (2)
NA2—found in California and Washington nurseries; and (3) NA1—found in
North American nurseries and in California and Oregon forests (Grünwald et al.,
2008).
An alternative explanation is that P. ramorum may have existed in California
for many years, but only recently emerged because of changes in the environment
(e.g., increased temperatures, fire suppression, modifications in land use patterns)
that have led to an increased prevalence and aggressiveness of the pathogen
(Rizzo and Garbelotto, 2003). Native P. ramorum or other Phytophthora sp. may
also have evolved into a more virulent form, may have undergone a change in
host specificity or preference, or may represent an entirely new hybrid species
(Rizzo and Garbelotto, 2003). It has been reported that a novel Phytophthora
hybrid—a cross between P. cambivora, an oak pathogen, and P. fragariae-like
isolates, a strawberry pathogen—emerged in Europe in the 1990s and has killed
thousands of alder trees (Alnus spp.) (Brasier et al., 1999). Given the large num-
ber of Phytophthora spp. in California agricultural and horticultural environ-
ments, a hybrid origin for P. ramorum is certainly a possibility; although other
explanations are also possible (Rizzo and Garbelotto, 2003; Tyler et al., 2006).
The Rapid Global Spread of Yellow “Stripe” Rust on Wheat
The presence of two virulent and highly aggressive yellow rust strains at high
frequencies at epidemic sites on five continents may represent the most rapid and
expansive spread ever of an important crop pathogen. This epidemic trend may
continue because the aggressive strains, which can tolerate higher temperatures,
are still evolving.
—Hovmøller et al. (2010, p. 369)
Yellow “stripe” rust on wheat has recently reemerged as a major threat

to global food security (Hovmøller et al., 2010). This destructive, cooler cli-
mate, wheat disease can spread rapidly via wind and human activities locally
and globally. Between 2000 and 2002, two new and highly aggressive65 strains
of the associated fungus (Puccinia striiformis f. sp. tritici) appeared on three
continents—North America, Australia, and Europe—causing record wheat crop
losses (Hovmøller et al., 2010; Milus et al., 2009). As of 2009 these new strains
of yellow rust had spread to major wheat-growing areas in the Middle East, North
65â•›â•›Speaker Mogens Hovmøller defined “aggressiveness” as the quantitative ability to cause more
disease, more quickly, on a susceptible host.

FIGURE WO-21â•‡ Wheat production regions worldwide. Each red dot represents 20,000
tons of wheat production.
Figure WO-21.eps
SOURCE: Trethowan et al. (2005). 3 bitmaps
and Eastern Africa, Western and Central Asia, and China (Hovmøller et al., 2010).
According to Hovmøller et al. (2010), this may be the most rapid and expansive
spread of an important crop pathogen ever documented.
Wheat is the most widely grown cereal crop, produced as food for humans
and as feed for livestock (Figure WO-21). Worldwide, wheat accounts for one
fifth of the total human caloric intake. In regions such as Western Asia, it can
account for as much as half of the daily calorie intake (Hovmøller, this volume;
Stone, 2010). Epidemics of rust disease have been held in check by rust-resistant
wheat cultivars developed in the mid-20th century, and other agricultural prac-
tices (Koerner, 2010). Since 2000, however, the natural history66 of yellow rust
appears to have changed. Disease is now emerging in regions previously consid-
ered inhospitable to this fungal pathogen (Hovmøller et al., 2008; Milus et al.,
2009).
Phenomenology
Puccinia striiformis f. sp. tritici (hereafter, P. striiformis) is an obligate,
basidomycetous, pathogen of wheat. It infects the green tissues of host plants,
causing damage to leaf blades, and reducing the yield and quality of produced
grains and seeds (Chen, 2005). Severe infections of yellow rust also stunt the
growth of wheat plants. Average reported yield losses due to yellow rust range
from 10 to 70 percent; yield losses in highly susceptible cultivars can reach 100
percent (Chen, 2005). Weather—including humidity, rainfall, temperature, and
66â•›â•›The natural development of something (as an organism or disease) over a period of time.
60 FUNGAL DISEASES
wind—is critical for developing favorable conditions for fungal infection and
growth (Chen, 2005).
Named for the yellow pustules of powdery spores (urediniospores) that ap-
pear as “stripes” on the leaf blades of infected plants, yellow “stripe” rust is an
important “cooler climate” wheat disease (Chen, 2005) (Figure WO-22). Disease
can occur early in the growing season, when temperatures are low. Areas affected
by this disease tend to be in temperate regions and high-elevation areas in the
tropics (Chen, 2005). P. striiformis depends on a living host for survival and
produces huge numbers of airborne spores that are carried by wind from one
susceptible host to another (Brown and Hovmøller, 2002).

The most widespread yellow rust epidemic in the United States occurred in
2000 (Milus et al., 2006). Before that time, yellow rust in the United States was
restricted to the temperate regions of California and the Pacific Northwest, where
cool and moist weather patterns prevail (Chen, 2005). Suddenly, “overnight, more
or less,” remarked Mogens Støvring Hovmøller of Aarhus University, the disease
appeared in the warmer and drier wheat belt. (Dr. Hovmøller’s contribution to
the workshop summary report can be found in Appendix A, pages 252–263.) For
the first time, severe epidemics were reported east of the Rocky Mountains—in
South Dakota, Nebraska, Kansas, Oklahoma, and Texas (Figure WO-23) (Milus
et al., 2009).
Similarly, between 2002 and 2003, yellow rust swept across Western Aus-
tralia, where yellow rust was absent until 2002—an area once considered too
warm for severe epidemics of yellow rust (Milus et al., 2009). In central and
northern Europe, epidemics on wheat at that time were less pronounced, the
majority remaining resistant to the disease. According to Hovmøller, two P. stri-
iformis strains of almost identical DNA-fingerprints were responsible for these
epidemics (Hovmøller et al., 2008). Human activity was likely the main driver
for pathogen introduction in North America and Australia, Hovmøller remarked,
given the rapid spread of the pathogen to distant continents in less than 3 years
and the indistinguishable DNA fingerprints of prevalent pathogen isolates. Once
the pathogen arrived, the disease spread rapidly via airborne spores, he said. Ac-
cording to Hovmøller, the presence of large areas of susceptible crop varieties
likely amplified and accelerated the spread of these yellow rust strains.
The emergence of yellow rust in new warmer regions concerns researchers
because it suggests a change in its epidemiology. Indeed, the strains of P. stri-
iformis associated with recent outbreaks of disease are more aggressive, 67 more
heat tolerant, and able to produce two to three times more spores in less time than
other strains (Milus et al., 2009). These adaptations appear to improve the fitness
67â•›â•›Able to cause more disease more quickly on susceptible host plants.

FIGURE WO-22â•‡ Yellow “stripe” rust on wheat.

SOURCE: Hovmøller (2010). Figure WO-22.eps
bitmap
62 FUNGAL DISEASES
MT ND
MN
ME
Before 2000 OR ID SD WI
MI
V
WY NH
NY TMA
IA
NE PA CT
NV UT IL IN OH MDNJ
CO KS MO WV DE
KY VA
CA OK TN NC
AZ NM AR SC
LAMSAL GA
TX
Trace – 20% FL
Severe (>20%)
WA MT ND
2001 and thereafter MN ME
SD WI VT
OR ID MI
WY IA NY NH
NE IA MA
NE PA CT
NV UT CO ILIN OH MD NJ
IL
KS
KS MO
MO WV DE
KY VA
CA OK AR TN NC
AZ NM AR NC
SC
MS AL GA
MSAL
TX LA
FL
FIGURE WO-23â•‡ Presence of “trace” and “severe” levels of yellow rust in North America
since 2000.
SOURCE: Adapted from Chen (2005).
Figure WO-23.eps
of these strains and may explain their rapid spread on a global scale (Hovmøller
et al., 2008). In Australia and the United States, these newer, more aggressive
strains appear to have replaced older strains and have continued to thrive in sub-
sequent seasons (Milus et al., 2009). According to Hovmøller, while the epidemic
has apparently slowed down in North America from 2006 to 2009, major yellow
rust epidemics occurred in northern and eastern Africa, Western and Central
Asia, China, and the Middle East (Hovmøller et al., 2010). In 2010, according to
Hovmøller, observers reported disease outbreaks in northern and eastern Africa,
Asia, and the Middle East, and the epidemics reappeared in the United States.
Origins
The exact geographic origin of these more aggressive strains of P. striiformis
is unknown, although phylogenetic analyses reveal that West and Central Asia
may be the evolutionary origin. Hovmøller remarked that the dramatic change in
phenotype (e.g., spore production, heat tolerance), coupled with the sudden ap-
pearance of these strains suggests a recombination event somewhere, rather than
evolution through a series of mutations.
Unlike many of the fungal diseases discussed at the workshop, yellow rust
is well known. Epidemics of this disease have plagued the world’s farmers for
centuries, and P. striiformis is familiar to plant breeders. In the 1940s, Norman
Borlaug68 developed new “rust-resistant” wheat strains that also dramatically
increased global crop yields (Rust in the bread basket, 2010). These advances in-
spired the “Green Revolution” that brought these techniques and disease-resistant
wheat into widespread use. According to Brown and Hovmøller (2002), these
techniques have helped to keep many crop diseases under control, but now,
relatively few crop varieties (with specific resistance genes) are sometimes used
across large areas at “continental scales.” A reduced crop diversity in modern
agriculture, according to Hovmøller, increases the potential impact posed by the
global movement of new, more virulent forms of plant pathogens (Brown and
Hovmøller, 2002; Stukenbrock and McDonald, 2008).69
SURVEILLANCE, DETECTION, AND RESPONSE

Fungal diseases are an emerging threat to human, animal, and plant health—
not simply because of the morbidity and mortality associated with these infec-
tions, but also because of the limited means and capabilities to rapidly detect
and diagnose these diseases and the lack of effective tools for disease mitigation
and treatment. Detection of many emerging fungal diseases—such as amphib-
ian chytridiomycosis, WNS, and C. gattii—has relied on the astute observer
in the field. However, once these diseases have become established in a new
environment, the spread of fungal pathogens is limited by the environmental
constraints imposed on them, such as temperature, humidity, drought, or mois-
ture. Therapeutic interventions and management strategies for these diseases
remain limited—underscoring the urgent need for active disease surveillance
and additional research to better understand and address fungal disease threats.
As observed by Forum member Kevin Russell of the Department of Defense’s
Global Emerging Infections Surveillance and Response System, the fungal world
68â•›â•›Norman Borlaug was an American agronomist. His work to develop disease-resistant crop strains
earned him the titles of Nobel laureate and “the father of the Green Revolution.” Borlaug was one
of only six people to have won the Nobel Peace Prize, the Presidential Medal of Freedom, and the
Congressional Gold Medal. He was also a recipient of the Padma Vibhushan, India’s second highest
civilian honor.
69â•›â•›In 1999 a new, highly virulent, strain of wheat stem “black” rust, Ug99, emerged in Uganda.
This pathogen was able to circumvent the genetic resistance of wheat hybrids developed during the
Green Revolution. This pathogen has now spread to Kenya, Ethiopia, Sudan, Yemen, and Iran (Vurro
et al., 2010). Researchers worry that Ug99 will soon spread to the major wheat growing regions of
Pakistan and India, which account for ~20 percent of the world’s wheat supply (Vurro et al., 2010).
64 FUNGAL DISEASES
is too huge, too unknown, and too threatening not to develop improved capacity
for detection, diagnosis, and response to emerging fungal pathogens and diseases.
David Blehert, joined by Forum member Russell and fellow Forum member
Jacqueline Fletcher of Oklahoma State University, emphasized the importance of
cross-disciplinary communication and a “One Health”70 approach for developing
a more robust capacity for global disease surveillance, detection, and response.
As noted by Bartlett, the detection and response to the emergence of C. gattii in
British Columbia and the U.S. Pacific Northwest in 1999 ultimately involved pro-
fessionals and expertise from the veterinary, medical, public health, and plant and
wildlife communities. Indeed, because of its close interactions with plants and
ability to cause disease in humans and animals, C. gattii was called the “poster
child” for a One Health approach.
The plant health community was considered by many at the meeting to be
an equal partner in a One Health approach to infectious disease detection and
response. Fungi can form associations with and, in some cases, be pathogenic to
humans, animals, and plants, Fletcher observed. She described the need to gain
a better understanding of how fungal pathogens might “jump” between plant and
animal or human systems. Others remarked on the indirect, but potentially sig-
nificant, impacts of plant pathogens on human health and well-being, including
threats to ecosystem stability or global food security. As Fletcher noted, however,
“plant pathogens have only been incorporated into current One Health initiatives
[in] a very minor way.”
Surveillance Networks
Over the past several decades, various systems for passive and active sur-
veillance for emerging and reemerging diseases of humans, animals, and plants
have been developed at regional, national, and international levels (GAO, 2010).
Systems supporting the surveillance and detection of emerging fungal plant
pathogens are the most sophisticated, possibly due to the historical importance
of fungal diseases on economically important foodstuffs, crops, and plants (IOM,
2007; Rossman, 2009).
Disease surveillance and detection in the United States is a shared responsi-
bility of various state and federal programs (Figure WO-24) (GAO, 2010). The
Centers for Disease Control and Prevention (CDC) and other federal agencies, in-
cluding the USDA, Department of Defense (DoD), and Department of the Interior
(DOI), independently gather and analyze national infectious disease surveillance
reports as well as morbidity and mortality data for humans, plants, livestock, and
70â•›â•›One
World, One Health® is a registered trademark of the Wildlife Conservation Society and
reflects the need to establish a more holistic approach to preventing epizootic disease and for main-
taining ecosystem integrity for the benefit of humans, their domesticated animals, and the foundation
biodiversity that supports us all. For more information, see http://www.oneworldonehealth.org/ (ac-
cessed April 11, 2011).
FIGURE WO-24â•‡ Roles and responsibilities for monitoring for pathogens in humans,
animals, plants, food, and the environment in the United States.
SOURCE: GAO (2010). Figure WO-24.eps
bitmap
wildlife. The CDC, USDA, DoD, and DOI independently fund and maintain both
domestic and international laboratory networks for infectious disease diagnostics
(Choffnes, 2008; GAO, 2010). Reporting and verification of outbreaks of specific
diseases of concern or of unusual symptoms or health disturbances takes place at
the state level. The findings are sent to federal agencies for further investigation,
and if appropriate, the coordination of a response to the potential disease threat
(GAO, 2010).
The DOI includes the U.S. Geological Survey (USGS) National Wildlife
Health Center (NWHC), which is tasked with providing information, technical
assistance, research, education, and leadership on national and international wild-
life health issues. According to Blehert, WNS surveillance is currently largely
opportunistic, based upon people making unusual observations in the field, then
66 FUNGAL DISEASES
sending bats to the NWHC or other laboratory for diagnostic investigation. Fed-
eral, state, and local government officials have taken several steps to try to curb
the spread of WNS and prevent additional bat deaths. In 2009, the U.S Fish and
Wildlife Service issued a cave advisory that established guidelines for entering
bat hibernacula, that issued recommendations for decontamination of caving gear,
and that asked researchers and spelunkers not to bring clothing or equipment that
has been used in caves from affected areas to caves in unaffected areas (FWS,
2011). Federal and state agencies have also closed caves on public lands in order
to prevent people from inadvertently spreading the fungus to new areas (FWS,
2011). Total funding for WNS research from federal and state agencies increased
from approximately $1.8 million to $10 million between fiscal year 2007 and
fiscal year 2010.
Formal global surveillance programs—tracking the emergence and reemer-
gence of microbial threats to human, animal, and plant health—are coordinated
by the World Health Organization (WHO), the World Organisation for Animal
Health (OIE), and the Food and Agriculture Organization (IOM, 2007). WHO
manages the Global Outbreak Alert and Response Network which partners with
120 “informal” (as discussed below) and “formal” (e.g., regional WHO offices,
government, military, or university research centers) information sources to iden-
tify and respond to disease outbreaks (Heymann and Rodier, 2004). OIE manages
an international reporting system on animal disease that includes reporting of
“exceptional epidemiological events” and periodic gathering of animal health in-
formation (Jebara, 2004). Forum member Peter Daszak of the EcoHealth Alliance
noted that the responsibility for infectious disease surveillance of wildlife could
be undertaken by the United Nation’s International Union for the Conservation
of Nature (IUCN). The IUCN has developed a working group of 400 wildlife
specialists from around the world.71
International Regulations and Coordination

International regulations that support infectious disease surveillance and
detection activities include: the International Health Regulations (IHRs) and
the Sanitary and Phytosanitary Measures (Baker and Fidler, 2006; Cash and
Narasimhan, 2000; MacLeod et al., 2010; WHO, 2008). In 2008, the OIE added
amphibian chytridiomycosis to its list of “notifiable”72 aquatic animal diseases,
(Schloegel et al., 2010). As several participants observed, however, this report-
71â•›â•›For more information, see http://www.iucn.org/about/work/programmes/species/about_ssc/

specialist_groups/specialist_group_pprofiles/veterinary_sg_profile/.
72â•›â•›Within the OIE, a notifiable disease is one whose “detection must, by mandate, be notified by the
competent veterinary authority to the OIE as required under Chapter 1.1 of the Aquatic Code. OIE
members are also required to report the presence or absence of each disease in their territory on a
semi-annual basis, and ensure disease surveillance programs are implemented to support any claims
of freedom from one or both diseases” (Schloegel et al., 2010, p. 4).
ing requirement only applies to OIE member states, and only to animals traded
internationally. Indeed, the effectiveness of these formal and informal reporting
regimes has yet to be demonstrated, and many have suggested that fear of ad-
verse economic consequences (e.g., trade and tourism restrictions) will limit their
usefulness as an early warning disease reporting network (Cash and Narasimhan,
2000; Fidler, in IOM, 2010; Hueston, in IOM, 2007; Perrings et al., 2010).
Guidelines on hygiene and quarantine procedures for captive and wild ani-
mals have also been developed by the conservation community to reduce the
spread of zoonotic diseases, but these guidelines are considered underused or dif-
ficult to enforce (Daszak et al., 2000). Two conventions developed to address the
international wildlife trade and the conservation of biodiversity, the Convention
for International Trade of Endangered Species and the Convention on Biologi-
cal Diversity are based entirely on voluntary agreement, noted speaker Weldon.
While these agreements reflect noble aspirational goals, according to Weldon,
there is limited opportunity to actually implement the measures.
Several participants emphasized the limitations of current capacity to detect
emerging pathogenic fungi. As speaker Fisher observed, national strategies are
limited by their focus on known threats to humans and agriculturally important
species, and international strategies are nearly nonexistent or very slow moving.
This is particularly true of wildlife surveillance, which Fisher said is “completely
under the radar.” Forum member Roger Breeze of Lawrence Livermore National
Laboratory agreed, noting that “we are not very good at looking for things we
know about, even those diseases that are economically important, such as foot
and mouth disease.” Breeze continued that “what we are talking about over the
last few days is broadening the number of organisms involved and the number of
areas of economic life that are involved.” He went on to note that many organisms
discussed during the workshop, such as ornamental plants, do not currently fall
under any one organization’s regulatory responsibility. “We have a huge interna-
tional failure in biosecurity,” according to Breeze, and the problem “needs to be
approached in a different manner.”
No single agency or multilateral organization is solely focused on infectious
diseases in humans, plants, and animals. Several workshop participants observed
that the creation of a single entity that was responsible for collecting and analyz-
ing data from across the “threat” spectrum and ensuring that disease interventions
are based on the input of professionals working with humans, domestic animals,
and wildlife could significantly enhance current disease surveillance and response
capabilities (Choffnes, 2008; GAO, 2010; Hueston, in IOM, 2007; Perrings et al.,
2010).
Improved coordination of disease surveillance and response activities,
Daszak noted, would “benefit all sectors—whether it is food production, travel
and trade, or human and environmental health.” Moreover, sectors may benefit in
unanticipated ways. Blehert remarked that “wildlife health is important to world
health. Not just with regard to disease surveillance, but also with regard to basic
68 FUNGAL DISEASES
research. There is much that we can learn from emerging diseases of wildlife such
as WNS or amphibian chytridiomycosis that likely have significant implications
with regard to ecosystem integrity and function. Only by incorporating domestic
animal health, wildlife health, and human health into the same model can we fully
understand the ecology of infectious disease.”
Several participants suggested that within the United States, an interagency
task force could link together the plant, animal, and human health communities.
Forum member Russell added that the Department of Defense is now one of
several interagency partners involved in a forum on emerging pandemic threats
as a sub-Interagency Policy Committee (IPC) of the U.S. government’s Global
Health Initiative (GHI). Among its other activities, this sub-IPC assembled an
interagency working group that developed a document detailing the U.S. response
to the revised IHRs. He suggested that this interagency forum could serve as an
effective model for coordinating the U.S. government activities in areas of com-
mon concern.
Forum member Edward McSweegan, from the National Institutes of Allergy
and Infectious Diseases, added that a previous interagency program that was
focused on international infectious diseases was orchestrated by the U.S. Depart-
ment of State and the Office of Science and Technology Policy (OSTP). Forum
Vice-Chair, James Hughes of Emory University, noted that this effort was estab-
lished under the aegis of the Committee on International Science, Engineering,
and Technology Policy of President Clinton’s National Science and Technology
Council and involved many agencies: the National Institutes of Health (NIH),
CDC, Food and Drug Administration, U.S. Agency for International Develop-
ment, and DoD, among others. He said that many of the recommendations from
their 1995 report are “still relevant to today’s world.”73 McSweegan also sug-
gested that the funding of cross-disciplinary research on emerging fungal diseases
might be modeled after the success of the NIH–National Science Foundation
(NSF) Ecology of Infectious Diseases Initiative, 74 perhaps as a collaboration of
the NIH, USGS, and USDA.
Informal Disease Reporting Networks

Informal disease reporting networks are an increasingly important compo-
nent of global disease surveillance. Examples include ProMED-mail, which is
administered by the International Society for Infectious Diseases, and Bd-Maps,
which was developed at Imperial College London. Both are platforms that allow
73â•›â•›For more information see http://clinton1.nara.gov/White_House/EOP/OSTP/CISET/html/exsum.

html#top.
74â•›â•›For more information on the joint NIH–NSF Ecology of Infectious Diseases Initiative, visit the
website http://www.fic.nih.gov/programs/research_grants/ecology/index.htm.
contributors anywhere in the world to report and access disease observations—

even via cell phone applications (Brownstein et al., 2009; Fisher et al., 2009).
Since its founding in 1994, ProMED-mail75 (Program for Monitoring Emerg-
ing Diseases, PMM) has served as an important platform for rapid communica-
tion about emerging infectious diseases of humans, animals, and plants (IOM,
2007). Speaker Larry Madoff, of the Massachusetts Department of Public Health
and University of Massachusetts Medical School, explained that in contrast to
the traditional, hierarchical approach to public health reporting, ProMED collates
information from a wide variety of unofficial or informal sources76 and distributes
reports to members in near real time (Brownstein et al., 2009; Madoff, 2004;
Morse et al., 1996). A recent quantitative assessment of the effect of informal
source reporting on the global capacity for infectious disease detection concluded
that ProMED-mail and other informal disease reporting resources improve the
timeliness of detection and reporting, although the effect varies geographically
(Chan et al., 2010).
ProMED-mail is a free service, with all reports screened by a panel of expert
moderators before being posted to over 54,000 subscribers from more than 180
countries. ProMED-mail emphasizes a One Health approach to disease surveil-
lance, Madoff remarked, by reporting on plant (mostly threats to food crops),
animal (both agricultural and zoonotic threats), and human pathogens to all
subscribers. Pointing to C. gattii as an example of a recently emerged fungal hu-
man pathogen with many non-human hosts, Madoff reminded the audience that
the risk of disease emergence in humans is greater among pathogens with many
non-human hosts (Woolhouse and Gowtage-Sequeria, 2005).
Informal efforts to aggregate information on the presence or absence of
disease in the field also contribute to improving the speed and broadening the
scope of current disease surveillance. Fisher described the Bd Global Mapping
Project77 (Bd-Maps) and its associated activity, RACE (Risk Assessment of Chy-
tridiomycosis to European amphibian biodiversity). Bd-Maps collects informa-
tion from groups of researchers in the field and national surveillance data from
several European countries, including the Netherlands, Spain, Switzerland, and
the United Kingdom, to create a shared database that provides information on
where Bd has been detected, globally and locally. Fisher hopes this information
will not only aid in the prediction of where Bd will likely emerge in the future,
but also encourage at-risk areas to implement appropriate biosecurity controls.
The Bd-Maps project has a public website with a map detailing the incidence
of positive Bd reports and, for each report, links to data. There is also an embar-
goed (private) website to encourage participation of scientists who prefer that
their data remain private. As of December 2010, the publicly available database
75â•›â•›See http://www.promedmail.org.
76â•›â•›Including clinician reports, blogs, chat rooms, websites, news media, YouTube videos, and other
Internet sources.
77â•›â•›See at www.bd-maps.net.
70 FUNGAL DISEASES
reported about 6,500 Bd-positive animals out of 30,000 sampled among 3,500
sites worldwide, with 49 of 74 countries and 440 species of amphibians with
known Bd infections. The data come from multiple sources, including contribu-
tions directly from the field using a smart phone application called EpiCollect. 78
Fisher observed that these data may be used to assess either global or country-
level trends and to detect broad-level associations (e.g., the data show that Bd is
present in many areas where species richness has declined without any [other]
explanation). Fisher noted that the project has provided a means for communicat-
ing important information rapidly among interested parties.
Predictive Modeling
Surveillance and response efforts could be better targeted to at-risk popula-
tions or circumstances through the use of mathematical models and Geographical
Information Systems (Weinberg, 2005). Several workshop participants described
the use of predictive modeling as a way to “get ahead” of the spread of invasive
fungal diseases into new and highly susceptible regions:
• Rödder et al. (2009) developed a model based on taxa susceptibility to

Bd, biogeographic, basic biology, environmental, and demographic data
to illustrate which regions of the world are more at risk for Bd among
amphibians than others.
• Meentemeyer et al. (2004) and Václavík et al. (2010) identified a number
of areas in California and Oregon, respectively, currently unaffected by
sudden oak death, but that are at high risk based on host species distribu-
tions, climate suitability for pathogen transmission (e.g., rain), and other
factors.
• Kelly et al. (2005) used the agreement of multiple models to develop a
risk map for the development of sudden oak death in the United States
based on information on nationwide vegetation/host (hardwood diversity
and hardwood density), topography, and climate (e.g., precipitation, frost
days, temperature, and many other layers) (Figure WO-25).
• Mak et al. (2010) demonstrated that data derived from environmental
sampling (in native vegetation, soil, air, and water), combined with animal
and human surveillance data, could be used to predict C. gattii occur-
rence. The methodology employed, ecological niche modeling, yielded
very accurate predictions for C. gattii in British Columbia, with animal
surveillance data in particular being a good indicator of C. gattii in an
area (Mak et al., 2010).
78â•›â•›EpiCollect allows global positioning systems-localized data to be submitted by phone to a com-
mon web database (see Aanensen et al., 2009).

FIGURE WO-25â•‡ Risk for sudden

Figureoak death in the continental United States based on
WO-25.eps
agreement among five spatially referenced models.
SOURCE: U.S. Department of Agriculture,bitmap
Forest Service (Kelly et al., 2005).
Daszak discussed the use of predictive models to get ahead of disease emer-
gence entirely by anticipating where viral pathogens of zoonotic origin are most
likely to emerge in the future. The PREDICT project is part of the U.S. Agency
for International Development Emerging Pandemic Threat program. 79 PREDICT
uses wildlife surveillance data and models to identify (1) geographic hot spots for
the emergence of infectious disease, and (2) species that may serve as reservoirs
of disease.80 Daszak noted that the prediction models developed for the PREDICT
project were grounded in wildlife surveillance data that included the active col-
lection of tens of thousands of samples from wildlife among 24 countries. Any
newly discovered viruses in these samples are deemed “high priority” if they ap-
pear to be closely related to other known viral pathogens. High-priority pathogens
are further characterized and, if appropriate, people who interact with the wild-
life that may be affected by these pathogens are educated and advised to avoid
contact. While PREDICT is currently focused only on viral pathogens, Daszak
observed that the same approach could be used for fungi and fungal pathogens.
79â•›â•›For more information, see: http://www.usaid.gov/our_work/global_health/home/News/ai_docs/

emerging_threats.pdf.
80â•›â•›See contributed manuscript by Daszak in Appendix A (pages 188–196).
72 FUNGAL DISEASES
Detection and Diagnosis

As many participants noted, improving the capacity for detecting fungal
disease threats relies on having trained and acute observers in the field as well
as a better understanding of the “baseline” of fungal diversity and distribution.
Methods for the “discovery” of fungi and fungal pathogens are also needed. As
speaker Rizzo noted, “We can put things on lists, but those are the things we
know about. The big problems are the ones we don’t know about.” Similar chal-
lenges were identified for disease diagnosis.
Astute Observers in the Field

Human capacity is needed for more effective surveillance and detection of
fungal and other infectious diseases of humans, animals, and plants, remarked
many participants. Weldon noted the importance of field biologists in discovering
the accelerated loss of amphibian biodiversity and in initiating investigations on
the possible causes—from climate change and habitat destruction to chemical
pollutants. Blackwell remarked that the causative agent of amphibian chytridio-
mycosis (Bd) was recognized as a fungus rather late in the epidemic by one of the
few mycologists who study the fungal phylum Chytridiomycota. Howlett urged
more training in classical mycology: “While molecular systematics and phyloge-
nomics has helped to advance understanding of mycology, these methods need
to be complemented by field studies and identification of the causative agent of a
disease by symptoms or pathogen morphology.” Forum member Fletcher agreed,
adding that more classically trained plant pathologists are needed: “These are the
scientists who can go out into the field and identify pathogens of any type, fungal
or otherwise.” Many senior plant pathologists are near retirement, but not many
younger pathologists have the skills and knowledge to take their place, she said.
Rizzo agreed, noting that in the 1980s, seven researchers in California specialized
in forest diseases; by the time that sudden oak death emerged as a major problem
in California, there were none. He further observed that agricultural departments
in colleges across the United States continue to be downsized.
Speakers Galgiani and Holland emphasized the importance of improved edu-
cation of physicians and other front-line healthcare workers in the diagnosis and
treatment of fungal diseases. Holland noted that fungal diseases due to previously
undiagnosed primary immune deficiencies are not frequent, but that a few cases
happen “every year, in every country.” He further cautioned that, “if you don’t
think about them and you don’t recognize these diseases as fungal, the patients
don’t survive.” Even in areas with endemic fungal disease, the correct differential
diagnosis is often missed, commented Galgiani. In the case of symptomatic Val-
ley Fever, which often initially presents as a community-acquired pneumonia,
specific antibody testing is required to discriminate Coccidioides infections from
other causes of pneumonias. Even then, Galgiani noted, with early infections
conventional serological testing produces false positives in an estimated one third
to two thirds of all infected patients.
Rapid and Accurate Tools for Detection and Diagnosis

The lack of sensitive and specific tools for the diagnosis of emerging diseases
limits the effectiveness of disease surveillance and treatment efforts (IOM, 2007).
In the ornamental plant trade, surveillance focuses on preventing the introduction
of plant pathogens. This occurs at the international, national, and regional levels,
Rizzo explained. In Europe and North America, strict controls have been placed
on nurseries to quickly contain and eradicate outbreaks of P. ramorum (Kliejunas,
2010). In the United States, plants in nurseries are subject to routine inspections,
and plants that are sold across state or county boundaries are closely monitored.
In states such as Oregon, Washington, and California, they are quarantined to
ensure they are free from infection (Kliejunas, 2010). Despite these measures, P.
ramorum continues to spread via ornamental plant trade pathways (see sudden
oak death case example). In part, this is due to the lack of effective detection
tools. Asymptomatic infections and the use of fungicides can limit the effective-
ness of quarantine protocols based only on visual inspection.
To track the spread of the C. gattii outbreak, public health officials in British
Columbia and the U.S. Pacific Northwest have listed C. gattii–associated disease
as a reportable disease. However, speaker Julie Harris of the CDC remarked that
C. gattii surveillance has been limited by overreporting of the most severe cases
and underreporting of all cases. In cases where samples are sent to the laboratory
for identification, not all labs are using the canavanine-glycine-bromothymol blue
(CGB) agar test81 or genetic sequencing82 that are needed to differentiate between
C. gattii and C. neoformans infections. Harris noted that underreporting is due to
a number of factors including the fact that many of the smaller laboratories across
Washington and Oregon:
• ay not be aware of the need for culture to make an accurate diagnosis;

m
• may not be aware that they should be sending isolates to their respective
state health departments for confirmatory testing; or
• may lack the capacity for fungal culture and testing.
Harris observed that labs with mycology capacity are not as common as labs with
viral or bacterial capacity, and requests for additional training or capacity need
to come from the local level. She noted that the C. gattii Public Health Working
Group, formed in 2008 by the CDC and state and local public health depart-
ments and laboratories and the British Columbian Centre for Disease Control,
is working to standardize surveillance by increasing clinician awareness of C.
81â•›â•›During a canavanine-glycine-bromothymol blue agar test, C. gattii grows and the medium
changes color; C. neoformans does not grow, and the medium remains a light green color.
82â•›â•›Bartlett pointed out that because of the close evolutionary relationship between the two species,
much of the early literature on the outbreak refers to the outbreak pathogen by its former name, C.
neoformans var. gattii.
74 FUNGAL DISEASES
gattii infection and working with global laboratories to characterize genetic and
phenotypic variety in C. gattii.
Padilla, from the Smithsonian Conservation Biology Institute (SCBI), iden-
tified fungal diagnostic capacity as a particularly challenging area of emerging
fungal disease threat management in wild animals and one in need of further
research. Too often, new fungal pathogens in wildlife are either misdiagnosed or
undiagnosed. The limited diagnostic capacity leads to the “clumping” of infor-
mation under known fungal disease syndromes, Padilla remarked, and this often
precludes the recognition of true emerging fungal diseases and prevents further
investigation of true host–pathogen dynamics. Fungal infections are often iden-
tified only to the genus, not the species level, making it difficult to understand
host–species relationships. The clumping of information could also result in
dangerous management decisions—when assumptions about one host are based
on what is known about another host. For example, he reported that preliminary
findings by researchers working at the SCBI suggested that the bacterium Janthi-
nobacterium lividum does not provide the same anti-chytridiomycosis protection
in Bd-infected captive-bred Panamanian golden frogs (Atelopus zeteki), as it does
in Bd-infected yellow mountain frogs (Rana muscosa). In fact, J. lividum seems
limited in its ability to colonize the skin of A. zeteki and thus is also limited in
its ability to play the same anti-fungal role that it does in R. muscosa. However,
the understanding of a bacterium–host system conferring anti-fungal protective
properties suggests that other species-specific host-adapted bacteria could confer
the same protection to A. zeteki. Padilla expressed hope that these findings will
lead to more appropriate treatments for this particular frog species.
The Fungal “Background”

Limited understanding of fungal biodiversity and biogeography can impede
surveillance, detection, and discovery efforts, noted Blehert. It has been reported
that an abundance of closely related Geomyces species have been found in the
same soil that harbors G. destructans (Lindner et al., 2010). These related species
are currently confounding the ability to conduct routine soil analysis as a mode of
surveillance for WNS, Blehert said. Rizzo added that “when we find something
new, it’s very difficult to know whether it is exotic and something to be concerned
about or an interesting new native organism that we weren’t aware of.”
Russell asked participants for their views on effective techniques for the
“discovery” of fungal pathogens (i.e., detection of an unknown disease agent).
Heitman suggested that the fungal nuclear ribosomal internal transcribed spacer
(ITS83) sequences might be useful for determining if genetic material isolated by
researchers is fungal in origin. Blackwell mentioned that mycologists now use
83â•›â•›ITS
sequences are sections of non-functional RNA that are highly variable even between closely
related species and are widely used for taxonomic purposes (e.g., Cendejas-Bueno et al., 2010; Garner
et al., 2010; Leaw et al., 2006).
non-culture–based molecular tools coupled with field explorations to identify new

fungal species (Jones et al., 2011; Jumpponen and Jones, 2009; Lara et al., 2010;
Porter et al., 2008; Schadt et al., 2003) and to characterize the distribution of
fungal species (Daughtrey et al., 1996). These techniques are increasingly being
used because many fungal species are not easily cultured. Culturing techniques
and pathology investigations, however, are still needed to characterize an organ-
ism: these are “hard and laborious things to do,” observed Daszak, but the “value
of the product is so much better, it is orders of magnitude better, because you can
do something with it.” You can “send it to others for consultation or determine an
organism’s biology or how it causes disease in host species,” Daszak said.
Blackwell noted that the NSF has had several programs in systematics84 and
biodiversity for some years. In 2004, the NSF created the “Assembling the Tree
of Life” program with the goal of constructing an evolutionary history for all
major lineages of life (See glossary for more information).These and other related
programs have increased support for research on the evolution and diversity of
Kingdom Fungi, which has been helpful for improving detection, diagnosis, and
discovery methods (see Blackwell et al., 2006; Hibbett et al., 2007). A database
of 40,000 fungal species (with an emphasis on fungal plant pathogens) developed
by the USDA Agricultural Research Service includes information such as host
range, geographic distribution, relevant scientific literature, and for some species,
descriptions and illustrations (Rossman and Palm-Hernandez, 2008).
Treatment and Response

Active surveillance and early detection of emerging fungal diseases are im-
portant and partially effective. However, due to the magnitude of trade in plants
and plant products, “[pathogens] are getting through, and they are going to get
through; we are not going to stop that” as Stack remarked. Attention is also
needed to developing ways to respond to fungal disease threats (e.g., effective
and economical treatment options) and to recover from emerging fungal diseases.
Responding to Fungal Diseases of Plants—from Agriculture to Landscapes

In plants, disease eradication strategies include clear-cutting or controlled
burning of infected plants (Rizzo et al., 2005). Fungicides are often used to
protect high-value plants, but their widespread and frequent use is often not eco-
nomically feasible (Rizzo et al., 2005; Scheffer et al., 2008). The development
of resistant cultivars or strains, which may take years to decades, is currently the
most successful disease control strategy in plants, particularly agricultural crops
(Vurro et al., 2010).
84â•›â•›The study of the general principles of scientific classification, and the classification of organisms
according to the presumed, natural, and evolutionary relationships among them.

76 FUNGAL DISEASES
Developing strains of wheat that are resistant to the newly emerging and
more aggressive forms of yellow rust is the primary strategy for limiting the
devastating effects of P. striiformis on wheat. In the meantime, early detection is
essential to reduce crop yield losses due to yellow rust, Hovmøller said. In the
short term, options for control are limited to fungicide sprays which may be un-
available or not affordable to farmers in the developing world. The replacement
of susceptible wheat with locally adapted, resistant, or less susceptible varieties
can also slow disease spread, he remarked.
As Hovmøller noted, when it comes to wheat rust, “what’s going on in
one continent may be your problem the following day.” To prevent long-term
damage, intensified international collaboration is needed to build wheat rust
surveillance, detection, and response capacity. Several promising developments
on the international scale were reported by Hovmøller. In 2008, a Global Rust
Reference Center (GRRC) for yellow rust was established to improve yellow rust
management in countries where facilities and expertise are scarce. GRRC is sup-
ported by Aarhus University in Denmark, the International Center for Agricultural
Research in the Dry Area, and the International Maize and Wheat Improvement
Center (CIMMYT). In 2011 the activities will be extended to wheat stem rust
(Puccinia graminis) via projects facilitated by the Borlaug Global Rust Initiative.
GRRC is complementing existing national diagnostic laboratories, which cannot
receive rust samples year round from all countries. The primary goals of GRRC
are to conduct virulence and race85 analyses, secure isolates for future resistance
breeding and research, facilitate research and training, and provide information
for a global wheat rust early warning system. The Borlaug Global Rust Initiative,
which was established in response to the stem rust Ug99 outbreak in East Africa,
now deals with all three wheat rusts.86
Disease management is considerably more difficult when dealing with plants
of limited economic value (i.e., non-timber and non-crop plants), despite their
significant ecological value, Rizzo noted. Management of sudden oak death
and ramorum blight, according to Rizzo, is “scale dependent.” One can manage
individual trees, landscapes, or entire regions (Rizzo et al., 2005). At the indi-
vidual level, fungicides are available that can be injected into a tree or sprayed
on the bark to prevent infection (Garbelotto et al., 2002). In forests, containment,
including cutting and controlled burnings in areas with infected trees, is the pri-
mary means of infection control. When asked whether there is a possibility for
developing treatments for sudden oak death that could be applied at the landscape
level, Rizzo said the options are limited. In Oregon, there have been attempts to
conduct aerial spraying with phosphonate (a chemical fungicide), but it is un-
likely that any type of aerial spraying would be acceptable in California. Rizzo
also cautioned that it took many decades to breed genetic resistance for Dutch
85â•›â•›A subspecies group of pathogens that infect a given set of plant varieties (Cornell University,
plant pathology glossary).
86â•›â•›For more information see www.globalrust.org.
elm disease. The complexity of oak genetics also makes it very challenging to
use breeding to develop oaks resistant to P. ramorum infection. Rizzo went on
to note that research on potential biocontrol agents, such as viruses, is at a very
early stage.
Landscape-level management also uses predictive modeling. For sudden oak
death, models based on host species distribution, climate, and other factors iden-
tify areas at risk for invasion by the pathogen. These areas can then be surveyed
using “aerial imaging, plot-based monitoring, and stream sampling to determine
the presence of P. ramorum or signs of infected trees” (IOM, 2008b). Eradication
methods are only effective if the disease is detected early enough. For areas where
the pathogen is established, management approaches seek to avoid negative eco-
logical consequences, such as the growth of invasive plant species. Ultimately,
Rizzo said, we are trying to develop methods to “live with the pathogen.”
Treatment Options for Fungal Diseases of Humans and Animals

Available antifungal therapies are generally of limited value due to toxicity
problems (Figure WO-26) (Ostrosky-Zeichner et al., 2010). The lack of accurate
diagnostics further limits the effectiveness of existing fungal treatments. Ap-
proaches using antibody therapy and vaccines (for certain endemic pathogens)
remain challenging due to the ongoing evolution of pathogens (Cox and Magee,
2004; Galgiani, 2007, 2008; Ostrosky-Zeichner et al., 2010). Overall, there are
few new therapeutic agents in the development pipeline with the potential for
broad antifungal effects (Ostrosky-Zeichner et al., 2010).
The development of new treatments for fungal diseases has also been slowed
by an underappreciation for the effects that fungal diseases can have on human
health, Galgiani asserted. In endemic areas of Arizona and California, about a
third of all coccidioidomycosis cases lead to illnesses requiring medical atten-
tion87 (Tsang et al., 2010). While oral therapy with azole antifungal drugs is safe
and convenient, many patients do not respond to treatment (20–40 percent failure
rate). Moreover, many patients who initially respond to treatment experience re-
lapses after treatment ends (Galgiani, 2007; Hector and Laniado-Laborin, 2005).
Galgiani reviewed current efforts at the University of Arizona to develop a new
antifungal known as nikkomycin Z, a competitive inhibitor of chitin synthase
that interferes with cell wall construction (Galgiani, 2007). Discovered in an an-
tifungal discovery program by Bayer in the 1970s, the compound demonstrated
antifungal activity in mice in the 1980s (see Hector et al., 1990). Only after the
87â•›â•›Valley fever is often dismissed as a self-resolving mild illness. In fact valley fever can be long-
lasting and have a tremendous impact on activity levels (see Galgiani, 2007). Recent surveillance
activities conducted by the Arizona Department of Health Services, in collaboration with the CDC,
reported that coccidioidal illness lasted an average of 6 months, with 75 percent of workers taking
more than one month of sick leave and 40 percent of infected persons requiring at least one night of
hospitalization at some point during the course of their illness (Tsang et al., 2010). Annual hospital
costs alone amount to nearly $90 million ($86 million in 2007; Tsang et al., 2010).
78 FUNGAL DISEASES
FIGURE WO-26â•‡ Mechanisms of action of selected antifungals. An illustration of the

mechanism of action of currentlyFigure WO-26.eps
available antifungals as well as selected antifungals un-
der development. (A) Echinocandins andbitmap
nikkomycin Z inhibit the formation of the fungal
cell wall. (B) Sordarins interfere with protein assembly. (C) Azoles, polyenes, and terbi-
nafine disrupt the fungal cell membrane. (D) Flucytosine interferes with DNA synthesis.
Antibodies and vaccines prevent fungal infection or block and/or destroy the fungal cells.
SOURCE: Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Drug
Discovery (Ostrosky-Zeichner, L., A. Casadevall, J. N. Galgiani, F. C. Odds, and J. H. Rex.
2010. An insight into the antifungal pipeline: Selected new molecules and beyond. Nature
Reviews Drug Discovery 9(9):719–727).
University of Arizona acquired the compound in 2005, have clinical trials re-
sumed (Galgiani, 2007).88
In the absence of a vaccine or other preventive measures for C. gattii infec-
tion of humans and animals, officials concede the public can do little to protect
themselves from infection (Knox, 2010). Additional research is needed to clarify
the epidemiology and drug susceptibilities of the various strains of C. gattii
present in the region to help inform treatment guidelines. Moreover, research-
ers need to learn more about the natural history and pathogenicity of the fungus
to further prevention, treatment, and intervention efforts (Datta et al., 2009a,b).
Heitman noted that research is ongoing to determine the nature of hypervirulence
(D’Souza et al., 2011); the differences between immune responses to C. gattii
and C. neoformans infections (Cheng et al., 2009); and why C. gattii can so read-
ily invade the cells of immunocompetent individuals (Kronstad et al., 2011; Ma
et al., 2009; Voelz and May, 2010).
88â•›â•›See conributed manuscript by Galgiani in Appendix A (pages 196–207).

FIGURE WO-27â•‡ Frogs in the Sierra Nevada region, being treated in baths containing a
fungicidal bacterium in hopes ofFigure
eliminating infection by the fungal pathogen (Bd) associ-
WO-27.eps
ated with the deadly disease: amphibian chytridiomycosis.
SOURCE: Photo by Anand Varma.
bitmap
The challenge of developing effective treatments for fungal diseases is com-

pounded by the scale and complexity of treating diseases of wildlife. Fungicidal
treatment protocols are being explored for amphibian chytridiomycosis and in-
clude methods to alter the skin microbiome89 (Fisher et al., 2009; Harris et al.,
2009) (Figure WO-27). For WNS, some researchers are investigating whether
treating bats with antifungal agents might improve their survival (Platt, 2010),
while others are exploring the possibility of developing a vaccine against this
fungal pathogen (Buchen, 2010). Adapting these protocols to large and dispersed
wild animal populations, while minimizing unanticipated ecosystem impacts, is
challenging and may continue to limit conservation efforts (Fisher et al., 2009). In
addition to captive breeding programs, conservation efforts target habitat preser-
vation, limiting the spread of infected species, and protecting endangered species.
Further research is also needed to fill in some gaps in knowledge that still exist.
Researchers need to gain a better understanding of the biology of both Bd and G.
destructans and their respective hosts to answer questions related to the determi-
nants of virulence, the hallmarks of effective immune response, and the specific
89â•›â•›Fungicides, and more recently cutaneous bacteria of amphibians (e.g., J. lividum) known to
produce an antifungal metabolite, are applied to the skin of amphibians.

80 FUNGAL DISEASES
mechanisms by which these pathogens kill their hosts. Added to this is the unique
challenge of managing disease in hibernating animals in delicate underground
ecosystems. Biology of infectious diseases, however, is not part of the traditional
wildlife ecology education curriculum, Blehert remarked. Nor are speleologists,
tourists, recreational cavers, or hikers required to have such knowledge.
Buying Time Through the Conservation of Threatened Wildlife Populations

Captive breeding programs at the SCBI90 have helped to rescue species that
were on the verge of extinction, endangered by habitat loss or by the introduction
of disease into areas with naïve and susceptible host populations. According to
Padilla, captive propagation programs can also serve two additional functions in
the management and mitigation of emerging fungal diseases91:
• Fungal diseases identified in a captive animal population can be an early

indication of an emerging threat in the wild. Based on Padilla’s experi-
ence, there is a wide range of opportunistic and primary fungal diseases
that have been observed in captive animals.
• Captive populations are also established for the purpose of studying a
fungal disease that would otherwise be difficult or impossible to study.
An example of this important work is the captive population of Japanese
giant salamanders (Andrias japonicus) established by the Smithsonian
National Zoological Park, in which the presence of Bd and the efficacy
of itraconazole treatment can be studies and monitored over time in ways
that could not be possible in their wild counterparts.
SCBI has used captive breeding to save several species from extinction. The
endangered black-footed ferret population was revived from just 18 individuals
in 1988 to a current population of 800 to 1,000 in the wild (Weidensaul, 2000).
Other animal species, including the golden lion tamarin, California condors,
Przewalski’s horses, and the scimitar-horned oryx, have also benefited from the
SCBI’s captive breeding program’s success.
Despite success with multiple species, establishing and maintaining captive
breeding programs is technically challenging. In the fall of 2010, SCBI devel-
oped a captive colony of the endangered Virginia big-eared bats (Corynorhinus
townsendii virginianus) in response to the threat posed by G. destructans. Spe-
cialist insect-eating bats, such as the Virginia big-eared bat, are notoriously dif-
ficult to keep in captivity. But, Padilla noted, in light of the possible extinction
of this endangered subspecies, SCBI decided to take on the “high risk” project
90â•›â•›Formerly known as the National Zoo’s Conservation and Research Center, the SCBI is an um-
brella organization for the Smithsonian’s global efforts to conserve species and train future genera-
tions of conservationists. See http://nationalzoo.si.edu/scbi/default.cfm.
91â•›â•›See contributed manuscript by Padilla in Appendix A (pages 296–312).
of developing a captive colony of these bats. However, Padilla said, although

the bats did not die of WNS, the colony of 40 bats experienced extremely high
mortality (90 percent) in the first 200 days of captivity.
The Amphibian Ark92 is a global network of captive breeding programs
working in the short term to protect amphibian species at immediate risk of
extinction (IUCN, 2005). The Smithsonian’s National Zoo currently houses a
fifth of the world’s Panamanian golden frog populations (Figure WO-28). It is
hoped that the Smithsonian’s expertise in captive breeding will contribute to the
preservation of amphibians and New World bats that are currently at risk of local
or global extinction.
Weldon highlighted a number of successful conservation programs that target
amphibian populations outside of captive breeding programs.93 These included:
• The population management of Alytes obstetricans in Peńalara Natural

Park, Spain, in response to annual Bd outbreaks;
• Australia’s Bd Threat Abatement Plan, which was initiated in 2006, with
the goal of preventing amphibian populations and regions that are cur-
rently free of chytridiomycosis from becoming infected; and
• Madagascar’s Early Detection Plan, which monitors high-risk areas (e.g.,
ports of importation, areas visited by tourists, areas of high biodiversity)
and builds facilities for captive breeding and research in the event that Bd
does reach the island (Weldon et al., 2008).
Importantly, Weldon noted, these programs include disease prevention as a

prioritized conservation measure.
Weldon also recounted one instance in which conservation efforts uninten-
tionally contributed to the spread of diseases. Population decline of amphibians
on the island of Mallorca were linked to Bd infection in 2008 (Walker et al.,
2008). The “source” of infection was traced to a project designed to boost popu-
lations of the island’s midwife toad (Alytes muletensis). Cross-contamination is
thought to have occurred between two species that were cohoused at the breeding
facility: the midwife toad and imported frogs from South Africa (Xenopus gilli)
that were infected with Bd. Captive midwife toads reintroduced into the wild
served as vectors that brought the pathogen to other amphibian populations on the
island (Rosenblum et al., 2009; Walker et al., 2008). Weldon said, “This illustrates
that if we are to proceed with the reintroduction programs, great caution should
be taken, because you could potentially be introducing the pathogens with the
species that you are trying to conserve.”
92â•›â•›The Amphibian Ark carries out the ex situ components of the Amphibian Conservation Action
Plan developed by the World Conservation Union. For more information, see http://www.amphibian
ark.org/pdf/ACAP.pdf and www.amphibianark.org.
93â•›â•›See contributed manuscript by Weldon and Fisher in Appendix A (pages 355–367).
82 FUNGAL DISEASES
FIGURE WO-28â•‡ Panamanian golden frog (Atelopus zeteki). The Smithsonian National
Figureprogram
Zoo has established a captive breeding WO-28.eps
to help rescue this critically endangered
species. bitmap
SOURCE: Photo by Brian Gratwicke, Wikimedia commons.
Prospects for Preventing and Managing Emerging Fungal Diseases

Although participants described a number of challenges in efforts to detect
and respond to emerging fungal pathogens, discussion also revealed many op-
portunities to better prevent and manage these threats. Daszak stressed the need
to “focus on the underlying causes, because they cross all the kingdoms. The driv-
ers of plant disease also drive the emergence of disease in wildlife and humans:
travel, trade, agriculture, deforestation, and other environmental disturbances.”
While improving capacity to prevent and manage disease emergence is an enor-
mous undertaking and a long-term endeavor, Daszak stressed that “the evidence
and data on steps that can be taken are there; it is just a matter of turning our
knowledge into action.”
To many participants, a better understanding of the ecology of fungi and
fungal disease seemed paramount (Rizzo, 2005). To better manage outbreaks
of infectious disease, scientists may also benefit from a greater understanding
of biological invasions in all their variety and complexity. The incipient “cross-
fertilization” of ecology and epidemiology offers support for such investigations,
as does the growing recognition of the interdependence of human, animal, and
plant health, and of the central importance of the environment in influencing
host–pathogen interactions (Scholthof, 2007). The following strategies and areas
of focus for preventing and managing all types of biological invasions, including
fungal pathogens, were discussed during the workshop as particularly promising:
• Anticipating invasions based on global and local trade patterns (Dybas,

2004);
• Identifying and interrupting routes of transport that represent high risk
for biological invasions of all kinds, rather than focusing on individual
species or known diseases (Rossman, 2009);
• Recognizing the importance of human travelers as disease couriers, trans-
mitters, and sentinels and, therefore, a critical target for infectious disease
surveillance and detection (Pimentel et al., 2005);
• Recognizing that domestic animals, wildlife, and plants can also serve as
important “asymptomatic” carriers or sentinels for disease and are also an
important target for disease surveillance and detection efforts;
• Prescreening imported plants and animals that are likely to become prob-
lematic invasive species (O’Donnell, 2006);
• Establishing the prevention of the spread of invasive species as an inter-
national public good, which requires coordination among nation states
(Keller and Perrings, 2010). Because such a system is only as strong as
the “weakest link,” efforts are also needed to assist developing nations
in establishing capacity for surveillance, detection, and prevention of
biological invasions (Keller and Perrings, 2010);
• Educating the public and inspectors at airports and seaports about the en-
vironmental and economic threats posed by invasive species, and the role
of tourism and travel in biological introductions (Pimentel et al., 2005);
• Focusing efforts on markets (e.g., wildlife markets) to regulate, reduce,
or eliminate trade that threatens the health of humans, domestic animals,
wildlife, and ecosystems (Karesh et al., 2005);
• Developing bioeconomic models to assess the economic impact of the
introduction of invasive species and of alternatives for their prevention
and mitigation (Evans, 2003);
• Increasing capacity for the early detection of, and rapid response to, bio-
logical invasions (Dybas, 2004); and
• Applying mathematical models to forecast the worldwide spread of infec-
tious diseases, identify endangered regions, and analyze potential control
strategies (Hufnagel et al., 2004; Weinberg, 2005).
Each of these approaches supports the overall goal of identifying and exploit-
ing common characteristics of invasive animals, plants, and microbes in order to
reduce their impact. To pursue this strategy requires “a new perspective, a new
thinking, a consideration of all alien introductions in a deliberate, truly compre-
hensive system,” ecologist Richard Mack has observed (Dybas, 2004, p. 618). “If
we do that, then we will have a sound science-based policy.”
84 FUNGAL DISEASES
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Appendix A
Contributed Manuscripts
A1
THE EMERGENCE OF CRYPTOCOCCUS GATTII IN BRITISH

COLUMBIA AND THE PACIFIC NORTHWEST1
Karen H. Bartlett, Sarah E. Kidd, and James W. Kronstad2
An unprecedented emergence of cryptococcal infections in animals and oth-

erwise healthy humans was recognized in 1999 on the east coast of Vancouver
Island, British Columbia. Unexpectedly, these infections were caused by Cryp-
tococcus gattii, a species closely related to the AIDS-associated fungal pathogen
Cryptococcus neoformans. Human cases have continued over the past 8 years
and now total approximately 170 with eight deaths. Extensive environmental
1â•›â•›Reprinted
with kind permission from Springer Science+Business Media: Current Infectious Dis-
eases Reports, The emergence of Cryptococcus gattii in British Columbia and the Pacific Northwest,
10, 2008, p. 108–115, Karen H. Bartlett, Sarah E. Kidd, and James W. Kronstad.
â•›â•›CurrentInfectious Disease Reports 2008, 10:58–65
â•›â•›CurrentMedicine Group LLC ISSN 1523-3847
â•›â•›Copyright © 2008 by Current Medicine Group LLC
â•›â•›Papers of particular interest, published recently, have been highlighted as:

â•›â•›+ Of importance
â•›â•›++ Of major importance
2â•›â•›Karen H. Bartlett, PhD, Sarah E. Kidd, PhD, and James W. Kronstad, PhD. Corresponding author:
James W. Kronstad, PhD, The Michael Smith Laboratories, University of British Columbia, 2185 East
Mall, Vancouver, BC, V6T 1Z4, Canada. Email: kronstad@interchange.ubc.ca.
101
102 FUNGAL DISEASES
sampling, coupled with detailed molecular typing of isolates, revealed areas

of permanent and transient colonization with primarily three genotypes of the
fungus. C. gattii was found in air, soil, water, and in association with numerous
tree species. Importantly, there is solid evidence for human-mediated dispersal
of the pathogen, and C. gattii has now been detected in the environment on the
mainland of British Columbia and in the Pacific Northwest. Associated animal
and human cases are now being reported and further spread of the pathogen may
be inevitable.
Introduction
The basidiomycetous yeast Cryptococcus neoformans has a global distribu-
tion and has achieved prominence in recent decades because of its propensity
to infect immunocompromised people (Casadevall and Perfect, 1998). In fact,
cryptococcosis is recognized as an AIDS-defining illness, and in the absence of
highly active antiretroviral therapy, the disease is a significant cause of death in
individuals with HIV infection (Bicanic and Harrison, 2005; Bicanic et al., 2005).
People and animals acquire the fungus via the inhalation of desiccated yeast
cells or basidiospores from environmental sources such as avian guano, soil, and
trees. Pulmonary infection often results in dissemination to the central nervous
system and C. neoformans is the leading cause of fungal meningitis (Casadevall
and Perfect, 1998).
Isolates of C. neoformans have previously been divided into three varieties
known as grubii, neoformans, and gattii and into serotypes (A–D and hybrids
such as AD) defined by antigenic differences in the capsular polysaccharide that
is the major virulence factor (Casadevall and Perfect, 1998). The gattii variety is
now recognized as a separate species based on phenotypic and molecular traits,
and mating (Kwon-Chung et al., 2002). Thus the current view is that the species
C. neoformans (var grubii and neoformans) contains strains of serotypes A, D,
and AD, and the distinct species C. gattii contains isolates of the B and C sero-
types (Kwon-Chung and Varma, 2006). An excellent review of the differences be-
tween C. gattii and C. neoformans has been published by Sorrell (Sorrell, 2001).
Extensive surveys have been performed over the past 10 years to characterize
the genotypes and distribution of C. neoformans and C. gattii isolates (Barreto de
Oliveira et al., 2004; Boekhout et al., 2001; Boukhout et al., 1997; Fraser et al.,
2005+; Kidd, 2003; Kidd et al., 2004 ++; Kidd et al., 2005+; Meyer et al., 1999;
Meyer et al., 2003). These surveys used a variety of DNA-based typing methods
to provide detailed classifications of isolates into molecular types. Thus, isolates
of C. neoformans var grubii (serotype A) are represented by the VNI, VNII, and
VNB (Litvintseva et al., 2006) molecular types, var neoformans (serotype D) is
represented by the VNIV type, and isolates of the AD hybrid serotype are the
VNIII type. Four molecular types are recognized for C. gattii isolates (designated
VGI–VGIV) and further divisions within the molecular types have been identified
APPENDIX A 103
FIGURE A1-1â•‡ Map of the forecasted ecologic niche and region of emergence of C. gattii
Figurepotential,
in British Columbia (BC). The optimal, A1-1.eps and unsuitable ecologic niches of C.
gattii in BC are indicated based on biogeoclimatic
bitmap data for the region (Mak, 2007). Note
that the distribution of human and animal cases and the locations of positive environmental
samples coincide primarily with the optimal ecologic niche. The information on human
and animal cases, and environmental sampling, from Washington (WA) is not included.
(Fraser et al., 2005+; Kidd et al., 2005+; Kidd et al., 2007++). For example, VGII
strains can be further classified into VGIIa and VGIIb subtypes, as well as other
less-well characterized subtypes (Kidd et al., 2004; MacDougall et al., 2007++).
There is currently an intense focus on C. gattii due to the unprecedented
emergence of the VGI, VGIIa, and VGIIb molecular types as primary pathogens
of humans and animals on Vancouver Island in British Columbia (BC) (Kidd
et al., 2004; MacDougall et al., 2007++) (Fig. A1-1). Remarkably, the major-
ity of human cases have occurred in people without recognized immunologic
defects, thus highlighting the unusual pathogenicity of C. gattii relative to C.
neoformans. The purpose of this review is to summarize recent progress in the
investigation of this fascinating emergence with regard to human and animal
exposure, environmental colonization, isolate characterization, and the potential
for further dispersal.
104 FUNGAL DISEASES
Overview of Veterinary and Clinical Aspects

of the Emergence of C. gattii in BC
Animal sentinels played a key role in the study of the emergence of C. gattii
in BC and in particular contributed to our understanding of the range of environ-
mental niches for the pathogen. A single veterinary pathology laboratory handled
clinical specimens from the majority of southern BC veterinary practices, and this
allowed early detection and monitoring of C. gattii in the animal population. In
addition, the BC Provincial Animal Health Branch Laboratory was able to per-
form necropsies on porpoises that were found stranded and dead on Vancouver
Island and nearby islands, and these became index cases (Stephen et al., 2002).
Beginning prior to the first documented human case in 1999 and continuing to the
present, veterinary cases have been diagnosed two to three times more frequently
than human cases (Lester et al., 2004); this disparity is likely an underestimate
given that only those animals seen by a veterinarian are diagnosed and that infec-
tions in wildlife are not considered. The diagnosed cases have primarily been in
companion animals (dogs, cats, and ferrets) but also include other domesticated
species such as llamas, horses, mink, and psittacine birds (Duncan et al., 2006b;
Lester et al., 2004; Stephen et al., 2002). Sampling in the environs of these animal
cases has been particularly productive for identifying sources of C. gattii (Kidd
et al., 2007a++; MacDougal et al., 2007++).
Unlike the colonized koalas of Australia (Krockenberger et al., 2002), no sig-
nificant wild animal host or reservoir has been identified in BC. Limited surveys
of wild animals were performed between 2003 and 2007 with the examination
of necropsy samples of nares, lung, anus or cloacae, and brain for C. gattii. In
two surveys, all fatally injured animals turned into rescue facilities were studied.
In the first study, 91 animals (14 species) were examined, and only two eastern
gray squirrels were positive (Duncan et al., 2006a). In the second study, only one
great blue heron was found to have a pulmonary C. gattii infection of 226 animals
necropsied (Bartlett, unpublished data). Additionally, 18 river otters were trapped
in early spring 2007, but none showed signs of disease or colonization with C.
gattii (Bartlett and Balke, unpublished data). Duncan et al. (2005b) established
sentinel veterinary practices in areas known to have exposure to airborne C. gattii
and found positive C. gattii cultures from nasal swabs of asymptomatic animals
in 4.3% of 94 cats, 1.1% of 280 dogs, and 1.5% of 351 horses. Additionally, six
cats and two dogs were found to have cryptococcal antigen titers of greater than
1:2. Of seven cats and five dogs that were selected from the asymptomatic but
culture- or antigen-positive cohorts and followed over 27 months, only two cats
progressed to clinical disease, suggesting that the majority of animals exposed to
C. gattii may naturally clear the organism (Duncan et al., 2005a).
In the first years of recognition of both the emergence of C. gattii disease
and the stability of the pathogen’s environmental niche, it appeared that all hu-
man and animal cases had some contact with Vancouver Island. MacDougall and
Fyfe (MacDougall and Fyfe, 2006) were able to identify human cases of disease
APPENDIX A 105
with historic travel to Vancouver Island and to determine a likely incubation

period (median 6–7 months) based on isolated exposure. In addition, Hoang
et al. (Hoang et al., 2004) performed a retrospective chart review examining all
cases of cryptococcosis identified between 1997 and 2002 at the largest teach-
ing hospital located on the BC mainland. They discovered that there had been a
sudden increase in cryptococcal cases of all origins (C. neoformans var grubii,
C. n. var neoformans, C. gattii, and C. laurentii), but all C. gattii cases (3/26
charts) reported travel history to Vancouver Island (Hoang et al., 2004). The first
cases of mainland-acquired C. gattii infection were identified in animals (ferret,
llama, and cats) in 2003, and three cases in cats in Washington were reported in
2005. Eight off-island human cases with no travel history to an endemic area
were documented (five in BC and two in Oregon) in 2004 to 2005 (MacDougall
et al., 2007++). Upton et al. (Upton et al., 2007+) recently reported the first con-
firmed human case in Washington presenting in 2006, and the Whatcom County
Public Health Department has now identified four additional cases diagnosed in
2007 (Stern, personal communication). Unlike in BC, cryptococcosis is not yet
a reportable disease in Washington, although public health officials are actively
soliciting case studies. The VGIIa genotype accounted for 78% of the examined
veterinary cases and 87% of the human cases; all off-island veterinary cases to
date had the VGIIa genotype (Bartlett, unpublished data) (MacDougall et al.,
2007++).
Environmental and Dispersal Studies on Vancouver Island

Competing theories have been proposed regarding the origin of C. gattii
on Vancouver Island (eg, recent introduction, long-term colonization, specific
imported vectors). Suffice it to say, the colonization pattern and dispersal of the
organism argues against a one-time introduction to Vancouver Island, particularly
if the timeline extends only to the first animal and human cases (1998–1999).
The first systematic sampling performed on Vancouver Island in 2002 mapped
the colonization of C. gattii along a 200 km north-south and a 40 km east-west
corridor. This study revealed that C. gattii is not homogeneously spread in the
environment, with central Vancouver Island having a higher percentage of colo-
nized trees and higher concentration of the organism in soil. The heterogeneous
pockets of colonization could explain why limited-sampling strategies may miss
the organism. Additionally, even though C. gattii has been found to be perma-
nently colonized in some areas, it appears to be transiently colonized in others.
The permanently colonized sites have yielded C. gattii repeatedly over the last 5
years, although transiently positive results may be due to limits of detection or
failure of the organism to establish true colonization (Kidd et al., 2007a++). As
well, sites that initially appeared to be negative for C. gattii have more recently
yielded positive environmental samples (Bartlett, unpublished data). It has been
shown that in addition to the airborne spread of propagules, wood products,
106 FUNGAL DISEASES
soil, water, vehicles, and shoes can act as dispersal mechanisms for the organ-
ism (Kidd et al., 2007a++). These mechanisms are consistent with the findings
of a veterinary case-control study, where statistically significant risk factors for
disease in cats and dogs related to soil disturbance within 10 km of cases, log-
ging within 10 km, travel to Vancouver Island, or owner hiking within 6 months
of diagnosis (Duncan et al., 2006c). Although limited environmental sampling
in the San Juan Islands, Olympic Peninsula, and Oregon has not yielded C. gat-
tii (Fraser et al., 2006; Kidd et al., 2007b++; Upton et al., 2007+). Kidd et al.
(2007a++,2007b++) reported finding positive environmental samples from is-
lands in the Georgia Strait and in northern Washington.
A rather surprising finding was that co-isolated C. gattii strains are heteroge-
neous. The first isolates distributed to the research community were mostly from
one sampling site (central Vancouver Island) and may have unduly influenced
our thinking about the composition of the BC outbreak strains (Kidd et al.,
2004++; Fraser et al., 2005+; Fraser et al., 2003). In the initial analysis of the C.
gattii isolates from this site, Kidd et al. (Kidd et al., 2004++) used polymerase
chain reaction (PCR)-fingerprinting to demonstrate that 5% represented the VGI
molecular type and 95% belonged to VGII (90% of these were VGIIa and 10%
were VGIIb based upon a one polymorphic band in the PCR-fingerprint profiles).
Subsequent work revealed that the composition of the C. gattii population var-
ies in different regions where detailed molecular subtyping of isolates has been
undertaken. In the southern extreme of Vancouver Island, VGIIa accounts for
91% of the isolates and the remainder are VGIIb, whereas at another site VGIIa
accounts for only 66% of the isolates, with VGIIb and VGI at 19% and 15%,
respectively (Bartlett and Kidd, unpublished data). Of course, the genotype
frequencies are likely to be dynamic, and repeated sampling is important. Also,
additional diagnostic tools sensitive enough to detect and differentiate isolates
directly in environmental samples (eg, PCR on soil samples) would facilitate a
better understanding of the population structure and mechanisms of spread of the
organism. Already heightened awareness of changing ecologic niches has resulted
in an expansion of knowledge of the environmental origins of other cryptococcal
species (Filion et al., 2006).
Molecular Characterization of Isolates from BC and the Pacific Northwest

Following the initial analyses of genotype frequency described above, Kidd
et al. (2005+) used multilocus sequence typing (MSLT) and gene genealogy
analyses with four genes to examine patterns of molecular variation as well
as population structure of the isolates from Vancouver Island compared with a
worldwide sample of C. gattii strains. This work demonstrated that the VGIIa
and VGIIb genotypes originally established by PCR-fingerprinting (Kidd et al.,
2004++) corresponded to specific MLST profiles. Similar MLST results with
additional genes were obtained by Fraser et al. (Fraser et al., 2005+). Of specific
APPENDIX A 107
interest from these studies was the identification of isolates from other areas of
the world with identical or similar genotypes to the VGIIa (as represented by
isolate A1MR265) and VGIIb (represented by isolate A1MR272) strains from
Vancouver Island. For example, the VGIIa genotype was also shared by the
NIH444 strain (from a patient in Seattle, ca 1971), CBS7750 (from a Eucalyp-
tus tree in San Francisco, ca 1990) and with isolates from other parts of North
America (KB10455 and KB9944) (Fraser et al., 2005+; Kidd et al., 2005+). A
Brazilian clinical isolate, ICB107, differed from the VGIIa genotype at only one
of 22 loci (Fraser et al., 2005+). The VGIIb genotype was also observed among
environmental isolates from Australia (eg, Ram002, Ram005, WM1008), clini-
cal isolates from Australia (eg, NT-6, NT-13), as well as a clinical isolate from
Thailand (MC-S-115) (Fraser et al., 2005+; Kidd et al., 2005+). A Caribbean
strain 99/473 of the VGIIb type was also found to differ at only one of 22 loci
(Fraser et al., 2005+). Intriguingly, two isolates from human cases in Oregon
(2004) were recently found to represent subtypes within the VGII genotype that
have not identified among any other strains to date (MacDougall et al., 2007++).
The VGIIa and VGIIb isolates from Vancouver Island have been obtained
from both clinical and environmental sources. However, the situation is more
complex for strains of the VGI genotype from clinical and environmental sources.
Specifically, Kidd et al. (2005+) characterized six VGI isolates from Vancouver
Island and identified four different genotypes by MLST analysis. Two of these
were environmental isolates with a different genotype from the clinical isolates.
Thus, in contrast to the VGII types, it was not possible to establish an epidemio-
logic link between environmental and clinical isolates of the VGI type. However,
recent analysis of further environmental VGI isolates from Vancouver Island
indicated that they were highly similar to a porpoise isolate (A1MF2863), being
identical at four MLST loci (Kidd and Bartlett, unpublished data). It is possible
that the clinical isolates of the VGI type represent strains acquired during travel
outside of Vancouver Island.
Overall, Kidd et al. (2005+) found that the Vancouver Island isolates were
part of a predominately clonal population with little evidence of sexual recom-
bination occurring between them. Fraser et al. (2005+) also presented evidence
that the VGIIa and VGIIb strains from Vancouver Island were related in that they
shared 14 identical loci out of the 30 examined and proposed that the genotypes
represent either siblings arising from a past mating event, or that one may be the
parent of the other, perhaps as the result of same-sex mating between MATα par-
ents. Selected isolates from Vancouver Island and other parts of the world have
been tested for mating competence. These studies revealed that the VGII isolates
are generally fertile whereas VGI strains are not (Campbell et al., 2005; Fraser
et al., 2003; Kidd et al., 2004++). In general, the ability of C. gattii isolates to
mate has implications for recombination events that might generate strains with
different virulence properties and environmental adaptability.
108 FUNGAL DISEASES
The Global Distribution of C. gattii

Prior to the emergence of C. gattii on Vancouver Island, it was commonly
accepted that this species was restricted to tropical and subtropical regions of
the world, and that infection was associated with exposure to Eucalyptus trees
(Ellis and Pfeiffer, 1990; Kwon-Chung and Bennett, 1984; Sorrell et al., 1996).
The idea of a limited geographic distribution came from a study that surveyed a
worldwide collection of clinical isolates (Kwon-Chung and Bennett, 1984). This
survey revealed that C. gattii was prevalent only in regions with tropical and
subtropical climates (22%–50% of isolates) relative to C. neoformans (50%–71%
of isolates). However, this study also reported that 13% of the strains from North
America, and 3.3% of the strains from Europe were C. gattii (without reference to
travel histories). More recent surveys have focused on identifying the molecular
types of C. gattii found in collections from various regions. In this regard, VGI
appears to be the most widely distributed type worldwide (Kidd, 2003; Meyer
et al., 2003), and this type is also found most frequently among clinical and en-
vironmental isolates in Australia (Campbell et al., 2005). Strains of the VGII type
are also found in parts of Australia as well as in North and South America (Fraser
et al., 2005+; Kidd, 2003; Kidd et al., 2004++; Kidd et al., 2005+; Meyer et al.,
2003). In a recent, large-scale study of IberoAmerican isolates, VGIII predomi-
nated, and this type has also been found in India and the United States (Kidd,
2003; Meyer et al., 2003). The VGIV type has been found in Central America
and South Africa (Kidd, 2003; Meyer et al., 2003). Notably, the VGIII and VGIV
types were not found in the collections from Vancouver Island suggesting that
these genotypes may have a more limited distribution.
More recently, Meyer et al. (2007) have surveyed 160 VGII strains recov-
ered globally since 1986 using PCR-fingerprinting, amplified fragment length
polymorphism analysis and MLST with eight loci. This work revealed that the
VGIIa genotype from Vancouver Island is also found among Brazilian isolates
and that Colombian isolates are closely related. Interestingly, the majority of the
latter isolates are mating type a in contrast to mating type α for the Vancouver
Island strains (Escandon et al., 2006), and mating was demonstrated between the
Colombian MATa strains and VGIIa MATα strains from Brazil and Vancouver
Island. This work suggests that the VGIIa genotype was present in South America
as early as 1986 and it sheds additional light on the potential mating interactions
for VGII types of C. gattii that may be relevant for the situation on Vancouver
Island.
Overall, these surveys provide an interesting view that the genotypes of C.
gattii (at least for VGI and VGII) are likely to have a worldwide distribution and
the concomitant potential for permanent colonization of suitable environments.
This view highlights the need for more extensive environmental sampling glob-
ally to generate a detailed picture of genotype frequency over time and location.
The most extensive view is now available from the work on Vancouver Island
and the lessons learned from this work can be applied in other locations (Kidd
APPENDIX A 109
et al., 2007a++), especially with regard to the need for extensive multisource
sampling over many years. The wide distribution of C. gattii genotypes should
also be considered in light of recent reports that infections with this species are
occurring in patients with AIDS (South Africa [Morgan et al., 2006], Southern
California [Chaturvedi et al., 2005a]). Therefore, it will be important to identify
the endemic areas for specific C. gattii genotypes in order to monitor human and
animal disease.
Origin of the C. gattii in BC and the Pacific Northwest:

Aboriginal Species or Landed Immigrant?
It is fun to speculate about the origin of the genotypes on Vancouver Island,
and this activity has consumed much energy in the research community. How-
ever, the extent of global strain dispersal has been demonstrated to be significant
(Kidd et al., 2005+, Xu et al., 2000), making it difficult to accurately determine
a specific origin of any given genotype. It is possible that the species has been a
long-term resident of BC and that changing conditions (eg, climate or land use) or
improved surveillance are responsible for the current level of awareness. Alterna-
tively, it has been suggested that the emergence is due to the recent introduction
of a particularly virulent genotype that may be well adapted to the local condi-
tions such that large numbers of infectious cells are propagated (Fraser et al.,
2005+). Although it may be difficult to garner strong evidence for a given theory,
it is clear that much more information is needed about the C. gattii genotypes
on Vancouver Island and worldwide and about the disease caused by C. gattii in
immunocompetent hosts. Below, we discuss some of the studies that are needed
to generate a more detailed view of C. gattii that may help in infection control.
Ecologic adaptability, colonization, and dispersal

The environmental sampling revealed a high level of soil colonization on
Vancouver Island, and it would be interesting to examine soil persistence and
competition in laboratory and field settings. These types of experiments may be
relevant to addressing how the fungus becomes aerosolized and the nature of the
infectious particle. An investigation of conditions required for the propagation of
the infectious particles in soil/trees would also be highly relevant to understand-
ing the factors that influence exposure of humans/animals.
It is likely that no one factor can explain the dramatic emergence of C.
gattii on Vancouver Island, and there may be interplay between soil conditions,
temperature, and moisture. Current weather station data are insufficient to ad-
equately describe the microclimates in areas colonized by the pathogen. Climate
oscillations driven by alternating El Niño and La Niña currents have produced
both drier and wetter than normal summer conditions in BC over the last few
decades. Outbreaks of another fungal disease, coccidioidomycosis, have been
110 FUNGAL DISEASES
shown to follow soil disruption in California (Zender and Talamantes, 2006).

Data gathered from the BC environment conclusively show that C. gattii is well
adapted to survive in dry, low nutrient soil and is more likely to be airborne dur-
ing dry summer weather (Kidd et al., 2007a++). The stability of the colonization
of soil and trees at permanently colonized sites suggests that the pathogen can
effectively compete with resident soil microflora. Longer cycles of meteorology
patterns and finer tools of climate measurement will be needed to understand the
complex relationship of microbe, climate, and ecologic niche.
Additional sampling around the world is needed to investigate predicted
favorable climate/soil/water conditions that might allow colonization by C. gat-
tii. Mak (2007) has recently developed ecologic niche models that predict the
probable extent of environmental colonization of C. gattii based on human, ani-
mal, and environmental data and climate projections for the Pacific Northwest
(Fig. A1-1). Areas that may eventually be impacted include the Lower Mainland
of BC with a population base of approximately 2 million people. These projec-
tions could be used by public health officials on both sides of the US-Canada
border to plan strategies for risk communication and anticipated morbidity and
mortality (Mak, 2007).
Clinical considerations
Perhaps the most relevant topics regarding the emergence of C. gattii have to
do with identifying risk factors for people, designing ways to limit exposure, and
developing effective methods to treat the infections that do occur. It is common
to see statements in the literature that C. gattii is a primary pathogen that infects
immunocompetent people, and that C. neoformans is an opportunistic pathogen
that infects immunocompromised people. The distinction may be less clear given
that C. gattii is now being found in AIDS patients and C. neoformans can infect
seemingly immunocompetent people (Chaturvedi et al., 2005a; Morgan et al.,
2006; Speed and Dunt, 1995). There is clearly a need for retrospective studies
of patients to determine host risk factors as well as prospective case studies to
determine efficacy of treatments. The number of cases continuing to occur on
Vancouver Island (and among tourists [Lindberg et al., 2007]) would allow this
type of investigation.
An interesting consideration in terms of exploring possible virulence differ-
ences for C. gattii versus C. neoformans is whether mouse virulence studies have
relevance for human disease. For example, the strains with the VGIIa and VGIIb
genotypes from Vancouver Island both cause disease in humans, but laboratory
studies revealed virulence differences between the two strains tested (Fraser et al.,
2005+). The more virulent strain, A1MR265, of the VGIIa genotype showed
equal virulence in the mouse model to strain H99 that is representative of the
most common VNI type of C. neoformans (var grubii). It is possible that these re-
sults reflect the fact that only one isolate of each genotype from Vancouver Island
APPENDIX A 111
was tested and the isolates selected may not be representative. It is clear, however,
that strains of C. gattii show virulence differences (Kronstad, unpublished data)
(Chaturvedi et al., 2005b; Fraser et al., 2005+) and that multiple isolates from
Vancouver Island and worldwide collections need to be tested. The same is true
for C. neoformans as demonstrated by the range of virulence detected by Clancy
et al. (2006). Thus, we need to develop better models to assess differences in
virulence and to explore possible differences that may be relevant to infection of
immunocompetent versus immunocompromised hosts.
Applications of genomic approaches to develop a detailed understanding of

C. gattii
The emergence of C. gattii provided the impetus to sequence the genomes
of isolates representing the VGI (WM276) and VGIIa (A1MR265) genotypes
(Michael Smith Genome Sciences Center, 2007++; The Broad Institute, 2007++).
These are important resources for the next steps in characterizing the virulence
of C. gattii, the genetic diversity of the species and the interactions of the fungus
with the environment. One can imagine, for example, using the genomes for
transcriptome and proteome studies to identify differences in expression for C.
gattii relative to C. neoformans. Some of these differences may reveal factors
that contribute to the primary pathogenesis of C. gattii relative to C. neoformans.
The two C. gattii genomes also provide a platform for more detailed analyses
of genotypes and comparative studies of genome variability. In the latter case,
comparative hybridization or genome resequencing approaches can be used to
study the microevolution of genomes in strains in the environment and clinical
strains during passage through human and animal hosts (eg, during relapse or
drug therapy). Comparative genome hybridization experiments with the VGI and
VGIIa genomes have been initiated to identify genomic changes in mutants that
have lost virulence and to examine genome variation in strains representing the
VGI, VGIIa, and VGIIb genotypes (Kronstad, unpublished data). The declining
cost of sequencing will also allow further genome-sequencing projects to provide
a deeper view of genome content and variability. The more detailed information
may eventually lead to the separation of the molecular types of C. gattii into
distinct varieties or species.
Media Coverage of the Emergence of C. gattii

Any emerging infectious disease represents a challenge to the public health
system. The system must respond to educate caregivers about appropriate in-
terventions while balancing the message to allow the public to make informed
choices. For example, the lay press recently reported concern by members of the
public in Alabama where experimental plots of genetically engineered Eucalyptus
trees will be grown; the fear being that C. gattii will be imported into the environ-
112 FUNGAL DISEASES
ment through the Eucalypts (United Press International, 2007), even though no
link to Eucalyptus was shown in the BC experience (Kidd et al., 2007a++). In
an examination of press coverage of C. gattii as an emerging infectious disease
agent, researchers at the University of BC Centre for Health and Environment
Research found that during the period 2001 to 2006, BC newspapers carried 422
articles warning the public about West Nile Virus (although no West Nile Virus
cases have been reported in BC) compared with 79 articles about C. gattii (170
human cases, eight deaths) (Nicol et al., unpublished data). The research group
concluded that because West Nile Virus is a public health risk with identifiable
precautionary actions in central Canada, newspapers were more likely to print
stock West Nile Virus stories. C. gattii was seen to be a local phenomenon with no
identifiable risk aversion strategies and to have potential economic repercussions
to the areas affected and so was less reported. There also seemed to be confusion
by news writers about the biology of Cryptococcus because the term “virus”
seems to be better understood as a pathogen compared to “yeast” (Nicol et al., un-
published data). Similarly, some news items labeled C. gattii as an “Australian”
fungus despite the body of literature cited above on the global distribution of the
pathogen. Overall, these observations demonstrate that effective education of the
media and the public is a critical component of the management of an emerging
infectious disease.
Conclusions
A great deal has been learned about the emergence of C. gattii in BC over
the past 8 years. We now have a clear picture of the environmental sources of
the pathogen and mechanisms of dispersal, we have an understanding of the
genotypes that are causing disease in humans and animals, and we have some
information about clinical presentation and treatment. Certainly, there is a great
deal more to investigate in terms of risk factors for the human population and
treatment outcomes. In this regard, the situation on Vancouver Island presents an
opportunity to develop a detailed view of an emerging infectious disease with
regard to environmental exposure, the role of sentinel animals in monitoring risk,
and the underlying factors that influence human susceptibility. This information
may prove useful for other emerging diseases and provide methods to manage
both the ongoing situation in BC and the apparent emergence of the disease in
the Pacific Northwest.
Acknowledgments
The authors thank the members of the BC Cryptococcal Working Group
(http://www.cher.ubc.ca/cryptococcus/) and the BC Centre for Disease Control
(http://www. bccdc.org/) for helpful discussions and Sunny Mak for the prepa-
ration of Figure A1-1. The authors are supported in part by grants from the US
APPENDIX A 113
National Institute of Allergy and Infectious Disease (Dr. Kronstad, award RO1-
AI-053721), the Canadian Institutes of Health Research (Drs. Kronstad and
Bartlett), British Columbia Lung Association (Dr. Bartlett), and WorkSafe BC
(Dr. Bartlett). Dr. Kronstad is a Burroughs Wellcome Fund Scholar in Molecular
Pathogenic Mycology, and Dr. Bartlett is a Michael Smith Foundation for Health
Research Scholar.
References and Recommended Reading

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114 FUNGAL DISEASES
+ Fraser JA, Giles SS, Wenink EC, et al.: Same-sex mating and the origin of the Vancouver Island
Cryptococcus gattii outbreak. Nature 2005, 437:1360–1364.
An extensive MLST analysis of C. gattii isolates from Vancouver Island and from around the
world. The authors found shared genotypes between the VGIIa and VGIIb strains from BC and
strains of these molecular types from other parts of the world. This study presents interesting
hypotheses about the origin of the VGIIa genotype in BC and reports the first virulence tests of
VGIIa and VGIIb strains from Vancouver Island.
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no evidence for the spread of the Vancouver Island Cryptococcus gattii outbreak to this locale.
FEMS Yeast Res 2006, 6:620–624.
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fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancou-
ver Island, Canada. Eukaryot Cell 2003, 2:1036–1045.
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and Health Sciences Centre (1997–2002): epidemiology, microbiology and histopathology. J
Med Microbiol 2004, 53:935–940.
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within the Cryptococcus neoformans complex [PhD thesis]. Sydney: University of Sydney;
2003.
++ Kidd SE, Chow Y, Mak S, et al.: Characterization of environmental sources of the human and
animal pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest
of the United States. Appl Environ Microbiol 2007a, 73:1433–1443.
This important study describes a systematic and thorough investigation of the environmental
colonization of C. gattii on Vancouver Island and the Pacific Northwest. Key findings include
the isolation of the pathogen from air, trees, soil, freshwater, and seawater, and the identification
of colonization hotspots. Additionally, this study identified characteristics of soil that may favor
C. gattii colonization.
++ Kidd SE, Bach PJ, Hingston AO, et al.: Cryptococcus gattii dispersal mechanisms, British Colum-
bia, Canada. Emerg Infect Dis 2007b, 13:51–57.
This study employed systematic environmental sampling strategies to document patterns of C.
gattii colonization on Vancouver Island and to obtain evidence for human-mediated dispersal
of the fungus.
+ Kidd SE, Guo H, Bartlett KH, et al.: Comparative gene genealogies indicate that two clonal lineages
of Cryptococcus gattii in British Columbia resemble strains from other geographical areas.
Eukaryot Cell 2005, 4:1629–1638.
This study employed MLST analysis and gene genealogy to reveal a predominantly clonal popu-
lation among the Vancouver Island isolates and to demonstrate that the genotypes of isolates
from BC resembled those of strains from other parts of the world.
++ Kidd SE, Hagen F, Tscharke RL, et al.: A rare genotype of Cryptococcus gattii caused the cryp-
tococcosis outbreak on Vancouver Island (British Columbia, Canada). Proc Natl Acad Sci USA
2004, 101:17258–17263.
This paper describes the results of the first marshaling of the expertise of the international
research community to tackle the analysis of the emergence of C. gattii in BC. The investiga-
tors described initial studies on the environmental source of the pathogen and identified the
molecular types of C. gattii that were responsible for the human and animal cases.
Krockenberger MB, Canfield PJ, Malik R: Cryptococcus neoformans in the koala (Phascolarctos
cinereus): colonization by C n var gattii and investigation of environmental sources. Med Mycol
2002, 40:263–272.
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neoformans. Am J Epidemiol 1984, 120:123–130.
APPENDIX A 115
Kwon-Chung KJ, Boekhout T, Fell JW, Diaz M: (1557) Proposal to conserve the name Cryptococcus
gattii against C. hondurianus and C. bacillisporus (Basidiomycota, Hymenomycetes, Tremel-
lomycetidae). Taxon 2002, 51:804–806.
Kwon-Chung KJ, Varma A: Do major species concepts support one, two or more species within
Cryptococcus neoformans? FEMS Yeast Res 2006, 6:574–587.
Lester SJ, Kowalewich NJ, Bartlett KH, et al.: Clinicopathologic features of an unusual outbreak of
cryptococcosis in dogs, cats, ferrets, and a bird: 38 cases (January to July 2003). J Am Vet Med
Assoc 2004, 225:1716–1722.
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Canada. Emerg Infect Dis 2007, 13:178–179.
Litvintseva AP, Thakur R, Vilgalys R, Mitchell TG: Multilocus sequence typing reveals three genetic
subpopulations of Cryptococcus neoformans var grubii (serotype A) including a unique popula-
tion in Botswana. Genetics 2006, 172:2223–2238.
MacDougall L, Fyfe M: Emergence of Cryptococcus gattii in a novel environment provides clues to
its incubation period. J Clin Microbiol 2006, 44:1851–1852.
++ MacDougall L, Kidd SE, Galanis E, et al.: Spread of Cryptococcus gattii in British Columbia,
Canada, and detection in the Pacific Northwest, USA. Emerg Infect Dis 2007, 13:42–50.
This paper describes the detection of C. gattii in three people and eight animals without a travel
history to Vancouver Island, and the detection of the pathogen in air, soil, water and on trees
from sites off the island. The study also reported locally acquired C. gattii infections in three
cats in Washington and two people in Oregon; interestingly, the genotypes of the strains from
the Oregon cases were VGIIa- and VGIIb-like, but MLST results indicated differences from the
isolates of the corresponding subtypes from Vancouver Island.
Mak S: Ecological niche modeling of Cryptococcus gattii in British Columbia [MSc thesis]. Vancou-
ver: University of British Columbia; 2007.
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mans isolates. Emerg Infect Dis 2003, 9:189–195.
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isolates traces the origin of the Vancouver Island outbreak to Latin America [abstract]. Presented
at the 24th Fungal Genetics Conference. Pacific Grove, CA; March 20–25, 2007.
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tococcus neoformans var neoformans by PCR-fingerprinting and RAPD—a pilot study to
standardize techniques on which to base a detailed epidemiological survey. Electrophoresis
1999, 20:1790–1799.
++ Michael Smith Genome Sciences Center: Cryptococcus Neoformans Summary. http://www.bcgsc.
ca/project/cryptococcus/summary/. Accessed July 9, 2007.
The sequences of the genomes of VGI and VGIIa strains are exceptional resources for detailed
investigations of the virulence properties of C. gattii. In addition, the sequences allow genome-
wide comparative studies with the genomes of C. neoformans var neoformans strains and a var
grubii strain.
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ology of cases identified in a South African province with high HIV seroprevalence, 2002–2004.
Clin Infect Dis 2006, 43:1077–1080.
Sorrell TC, Brownlee AG, Ruma P, et al.: Natural environmental sources of Cryptococcus neoformans
var gattii. J Clin Microbiol 1996, 34:1261–1263.
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coccus neoformans. Clin Infect Dis 1995, 21:28–34.
Stephen C, Lester S, Black W, et al.: Multispecies outbreak of cryptococcosis on southern Vancouver
Island, British Columbia. Can Vet J 2002, 43:792–794.
++ The Broad Institute: Cryptococcus neoformans Serotype B Database. http://www.broad.mit.edu/
annotation/genome/ cryptococcus_neoformans_b. Accessed July 9, 2007.
116 FUNGAL DISEASES
The sequences of the genomes of VGI and VGIIa strains are exceptional resources for de-
tailed investigations of the virulence properties of C. gattii. The genome sequence of a C.
neoformans var grubii strain is also available at the Broad Institute.United Press International:
GE eucalyptus tree investigation urged. http://www.sciencedaily.com/upi/index.php?feed=Sci
ence&article=UPI-1-20070614-13565200-bc-us-eucalyptus.xml. Accessed June 17, 2007.
+ Upton A, Fraser JA, Kidd SE, et al.: First contemporary case of human infection with Cryptococcus
gattii in Puget Sound: evidence for spread of the Vancouver Island outbreak. J Clin Microbiol
2007, In press.
This report and MacDougall et al. (2007) document the recent emergence of C. gattii outside
of BC.
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human pathogenic fungus Cryptococcus neoformans. Mol Ecol 2000, 9:1471–1481.
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J Biometeorol 2006, 50:174–182.
A2
THE GOOD, THE BAD, AND THE UGLY:

FUNGI MOLD YOUR WORLD
Meredith Blackwell3
Fungi4 are important members of many ecosystems. As heterotrophs they

are involved in nutrient cycles, especially of carbon, nitrogen, and phosphorus.
The effects of fungi were observed in prehistoric times, and their part in causing
plant disease was understood before the germ theory was advanced. Today fungi
are featured in the popular press and science Internet postings, indicating that
they are of increasing interest and importance. Molecular methods have helped to
popularize fungi by bringing rapid progress to fungal classification and discovery
and have enhanced understanding of their biology. Fungi are associates of all
major groups of organisms and are especially well known for their interactions
with plants and insects. Fungi also are economically important and provide drugs,
foods, and fermented beverages. The value of fungal activities and products far
exceeds the costs of the diseases they cause.
Introduction
Human beings were aware of fungal fruiting bodies in prehistoric times, and
the sudden appearance of mushrooms after rain awed those who did not com-
prehend the fungus lifecycle. Lowy (1974) wrote that the sudden appearance of
mushrooms of Amanita muscaria was believed to have been caused by thunder-
3â•›â•›Louisiana
State University.
4â•›â•›In
addition to members of Kingdom Fungi, several other organisms of the fungus-like group
Oomycota (Phytophthora) are included.
APPENDIX A 117
bolts as they struck the ground, a belief held independently in Roman, Hindu, and
Mayan cultures. Humans endowed mushrooms with magical properties (Wasson,
1968), and evidence of early fungal use exists in many parts of the world. Grave
guardians, masks, clothing ornaments, and other artifacts were made from the
fruiting bodies of wood-decaying basidiomycetes such as Fomitopsis officinalis
and Haploporus odorus (Blanchette, 1997; Blanchette et al., 1992a, 2002). A
surviving mushroom culture centered on magic mushrooms existed in Oaxaca
for many years, and the celebrated curandera, Maria Sabina, was visited by a
number of prominent individuals and notable musicians who sought her spiritual
guidance (Wasson, 1957, 1976). Although yeasts themselves were not known,
evidence of their activity comes from residues in nine millennia-old Neolithic
vessels (Vouillamoz et al., 2006).
Plant pathogenic fungi also were known in ancient times. Three centuries
BC, Theophrastus recognized fungi as the cause of certain diseases of crops,
but by the first century, the knowledge had been lost, and Pliny attributed lost
yields to the gods or stars (Carefoot and Sprott, 1969). Fungal effects such as
disease were not understood by many until the observations and experiments of
Miles Joseph Berkeley and Anton de Bary around the time of the Irish potato
famine of 1845–1846. This work actually came before the general acceptance
of the germ theory. The contribution of de Bary also argued strongly against
a lingering belief in spontaneous generation (Matta, 2010). Fungi continue to
appear suddenly as they invade natural landscapes to cause diseases of plants
and animals. The invading organisms often are not noticed until they encounter
naïve hosts in new regions where they cause devastating diseases. Earlier inva-
sions included the chestnut blight fungus and several waves of Dutch elm disease
fungi (Alexopoulos et al., 1996). The papers in this volume, Fungal Diseases: An
Emerging Threat to Human, Animal, and Plant Health (IOM, 2011), cover the
newest waves of invasive fungal diseases and their attack on naïve hosts.
More important, however, is the realization that fungi are essential for life on
Earth. Fungi are decomposers that destroy plant and animal bodies and return car-
bon, nitrogen, phosphorus, and other minerals to nutrient cycles. Compatible with
their primary role in decomposition, fungi interact with other living organisms
in nutritional relationships, and their secondary metabolites and enzymes sup-
ply medicines, food and drink, and industrial products for profitable enterprises.
Fungi appear regularly in newspapers and magazines. Over the past year, the
New York Times featured fungi prominently. Articles have included reports of the
identification of a microsporidian fungus partly responsible for colony collapse
disorder of bees, a chytrid responsible for global amphibian decline, Geomyces
destructans of bats, and pathogens of home garden vegetables. Ecological topics
included interactions between bark beetles and fungal symbionts, mycorrhizal as-
sociations, sexual reproduction in truffles, a fungus that exerts selective pressure
on rotifers, and fungal function in the environment. Fungi also have been covered
in the Wall Street Journal as food items, inhabitants of saunas, and the “Torula
118 FUNGAL DISEASES
of Cognac,” Baudoinia compniacensis, the fungus that grows on walls of wine

cellars in mists of alcoholic vapors. One fungus was reported widely because it
prompted a murder investigation in a German forest when its sulfurous odor of
decay was mistaken for that of a dead body (Anonymous, 2005). Coverage of
a broad range of fungal topics also can be found in science blogs and Internet
postings with reports of jet lag expressed in circadian rhythms of fungi; wood
decay; the evolutionary arms race between a smut fungus and maize; a new spe-
cies of introduced, beetle-associated fungus that kills plants in the Lauraceae;
and yeast genome sequencing leading to improved bioethanol production. Na-
tional Geographic News also reviews interesting fungal topics, including stories
on endophyte biology and “bringing order to the fungus among us,” describing
the Assembling the Fungal Tree of Life project (see below). Only 2 days before
this meeting (December 12, 2010), USA Today published an article by Elizabeth
Weise, “Why it’s cool to have a fungus among us.” The informative article could
have been the basis for this talk—if only it had appeared earlier. The range of
examples cited indicates a growing interest in and knowledge of fungi.
Fungi influence our daily lives in ways we seldom appreciate. Several en-
trenched fungal-influenced cultural practices are the result of fungal plant dis-
eases. These include tea drinking in the United Kingdom, a switch imposed by
devastation of coffee plants in Ceylon (present-day Sri Lanka) by the coffee
rust fungus (Hemileia vastatrix) in the late 1800s (Horsfall and Cowling, 1978);
consumption of cornbread as a staple in the southern United States colonies was
imposed because the wheat rust fungus prevented wheat cultivation in the humid
South (Horsfall, 1958); and the enjoyment of gingerbread comes from the time
when the effects of stinking smut of wheat were masked by molasses and ginger
(Carefoot and Sprott, 1969). We rely on fungi for clothing fads such as use of
cellulase enzymes of species of Trichoderma to speed the “stone washing” of our
blue jeans (Bhat, 2000). Perhaps, fungi may make us more beautiful when certain
“integrative approaches to better skin”5 are followed using a blend of fungi that
includes Cordyceps, reishi mushrooms, and other ingredients.
The Classification and Discovery of New Species of Fungi

Early phylogenetic studies based on DNA sequences defined a monophy-
letic6 group of Fungi. Oomycota and relatives, various slime mold clades, and
several other groups previously considered as zygomycetes have been excluded
from the monophyletic Fungi. Asexual and sexual fungi could be combined on the
basis of their genetic relationships, and asexual groupings of asexual fungi were
abandoned (Alexopoulos et al., 1996). More recently, mycologists have increased
5â•›â•›Several brands of skin creams include a variety of basidiomycete fruiting bodies as ingredients
that are said to provide for skin relief and other effects (e.g., Dr. Weil’s Mega-Mushroom lotions,
cleansers, and serums).
6â•›â•›A group of taxa containing an ancestor and all its descendants.
APPENDIX A 119
the number of DNA markers and taxa in diverse clades to produce increasingly
well-resolved phylogenies,7 the basis of predictive classifications (Figure A2-1)
(Hibbett et al., 2007; James et al., 2006; White et al., 2006). An issue of the jour-
nal Mycologia (98:829–1103, 2006) was devoted to the phylogenetics of many
major groups of fungi. Recent phylogenetic studies have provided new insights
into fungal relationships and show that the earliest diverging lineages of zoo-
sporic8 and zygosporic9 groups are not monophyletic as previously assumed on
the basis of morphological characters and that they are more diverse than previ-
ously understood. Other findings provide data to include microsporidia within or
very near the fungi (Lee et al., 2010). The new phylogenetic studies are largely
the result of several National Science Foundation projects (Research Coordina-
tion Networks: A Phylogeny for Kingdom Fungi [Deep Hypha] and Assembling
the Fungal Tree of Life 1 and 2) that involved more than 100 biologists from
about 20 countries (Blackwell et al., 2006; Hibbett et al., 2007). Current projects
under way include adding taxa to expand the fungal tree of life and pursuing an
increasing number of genomics projects.
About 100,000 species of fungi have been described, but a conservative
estimate suggests that there are 1.5 million fungi on Earth (Hawksworth, 1991,
2001). The estimate has spurred exploration for the million fungi that remain
undiscovered (Figures A2-2A through A2-2D).
More recently the 1.5 million estimate was surpassed by a higher estimate of
3.1 to 5.1 million species based on the use of molecular methods, including high-
throughput sequencing (O’Brien et al., 2005). Because of the great discrepancy
between known and estimated fungal species numbers, mycologists have a re-
newed interest in fungal discovery. Many have wondered, where are the missing
fungi (Hyde, 2001)? If the higher estimates are realistic, the number of fungi is
equal to the number of animal species and may exceed the number of plants by
10:1. Abundant evidence shows that many tropical fungi remain to be discovered
based on species accumulation curves of fungi collected in plots (Aime et al.,
2010). Other habitats reporting large numbers of fungi include living leaves of
tropical trees (Arnold, 2007), soil fungi (O’Brien et al., 2005; Taylor et al., 2010),
and even the fungi in the buildings in which we spend most of our time (Amend
et al., 2010). Many fungi, however, remain to be discovered in northern temperate
regions, including far northeastern Asia (Petersen and Hughes, 2007). We do not
have to look for undescribed fungi in completely new places or tropical regions,
however, because they may be in our backyards. My colleagues and I look for
new species among the yeasts and other microscopic fungi that are difficult to see
7â•›â•›A phylogeny is an inferred history of evolutionary relationships of organisms; often depicted in
a tree diagram.
8â•›â•›Zoospores are flagellated cells of certain fungi (see Figure A2-1) that are produced in sporangia
in asexual reproduction.
9â•›â•›Zygospores are thick-walled spores produced in some fungi (see Figure A2-1) resulting from the
fusion of like gametes.

120 FUNGAL DISEASES
Ascomycota (64 056 species)

Taphrinomycotina ex. Schizosaccharomyces (140 spp.)
Saccharomycotina ex. Saccharomyces, Candida (915 spp.)
Pezizomycotina ex. Neurospora (63 011 spp.)
Basidiomycota (31 503 species)

Pucciniomycotina ex. Rust fungi (8016 spp.)
Ustilaginomycotina ex. smut fungi (1113 spp.)
Agaricomycotina ex. mushrooms (20951 species)
Glomeromycota ex AM fungi (169 species)
“Zygomycetes”
Mucoromycotina ex. Mucor (327 species)
Zoopagomycotina ex. Zoopage (190 species)
Kickxellomycotina (269 species)
Entomophthoromycotina ex. Conidiobolus (277 species)
Olpidium (30 species)

Blastocladiomycota ex. Allomyces (179 species)
Chytridiomycota
Chytridiomycetales ex. Batrachochytrium (499 spp.)
(706 species)
Spizellomycetales ex. Rhizophlyctis (27 spp.)
Monoblepharidales ex. Gonapodya (26 spp.)
Neocallimastigomycota ex. Orpinomyces (20

spp.)
Microsporidia ex. Nosema (1300 spp.)
Rozella (22 species + environmental samples)
Animalia ex. humans (1.2 million spp.)
FIGURE A2-1â•‡ Diagrammatic representation of relationships of fungal taxa, examples

(ex.), and approximate number of species in each group. Zoosporic and zygosporic fungi
are more diverse than previously recognized
Figure on the basis of morphological traits, and
A2-1.eps
they are not monophyletic. Two flagellated taxa, Rozella and Olpidium, are of uncertain
taxonomic placement. Evidence from multilocus sequencing and genomics reveals that
microsporidians branch within or near fungi. Ascomycota and Basidiomycota, the most
speciose phyla, are each divided into three subphyla. The largest number of fungal species
are classified in the subphyla Pezizomycotina and Agaricomycotina.
APPENDIX A 121
FIGURE A2-2â•‡ Images of representative fungal groups. (A) Hyphae of the blastocladia-
Figure
lean fungus, Allomyces sp. Note A2-2A-D.eps
a terminal zoosporangium (ZS) containing zoospores. The
spiny, dark, thick-walled resting sporesbitmap
(RS) within the hyphae are those of a zoosporic
fungal parasite, Rozella allomycis, of uncertain taxonomic placement. Bar = 10 µm. (B)
Lobosporangium transversale. The zygosporic fungus in the Mortierellales has unusual
spiny lobed sporangia (Benny and Blackwell, 2004). Bar = 50 µm. (C) Sarcoscypha coc-
cinea. The several cm diameter fruiting body of the scarlet cup ascomycete, Sarcoscypha
coccinea. Ascospores are formed within asci on the inner surface of the cup. (D) Mutinus
sp. A stinkhorn similar to one mentioned in the text that caused a search for a dead body
(Anonymous, 2005). Stinkhorns produce noxious compounds that attract insect spore
dispersers. The dark slimy mass of spores has been partially removed by flies.
SOURCES: (A) photo courtesy of Timothy Y. James, provided by Meredith Blackwell
(2009). (B) micrograph courtesy of Kerry O’Donnell, provided by Meredith Blackwell
(2004). (C) photo courtesy of eriotropus/coqui, provided by Meredith Blackwell (2002).
(D) photo courtesy of Nhu H. Nguyen, provided by Meredith Blackwell (2005).
122 FUNGAL DISEASES
with the unaided eye (Boekhout, 2005; Suh et al., 2005), and members of early
diverging lineages that often are difficult to isolate and culture. Ascomycetes and
basidiomycetes are expected to provide the greatest diversity of additional taxa
based on numbers of currently known fungi, but certainly the developing methods
using high-throughput sequencing of DNA will lead to the discovery of more of
the early diverging groups (Figure A2-1) (Kirk et al., 2008).
Examples of large numbers of species isolated into culture from certain sub-
strates include the finding of 418 unique morphotypes of endophytic fungi from
83 leaves in Panama (Arnold, 2007), 257 fungal endophyte genotypes in coffee
plants (Vega et al., 2010), and 650 yeast isolates representing 290 genotypes of
nearly 200 undescribed taxa from the gut of beetles (Suh et al., 2005). Acquiring
cultures and specimens will remain important in cases when fungi and cultures
are needed for certain purposes, including population studies, environmental
remediation, and secondary metabolites. Taylor and his colleagues (2010) used
high-throughput sequencing to estimate the presence of more than 200 taxa in a
0.25 g soil sample with only 14 percent overlap in taxa in a sample taken a meter
away. If we are to determine the number of fungi on Earth, environmental se-
quencing will be necessary to speed fungal exploration and discovery. In addition
to new species, entire lineages, some probably at the level of subphylum, may be
recognized by DNA sequences such as Soil Clone Group 1 (Porter et al., 2008;
Rosling et al., 2010). More work will be needed to determine geographical and
substrate ranges in order to obtain more accurate estimates of species numbers.
Species discovery is relevant to the topic of this workshop because previ-
ously unknown plant and animal pathogenic fungi have been introduced into the
United States many times. These fungi probably caused few symptoms and went
unnoticed in their native hosts. Devastation of naïve hosts, however, led to their
recognition and subsequent description as new species. This scenario certainly
is repeated by the fungi discussed in this meeting, including Batrachochytrium
dendrobatidis, the pathogenic chytrid of amphibians spread around the world;
Geomyces destructans, the pathogen of bats in North America; and Phytophthora
ramorum, causing declines of certain plants in North America and Europe. Prior
invasions have included several fungal agents of Dutch elm disease; the chest-
nut blight fungi; the newly arrived agent of the laurel wilt delivered within the
mycangia10 of its ambrosia beetle vector; and Discula destructans, a pathogen of
North American dogwoods (Alexopoulos et al., 1996; Harrington and Fraedrich,
2010; Zhang and Blackwell, 2001). Recently, a new approach to discovering
the native ranges of certain fungi has been profitable. Ning Zhang (Personal
communication, Rutgers University, December 10, 2010) designed an efficient
assay method using specific primers to detect the dogwood pathogenic fungus in
herbarium specimens. The method promises to greatly reduce the time involved
in determining geographical and host ranges and is ideal for working with col-
10â•›â•›Mycangia
are pouch-like invaginations in the cuticle of certain insects used to transport cells and
spores of symbiotic fungi, found especially in some species of bark and ambrosia beetles as well as
a few other groups of insects.
APPENDIX A 123
laborators at herbaria throughout the world. Because patterns of introduction of

pathogens may exist, determination of native ranges is essential in combating
invasive organisms.
Supermodels
Fungi are important as model systems in research. Saccharomyces cerevisiae
(Figure A2-3) is a supermodel known for its baking and brewing prowess and as
the first eukaryote to have its entire genome sequenced.
FIGURE A2-3â•‡ Saccharomyces cerevisiae (Y-2235), baker’s yeast and model organism.
Figure
Note the many budding cells in the stained A2-3.eps
preparation. Bar = 5 µm.
bitmapprovided by Meredith Blackwell (2008).
SOURCE: Photo courtesy of Cletus P. Kurtzman,
124 FUNGAL DISEASES
In addition to S. cerevisiae, three other fungi that have been important in

research and were the subjects of Nobel Prize–winning research are Schizosac-
charomyces pombii, another fast-growing organism with a yeast growth form;
Penicillium crysosporium, producer of the first effective antibiotic; and Neuros-
pora crassa. In his Nobel Prize acceptance speech, Tatum (1958) acknowledged,
among others, “B.O. Dodge for his establishment of this Ascomycete as a most
suitable organism for genetic studies.” Beadle (1958) also spoke of Neurospora
crassa and pointed out that “Dodge was an enthusiastic supporter of Neurospora
as an organism for genetic work. ‘It’s even better than Drosophila,’ he insisted to
Thomas Hunt Morgan, whose laboratory he often visited. He finally persuaded
Morgan to take a collection of Neurospora cultures with him from Columbia
University to the new Biology Division of the California Institute of Technology,
which he established in 1928.” This was the beginning of the development of
Neurospora crassa in genetics research.
As mentioned above, S. cerevisiae was the first eukaryotic organism to have
its entire genome sequenced. This yeast and other species in the Saccharomy-
cotina have relatively small genomes that make them economical candidates for
sequencing (Mewes et al., 1997). In addition, yeasts and other model fungi are
easy to grow and complete their lifecycles in culture in a few days; because they
are haploid throughout most of their lifecycle, induced mutations are expressed
rapidly. Many fungi, including some yeasts, also have a sexual state from which
all products of meiosis can be isolated in addition to asexual spores and somatic
cells from which uniform populations can be established. They also are excellent
organisms for population studies (Anderson et al., 2010). Some fungi, including
S. cerevisiae, have morphological cues that indicate the occurrence of certain
cell cycle events, and a large body of background information is available for
previously established model fungi studies. Improvements in genome sequencing
have made it possible to develop many new “models,” including plant and animal
pathogens and their hosts. For example, yeasts from the gut of wood-feeding
beetles have been of particular interest because many of them ferment xylose, a
requirement for efficient digestion of lignocellulose in biofuel production. These
species have undergone biochemical and metabolic engineering to obtain more
information on xylose fermentation pathways, and genome sequencing is im-
portant toward this end (Jeffries et al., 2007; Joint Genome Institute, 2007; Van
Vleet and Jeffries, 2009).
Fungi Make Money: Useful Fungal Products

Humans have used a variety of fungal products for different purposes, in-
cluding cures. In fact some of the magical fungi mentioned above also have been
used for their medicinal properties, which may have been known since prehistoric
times. Evidence exists for the use of fungi by early humans. Ötzi the Iceman
lived about 5,300 years ago, and his mummified body was discovered in 1991
APPENDIX A 125
on the border of Italy and Austria. He carried pieces of the fruiting bodies from
two species of wood-rotting basidiomycetes, Piptoporus betulinus and Fomes
fomentarius, perhaps for medicinal uses (Peinter et al., 1998). Other writers
have suggested that one of the fruiting bodies was used as a strop for sharpening
knives and tools, but whatever their use, fungi appear to have been important to
Copper Age Europeans.
Some basidiomycetes have been used medicinally in more recent times.
Extracts of Inonotus obliquus was used in Europe as a treatment for cancer, and
the fruiting bodies of Fomitopsis officinalis (the quinine conk), mentioned earlier
as grave guardians in the Pacific Northwest, were also harvested for medicinal
properties. A different kind of medicinal use by foresters was the application of
sheets of mycelium on ax injuries to stop bleeding (Gilbertson, 1980). The spore
masses of giant puffballs that were discovered stockpiled along Hadrian’s Wall
(in Northern England) also have been used as a styptic (Personal communication,
Roy Watling, former Head of Mycology and Plant Pathology, Royal Botanic Gar-
den Edinburgh, August 27, 1977), and spores of unspecified puffballs also were
widely used as a styptic by natives of North America as well (Blackwell, 2004).
Certain ascomycete fungi, previously known as species of Cordyceps, have
been used in Asian traditional medicine for several centuries (Spatafora et al.,
2007). One of these fungi, a parasite of caterpillars, known as Cordyceps sinensis
since 1878, now is Ophiocordyceps sinensis based on a phylogenetic study (Sung
et al., 2007). Recent interest in the fungus has provided evidence that it may be
effective in the treatment of certain tumors (Spatafora et al., 2007). The revision
of the entire group of insect–pathogenic fungi previously placed in the genus
Cordyceps has resulted in the placement of species in three different families
(Sung et al., 2007). This is an important development because phylogenies are
predictive of traits common to closely related fungi, and other Ophiocordyceps
species may be targeted for the mining of metabolites. The efforts to develop
penicillin for the treatment of bacterial infections at the beginning of World War
II resulted in the discovery of a long-sought magic bullet and hastened the rise
of the modern pharmaceutical industry. In addition to the fungus-derived drug
penicillin, three statin drugs for lowering cholesterol levels (e.g., Lipitor®) and
the immune suppressant cyclosporine each have earned more than a billion dol-
lars annually. Cyclosporine, once critical to transplant surgery, is today used to
treat dry eye as well as more serious conditions (Blackwell, 2011).
Fungi also are big business in the food and beverage industries. In addition to
the usual fresh fruiting bodies of basidiomycetes (mushrooms) and a few highly
favored ascomycetes (truffles and morels), other fungi, such as cuitlacoche (corn
smut) and rice smut, are eaten in Mexico and Asia, respectively. Processed foods
also are made from fungi. These include yeast extract spreads such as marmite
and vegemite and the meat substitute, Quorn™, a product of hyphae of an as-
comycete, a species of Fusarium. Several species of Aspergillus are used in the
processing of soy sauce, and fungi play a part in the flavoring process of cheeses.
126 FUNGAL DISEASES
Throughout the world many fermented foods rely on fungi at least in part to in-
crease nutritional value, improve texture and flavor, and preserve the foodstuff. In
one short street block in Brussels, I examined shop windows to count the many
products that had been touched by fungi: coffee, certain teas, chocolate, cheeses,
bread, salami and dry-cured hams, and numerous fermented beverages (Tamang
and Fleet, 2009). Many African and Asian foods, including miso, ontjom, and
tempeh, are the products of fermentation (Nout, 2009; Rodríguez Couto and
Sanromán, 2006).
As in the case of other fungal products, the making of alcoholic beverages
almost certainly was discovered millennia ago, found accidently in prehistoric
times when wild yeasts settled into a sugary beverage. Yeasts are essential to
the multibillion-dollar alcoholic beverage industry. In the United States, sales
of beer, spirits, and wine were $116 billion in 2003 (Library Index, 2011). The
yeasts involved in brewing were first isolated into pure culture by Emil Hansen
at the Carlsberg Brewery in Copenhagen, and the brewery lab became an im-
portant site of classic yeast genetics and biotechnology research (Hansen and
Kielland-Brandt, 2003). Pretorius (2000) suggested that many additional yeast
species might be used in winemaking. In this context my colleagues and I have
discovered nearly 300 previously unknown yeasts, many of which have the abil-
ity to ferment a variety of sugars, yet are untried for making beverages (Suh et al.,
2005; Urbina and Blackwell, unpublished). In addition to its significance in brew-
ing and bread making, S. cerevisiae, of course, has been extremely important in
industrial biotechnology because of the development of efficient transformation
methods and specialized expression vectors, and for a variety of other genetics
tools (Nevoigt, 2008).
Fungi Interact with Other Organisms

Fungi interact with all major groups of organisms. Specific interactions with
photosynthetic organisms are generally well known (Table A2-1). About 80 per-
cent of all plant species and 92 percent of plant families form close associations
with fungi known as mycorrhizae (Smith and Read, 2008; Trappe, 1987). Fungi
and plant roots or underground stems form several kinds of mycorrhizae that are
classified by the morphology of the interacting fungus in relation to the root. The
associations are important for carbon, mineral, and water exchange, with carbon
generally transferred from the plant to the fungus.
Arbuscular mycorrhizal (AM) fungi are known from the 400 million-year-old
Rhynie chert. The fungi penetrate the plant cell wall and form a highly branched
arbuscule that invaginates the plasma membrane of the root cortex cells. The
200 members of the asexually reproducing phylum Glomeromycota are obligate
fungal partners of about 60 percent of all plant species. Hosts include a variety
of crop and forage plants such as maize, rice, alfalfa, and citrus, as well as many
non-cultivated plants. Molecular methods have detected previously unknown host
APPENDIX A 127
TABLE A2-1â•‡ Examples of Fungal Associations with Plants

Association Plants Fungus Reference
AM Mycorrhizae 60% of all species Glomeromycota Selosse et al. (2006)
Ectomycorrhizae 2,000 species ~5,000 species of Smith and Read (2008)
basidiomycetes,
ascomycetes,
Endogenales
Endophytes 95% of all plants Many groups of Rodriguez et al. (2009)
ascomycetes and some
basidiomycetes
Lichens ~100 species of ~32,000 ascomycetes Schoch et al. (2009)
photobionts (green (Leotiales, Dothideales,
algae, blue/green and Pezizales), a few
bacteria) basidiomycetes
specificity in some cases (Selosse et al., 2006). Ectomycorrhizal fungi (Figure

A2-4) are associated with fewer hosts, including certain dominant forest trees
such as birch, dipterocarp, eucalyptus, oak, and pine. Greater ectomyccorhizal
fungal diversity is evident, and basidiomycetes, ascomycetes, and a few zygo-
mycetes are involved in these associations. Many of the fungi are generalists, but
more specificity occurs than among AM associates. The fungi produce an external
mantle over young roots and often cause dramatic shortening and dichotomous
branching of the mycorrhizal root (Smith and Read, 2008).
Endophytes are fungi that usually grow within above-ground plant parts
without causing disease symptoms in about 95 percent of all plants examined
(Arnold, 2007). The fungi that form the associations have been placed in four
groups, depending on host specificity, tissues colonized, and amount of coloni-
zation within the plant (Rodriguez et al., 2009). Hypocrealean endophytes of
grasses and sedges produce alkaloids that have been suggested to deter feeding
by insects and vertebrates. Endophyte-infected grasses have enhanced growth and
drought resistance (Rodriguez et al., 2009). A different group of endophytes is
more taxonomically diverse and has broad plant host range with restricted growth
within the plant, often occupying only a single cell. Some of these horizontally
transmitted endophytes convey protection from plant pathogens (Arnold et al.,
2003; Rodriguez et al., 2009). An endophyte was reported to convey heat toler-
ance to its grass host near a hot springs in Yellowstone National Park, but ad-
ditional research has shown that only virus-infected endophytes convey thermal
tolerance, a sign of the complexity of such associations (Márquez et al., 2007).
About half of the estimated 64,000 ascomycetes (e.g., Leotiales, Dothideales,
and Pezizales) and a few basidiomycetes are the fungal associates (mycobionts)
of about 100 species of photosynthetic organisms (photobionts) to form lichens
128 FUNGAL DISEASES
FIGURE A2-4â•‡ Anaptychia ciliaris. Small A2-4.eps

Figure colonies of the lichen-forming fungus on agar
medium after 3 months of growth. Bar = 200 µm.
SOURCE: Photo courtesy of Ning Zhang, bitmap
provided by Meredith Blackwell (2010).
(Schoch et al., 2009). Lichens have been used as indicators of pollution. In

addition to the photosynthetic partner, usually a green alga, a photosynthetic,
nitrogen-fixing blue/green bacterium also may occur in a tripartite association in
the lichen. Although the fungal associate can be grown on artificial media, they
usually grow very slowly (Figure A2-5). Lichens are hosts for pathogenic fungi
as well as endolichenic fungi, the lichen equivalent of endophytes. Each partner
in the lichen has a scientific name, but the name of the lichen as a whole is that
of the fungus (Ahmadjian, 1993; Nash, 2008).
Wood-Decaying Fungi
Fungi are heterotrophic and their ability to degrade organic materials and
return them to nutrient cycles is an essential activity in almost all ecosystems.
The ability of a fungus to degrade specific substrates depends on the enzymes it
produces, and certain fungi are especially important in forest ecosystems where
they are the primary decomposers of wood. Basidiomycetes and some ascomy-
cetes are the primary decomposers of plant cell wall carbohydrates (cellulose and
APPENDIX A 129
FIGURE A2-5â•‡ Ectomycorrhizal root. The hyphae of Rhizopogon rubescens enveloping

the young roots of a Virginia pine seedling. The mycelium extends from the roots into the
surrounding environment.
Figure A2-5.eps
SOURCE: Photo by J. B. Anderson, providedbitmapby Meredith Blackwell (1996).
hemicellulose) and lignin polymers (Gilbertson, 1980). Some wood-decaying

fungi invade living trees and attack non-functional tissues, especially heartwood,
the non-conducting vascular tissue in the center of a cross section of the trunk.
Few wood-decaying fungi actually cause diseases and most of the damage comes
from the weakening of tree trunks so that they fall in wind or ice storms. The
loss of weakened trees is a natural process that culls branches and entire trees to
create clearings in older forests (Gilbertson, 1980). Aldo Leopold recognized the
value of wood decay for wildlife in the chapter “November” of A Sand County
Almanac and Sketches Here and There. He referred to his woodlot as “a mighty
fortress that fell heir to all the diseases of plants” known to humankind. The im-
portance of wood-decaying fungi in the formation of nesting holes for wildlife
is well known (Gilbertson, 1980). The red-cockaded woodpecker prefers to nest
in mature pines about 60 years old that have been rotted by the basidiomycete
Phellinus pini. Old pine stands are a diminishing habitat in regions where pines
are grown in plantations on a 15-year rotation or less for commercial use. The
ivory-billed woodpecker may be extinct because the extensive old-growth, bot-
tomland hardwood forests the species inhabited have been lost (Gilbertson, 1980).
A less significant but interesting use of wood decay is the creation of wooden
objects that have been modified by wood-decaying fungi. Spalted wood is distin-
guished by zone lines, the dark lines formed by oxidation at the points of contact
between closely related fungal colonies. The patterned wood is often favored by
130 FUNGAL DISEASES
collectors and increases the cost of hand-turned bowls at craft fairs. These fungal
effects include the deep blue/green stain of an ascomycete fungus that remains
green in intarsia of fine Italian furniture and the inlay of Tunbridge Ware objects
(Blanchette et al., 1992b). Even Stradivarius violins may have been made more
resonant by the partial decay of the wood (Schwarze et al., 2008).
Insects Associated with Fungi and Vertebrates

The importance of many insects in the ecosystem is overlooked, but many
of them are important in degradation of course particles, dispersal of bacteria
and fungi, and, as is well known, as agents of fungal fertilization. Fungi clearly
provide benefits for insects, although the exact advantages to the fungi beyond
providing habitat and a means of dispersal often are not clear (Buchner, 1965;
Gilbertson, 1984; Mueller et al., 2005). Few animals have the enzymes neces-
sary to digest refractory plant cell wall materials or to synthesize vitamins. Fungi
also may detoxify plant toxins and produce pheromones for insects (Table A2-2)
(Dowd, 1991; Vega and Dowd, 2005; Wheeler and Blackwell, 1984; Wilding
et al., 1989). The best known fungus–insect associations include the farming in-
teractions of basidiomycetes with Old World termites (Macrotermitinae) (Aanen
TABLE A2-2â•‡ Examples of Fungal Associations with Insects

Insect Group Fungi Reference
Macrotermitinae Termitomyces spp. Aanen et al. (2002)
Formicidae: Attini (derived clades) Leucocoprinus spp. Mueller et al. (2005)
Formicidae: Attini (most in Pterulaceae spp. Munkacsi et al. (2004)
Apterostigma pilosum clade)
Scolytinae and Platypodinae Ophiostomatoid ascomycetes Farrell et al. (2001);
(Bark and ambrosia beetles) Harrington (2005)
Siricidae (wood wasps) Certain species of Amylostereum, Martin (1992)
Stereum, and Daedalea
Passalidae (bess beetles) Several clades of xylose- Suh et al. (2003)
fermenting yeasts
Mushroom-feeding beetles Candida tanzawaensis clade Suh et al. (2005)
yeasts
Drosophila in cacti Various yeasts Starmer et al. (2006)
Nectar feeding beetles Various yeasts Lachance et al. (2001)
Coccidae Septobasidium Henk and Vilgalys (2007)
Certain insects, especially aquatic Harpellales, Asellariales Lichtwardt et al. (2001)
larvae
APPENDIX A 131
et al., 2002) and attine ants (Figure A2-6) (Formicidae: Attini) (Mueller et al.,
2005) and of ascomycetes by bark and ambrosia beetles (Scolytinae and Platy-
podinae) (Harrington, 2005). The females of another insect group, siricid wood
wasps (Siricidae), are less well studied, but they have been considered by some
to form farming interactions with fungi (see Gilbertson, 1984). The interaction,
however, does not meet all the criteria established for what has been defined as
“agriculture” (Mueller et al., 2005).
The farming association of the basidiomycete Termitomyces with Old World
macrotermitine termites arose once in Africa. Since that event no additional fun-
gal lineages have been domesticated and no reversals of the fungus to a free-living
state have been found. Repeated host switching, however, has occurred within
termite clades as reflected in the phylogenetic trees of termites and associated
fungi (Aanen et al., 2002). Nest initiation by both males and females of certain
species has been suggested to have influenced the mode of transmission of the
fungus, usually acquired from the environment or some source other than a parent
(horizontal transmission) (Aanen et al., 2002). In the New World it is not termites,
but attine ants that are involved with basidiomycetes in farming interactions, and
Aanen and his colleagues (2002) compared the associations. The attines have
become associated with several clades of fungi, and in contrast to termite trans-
mission, transmission of the fungi is usually directly from parent to offspring
(vertical) except in the early diverging ant lineages. Another important difference
is that the ant-associated fungi apparently do not reproduce sexually. The work
on the fungus–attine ant associations have revealed that ants have evolved with
several groups of fungi on several different occasions. Although the best-known
fungal mutalists are species of Leucocoprinus, other fungal groups, including
certain species of Pterulaceae, have an association with ants in the Apterostigma
pilosum clade (Munkacsi et al., 2004). The intensive studies of the fungi and at-
tine ant associations have led to the discovery of other organisms that participate
in the complex interactions. Species of hypocrealean ascomycetes in the genus
Escovopsis are parasites of the cultivated fungus. Actinomycete associates of the
ants produce antibiotics that have been reported to be specific in inhibiting Es-
covopsis (Currie et al., 1999), but more recently Sen et al. (2009) found that the
bacteria they isolated had more generalized antibiotic activity, including activity
against the cultivated fungus. The association of a fourth component of the as-
sociation is black yeasts that apparently reduce the efficiency of the antibiotics
(Little and Currie, 2008). This attine and—cultivated fungus—Escovopsis para-
site associations provide the best example of coevolution, in this case tripartite
association, among fungi and associates (Currie et al., 2003).
Unlike the termite and ant interactions, fungus-beetle associations have
arisen multiple times. Some bark and ambrosia beetles have mycangia already
mentioned above in which they carry inoculum of certain fungi (Malloch and
Blackwell, 1993). The fungi, often Ceratocystis and Ophiostoma or relatives,
may be the agents of plant diseases, and some of the fungi have been introduced
132 FUNGAL DISEASES
FIGURE A2-6â•‡Excavation of deeply entrenched nest of the ant Atta texana requires
heavy equipment or, alternatively, ground-penetrating radar to map such nests. The ant is
native to adjacent parts of Texas and Louisiana, and the nests are said to be able to contain
a three-story house. Visual materials based on a ground-penetrating radar nest model are
available on the Internet from Carol LaFayette, Department of Visualization, Texas A&M.
http://www.viz.tamu.edu/faculty/lurleen/main/attatunnel/tunnel.html.
Figure A2-6.eps
SOURCE: Photo courtesy of John Moser, provided by Meredith Blackwell (2009).
bitmap
APPENDIX A 133
with the beetles as in the case of Raffaelea laurelensis, the agent of laurel wilt
disease (Harrington, 2005; Harrington and Fraedrich, 2010). Ophiostoma ulmi
and similar fungi have been introduced into the United States, where they are
virulent pathogens of trees, including American elms. The most efficient dispers-
ers of some of these fungi actually were introduced before the fungus, Ophios-
toma ulmi (Alexopoulos et al., 1996). In this discussion of beneficial fungi, these
interactions benefit the insects and call attention to potential devastating effects
of efficient insect dispersal in the context of emerging plant diseases.
Other beneficial fungal associates of insects involve siricid wood wasps and
wood-decaying basidiomycetes, species of Amylostereum, Stereum, and Daeda-
lea. The wasps lay their eggs through long ovipositors, tube-shaped organs at
the posterior of the abdomen, and the larvae probably rely on fungal enzymes
to decompose and detoxify the wood they ingest (Gilbertson, 1984; Martin,
1992). Many more fungi are associated with insects as necrotrophic parasites
(Figure A2-7), and some of these deadly fungi have potential for development
as biological control agents (Vega et al., 2009). In addition, many of about 1,000
described yeast species have close associations with insects (Table A2-2), and
the yeasts provide important services to the insects (Vega and Dowd, 2005). Cer-
FIGURE A2-7â•‡ Hirsutella citriformis

Figure (Ophiostomataceae)
A2-7.eps on a delphacid planthopper.
The asexual fruiting structure of this fungus erupted through the cuticle of the parasitized
insect soon after its death.
bitmap
SOURCE: Photo courtesy of Jennifer Luangsa-ard, provided by Meredith Blackwell
(2010).
134 FUNGAL DISEASES
tain clades of gut yeasts appear to have diversified with insect hosts into certain
habitats, and the yeasts provide basic resources for the insects to survive when
subjected to new nutritional situations (Suh et al., 2003, 2006). About 200 species
of Septobasidium in the Septobasidiales are known as associates of scale insects;
only a few related species of Pachnocybe grow on wood (Henk and Vilgalys,
2007). The use of insect hosts is unusual for fungi that are related to the plant
pathogenic rust fungi. The fungi are parasites of a few of the scale individuals,
but in general benefit the entire insect colony by providing a protective covering
against parasitic wasps (Henk and Vilgalys, 2007). Two orders of zygomycetes,
Harpellales and Asellariales, were previously placed in a polyphyletic group
known as Trichomycetes. The results of several studies indicate that these gut
fungi produce vitamins and perhaps other benefits for their aquatic insect hosts
(Lichtwardt et al., 2001). One species is known to parasitize simulid black flies
(Lichtwardt et al., 2001), potentially a benefit to those who engage in outdoor
activities.
Another nutritional interaction between fungi and animals is only briefly
noted here, but is extremely important. An early diverging lineage of obligately
anaerobic multiflagellated fungi, the Neocallimastigomycota, and vertebrate her-
bivores are closely associated (Griffith et al., 2010). The fungi reside in the host
rumen or another anaerobic part of the gut, where they are important in supplying
cellulases and other enzymes for the degradation of the large quantities of cel-
lulose ingested by the herbivore (James et al., 2006).
Conclusion
Many fungi are obligate, beneficial associates of other groups of organisms.
These are the “good fungi” of this article, and we often fail to appreciate their
value because the fungi usually are unseen within their substrates unless they
form macroscopic fruiting bodies. More often it is the effects of the fungi that we
observe when they ferment fruit juice, or fitting to this volume, cause dramatic
new outbreaks of disease. The Robert Frost poem quoted in the prologue of this
publication describes the costs of the introduction of the disease caused by the
chestnut blight fungus, Cryphonectria parasitica. The poem predicts that the
disease will ravage until a new pathogen comes to kill the fungus, and in fact a
virus did appear to suppress the fungus. In 1974, however, yet another pathogen,
the oriental chestnut gall wasp, was introduced to attack the trees, an additional
turn not predicted by the verse.
Today, as one out of every six or seven humans on Earth is reported to be
malnourished or hungry (FAO, 2010), the war against pathogenic diseases of
plants and animals is as important as ever. An earlier writer, Jonathan Swift
(1667–1745) addressed the topic of hunger with his essay, A Modest Proposal,
written to bring attention to the starvation of Irish tenant farmers during the po-
tato famine. In Gulliver’s Travels he wrote directly of the importance of increas-
ing agriculture yields:
APPENDIX A 135
And he gave it for his opinion, “that whoever could make two ears of corn, or
two blades of grass, to grow upon a spot of ground where only one grew before,
would deserve better of mankind, and do more essential service to his country,
than the whole race of politicians put together.”
—Jonathan Swift, Gulliver’s Travels, Part II, Voyage to Brobdingnag,
first published in 1726–1727.
This volume, Fungal Diseases: An Emerging Threat to Human, Animal,

and Plant Health, provides a discussion of new fungal diseases of plants and the
animals that we strive to overcome at a time when introduced diseases contribute
to hunger.
Acknowledgments
I am grateful to Dr. Fernando Vega, who improved the original manuscript
through his careful editing. Several colleagues provided images, and Dr. Matthew
Brown kindly prepared the plate. I acknowledge support from the National Sci-
ence Foundation (NSF-0732671 and DEB-0417180) and the Louisiana State
University Boyd Professor support fund.
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A3
THE FUNGI: 1, 2, 3 … 5.1 MILLION SPECIES?11,12,13

Meredith Blackwell14,15
Premise of the Study

Fungi are major decomposers in certain ecosystems and essential associates
of many organisms. They provide enzymes and drugs and serve as experimental
organisms. In 1991, a landmark paper estimated that there are 1.5 million fungi
on the Earth. Because only 70000 fungi had been described at that time, the
estimate has been the impetus to search for previously unknown fungi. Fungal
habitats include soil, water, and organisms that may harbor large numbers of
understudied fungi, estimated to outnumber plants by at least 6 to 1. More recent
estimates based on high-throughput sequencing methods suggest that as many as
5.1 million fungal species exist.
Methods
Technological advances make it possible to apply molecular methods to
develop a stable classification and to discover and identify fungal taxa.
11â•›â•›Reprinted with kind permission from the Botanical Society of America, www.amjbot.org.
12â•›â•›Manuscript received 10 August 2010; revision accepted 19 January 2011.
â•›â•›The author thanks N. H. Nguyen, H. Raja, and J. A. Robertson for permission to use their photo-
graphs, two anonymous reviewers who helped to improve the manuscript, and David Hibbett, who
graciously provided an unpublished manuscript. She acknowledges funding from NSF DEB-0417180
and NSF-0639214.
13â•›â•›Key words: biodiversity; fungal habitats; fungal phylogeny; fungi; molecular methods; numbers
of fungi.
14â•›â•›Department of Biological Sciences; Louisiana State University; Baton Rouge, Louisiana 70803
USA.
15â•›â•›Author for correspondence (e-mail: mblackwell@lsu.edu) doi:10.3732/ajb.1000298.
APPENDIX A 141
Key Results
Molecular methods have dramatically increased our knowledge of Fungi in
less than 20 years, revealing a monophyletic kingdom and increased diversity
among early-diverging lineages. Mycologists are making significant advances in
species discovery, but many fungi remain to be discovered.
Conclusions
Fungi are essential to the survival of many groups of organisms with which
they form associations. They also attract attention as predators of invertebrate
animals, pathogens of potatoes and rice and humans and bats, killers of frogs and
crayfish, producers of secondary metabolites to lower cholesterol, and subjects
of prize winning research. Molecular tools in use and under development can be
used to discover the world’s unknown fungi in less than 1000 years predicted at
current new species acquisition rates.
What are Fungi?

Fungal biologists debated for more than 200 years about which organisms
should be counted as Fungi. In less than 5 years, DNA sequencing provided a
multitude of new characters for analysis and identified about 10 phyla as mem-
bers of the monophyletic kingdom Fungi (Fig. A3-1). Mycologists benefited
from early developments applied directly to fungi. The “universal primers,” so
popular in the early 1990s for the polymerase chain reaction (PCR), actually
were designed for fungi (Innis et al., 1990; White et al., 1990). Use of the PCR
was a monumental advance for those who studied minute, often unculturable,
organisms. Problems of too few morphological characters (e.g., yeasts), noncorre-
sponding characters among taxa (e.g., asexual and sexual states), and convergent
morphologies (e.g., long-necked perithecia producing sticky ascospores selected
for insect dispersal) were suddenly overcome. Rather than producing totally new
hypotheses of relationships, however, it is interesting to note that many of the new
findings supported previous, competing hypotheses that had been based on mor-
phological evidence (Alexopoulos et al., 1996; Stajich et al., 2009). Sequences
and phylogenetic analyses were used not only to hypothesize relationships, but
also to identify taxa rapidly (Kurtzman and Robnett, 1998; Brock et al., 2009;
Begerow et al., 2010).
Most fungi lack flagella and have filamentous bodies with distinctive cell
wall carbohydrates and haploid thalli as a result of zygotic meiosis. They interact
with all major groups of organisms. By their descent from an ancestor shared with
animals about a billion years ago plus or minus 500 million years (Berbee and
Taylor, 2010), the Fungi constitute a major eukaryotic lineage equal in numbers to
animals and exceeding plants (Figs. A3-2–10). The group includes molds, yeasts,
mushrooms, polypores, plant parasitic rusts and smuts, and Penicillium chrysoge-
142 FUNGAL DISEASES
[sic]
Figure A3-1.eps
FIGURE A3-1â•‡ Fungal phyla and approximate number of species in each group (Kirk
et al., 2008). Evidence from gene order conversion and multilocus sequencing indicates
bitmap
that microsporidians are Fungi (see below; Lee et al., 2010). Note also that zoosporic and
zygosporic fungal groups are not supported as monophyletic. Tree based on Hibbett et al.
(2007), White et al. (2006), and James et al. (2006).
num, Neurospora crassa, Saccharomyces cerevisiae, and Schizosaccharomyces

pombe, the important model organisms studied by Nobel laureates.
Phylogenetic studies provided evidence that nucleriid protists are the sister
group of Fungi (Medina et al., 2003), nonphotosynthetic heterokont flagellates
are placed among brown algae and other stramenopiles, and slime mold groups
are excluded from Fungi (Alexopoulos et al., 1996). Current phylogenetic evi-
dence suggests that the flagellum may have been lost several times among the
early-diverging fungi and that there is more diversity among early diverging
APPENDIX A 143
zoosporic and zygosporic lineages than previously realized (Bowman et al., 1992;
Blackwell et al., 2006; Hibbett et al., 2007; Stajich et al., 2009).
Sequences of one or several genes are no longer evidence enough in phylo-
genetic research. A much-cited example of the kind of problem that may occur
when single genes with different rates of change are used in analyses involves
Microsporidia. These organisms were misinterpreted as early-diverging eukary-
otes in the tree of life based on their apparent reduced morphology (Cavalier-
Smith, 1983). Subsequently, phylogenetic analyses using small subunit ribosomal
RNA genes wrongly supported a microsporidian divergence before the origin of
mitochondria in eukaryotic organisms (Vossbrinck et al., 1987). More recent mor-
phological and physiological studies have not upheld this placement, and analy-
ses of additional sequences, including those of protein-coding genes, support the
view that these obligate intracellular parasites of insect and vertebrate hosts are
members of the Fungi (Keeling, 2009; Corradi and Keeling, 2009). Additional
evidence from genome structure as well as phylogenetic analyses, supports the
inclusion of microsporidians within the Fungi and indicates that comparison of
whole genomes contributes to the solution of challenging phylogenetic problems
(Lee et al., 2010).
The level of resolution and sophistication of systematics studies made pos-
sible by molecular markers and phylogenetic analyses put mycologists on equal
footing with other biologists for competitive funding, and they joined in several
community-wide efforts to organize fungal diversity within a phylogenetic classi-
fication. Three projects funded by the National Science Foundation were initiated,
including the Research Coordination Network: A Phylogeny for Kingdom Fungi
(Deep Hypha) and successive Tree of Life projects, Assembling the Fungal Tree
of Life (AFTOL-1) and a second ongoing project (AFTOL-2) (Blackwell et al.,
2006). A major product of the Deep Hypha project was the publication of 24
papers on fungal phylogeny in a single journal issue (Mycologia 98: 829–1103).
The papers included an introduction to progress in fungal phylogeny, a paper on
dating the origin of Fungi, one on the evolution of morphological traits, and 21
articles with multilocus phylogenies of most major groups. Participants included
156 authors with some involved in more than one paper; only 72 of the authors
were originally from North America. The multi-investigator AFTOL-1 publica-
tion (Hibbett et al., 2007) included a widely used and often cited phylogenetic
classification to the level of order (e.g., Kirk et al., 2008; The NCBI Entrez
Taxonomy Home-page, http://www.ncbi.nlm.nih.gov/taxonomy; Science Watch,
http://sciencewatch.com/dr/nhp/2009/09jannhp/09jannhpHibb). The paper in-
cluded 68 authors from more than 20 countries.
It is important to note that there was broad participation and, essentially,
global involvement on these projects, emphasizing that studies of biodiversity are
indeed global endeavors. Additional pages were contributed to the Tree of Life
web project (http://www.tolweb.org/Fungi/2377) to make information on fungi
more accessible to students and the general public. Two objectives of the ongo-
ing AFTOL-2 project include increased taxon sampling of fungi for molecular
144 FUNGAL DISEASES
Figure A3-2-10.eps
bitmap
APPENDIX A 145
FIGURES A3-2–10â•‡ Examples of fungal diversity. 2. Lemonniera sp. Tetraradiate conidia

developed on a submerged leaf in a well-aerated freshwater stream surrounded by lush
vegetation. This type of aquatic species, an Ingoldian ascomycete, is named for C. T.
Ingold, who pioneered the study of these fungi, that are characterized by highly branched
conidia. Photo courtesy of H. Raja. 3. The aero-aquatic ascomycete Helicoon gigantispo-
rum produces distinctive tightly coiled conidia. As the spore develops air is trapped in the
coil and causes it to be buoyant. This feature is an adaptation for the polyphyletic aero-
aquatic fungi that grow on leaves in slow-moving or stagnant freshwater. Photo courtesy of
H. Raja. 4. The smut Testicularia sp. develops in the ovary of grasses and (as shown here)
sedges. The spores mature sequentially, with the dark spores being more mature. A plant
taxonomy student once thought he had discovered a new species of Leersia, distinguished
by large ovaries of ca. 1 cm, only to be disappointed that the enlargement was caused by
a fungus. It is helpful to mycologists when plant taxonomists collect and accession fungal
diversity by selecting some diseased plant specimens, an activity that should be encour-
aged. 5. Perithecia of Pyxidiophora sp. (Laboulbeniomycetes) developed in moist chamber
on moose dung from Meredith Station, New Brunswick, Canada. The 150 µm long asco-
spores are seen at the tip of the perithecium neck in the center. Spores adhere to phoretic
mites that are carried by dung beetles to fresh dung piles. Some fungi have complex animal
dispersal systems. Pyxidiophora species are usually mycoparasites that grow on fungi in
dung or other substrates including wrack washed up on beaches. The genus is a “missing
link” and provided clues to confirm that Laboulbeniomycetes are ascomycetes and not
other kinds of fungi or floridian red algae. 6. The ca. 8 cm wide basidiomata of Pycnopo-
rus sp., a wide-ranging, brightly colored, wood-decaying polypore, photographed at Barro
Colorado Island, Panama. Some collectors have referred to basidiomycetes that produce
colorful basidiomata as charismatic megamycota of the fungus world. 7. Peniophorella
baculorubrensis, a bark-decaying basidiomycete common on and restricted to living live
oak (Quercus virginiana), decays the bark and changes its water-holding capacity. The ef-
fect of decay on bryophyte communties by this fungus was first studied by ecologists (Pen-
found and Mackaness, 1940) more than 70 yr ago but was not described until a specialist
on wood-decaying fungi happened to notice it on the Louisiana State University campus,
Baton Rouge (Gilbertson and Blackwell, 1984). The inconspicuous basidiomata are shown
growing on the lower side of a 7 cm long bark segment aimed downward for basidiospore
discharge in response to gravity. 8. Basidiomata of Perenniporia phloiophila on the bark
of living Quercus virginiana. Although the basidiomata are obvious against the darker
bark, this species was not described until it was discovered at the same time and often on
the same trees as Peniophorella baculorubrensis. Although the fungus usually rots only
the outer bark, it will invade and decay wood whenever the vascular cambium is broached
by a bird or insect. In addition to the two species on live oak, six other species have been
described from the campus, illustrating the need for specialists to study noncharismatic
fungi. 9. A basidioma (8 cm diameter) of the wood-decaying fungus, Favolus tenuiculus,
a favorite food of several species of mushroom-feeding beetles (see Fig. A3-10). Photo
courtesy of N. H. Nguyen. 10. The small (>10 mm long) brightly colored beetle, Myco-
tretus sp. (Erotylidae), was collected at Barro Colorado Island, Panama. Many erotylid
beetles have specialized yeast-packed pouches at the anterior end of the midgut. More than
200 novel yeasts have been isolated from the gut of ca. 15 families of mushroom-feeding
beetles (Suh et al., 2005). Photo courtesy of James A. Robertson.
146 FUNGAL DISEASES
data and the discovery of correlated morphological and biochemical characters

(AFTOL Structural and Biochemical Database, https://aftol.umn.edu; Celio et al.,
2006).
Known Fungal Species

The Dictionary of Fungi (Kirk et al., 2008) reported 97330 species of de-
scribed fungi at the “numbers of fungi” entry. The addition of 1300 microsporid-
ians brings the total of all described fungi to about 99000 species (Fig. A3-1). The
Dictionary’s estimate of known species has almost tripled in the period between
the first edition in 1943 (38000 described species) and now, amounting to an in-
crease of more than 60000 described species over the 65-yr period (Fig. A3-11).
Factors such as difficulty of isolation and failure to apply molecular methods may
contribute to lower numbers of species in certain groups, but there cannot be any
doubt that ascomycetes and basidiomycetes comprise the vast majority of fungal
diversity (Fig. A3-1).
FIGURE A3-11â•‡ Numbers of known fungi from the Dictionary of the Fungi (editions
1–10, 1950–2008). Authors state that the large increase in species numbers in the 10th
edition may be inflated becauseFigure A3-11.eps
asexual and sexual forms were counted separately and
bitmap
molecular techniques that distinguish close taxa have been used.
APPENDIX A 147
Estimated total fungal numbers

In 1991, a landmark paper provided several qualified estimates of the number
of fungi on the Earth based on ratios of known fungi to plant species in regions
where fungi were considered to be well-studied (Hawksworth, 1991). “Estimate
G” of 1.5 million species was accepted as a reasonable working hypothesis based
on a fungus to plant ratio of 6:1, in contrast to the much lower 50–60-yr-old es-
timates by Bisby and Ainsworth (1943) of 100000 fungal species and by Martin
(1951) of 250000 species based on one fungus for every phanerogam known at
the time. A more recent estimate of the total number of fungi, 720 256 (Schmit
and Mueller, 2007), is also low compared to present estimates that include envi-
ronmental samples.
Hawksworth’s (1991) estimate now is considered to be conservative by
many, including Hawksworth (Hawksworth and Rossman, 1997), because numer-
ous potential fungal habitats and localities remain understudied (Hawksworth,
2001). Furthermore, the use of molecular methods had not yet been considered
as a means of species discovery. For example, analysis of environmental DNA
samples from a soil community revealed a high rate of new species accumula-
tion at the site, and these data supported an estimate of 3.5 to 5.1 million species
(O’Brien et al., 2005). Using the present discovery rate of about 1200 fungal
species per year based on the last 10 years, Hibbett and his colleagues (in press)
estimated that it would take 1170 years to describe 1.4 million fungi (based on
Estimate G of Hawksworth [1991]) and 2840 to 4170 yr to describe 3.5 to 5.1
million (based on O’Brien et al., 2005).
Using present higher estimates of land plant numbers as somewhat under
400000 species (Paton et al., 2008; Joppa et al., 2010) fungal species numbers
now are expected to outnumber land plants by as much as 10.6:1 based on
O’Brien et al. (2005). Even higher ratios have been predicted using data from
highthroughput sequencing of clone libraries, although individual ecosystems
will vary (L. Taylor, University of Alaska, Fairbanks, personal communication,
January 2011). The large gap between known and estimated species numbers has
led to a series of papers and symposia (e.g., Hawksworth and Rossman, 1997;
Hawksworth, 2001; Hyde, 2001; Mueller and Schmit, 2007) attempting to answer
the question “Where are the missing fungi?”
How to Discover New Fungi

Collecting and culturing fungi from the environment will remain important
because of the need to identify specimens, revise taxonomy, assess the roles in the
environment, and provide strains for biological control, environmental remedia-
tion, and industrial processes. A physical specimen, including an inert culture,
is still required as a type specimen (but see Conclusions later), and vouchers
of known fungi are used for documenting DNA sequences deposited in some
databases (Nilsson et al., 2006). For example, the current AFTOL project has
148 FUNGAL DISEASES
a requirement that each sequence deposited as part of the project be linked to a

specimen, including a culture.
All taxa biological inventories (ATBIs) attempt to survey organisms within
particular geographical regions by collection of specimens and culture of sub-
strates. One of these, Discover Life in America, All Taxa Biological Inventory,
seeks to survey an estimated 50000 to 100000 species of organisms in the Great
Smoky Mountains National Park. Karen Hughes and Ronald Petersen have been
successful in collecting more than 3000 species of fungi, mostly agarics housed
in the University of Tennessee Fungal Herbarium (http://tenn.bio.utk.edu/fungus/
database/fungus-browse-results.asp?GSMNP=GSMNP), out of about 17000 spe-
cies of all taxa that have been collected by others in the park (Biodiversity Sur-
veys and Inventories: Agaric Diversity in the Great Smoky Mountains National
Park, NSF DEB 0338699). All fungal specimens have been identified, and the
agarics have been studied to the extent that a culture, ITS barcode sequence,
and genetic analysis are available for many species. This successful project has
required hours of time over a number of years and costly resources for studying
the material, but it serves as an example of the commitment needed to acquire
specimen-based information on fungi.
DNA methodology makes it possible to use independent sampling methods
to discover the presence of organisms without ever seeing a culture or a specimen.
Several new methods significantly outperform previous automated sequencing
methods (e.g., Jumpponen and Jones, 2009; Metzker, 2010). Although there
may be certain limitations and biases for the different methods (Amend et al.,
2010a; Tedersoo et al., 2010), mycologists have been quick to embrace them in
ecological and biodiversity studies. O’Brien and colleagues (2005) pointed out
that collection and culture methods revealed numbers of fungi similar to those
acquired by sampling environmental DNA. Hibbett et al. (in press), however,
used data from GenBank to show that by 2008 and 2009 the number of envi-
ronmental samples, excluding overwhelming numbers of sequences discovered
by pyrosequencing, exceeded the accessions of specimen-based sequences. The
rapid development of automated, high-throughput methods also has made it pos-
sible to acquire whole genome sequences for population level studies (Liti et al.,
2009; Neafsey et al., 2010).
Which Regions of the Earth Harbor Fungal Diversity?

Fungi grow in almost all habitats on Earth, surpassed only by bacteria in their
ability to withstand extremes in temperature, water activity, and carbon source
(Raspor and Zupan, 2006). Tropical regions of the world are considered to have
the highest diversity for most groups of organisms (Pianka, 1966; Hillebrand,
2004), and this is generally true for fungi as well (Arnold and Lutzoni, 2007).
A group of researchers are studying the diversity of the Guyana Shield.
For the last 11 years, Terry Henkel and Cathie Aime and their colleagues have
studied the fungi in six 1-km2 plots—three in a Dicymbe corymbosa-dominated
APPENDIX A 149
forest and three in a mixed tropical forest. Their current collections contain 1200
morphospecies, primarily basidiomycetes. Approximately 260 species were col-
lected repeatedly only in the Dicymbe plots. Thus far, two new genera and ca.
50 new species have been described. On the basis of groups already studied,
Aime estimated that ca. 120 new ectomycorrhizal taxa have been discovered.
Including novel saprobes as well as ectomycorrhizal fungi, ca. 500 new species
are expected among the 1200 taxa collected. It is clear, however, that these are
not simply high numbers of new taxa, but biologically interesting fungi as well
(Aime et al., 2010). One species is so unusual, that a reviewer of the original
report called it “the find of the century” (Redhead, 2002). As Aime has quipped
“if one were to compare the ratio of fungi to plants in the Dicymbe plots as did
Hawksworth (1991), the ratio would be 260 to 1, obviously an overestimate but
also a cautionary exercise in basing any estimate on a single ecotype” (M. C.
Aime, Louisiana State University, personal communication, August 2010).
Many fungi have in fact come from temperate regions, and some studies
report a high diversity of fungi. For example, in a study of indoor air from build-
ings using culture-independent sampling methods, diversity was found to be
significantly higher in temperate sites independent of building design or use. The
authors also alluded to the possibility that previous studies of certain mycorrhizal
fungi showed similar trends (Amend et al., 2010b). More investigation in this
area is needed, but it is clear that many undescribed fungi are present in temper-
ate regions. Popular literature often rationalizes the need to save the rainforests,
not because of their intrinsic value, but because of the potential drug-producing
organisms that may be found there. Many of the commercially most success-
ful fungal drugs, however, come from temperate fungi. Penicillium chrysoge-
num, producer of penicillin, was found in a northern temperate city. Another
remarkable fungus, Tolypocladium infl atum from Norwegian soil, synthesizes
cyclosporine, an immune-suppressant drug that revolutionized organ transplants
(Borel, 2002); the sexual state of this fungus was collected in New York, USA
(Hodge et al., 1996). Today the drug is commonly used to treat dry eye (Perry
et al., 2008), as well as many serious conditions. Statins produced by fungi such
as Aspergillus terreus from temperate regions, combat high cholesterol levels,
as well as providing other benefits (Vaughan et al., 1996; Askenazi et al., 2003;
Baigent et al., 2005).
In temperate deserts, mycorrhizal boletes, agarics, and rust and smut fungi,
are common. A surprising number of wood-decaying basidiomycetes have been
discovered on living and dead desert plants, including cacti and are in the Univer-
sity of Arizona, Robert L. Gilbertson Mycological Herbarium (http:// ag.arizona.
edu/mycoherb/herbholdings). When a noted mycologist moved to Arizona early
in his career, he became excited about the new and unreported fungal diversity
found in the desert. His proposed study of the wood-decaying fungi of the So-
noran Desert was poorly received with a comment that wood-decaying fungi
were not present in the desert (R. L. Gilbertson, University of Arizona, personal
communication, August 1979). The Sonoran Desert, however, has many plants
150 FUNGAL DISEASES
(e.g., cacti, ocotillo, and mesquite and other desert legumes) that are substrates
for polypores and resupinate basidiomycetes (e.g., Gilbertson and Ryvarden,
1986, 1987).
Fungi also grow at low temperatures. An example involves fungal deteriora-
tion of historic huts built between 1901 and 1911 for use by Antarctic explorers
including Robert Scott and Ernest Shackleton, and although there are not large
species numbers, it is important not to overlook this fungal habitat in diversity
studies (Held et al., 2005). Lichens have often been reported to be common in
Arctic and Antarctic regions (Wirtz et al., 2008), and yeasts are active under
frozen conditions in the Antarctic (Vishniac, 2006; Amato et al., 2009). In some
cases, a yeast isolated from the Antarctic (based on 28S rDNA barcoding) also
has been reported from varied habitats, including human infections, the gut of
insects, deep seas, and hydro-carbon seeps (Kurtzman and Fell, 1998; Bass
et al., 2007; personal observation). Although some fungi are specialized for cold
regions, others simply occupy a wide variety of environmental conditions.
Many regions and habitats of the world need to be included in fungal dis-
covery. In general, microscopic fungi and those that cannot be cultured are very
poorly known. Parts of Africa remain to be collected for many, although not all,
fungal groups (Crous et al., 2006). Fungi are important as symbionts, and they
are associated with every major group of organisms, bacteria, plants and green
algae, and animals including insects. Because certain under-studied symbiotic
associations are known to include large numbers of fungi, these are a good place
to search for new taxa. The associated organisms also allow for resampling, a
quick way to obtain data about host specificity. Targeting hosts also is a produc-
tive method for discovering fungal fossils, such as those associated with plants of
the Rhynie Chert (Taylor et al., 2004). Examples of diversity in particular fungal
habitats are reviewed in the following sections.
Fungi and Plant Roots

Mycorrhizal plants and their fungal partners have been studied by a number
of mycologists (Trappe, 1987; Smith and Read, 2008). The fungi often are es-
sential to their plant hosts because they take up water, nitrogen, phosphorus, and
other nutrients from the soil and transfer them to the plant roots. Some of these
fungi may not prosper or even grow without the host. In addition to flowering
plants and conifers, many bryophytes and ferns are mycorrhizal (Pressel et al.,
2010). Certain mycorrhizal fungi specialize on orchids and ericoid plants, and
some are known to have invaded new habitats with successful invasive plants
(Pringle et al., 2009).
There are two main types of mycorrhizal fungi, arbuscular mycorrhizae
(AM) and ectomycorrhizae. AM associations are more common and occur with
up to 80% of all plant species and 92% of plant families. AM fungi are all mem-
bers of the phylum Glomeromycota, a less diverse group than ectomycorrhizal
APPENDIX A 151
fungi with about 250 described species in a variety of taxa (Gerdemann, 1968;
Schüssler and Walker, 2011; Wang and Qiu, 2006). Evidence from recent mo-
lecular studies, however, indicates that cryptic species with higher levels of host
specificity than previously realized will increase the number of known AM fungi
(Selosse et al., 2006; Smith and Read, 2008). More than 6000 species, mostly of
mushroom-forming basidiomycetes, form ectomycorrhizae with about 10% of
all plant families. Greater host specificity usually occurs in the ectomycorrhizal
fungus–plant associations than in AM associations (Smith and Read, 2008). Vast
parts of the world remain to be sampled (Mueller et al., 2007), and it is expected
that barriers to inter-breeding have led to high genetic diversity among these fungi
(Petersen and Hughes, 2007).
Inside Plant Leaves and Stems

Almost all plants on Earth are infected with endophytes, fungi that do not
cause disease symptoms (Saikkonen et al., 1998). Endophytes occur between
the cells, usually of above ground plant parts, and represent a broad array of
taxonomic groups (Arnold, 2007; Rodriguez et al., 2009). The earliest studies of
endophytes were of those associated with grasses (Diehl, 1950). Some grass en-
dophytes are specialized members of the Clavicipitaceae, relatives of insect and
fungal parasites in the Hypocreales, and many species produce alkaloid toxins
effective against insects, other invertebrate animals, and vertebrates (Clay et al.,
1993). Some grass endophytes are transmitted to the host offspring in seeds, and
others inhibit sexual reproduction in the host and are dispersed within plant parts
such as leaf fragments. For grass endophytes that reproduce sexually, fertilization
may occur by insect dispersal. Water intake is increased in infected hosts, and
these plants often grow taller than uninfected hosts.
A much more diverse group of endophytic fungi are associated with plants
in addition to grasses, including a variety of dicots and conifers (Carroll, 1988;
Rodriguez et al., 2009). In some tropical forests considered to be diversity hot-
spots for endophytes, there are extremely large numbers of the fungi, sometimes
with hundreds reported from a single tree species, judged by both cultural and
molecular methods of discovery and identification (Arnold et al., 2001; Arnold
and Lutzoni, 2007; Pinruan et al., 2007; Rodriguez et al., 2009). In one study,
more than 400 unique morphotypes were isolated from 83 leaves of two species
of tropical trees. A subset of the fungi was distributed among at least seven orders
of ascomycetes (Arnold et al., 2000). Leaves usually acquired multiple infections
as they matured, and there was strong evidence that the endophytes protected
leaves of plants, such as Theobroma cacao, from infection when they were chal-
lenged with pathogens (Arnold et al., 2003). Vega and colleagues (2010) also
found high diversity of endophytes in cultivated coffee plants. Interestingly, some
of these were insect pathogens and experiments are being conducted to develop
endophytes as biological control agents of insect pests.
152 FUNGAL DISEASES
Plant Pathogens
Plant pathogens differ from endophytes in that they cause disease symptoms.
Although some zoosporic and zygosporic fungi are plant pathogens, most plant
pathogens are ascomycetes and basidiomycetes. A large number of ascomycetes
and ca. 8000 species of basidiomycetes are plant pathogens. In addition to crop
pathogens, it is important to remember that many pathogens are numerous and
important in natural ecosystems (Farr et al., 1989; Burdon, 1993). Nonpathogenic
phylloplane yeasts occupy leaf surfaces of many plants and are increasingly rec-
ognized for their control of potential leaf pathogens (Fonseca and Inácio, 2006).
In addition to the thousands of native fungi that parasitize plants in the United
States, pathologists are constantly on the lookout for introduced pathogens that
often are undescribed when they arrive to decimate naïve native plant popula-
tions. For example, invasive fungi such as those grouped as Dutch elm disease
fungi, chestnut blight fungus, dogwood anthracnose fungus, and redbay wilt
fungus, were all unknown until they were observed soon after their introduction
(Alexopoulos et al., 1996; Zhang and Blackwell, 2001; Harrington et al., 2008).
Exotic localities will need to be searched for undescribed fungi that probably go
largely unnoticed on their native hosts. It is important to note that although fungi
may cause only minor symptoms to hosts in their native habitats, one of these
may have the potential to be the next destructive disease after introduction to a
new region.
Molecular methods have helped to clarify limits of closely related species
and to establish host ranges (e.g., Crous et al., 2008). In a study of 26 leaf spot
fungi in Australia, three genera of Myrtaceae, including Eucalyptus, were hosts
for three new genera and 20 new species (Cheewangkoon et al., 2009). Although
the authors acknowledged the high level of new taxa discovered, they pointed
out that the potential for host shifts within plantations might lower estimates of
fungal species numbers worldwide. Host or substrate specificity is a concept that
can be applied to fungal groups that are closely associated with hosts such as en-
dophytes, pathogens, and mycorrhizal fungi but not usually for saprobic species
(Zhou and Hyde, 2001). In the past species of plant pathogens often were based
on host identity, a practice that is not always effective because some groups are
host-specific while others are not.
Lichens and Lichenicolus Fungi

About 20% of all fungi and 40% of the ascomycetes (13500 species) are
lichen-forming fungi (Lutzoni and Miadlikowska, 2009). Lichenicolous fungi,
parasites, and other associates of lichens are not well collected, but an esti-
mate for the combined lichens and lichenicolous fungi is about 20000 species
(Feuerer and Hawksworth, 2007). Lichens and lichenicolous fungi are polyphy-
letic, and several different groups of ascomycetes and a few species of basidio-
mycetes have become associated with green algae and cyanobacteria (Lutzoni and
APPENDIX A 153
Miadlikowska, 2009). Feuerer (2010) can be consulted for information on lichen

diversity worldwide. This checklist also highlights the absence of collections in
certain regions.
Deserts are rich in lichens. Of 1971 lichen species and associated fungi
reported from the Sonoran Desert, about 25% studied since 1990 are new. Three
volumes on lichens of the greater Sonoran Desert region have been published
(Nash et al., 2002, 2004). Other habitats of high lichen diversity are Arctic and
Antarctic regions (Feuerer, 2010).
Fungi From Arthropod and Invertebrate Animals

There is a need for more information on arthropod- and insect-associated
fungi. As was mentioned earlier, estimates of global fungal diversity usually omit
insect-associated species because they are so poorly known (Hawksworth, 1991;
Rossman, 1994; Mueller and Schmit, 2007; Schmit and Mueller, 2007). Several
post-1991 estimates of insect-associated fungi suggested that 20Â€000–50Â€000 spe-
cies exist (Rossman, 1994; Weir and Hammond 1997a, b; Schmit and Mueller,
2007). Some parasites are biotrophic, associated with living insects, and many
do not grow in culture. These also usually require special methods for removal
and mounting, and few mycologists or entomologists have ever seen members
of the Laboulbeniomycetes or the fungal trichomycetes, Asellariales and Harpel-
lales (Lichtwardt et al., 2001; Cafaro, 2005). Laboulbeniomycetes are seta-sized,
ectoparasitic ascomycetes of insects, mites, and millipedes (Weir and Blackwell,
2005). All 2000 known species have distinctive life cycles with determinate thalli
arising from two-celled ascospores. About 90% of the species have been found
on adult beetles (12 of 24 superfamilies) or on flies. New arthropod hosts at the
level of family are still being discovered (Weir and Hammond, 1997a, b; Rossi
and Weir, 2007), and there is an indication that there is some degree of host speci-
ficity (De Kesel, 1996). In the future, increased use of molecular methods will
make it possible to determine the degree of species level host specificity, but the
information is not available now. Septobasidiales, relatives of the basidiomycete
rust fungi are associated with scale insects, and their felty basidiomata presum-
ably protect the insects from parasitoid wasps. Many microsporidians also are
parasites of a broad group of host insects.
Necrotrophic parasites of insects include some members of Chytridiomycota,
Blastocladiales (Coelomomyces), Entomophthorales, and Tubeufiaceae (Podo-
nectria) (Benjamin et al., 2004). About 5000 members of three families of Hypo-
creales are necrotrophic parasites of arthropods (Spatafora et al., 2007, 2010).
These species show an evolutionary pattern of host shifting among plants, fungi,
and insects in addition to displaying a high level of host specificity.
Fungi also occur in ancient, obligate gardening associations with bark and
ambrosia beetles, attine ants, and Old World termites, and new species are still
being discovered in these groups (Benjamin et al., 2004; Little and Currie, 2007;
154 FUNGAL DISEASES
Harrington et al., 2008; Aanen et al., 2009). Many yeasts are associated with
insects, particularly insects that feed on nectar (Lachance, 2006; Robert et al.,
2006).
Other insects contain gut yeasts, a habitat where few have looked for them.
Isolations from the gut of mushroom-feeding beetles yielded up to 200 new spe-
cies of yeasts (Suh et al., 2004, 2005; see also Lachance et al., 2010). Because
only about 1500 ascomycete yeasts (Saccharomycotina) have been described, the
gut yeasts represent a dramatic increase in diversity from a limited geographical
range (Boekhout, 2005; C. Kurtzman, USDA-ARS, personal communication,
July 2010). In fact, the estimated total number of yeast species worldwide could
be increased by as much as 50% by simply recollecting in previously collected
sites from the study (Suh et al., 2005). As Lachance (2006) pointed out, based
on predictions of yeast numbers using data from species in slime fluxes and in
associations with flower-visiting insects, it is necessary to obtain more informa-
tion on specificity and geographical ranges before better estimates can be made.
Although not all insects harbor large numbers of yeasts in their guts, those with
restricted diets in all life history stages such as mushrooms or wood are often
associated with yeasts. Host insects may acquire digestive enzymes or vitamins
from the yeasts. This contention is supported by the fact that unrelated insects
feeding on mushrooms (e.g., beetles in different lineages, lepidopteran larvae) all
have gut yeasts with similar assimilative capabilities and vitamin production. The
high rate of discovery of yeasts in under-collected habitats and localities suggests
that far more taxa await discovery (Suh et al., 2005), and the gut habitat has been
considered a yeast diversity hotspot (Boekhout, 2005).
Insects may be food for fungi, especially in low nitrogen environments. The
mycelium of Pleurotus ostreatus, a favorite edible species for humans, secretes
toxic droplets that kill nematodes. A study involving the mushroom-producing,
ectomycorrhizal basidiomycete, Laccaria bicolor, was designed to determine
the amount of predation by springtails on the fungal mycelium. The study led
to the surprise discovery that the fungus was not insect food, but rather, it, and
indirectly, the host tree benefited by obtaining substantial amounts of nitrogen
from the insects (Klironomos and Hart, 2001). The predatory habit has arisen
independently on several occasions in at least four phyla of fungi and oomyce-
tes. Predaceous fungi such as species of Arthrobotrys and Dactylella lure, then
trap, snare, or grip nematodes and other small invertebrate animals in soils and
in wood (Barron, 1977).
Ødegaard (2000) revised global estimates of arthropods downward from 30
million to 5–10 million. Not all insects and arthropods are tightly associated with
fungi, but even the revised species estimates indicate that the numbers of insect-
associated fungi will be very high.
APPENDIX A 155
Soil Fungi
Soil is a habitat of high fungal diversity (Waksman, 1922; Gilman, 1957;
Kirk et al., 2004; Domsch et al., 2007). Soil fungi and bacteria are important
in biogeochemical cycles (Vandenkoornhuyse et al., 2002), and the diversity of
soil fungi is highest near organic material such as roots and root exudates. Per
volume, large numbers of microscopic fungi occur in pure soil, and these are
largely asexual ascomycetes and some zygomycetes, including animal-associated
Zoopagales. Gams (2006) estimated that 3150 species of soil fungi are known,
and ca. 70% are available in culture. There presently is a high rate of new spe-
cies acquisition, and the group appears to be better known than most ecologically
defined groups. Molecular studies, however, are predicted to increase the total
number (Bills et al., 2004). In fact a study of soil communities in several for-
est types at the Bonanza Creek Long Term Ecological Research site, Fairbanks,
Alaska, United States, revealed not only seasonal changes in community compo-
sition but also in dominance of fungi over bacteria. The data acquired by several
molecular methods including high-throughput sequencing greatly increased the
total number of fungal sequences in GenBank at the time (Taylor et al., 2010).
Taylor and his colleagues found more than 200 operational taxonomic units in a
0.25 g soil sample with only 14% overlap in a sample taken a meter away. This
study is not directly comparable with the soil fungi reported by Gams (2006)
because Gams’ figures excluded fungi such as mycorrhizal species.
Another study of soil fungi based on environmental DNA sequences showed
an unexpected distribution of a group of zoosporic fungi, Chytridiomycota. The
chytrids, were found to be the predominate group of fungi in nonvegetated,
high-elevation soils at sites in Nepal and in the United States in Colorado, where
more than 60% of the clone libraries obtained were from chytrids. A phylogenetic
analysis of the sequences compared with those of a broad selection of known
chytrids, indicated that a diverse group of Chytridiomycota representing three
orders was present (Freeman et al., 2009).
Most major fungal lineages are known from cultures and specimens, but
there have been a few surprises even in well-sampled habitats such as soil. Soil
clone group I (SCGI) represents a major lineage of fungi that occurs in temperate
and tropical soils on three continents, but no one has ever seen or isolated any of
the species into culture (Schadt et al., 2003; Porter et al., 2008).
The phylogenetic position of this lineage, perhaps a new phylum, appeared
as a sister group to the clade of Pezizomycotina–Saccharomycotina (Porter et al.,
2008). Other unexpected higher taxonomic level fungal clades have been detected
from environmental DNA sequences (Vandenkoornhuyse et al., 2002; Jumpponen
and Johnson, 2005; Porter et al., 2008). Another lineage detected by environ-
mental sequences was subjected to fluorescent in situ hybridization (FISH). The
outline of a single-celled, flagellated organism was detected (Jones and Richards,
2009), but apparently none of these fungi has been cultured either. Higher-level
156 FUNGAL DISEASES
bacterial taxa have been discovered by environmental sampling, but this is a far
less common occurrence for fungi (Porter et al., 2008).
Fungi form crusts that stabilize desert soils. Crusts usually are made up
of darkly pigmented ascomycetes, lichens, and nitrogen-fixing cyanobacteria
(States and Christensen, 2001). Rock-inhabiting fungi occur in the surface and
subsurface layers of desert rocks. These darkly pigmented ascomycetes are mem-
bers of the classes Dothideomycetes and Arthoniomycetes, but basidiomycetes
and bacteria may occur in the associations (Kuhlman et al., 2006; Ruibal et al.,
2009). Easily cultured asexual ascomycetes and other fungi also occur in desert
soils, and these include an unusual zygomycete, Lobosporangium transversale
(Ranzoni, 1968), known only from three isolations including Sonoran Desert soil.
Yeasts are well known from American deserts in association with cacti and flies
where they detoxify plant metabolites (Starmer et al., 2006).
Freshwater Fungi
Certain fungi are adapted for life in fresh water. More than 3000 species of
ascomycetes are specialized for a saprobic life style in freshwater habitats where
they have enhanced growth and sporulation (Shearer et al., 2007; Kirk et al.,
2008; Shearer and Raja, 2010). The asci are evanescent, and ascospores have ap-
pendages and sticky spore sheaths, that anchor the spores to potential substrates
in the aquatic environment. Conidia have several dispersal strategies, and these
are designated as Ingoldian (Fig. A3-2) and aero-aquatic (Fig. A3-3) conidia.
Ingoldian conidia are sigmoidal, branched, or tetraradiate and attach to plants
and other material in the water. The conidia float on foam that accumulates at the
banks of streams, especially during heavy runoff, and when the bubbles burst, the
spores may be dispersed for great distances from the water and into trees, where
they can be isolated from water-filled tree holes (Bandoni, 1981; Descals and
Moralejo, 2001; Gönczöl and Révay, 2003). Aero-aquatic fungi have multicel-
lular, often tightly helical conidia with air spaces to make them buoyant on the
surface of slower-moving waters (Fisher, 1977).
Other, less obviously modified fungi are present in water, and some of
these are active in degrading leaves in streams after the heavy autumn leaf fall.
A few specialized freshwater basidiomycetes also are known, and several have
branched conidia similar to those of the Ingoldian ascomycetes. Flagellated fungi
occur in aquatic habitats, including Chytridiomycota, Blastocladiomycota, and
Monoblepharomycota (James et al., 2006). Batrachochytrium dendrobatidis, the
recently described amphibian killer, is an aquatic chytrid (Longcore et al., 1999).
Members of Neocallimastigomycota also live in a specialized largely aquatic
environment, the gut of vertebrate herbivores, where they are essential for diges-
tion of cellulosic substrates.
APPENDIX A 157
Marine Fungi
Marine waters provide a habitat for certain specialized fungi (Kohlmeyer
and Volkmann-Kohlmeyer, 1991), and Hyde et al. (1998) estimated that more
than 1500 species of marine fungi occur in a broad array of taxonomic groups.
Many of these fungi are distinct from freshwater aquatic species, and they may be
saprobic on aquatic plant substrates. Some species have characters such as sticky
spore appendages, indicators of specialization for the marine habitat (Kohlmeyer
et al., 2000).
It is interesting that few fungi from early-diverging lineages have been
reported from marine environments, perhaps in part because mycologists study-
ing these groups sampled more often from fresh water habitats. More recently,
an investigation of deep-sea hydrothermal ecosystems revealed not only novel
species of ascomycetes and basidiomycetes, but also what may be a previously
unknown lineage of chytrids (Le Calvez et al., 2009).
Most marine fungi are ascomycetes and basidiomycetes, and these include
ascomycete and basidiomycete yeasts (Nagahama, 2006). Some of the yeasts
degrade hydrocarbon compounds present in natural underwater seeps and spills
(Davies and Westlake, 1979). Certain ascomycetes are specialists on calcareous
substrates including mollusk shells and cnidarian reefs. Even a few mushroom-
forming basidiomycetes are restricted to marine waters (Binder et al., 2006).
Some fungi use other marine invertebrates as hosts (Kim and Harvell, 2004),
including antibiotic producers that live in sponges (Bhadury et al., 2006; Pivkin
et al., 2006; Wang et al., 2008). A wide variety of fungi considered to be terres-
trial also are found in marine environments. Basidiomycete (i.e., Lacazia loboi)
and ascomycete yeasts, and other fungi including Basidiobolus ranarum, may oc-
cur in marine waters where they infect porpoises and other vertebrates (Kurtzman
and Fell, 1998; Murdoch et al., 2008; Morris et al., 2010).
Fungal Species
Currently, molecular methods provide large numbers of characters for use
in phylogenetic species discrimination (e.g., Kohn, 2005; Giraud et al., 2008). In
the past, biologists relied primarily on phenotype for species delimitation, and
most of the formally described species known today were based on morphology.
In addition, mating tests have been used to distinguish so-called biological spe-
cies, especially among heterothallic basidiomycetes (Anderson and Ullrich, 1979;
Petersen, 1995). The ability to mate, however, may be an ancestral character.
For example, Turner et al. (2010) found evidence that fungi have evolved strong
barriers to mating when they have sympatric rather than allopatric distributions.
Distant populations would not have had strong selective pressure against hybrid-
ization, thereby avoiding production of progeny less fit than conspecific progeny
(e.g., Garbelotto et al., 2007; Stireman et al., 2010). This phenomenon, known
as reinforcement, helps to explain how fungi from different continents can mate
158 FUNGAL DISEASES
in the laboratory but never in nature and is an argument in favor of recognizing

species by phylogenetics. A number of researchers have recognized species using
“phylogenetic species recognition” criteria (Taylor et al., 2000). The operational
phylogenetic method is based on a “concordance of multiple gene genealogies,”
and in addition to discriminating species, the method indicates whether fungal
populations actually exchange genes in nature (Taylor et al., 2000; Fisher et al.,
2002; Dettman et al., 2006; Jacobson et al., 2006).
The use of phylogenetic species criteria results in recognition of more spe-
cies than those delimited by morphological characters. For example, work on
Neurospora species resulted in the discovery of 15 species within five previously
recognized species (Dettman et al., 2006; Villalta et al., 2009). There are many
such examples among other groups of fungi, and eventually these may be a sig-
nificant source of new species discovery in the effort to discover 5 million fungi.
Fungal species recognized in this way may be described without a phenotypic
diagnosis, but it is not uncommon for distinguishing characters to be found with
guidance from the phylogenetics study (e.g., Otrosina and Garbelotto, 2010).
Conclusions
Until recently, estimates of numbers of fungi did not include results from
large-scale environmental sequencing methods. Newer estimates based on data
acquired from several molecular methods, however, have predicted as many as
5.1 million species of fungi (O’Brien et al., 2005; Taylor et al., 2010). Mycolo-
gists also are beginning to use high-throughput methods to gain insight into ques-
tions including geographical ranges and host and substrate specificity, topics that
have direct bearing on species numbers (Lumbsch et al., 2008). For example,
high-throughput methods have been used to determine the amount of overlap
between species within a given region by comparing soil samples a meter apart
to find only 14% species overlap (Taylor et al., 2010).
A better estimate of fungal numbers also can be speeded by enlisting more
biologists to accomplish the goal. When amphibian populations first were ob-
served to be dwindling and some species were determined to have disappeared
almost 20 yr earlier, a number of causes, all nonfungal, were suggested as the
explanation. The revelation that a chytrid was involved brought to mind that there
were probably fewer than 10 mycologists in the world who could collect, isolate,
culture, and identify the novel flagellated fungus, Batrachochytrium dendrobati-
dis (Longcore et al., 1999). Since that time interest in and publications on chytrids
have increased dramatically (e.g., Freeman et al., 2009; LeCalvez et al., 2009).
The interest in amphibian disease was in part the impetus for a large number of
recent publications on amphibian decline, but amphibian decline also justified
other projects, including training new chytrid systematists in monographic work.
This effort has resulted in the discovery of many new chytrid species and the
description of five new orders between 2008 and 2010. The rise of AIDS and the
APPENDIX A 159
accompanying large number of fungal infections brought about a similar interest

in medical mycology several decades ago.
In addition to any sudden influx of biologists to obtain better estimates of
fungal numbers, a new approach clearly is needed. In a thoughtful paper, Hib-
bett and colleagues (in press) called for obtaining clusters of similar sequences
and assigning Latin binomials to these molecular operational taxonomic units
(MOTUs). The names would allow the sequences to be integrated into a speci-
men-based taxonomic data stream. They considered inclusion of the sequence-
based taxa among all taxa to be a better alternative than the candidate taxon
status used by bacteriologists. Changes in the International Code of Botanical
Nomenclature would be needed if sequence-based materials were to be allowed as
nomenclatorial types. This proposal seems to be a practical approach to handling
the overwhelming fungal diversity being discovered.
Recent experience in working as a broadly inclusive group to plan and
produce a phylogenetic classification, the development of freely accessible data-
bases, and the use of new tools to survey fungi in ecological studies has prepared
the mycological community to accomplish a number of new goals, including the
discovery of millions of fungi.
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A4
BAT WHITE-NOSE SYNDROME IN NORTH AMERICA16

David S. Blehert, Jeffrey M. Lorch, Anne E. Ballmann, Paul M. Cryan, and
Carol U. Meteyer17
Since 2007, infections by a previously unrecognized, perhaps imported
fungus killed an estimated 1 million bats in North America.
Summary
• The newly described fungus, Geomyces destructans, causes an invasive
skin infection in bats and is the likely agent of white-nose syndrome
(WNS).
16â•›â•›Reprinted with permission from the American Society for Microbiology (Microbe, June 2011,
pp. 267–273).
17â•›â•›David S. Blehert is the head of the diagnostic microbiology laboratory at the U.S. Geological
Survey (USGS)–National Wildlife Health Center, Madison, Wis. (dblehert@usgs.gov), Jeffrey M.

Lorch is a graduate student with the Molecular and Environmental Toxicology Center, University
of Wisconsin–Madison, Medical Sciences Center, Madison, Wisconsin (jmlorch@wisc.edu), Anne
E. Ballmann is a wildlife disease specialist at the USGS–National Wildlife Health Center, Madison,
Wis. (aballmann@usgs.gov), Paul M. Cryan is a bat ecologist at the USGS–Fort Collins Science
Center, Fort Collins, Colo. (cryanp@usgs.gov), and Carol U. Meteyer is a wildlife pathologist at the
USGS–National Wildlife Health Center, Madison, Wis. (cmeteyer@usgs.gov).
168 FUNGAL DISEASES
• With immune system functions and body temperatures reduced dur-

ing hibernation, bats may be unusually susceptible to a pathogenic
fungus such as G. destructans.
• WNS was first observed in a popular show cave near Albany, New
York, leading some investigators to suspect that a visitor inadver-
tently introduced G. destructans at this site, triggering a wider WNS
outbreak in North America.
• Biologists trying to manage WNS within North American bat popula-
tions face major challenges, including the variety of susceptible host
species, incredible dispersal capabilities of bats, difficulties in treating
such populations, and persistence of the pathogen in their vulnerable
underground habitats.
In 2007 bats in eastern North America began dying in unprecedented num-

bers from a previously undocumented disease, now called white-nose syndrome
(WNS). Although the ecological and economic impacts of this disease are not
fully elucidated, this severe loss of insectivorous bats threatens decreased crop
yields, forest defoliation, and a rise in insect-borne diseases. The recent emer-
gence of WNS in bats of eastern North America, its rapid spread, and the severity
of the outbreak highlight the importance of wildlife disease as an integral com-
ponent of ecosystem health.
Biologists with the New York State Department of Environmental Conserva-
tion first recognized WNS as a problem in late winter 2007 at five hibernation
sites near Albany, N.Y. Subsequently, a recreational caver furnished a photograph
from February 2006 in nearby Howes Cave depicting bats with clinical signs of
WNS, implicating this location as the likely index site and suggesting disease
emergence the winter before New York state biologists drew public attention to
the disease. By 2011 WNS had spread south along the Appalachian Mountains
into eastern Tennessee, as far west as southern Indiana and western Kentucky,
and north into the Canadian provinces of Quebec, Ontario, and New Brunswick
(Figure A4-1). Experts estimate that more than 1 million bats have died from
WNS thus far. Modeling studies show that, if such mortality trends continue, one
of the most abundant bat species in eastern North America, the little brown bat
(Myotis lucifugus), could disappear from this region within 16 years. Sustained
killing of this magnitude from an infectious disease is unprecedented among the
approximately 1,100 species of bats known worldwide.
The Host, Pathogen, and Environment

The likely agent of WNS is a newly described fungus, Geomyces destruc-
tans, which causes an invasive skin infection that is the hallmark of this disease
(Figure A4-2). G. destructans belongs to the order Helotiales within the phylum
Ascomycota. Characteristics that distinguish it from other Geomyces spp. include
APPENDIX A 169
FIGURE A4-1â•‡Occurrence of white-nose syndrome and/or Geomyces destructans in

the United States (by county) and Canada (by county or district) from winter 2005–2006
through April 2011. Figure A4-1.eps
bitmap
curved conidia (Figure A4-2), slow growth on laboratory medium, cold adapta-
tion, and pathogenicity to bats. Species of Geomyces exist in soils worldwide,
especially in colder regions.
Any infectious disease involves interactions among a susceptible host, patho-
gen, and the environment. To comprehend the ecology of WNS, we must consider
the physiological and behavioral aspects of bats that make them susceptible to the
disease, the characteristics of the fungus that allow it to act as a pathogen, and
the role of underground sites (hibernacula) such as caves and mines in provid-
ing conditions conducive to maintaining this pathogen and enabling it to infect
these hosts.
WNS appears to occur only in bats, suggesting they possess unique traits
that make them a suitable host. Bats are nocturnal and the only mammals capable
of powered flight. Their forelimbs are highly modified, consisting of elongated
phalanges connected by a thin layer of skin to form wings. This body plan pro-
vides bats with selective advantages that allow them to dominate the night skies,
making them the second most diverse group of mammals, accounting for ap-
proximately 1,100 of 5,400 mammalian species. Of 45 bat species in the United
170 FUNGAL DISEASES
FIGURE A4-2â•‡ Micrograph of Geomyces destructans showing distinctive asymmetrically

curved conidia either free or borne singly at the tips and sides of branched conidiophores
(bar, 10 µm).
Figure A4-2.eps
bitmap
States, at least 6 of the approximately 25 that hibernate have been documented
with WNS, including the little brown bat, the northern long-eared bat (M. sep-
tentrionalis), the eastern small-footed bat (M. leibii), the endangered Indiana
bat (M. sodalis), the tricolored bat (Perimyotis subflavus), and the big brown
bat (Eptesicus fuscus). All six of those species are insectivorous and cope with
winter food shortages by hibernating in cold and humid, thermally stable caves
and mines. When hibernating, the animals typically congregate in large numbers,
dramatically reduce metabolic functions, and assume a body temperature close to
that of their surroundings (2–7°C). These physiological adaptations and behaviors
likely predispose bats to infection by G. destructans and consequent development
of WNS. Because approximately half the bat species of the United States are ob-
ligate hibernators, another 19 species are at risk for infection by G. destructans
if it spreads beyond its current range.
G. destructans colonizes the skin of bat muzzles, wings, and ears, then
erodes the epidermis and invades the underlying skin and connective tissues. This
pattern is distinctive and is more severe than that caused by typical transmissible
dermatophytes. Although the disease was named for the characteristic white
growth visible around an infected animal’s nose, the primary site of infection is
APPENDIX A 171
the wing (Figure A4-3A). Gross damage to wing membranes such as depigmenta-
tion, holes, and tears are suggestive of WNS, but these lesions are nonspecific,
and histopathologic examination is necessary to diagnose the disease.
Specifically, fungal invasion of wing membranes ranges from characteristic
cup-like epidermal erosions filled with fungal hyphae to ulceration and invasion
of underlying connective tissue, with fungal invasion sometimes spanning the full
thickness of the wing membrane (Figure A4-3B). Fungal hyphae can also fill hair
follicles and destroy skin glands and local connective tissue. Bat wings play an
important role in the pathogenesis of WNS by providing a large surface area for
the fungus to colonize. Once infected, the thin layer of skin that composes the
bat wing is vulnerable to damage that may catastrophically disrupt homeostasis
during hibernation.
In North America, bat hibernacula range in temperature from approximately
2–14°C, temperatures all permissive to growth of G. destructans. Within this
temperature range, G. destructans exhibits increasing growth rates with increas-
ing temperature (Figure A4-4), but the fungus does not grow at temperatures
of approximately 20°C or higher. This temperature sensitivity helps to explain
why WNS is observed only among hibernating or recently emerged bats and
why the disease is not diagnosed in bats during their active season when body
temperatures are consistently elevated above those permissive to growth of G.
destructans.
Looking for Other Host and Environmental Susceptibility Factors

Hosts with impaired immune functions tend to be susceptible to opportu-
nistic fungi in their environments. Guided by this concept, some investigators
suspected that insults such as exposure to environmental contaminants or infec-
tions by viral pathogens compromised bat immunity and made them vulnerable
to G. destructans. However, neither contaminant exposure nor viral coinfections
can be consistently identified in bats infected with that fungus.
Hibernating bats with WNS generally do not exhibit signs of an inflam-
matory response. However, severe inflammation typifies fungal skin infections
of bats aroused from hibernation, providing evidence that such animals are not
immunocompromised. Although studies of bat immune functions are in their
infancy, studies of other mammalian species indicate that their immune func-
tions are naturally suppressed during hibernation. Thus, rather than suggest-
ing immune-function impairment, the lack of inflammatory response to fungal
infection by hibernating bats may reflect an immune suppression that is part of
hibernation physiology.
In addition, the body temperature of hibernating bats drops dramatically,
providing another vulnerability to infection by G. destructans. Fatal fungal dis-
eases are relatively rare among endothermic, or warm-blooded, animals because
their tissues are too warm to support the growth of most fungal species. However,
172 FUNGAL DISEASES
(A)
(B)
Figure A4-3A.eps
bitmap
FIGURE A4-3â•‡(A) Three little brown bats (Myotis lucifugus) photographed by Alan
Hicks (New York State Department of Environmental Conservation) in Graphite Mine,
New York, in November, 2008. Note the white fungus colonizing the muzzles and nostrils
Figure fungal
of all three bats. Also note the extensive A4-3B.eps
colonization of the skin of the ears and
bitmap
wings of the bat pictured on the right; (B) Periodic acid-Schiff (PAS) stained microscopic
section of wing membrane from a little brown bat with white-nose syndrome collected in
Pennsylvania in February, 2009. Dense colonies of fungal hyphae erode skin and fill the
cup-shaped depressions (arrow). Ulceration of epidermis with penetration and replacement
of subcutaneous tissue (arrow heads) dramatically alters the integrity of wing membrane.
Bar = 25 µm.
APPENDIX A 173
0.8
0.7
Colony Expansion (mm/day)
0.6
0.5
0.4
0.3
0.2
0.1
0
0 5 10 15 20 25
Temperature
FIGURE A4-4â•‡ Colony expansion rates of Geomyces destructans when grown on corn-
meal agar at 3, 7, 14, and 20°C. The trend line estimates colony expansion rates at tem-
peratures ranging from 3–20°C.
Figure A4-4.eps
redrawn
fungi are more apt to cause fatal diseases in ectothermic, or cold-blooded, organ-
isms such as insects, fish, amphibians, and plants. Bats and other mammals that
hibernate are unique in that they are warm-blooded when metabolically active,
but cold-blooded during hibernation—a period when their metabolism and body
temperatures are dramatically suppressed. Although lowered body temperatures
may predispose torpid bats to infection by G. destructans, the mechanism en-
abling this specific fungus to be a pathogen for bats while other cave-associated
fungi remain innocuous is not known.
How G. destructans kills bats is under active investigation. One possibility
is that fungal infection disrupts how bats behave while hibernating, leading to
more frequent or longer arousals from torpor and thus accelerating usage of fat
reserves. However, fat depletion is not consistently observed among all bats with
WNS. Infected bats also may exhibit other aberrant behaviors midway through
the hibernation season, such as shifting from thermally stable roost sites deep
within hibernacula to areas with more variable temperatures near entrances.
174 FUNGAL DISEASES
Sometimes, they depart early from hibernacula. Thus, exposure to cold could
account for some WNS-associated mortality.
Further, fungal damage to wing membranes, which can account for more
than 85% of the total surface area of a bat, may increase fatality rates. In addition
to the key role that wings play in flight, wing membrane integrity is essential for
maintaining water balance, temperature, blood circulation, and cutaneous respi-
ration. Disrupting any of these functions could increase WNS mortality rates.
As with so many other diseases, the environment affects the progress and
transmission of WNS. Some pathogenic fungi such as Histoplasma capsulatum,
Cryptococcus spp., and Batrachochytrium dendrobatidis can persist in the envi-
ronment without an animal host for survival. This independence contrasts with
host-requiring viruses or other pathogens for which transmission dynamics tend
to moderate as infected hosts are removed from a population. G. destructans
likely does not require bat hosts to survive and can persist in caves by exploiting
other nutrients.
The cool and humid conditions of underground hibernacula provide ideal
environmental conditions for G. destructans or other fungal growth. While most
G. destructans isolates were cultured from skin or fur of bats collected in or near
underground hibernacula during winter, DNA from the same fungus is found
in soil samples from several hibernacula that harbor WNS-infected bats in the
northeastern US. Also, G. destructans has been cultured from soil samples from
hibernacula in three states where WNS occurs, supporting the hypothesis that bat
hibernacula are reservoirs for this pathogen and that bats, humans, or fomites may
transport G. destructans between hibernacula. How temperature and humidity
differences among hibernacula influence G. destructans and WNS is not known.
Uncertainties about WNS Emergence

What caused WNS to emerge in a North American cave during the winter of
2005 to 2006? Bats with clinical signs consistent with WNS were first observed in
Howes Cave, a hibernaculum connected to a popular North American show cave.
Because of its high human traffic, a tourist might have inadvertently introduced
G. destructans at this site.
Europe might be the source for the fungus causing WNS. Reports dating
back several decades describe hibernating bats in Germany with white muzzles
resembling bats with WNS in North America. Recent culture and PCR surveys in-
dicate that G. destructans is widespread in Europe, including among hibernating
bats in hibernacula in the Czech Republic, France, Germany, Hungary, Slovakia,
and Switzerland. Unlike in North America, however, mortality rates and popula-
tion declines remain normal among European bat species. This sharp contrast
between disease manifestation among bats in Europe and North America provides
an opportunity to investigate how bat species may differ in terms of their suscep-
tibilities to fungal infection, continental variability among fungal strains, and the
influence of environmental conditions and bat behavior on this fungal disease.
APPENDIX A 175
Challenges in Managing WNS, Conserving Bat Populations

Bat conservation efforts have historically focused mainly on reducing hu-
man causes of bat mortality, including habitat destruction, detrimental intrusions
into roosts, and intentional extermination of colonies. Bat census figures prior
to the emergence of WNS in North America indicate many populations of cave-
hibernating bats were stable or increasing. However, the current WNS outbreak
brings an even more serious threat to bat populations of North America, confront-
ing biologists with a new set of conservation and management challenges.
Mitigating diseases in free-ranging wildlife populations requires very dif-
ferent approaches from those applied in agriculture for domestic animals. Once
established, diseases in free-ranging wildlife are rarely, if ever, eradicated. Biolo-
gists trying to manage WNS within bat populations face multiple challenges, in-
cluding the need to deal with numerous host species, long-distance migrations of
infected hosts, poor access to some host populations, impracticalities associated
with treating individual wild animals, infected hosts that are sensitive to being
disturbed and that inhabit fragile ecosystems, and environmental persistence of
the pathogen.
The guiding principle for physicians and veterinarians, “first, do no harm,”
will help to prevent WNS management efforts from having unintended adverse
consequences. For example: depopulating an infected colony would not be ef-
fective unless all infectious animals are removed and all hibernacula used by the
population are decontaminated—conditions unlikely to be achieved among free-
ranging wildlife; using disinfectants to decontaminate hibernacula could have
toxic effects on other organisms reliant on those environments; treating individual
bats with antifungal agents is labor intensive, is not self-sustaining, and could
be toxic for treated animals or their symbionts; and careless intervention could
disrupt natural selective processes that might yield behaviorally or immunologi-
cally resistant bats.
However, “first, do no harm” does not mean “do nothing.” State and federal
agencies already are taking measures to combat WNS, including closing caves
and mandating decontamination procedures. Such steps are intended to prevent
people from disturbing hibernating bats and to reduce the chance that intruding
humans will transfer G. destructans from one hibernaculum to another. For ex-
ample, taking a proactive approach prior to the appearance of WNS, state wildlife
officials in Wisconsin conferred threatened status on four cave bat species that
hibernate within its borders and designated G. destructans a prohibited invasive
species providing state resource managers with legal authorities to take disease
management actions.
Since the first description of G. destructans in 2008, its genome has been
sequenced, and WNS pathology has been more fully defined. Additionally, hi-
bernacula are being surveyed internationally, and ongoing analyses are revealing
much about the biodiversity of fungi associated with bat hibernacula. With these
and other advances in understanding WNS, opportunities will arise to better
176 FUNGAL DISEASES
manage the disease cycle. The sudden and unexpected emergence of WNS exem-
plifies the importance of monitoring, investigating, and responding to emerging
wildlife diseases and the ecological and societal threats that they present.
SUGGESTED READING
Blehert, D. S., A. C. Hicks, M. Behr, C. U. Meteyer, B. M. Berlowski-Zier, E. L. Buckles, J. T.
H. Coleman, S. R. Darling, A. Gargas, R. Niver, J. C. Okoniewski, R. J. Rudd, and W. B.
Stone. 2009. Bat white-nose syndrome: an emerging fungal pathogen? Science 323:227.
Casadevall, A. 2005. Fungal virulence, vertebrate endothermy, and dinosaur extinction: Is there a
connection? Fungal Genet. Biol. 42:98–106.
Cryan, P. M., C. U. Meteyer, D. S. Blehert, and J. G. Boyles. 2010. Wing pathology of white-nose
syndrome in bats suggests life-threatening disruption of physiology. BMC Biol. 8:135.
Desprez-Loustau, M-L., C. Robin, M. Buée, R. Courtecuisse, J. Garbaye, F. Suffert, I. Sache,
and D. M. Rizzo. 2007. The fungal dimension of biological invasions. Trends Ecol. Evol.
22:472–480.
Butchkoski, and T. H. Kunz. 2010. An emerging disease causes regional population collapse
of a common North American bat species. Science 329:679–682.
Gargas, A., M. T. Trest, M. Christensen, T. J. Volk, and D. S. Blehert. 2009. Geomyces destructans
Kunz, T. H. and M. B. Fenton (ed.). 2003. Bat ecology. University of Chicago Press, Chicago.
Lindner, D. L., A. Gargas, J. M. Lorch, M. T. Banik, J. Glaeser, T. H. Kunz, and D. S. Blehert.
2010. DNA-based detection of the fungal pathogen Geomyces destructans in soil from bat
hibernation sites. Mycologia 103:241–246.
Meteyer, C. U., E. L. Buckles, D. S. Blehert, A. C Hicks, D. E. Green, V. Shearn-Bochsler, N.
J. Thomas, A. Gargas, and M. J. Behr. 2009. Pathology criteria for confirming white-nose
syndrome in bats. J. Vet. Diag. Invest. 21:411–414.
Wibbelt, G., A. Kurth, D. Hellmann, M. Weishaar, A. Barlow, M. Veith, J. Prüger, T. Görföl,
T. Grosche, F. Bontadina, U. Zöphel, H.-P. Seidl, P. M. Cryan, and D. S. Blehert. 2010.
White-nose syndrome fungus (Geomyces destructans) in bats, Europe. Emerg. Infect. Dis.
16:1237–1242.
APPENDIX A 177
A5
MAMMALIAN ENDOTHERMY OPTIMALLY

RESTRICTS FUNGI AND METABOLIC COSTS18,19
Aviv Bergman20 and Arturo Casadevall21
Abstract
Endothermy and homeothermy are mammalian characteristics whose evolu-
tionary origins are poorly understood. Given that fungal species rapidly lose their
capacity for growth above ambient temperatures, we have proposed that mamma-
lian endothermy enhances fitness by creating exclusionary thermal zones that pro-
tect against fungal disease. According to this view, the relative paucity of invasive
fungal diseases in immunologically intact mammals relative to other infectious
diseases would reflect an inability of most fungal species to establish themselves
in a mammalian host. In this study, that hypothesis was tested by modeling the
fitness increase with temperature versus its metabolic costs. We analyzed the trad-
eoff involved between the costs of the excess metabolic rates required to maintain
a body temperature and the benefit gained by creating a thermal exclusion zone
that protects against environmental microbes such as fungi. The result yields an
optimum at 36.7°C, which closely approximates mammalian body temperatures.
This calculation is consistent with and supportive of the notion that an intrinsic
thermally based resistance against fungal diseases could have contributed to the
success of mammals in the Tertiary relative to that of other vertebrates.
Importance
Mammals are characterized by both maintaining and closely regulating high
body temperatures, processes that are known as endothermy and homeothermy,
18â•›â•›Originally published as: Bergman, A. and A. Casadevall. 2010. Mammalian Endothermy Op-
timally Restricts Fungi and Metabolic Costs. mBio 1(5): e00212-10. doi:10.1128/mBio.00212-10.
19â•›â•›Received 17 August 2010 Accepted 11 October 2010 Published 9 November 2010 Citation
bolic costs. mBio 1(5):e00212-10. doi: 10.1128/mBio.00212-10. Editor Françoise Dromer, Institut
Pasteur Copyright © 2010 Bergman and Casadevall. This is an open-access article distributed under
the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License,
which permits unrestricted noncommercial use, distribution, and reproduction in any medium, pro-
vided the original author and source are credited. Address correspondence to Arturo Casadevall,
arturo.casadevall@einstein.yu.edu.
20â•›â•›Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx,
New York, USA.

21â•›â•›Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx,
New York, USA.

178 FUNGAL DISEASES
respectively. The mammalian lifestyle is energy intensive and costly. The evolu-
tionary mechanisms responsible for the emergence and success of these mamma-
lian characteristics are not understood. This work suggests that high mammalian
temperatures represent optima in the tradeoff between metabolic costs and the
increased fitness that comes with resistance to fungal diseases.
Endothermy and homeothermy are fundamental aspects of mammalian phys-
iology whose evolutionary origin remains poorly understood. Although many ex-
planations have been suggested for the origins of endothermy and homeothermy,
none are fully satisfactory given their high metabolic costs (Kemp, 2008; Ruben,
1995). Furthermore, the factors responsible for the mammalian set point remain
unknown, posing the additional question of why mammals are so hot. Recently,
the observation that fungal diseases are common in plants and insects but rare in
mammals, combined with the thermal susceptibility of fungi, led to the proposal
that mammalian endothermy and homeothermy create a thermal exclusionary
zone that protects mammals against mycoses (Robert and Casadevall, 2009).
Endothermy was also suggested to have provided a fitness advantage in the
fungal bloom that followed the end of the Cretaceous such that it could have
contributed to the success of mammals in the Tertiary (Casadevall, 2005; Robert
and Casadevall, 2009).
Assuming that a relationship exists between endothermy and reduced sus-
ceptibility to certain classes of microbes, we hypothesized a tradeoff relationship
whereby the high costs of endothermy were mitigated by protection against in-
fectious diseases. In other words, we posited that increases in body temperature
would protect against microbes by creating a thermal exclusionary zone but that
such increases would be increasingly costly with regard to metabolic rates as the
host body temperature diverged from ambient temperatures. Given that there is
robust information on fungal thermal tolerances (Robert and Casadevall, 2009),
we decided to test this hypothesis by attempting to identify body temperatures
that confer maximal fitness for certain metabolic rates.
To address this question, we propose a first-order model wherein a tradeoff
exists between the excess metabolic rates required to maintain a body tempera-
ture, T, and the benefit gained by protection against deleterious microbes because
of the creation of a thermal exclusion zone. Metabolism, the exchange of energy
between the organism and its environment, as well as the transformation of that
energy to material within an organism, is affected by two main factors, body
mass, M, and body temperature, T. Due to the fractal nature of transport networks,
that is, vessel architecture and branching (Gillooly et al., 2001; Savage et al.,
2008), over ontogeny, the resting metabolic rate, Brest, scales with body mass, m,
as Brest = B0m3⁄4, where B0 is a normalization constant for a given taxon. Also, the
normalization coefficient, B0, exponentially increases with body temperature B0
~ e–E0/KT, where E0 is the average activation energy for the rate-limiting enzyme-
catalyzed biochemical reactions of metabolism (ca. 0.65 eV), K is Boltzmann’s
constant (8.62 × 10–5eV/K), and T is body temperature (Brown et al., 2004;
APPENDIX A 179
Gillooly et al., 2001). The scaling relationship between resting metabolic rate
and body mass, ∝m3⁄4, has been predicted from allometric theories and supported
by data on a diverse set of organisms, including mammals, birds, fish, and mol-
lusks (Brody, 1964; Moses et al., 2008; Savage et al., 2004; West et al., 1997).
As can be seen from the formulas above, body temperature affects the metabolic
rate through its effects on rates of biochemical reaction kinetics according to
Boltzmann’s factor, e–Ei⁄kT, where T is measured in kelvins (absolute temperature).
The resting metabolic rate, Brest, is proportional to the product of these two ef-
fects and again has been shown to be well approximated, within a biologically
relevant temperature range (0°C to 40°C), as B(T) ∝e–Ei⁄kTm3/4 (Gillooly, 2001).
The first part of our analysis examined the excess cost for an organism of body
mass m to maintain a body temperature T (assuming no dependence of body mass
on temperature).
In the second part of our analysis, the benefit, noted here as F(T), is calcu-
lated as the reduction in the number of fungal species capable of infecting a host;
this number is reduced approximately by s ≈ 6% for every degree Celsius in the
temperature range of 27°C to 40°C (Robert and Casadevall, 2009). The increased
benefit of the successive elimination of fungal species can thus be expressed as
F(T) ∝F0[1 – (1 – s)T], where F0 is a constant scaling factor. The quantity W(T)
= F(T)⁄B(T) can represent the balance between cost and benefit; thus, W(T) can be
viewed as the total fitness of an organism as a function of its body temperature.
Within the biologically relevant temperature range, the proposed fitness measure
reaches its maximum at approximately 37°C (Fig. A5-1). Note that in this for-
mulation, the optimal body temperature, where W(T) attains its maximum value,
does not depend on the organism’s body mass. Furthermore, the one parameter
that is determined from biological observation is the reduction in the number of
fungal species capable of infecting a host; thus, to determine our model’s de-
pendence on this parameter, we calculated the optimal temperature over a wide
range of possible reduction percentages, i.e., 4% to 8%. In this range, the optimal
temperature was found to remain in a tight range of less than 2°C, from 37.7°C to
35.9°C, respectively, which is still within the biologically relevant range of mam-
malian body temperatures. The insensitivity of the model to its only parameter
further strengthens our hypothesis.
In summary, we present a minimal, parsimonious model to account for the
cost of maintaining a high body temperature in mammalian organisms. A body
temperature of 36.7°C maximizes fitness by restricting the growth of most fun-
gal species relative to its metabolic cost. Our model suggests that no additional
elaborations are required to explain the evolution of endothermy other than the
tradeoff between protection against environmentally acquired microbial dis-
eases and the cost of metabolism. Although we cannot rule out the possibility
that this body temperature optimum arose by some remarkable coincidence, we
think this highly unlikely because it emerges from considering two unrelated
processes, fungal thermal tolerance and mammalian metabolic costs. Nonethe-
180 FUNGAL DISEASES
FIGURE A5-1â•‡ Organism fitness as a function of body temperature. We normalized fit-

ness, W(T), to attain a maximum Figure
value of 1A5-1.eps
and plotted body temperature in degrees Cel-
bitmapa maximum value at WMAX(T) ≈ 36.7°C.
sius over a range of 27°C to 60°C. Fitness reaches
less, we acknowledge that similar temperature optima might emerge from other
considerations. For example, the specific heat capacity of water has a minimum
at 36°C, and if the efficiency of metabolic processes is related to heat capacity,
then using this parameter as the optimality criterion may result in a similar range
of solutions. Nevertheless, we note the internal consistency in the theme that
fungal diseases are rare in immunologically intact mammals and the tradeoff
between increased fitness and metabolic costs closely approximates mammalian
body temperatures.
Acknowledgements
Aviv Bergman is supported by 5P01AG027734-04 and 5R01AG028872-04.
Arturo Casadevall is supported by AI33774-11, HL59842-07, AI33142-11,
AI52733-02, and U54-AI057158-Lipkin.
References
Brody, S. 1964. Bioenergetics and growth. Hafner, New York, NY.
Brown, J. H., J. F. Gillooly, A. P. Allen, V. M. Savage, and G. B. West. 2004. Toward a metabolic
theory of ecology. Ecology 85:1771–1789.
APPENDIX A 181
Casadevall, A. 2005. Fungal virulence, vertebrate endothermy, and dinosaur extinction: is there a
connection? Fungal Genet. Biol. 42:98–106.
Gillooly, J. F., J. H. Brown, G. B. West, V. M. Savage, and E. L. Charnov. 2001. Effects of size and
temperature on metabolic rate. Science 293:2248–2251.
Kemp, T. S. 2008. The origin of mammalian endothermy: a paradigm for the evolution of complex
biological structure. Zool. J. Linn. Soc. 147:473–488.
Moses, M. E., C. Hou, W. H. Woodruff, G. B. West, J. C. Nekola, W. Zuo, and J. H. Brown. 2008.
Revisiting a model of ontogenetic growth: estimating model parameters from theory and data.
Am. Nat. 171:632–645.
gens. J. Infect. Dis. 200:1623–1626.
Ruben, J. 1995. The evolution of endothermy in mammals and birds: from physiology to fossils.
Annu. Rev. Physiol. 57:69–95.
Savage, V. M., E. J. Deeds, and W. Fontana. 2008. Sizing up allometric scaling theory. PLoS Comput.
Biol. 4:e1000171.
Savage, V. M., J. F. Gillooly, W. H. Woodruff, G. B. West, A. P. Allen, B. J. Enquist, and J. H. Brown.
2004. The predominance of quarter-power scaling in biology. Funct. Ecol. 18:257–282.
West, G. B., J. H. Brown, and B. J. Enquist. 1997. A general model for the origin of allometric scaling
laws in biology. Science 276:122–126.
A6
VERTEBRATE ENDOTHERMY RESTRICTS MOST

FUNGI AS POTENTIAL PATHOGENS22,23
Vincent A. Robert24 and Arturo Casadevall25
The paucity of fungal diseases in mammals relative to insects, amphibians,

and plants is puzzling. We analyzed the thermal tolerance of 4802 fungal strains
from 144 genera and found that most cannot grow at mammalian temperatures.
Fungi from insects and mammals had greater thermal tolerances than did isolates
from soils and plants. Every 1°C increase in the 30°C–40°C range excluded an
22â•›â•›Vincent A.Robert and Arturo Casadevall, “A Vertebrate Endothermy Restricts Most Fungi as Po-
tential Pathogens”, Journal of Infectious Diseases, 2009, Vol. 200, Iss. 10, pp. 1623–1626. Reprinted
by permission of Oxford University Press.
23â•›â•›Received 23 May 2009; accepted 18 June 2009; electronically published 14 October 2009. Po-
tential conflicts of interest: none reported. Financial support: National Institutes of Health (awards
5R01AI033774, 5R01HL059842, and 2U54AI057158). Reprints or Correspondence: Dr Arturo Casa-
devall, Department of Medicine, Albert Einstein College of Medicine, Yeshiva University, 1300
Morris Park Ave, Bronx, NY 10461 (arturo.casadevall@einstein.yu.edu).
â•›â•›The Journal of Infectious Diseases 2009;200:000–000
â•›â•›© 2009 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2009/
20010-00XX$15.00
â•›â•›DOI: 10.1086/644642
24â•›â•›Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands.
25â•›â•›Department of Microbiology and Immunology and Division of Infectious Diseases, Department
of Medicine, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York.
182 FUNGAL DISEASES
additional 6% of fungal isolates, implying that fever could significantly increase

the thermal exclusion zone. Mammalian endothermy and homeothermy are po-
tent nonspecific defenses against most fungi that could have provided a strong
evolutionary survival advantage against fungal diseases.
Of the 1.5 million fungal species, only a few hundred are pathogenic to
mammals (Kwon-Chung and Bennett, 1992). Fungal diseases in mammals often
reflect impaired immune function, and fungi did not emerge as major pathogens
for humans until the late 20th century. For example, candidiasis was uncommon
until the 1950s, when thrush was associated with the introduction of antibiot-
ics that disrupted bacterial flora. Similarly, diseases such as cryptococcosis,
aspergillosis, and histoplasmosis were rare until recently, when their prevalence
increased with the human immunodeficiency virus epidemic and the development
of immunosuppressive therapies. In contrast, the number of fungal species patho-
genic to plants and insects is estimated to be 270,000 and 50,000, respectively
(Hawksworth and Rossman, 1997). Amphibians are particularly vulnerable to
certain fungal infections, as evidenced by the current catastrophic epidemic of
chytridiomycosis in frogs.
The resistance of mammals with intact immune systems to systemic fungal
diseases, coupled with their endothermic and homeothermic lifestyles, suggested
that these costly physiological adaptations were evolutionarily selected because
they conferred a survival advantage by protecting against environmental patho-
gens (Casadevall, 2005). However, testing this hypothesis was difficult because
knowledge of fungal thermal tolerance is largely anecdotal. Consequently, we
evaluated the thermal growth tolerances of fungal species in a reference collection
and compared them to mammalian temperatures.
Methods
A total of 4802 fungal strains belonging to 144 genera in the Centraalbureau
voor Schimmelcultures (Utrecht) collection were tested for growth at 4°C, 12°C,
15°C, 18°C, 21°C, 25°C, 30°C, 35°C, 37°C, 40°C, 42°C, and 45°C. Strains were
grown for times ranging from a few days to a few weeks on the most suitable
medium, generally glucose–peptone–yeast extract agar, potato-dextrose agar, or
yeast extract–malt extract agar. Growth was considered positive when a colony
was visible without magnification. The strain set included Ascomycetes and
Basidiomycetes but excluded Zygomycetes, which is not in the yeast database.
The culture deposit records were reviewed to identify the isolation source.
Fungi isolated from flowers, grains, and herbal exudates were grouped under
plant isolates. Animal isolates were classified depending on whether they origi-
nated from endothermic (mammals and birds) or ectothermic (insects, nematodes,
fishes, and crustaceans) species. Another group comprised isolates from nonliving
environmental sources, which included predominantly soils; this group is referred
to as soil isolates. These groups were compared for thermal tolerance at 2 tem-
APPENDIX A 183
peratures, 25°C and 37°C, which reflect ambient and mammalian temperatures,
respectively.
To test the significance of the difference in growth patterns between fungal
strains isolated from different groups, we calculated the test statistics
) )
z = ( p 1 − p2 / P × (1 − P ) × (1 / n1 + 1 / n 2 ,
where p1 and p2 are the observed sample proportions for each group of fungal
strains at a given temperature, n1 and n2 are the size of the 2 groups under com-
parison, and
P = (p1 × n1 + p2 × n2)/(n1 + n2).
The statistic z was assumed to be distributed normally. The 2-tailed probability

from the absolute z score to infinity on both tails of the distribution was calcu-
lated (http://www.danielsoper.com/statcalc/calc21.aspx) and confirmed using the
NORMSDIST function in Excel (Microsoft) to assess the significance of differ-
ences in growth between groups at different temperatures.
Results and Discussion

Knowledge of fungal thermal tolerance is limited to a few species because
the subject has not been systematically studied. In fact, such studies may be
very difficult to do, and a comprehensive prospective study of fungal thermal
tolerance would require a gargantuan effort. However, culture collections pro-
vide an attractive alternative for initial explorations of this subject. Culture
collections store and maintain fungal strains and record basic nutritional needs
and temperature tolerances. This information, when accessed and analyzed with
bioinformatics tools, provides a useful starting point for the analysis of fungal
thermal tolerances.
Our results show that most strains grew well in the 12°C–30°C range, but
there was a rapid decline in thermal tolerance at temperatures >35°C (Fig-
ure A6-1). A plot of the fraction of fungal strain that grew versus temperature
in the 30°C–42°C range revealed a linear relationship with an equation of y =
-0.0166x × 2.7911, such that for every 1° increase in temperature >30°C, ~6%
fewer strains could grow.
For 3020 strains, there was information on both source isolation and tem-
perature tolerance. This group included isolates from the environment (primarily
soils), plants, ectothermic animals, and endothermic animals. The majority of
these isolates grew at 25°C regardless of their source (Table A6-1). Nevertheless,
the proportion growing at 25°C was significantly greater for isolates recovered
from living hosts than from soils, irrespective of whether the hosts were ecto-
thermic plants and animals or endothermic animals. At 37°C, the proportion of
184 FUNGAL DISEASES
FIGURE A6-1â•‡ Frequency histogram of thermal growth tolerance for 4802 fungal strains
(bars). Lines connect percentagesFigure
for 49 mammalian
A6-1.eps (blue) and 12 bird (red) species core
temperatures. Obtained from McNab (1970).
bitmap
TABLE A6-1â•‡ Growth Tolerances for Fungi from Soils, Animals, and Plants at
2 Temperatures
Isolate Growth P valuesb
Origin, host type Yes No Unknowna Total P1 P2 P3
at 25°C
Soils, NA 657 42 7 706
Plant, ectotherm 1108 30 5 1143 <.001
Animal
Ectotherm 490 0 6 496 <.001 .29
Endotherm 661 5 9 675 <.001 .214 .263
at 37°C
Soils, NA 146 535 15 706
Plant, ectotherm 304 871 22 1143 .292
Animal
Ectotherm 193 284 19 496 <.001 .004
Endotherm 466 202 7 605 <.001 <.001 <.001
NOTE. NA, not applicable
a Refers to a small no. of isolates for which the temperature growth data was not complete.
b P1 refers to the comparison of isolates from soils, P2 refers to the comparison versus plant iso-
lates, and P3 refers to the comparison between isolates from ectothermic and endothermic animals.
APPENDIX A 185
fungi that grew was much higher for isolates from endothermic animals than
from ectothermic animals. The proportions of Ascomycetes and Basidiomycetes
fungi in each group were comparable, except for ectothermic hosts, which yielded
predominantly Ascomycetes fungi.
Isolates from ectothermic hosts (such as plants and insects) were significantly
more thermotolerant than isolates from soils. A significantly greater percentage of
fungal strains from insects grew at 37°C relative to those recovered from plants,
possibly reflecting the fact that insects can increase their temperature through
behavioral fevers that increase survival after fungal infection (Thomas and Blan-
ford, 2003). However, this explanation is unlikely to apply to plants, which have
much lower metabolic rates. Since thermal tolerance must be associated with nu-
merous metabolic changes that mitigate fungal damage, the association between
greater thermotolerance and plant pathogenicity could mirror adaptation to sur-
vival in a host with potent antifungal defenses, raising the tantalizing possibility
that selection pressures by virulence may contribute to thermal stability and vice
versa. In this regard, we note that Hsp90 orchestrates morphogenesis in Candida
albicans (Shapiro et al., 2009), thus providing a molecular association for heat
shock and a virulence-related phenotype that may be conserved in other fungi.
A survey of the fungal genera represented in our sample collection revealed
differences in the percentage of isolates capable of growth at 37°C. All genera
studied included some thermotolerant species, as defined by their ability to grow
at 37°C, but there were large differences in the percentage of species within
each genera. Thermotolerant genera included those from both Ascomycetes and
Basidiomycetes, but basidiomycetous genera were disproportionately more com-
mon among the thermotolerant genera (P < .001, Fisher exact test). The strains
grouped within the sexually related basidiomycetous genera Filobasidiella (a
telemorph of Cryptococcus) and Cryptococcus (an anamorph of Filobasidiella)
included comparable numbers of thermotolerant species (61% among 116 strains
and 53% among 287 strains, respectively). These data suggest an association
between phylogeny and thermotolerance.
The capacity for thermotolerance was interspersed among Ascomycetes and
Basidiomycetes, suggesting that it may have emerged independently several
times in evolution. Alternatively, thermotolerance may be an ancient fungal trait
that was lost by those species that cannot grow at 37°C. In this regard, we note
that the climate for much of Earth’s history was much warmer than in recent
geologic epochs, having cooled by ~5°C during the Eocene-Oligocene transition
~34 million years ago (Liu et al., 2009). The fact that thermotolerance is a com-
plex trait that can be lost by a single mutation, as demonstrated by laboratory-
generated temperature-sensitive mutants, makes the explanation of a retained
phenotype attractive.
Our results may be relevant to the ongoing debate on the origin and func-
tion of endothermy, homeothermy, and fever, each a major unsolved problem in
vertebrate physiology (Kemp, 2008; Ruben, 1995). There is no consensus as to
186 FUNGAL DISEASES
why mammals have adopted such an energetically costly lifestyle. Endothermy

is associated with certain metabolic benefits and thermodynamic efficiency, but
these benefits come at a high cost since endothermic vertebrates require ~10 times
more oxygen to support metabolism than do ectothermic vertebrates (Ruben,
1995). Our analysis suggests that part of the cost is mitigated by the creation of
a thermal exclusionary zone that can protect against environmental microbes.
Given the high metabolic cost of endothermy, the core temperatures of individual
mammal and bird species are likely to be a compromise between its benefits and
costs. If endothermy was selected for protection against infectious disease, then
a case could be made that endothermy preceded homeothermy. Similarly, if one
considers fever as a mechanism to extend the thermal exclusionary zone against
environmental microbes such as fungi, increases in temperature of only 1°–3°
can significantly reduce the proportion of such microbes that can inhabit the host.
The benefits of endothermy and homeothermy in protection against microbes
do not appear to have been previously considered as mechanisms for evolutionary
selection, possibly because most of the viral and bacterial diseases that currently
plague animals are often acquired from other warm hosts, and these necessarily
involve thermotolerant microbes. However, the perspective is very different when
one focuses on environmentally acquired microbes and the fungi in particular.
Pathogenic microbes are a very small subset of the total terrestrial microbial
flora, and these can be divided as to whether they are acquired from other hosts
or directly from the environment (Casadevall and Pirofski, 2007). For mammals,
pathogenic microbes acquired from other hosts are usually adapted to mammalian
temperatures, but microbes acquired directly from the environment would not be
subject to such selection pressures. Hence, the potential benefit of endothermy
and homeothermy to host defense may become apparent only when one consid-
ers the entire microbiota and that subset of pathogenic microbes that is acquired
directly from the environment. In support of this notion, we note that bats become
susceptible to a cold-loving fungus when hibernation greatly reduces their body
temperatures (Blehert et al., 2009) and that primitive mammals (such as the egg-
laying platypus, which has a body temperature of 32°C) are susceptible to fungal
diseases (Obendorf et al., 1993). An epidemiological observation consistent with
the protective function of endothermy comes from the observation that serotype
D Cryptococcus neoformans are less thermotolerant (Martinez et al., 2001) than
other varieties and are associated with cutaneous cryptococcosis (Dromer et al.,
1996). Experimental support for the notion that endothermy restricts fungal in-
fection comes from the observation that rabbits, which have core temperatures
of 38°C–39°C, are notoriously resistant to cryptococcosis, and infection can be
induced only in cooler organs, such as testes (Perfect et al., 1980). However, the
same system also shows that mammalian immune systems also make a decisive
contribution to host defense against fungi since systemic cryptococcosis can be
induced in rabbits after corticosteroid administration (Perfect et al., 1980).
This study reflects the power of a bioinformatics analysis of archival data
APPENDIX A 187
from culture collection, which allows comparison of temperature growth data

on thousands of isolates. However, there are certain limitations that should be
considered in evaluating the data. Strains from plants and insects were dispropor-
tionately represented in the collection, and this may introduce certain biases. The
relative paucity or absence of strains from certain sources and taxonomic groups
could contribute to bias in the statistical analysis. For example, there were rela-
tively few isolates from birds and ectothermic animals other than insects, no Zy-
gomycetes fungi, and only a few filamentous fungi. Furthermore, the catalogued
information was insufficiently detailed to distinguish between skin and systemic
isolates from endotherms, which could differ in thermotolerance.
The discovery of fossilized fungal proliferation at the Cretaceous-Tertiary
boundary was proposed to contribute to extinction events at the end of the Creta-
ceous epoch that replaced reptiles with mammals as the dominant large animals
(Casadevall, 2005). Thermal tolerance is a necessary, but not sufficient, charac-
teristic of microbes being capable of causing invasive disease in mammals. Since
thermal tolerance almost certainly involves many genes and biochemical pro-
cesses, it is unlikely that this trait can be rapidly acquired by any one microbial
species. Consequently, new human pathogenic fungi are likely to emerge from
genera that are already tolerant to higher temperatures; such species may warrant
special attention given likely climatic changes in the years ahead that could alter
patterns of fungal prevalence.
References
Blehert DS, Hicks AC, Behr M, et al. Bat white-nose syndrome: an emerging fungal pathogen? Sci-
ence 2009; 323:227.
Casadevall A. Fungal virulence, vertebrate endothermy, and dinosaur extinction: is there a connec-
tion? Fungal Genet Biol 2005; 42:98–106.
Casadevall A, Pirofski LA. Accidental virulence, cryptic pathogenesis, martians, lost hosts, and the
pathogenicity of environmental microbes. Eukaryot Cell 2007; 6:2169–74.
Dromer F, Mathoulin S, Dupont B, Letenneur L, Ronin O. Individual and environmental factors
associated with infection due to Cryptococcus neoformans serotype D. Clin Infect Dis 1996;
23:91–6.
Hawksworth DL, Rossman AY. Where are all the undescribed fungi? Phytopathology 1997; 87:888–91.
Kemp TS. The origin of mammalian endothermy: a paradigm for the evolution of complex biological
structure. Zool J Linn Soc 2008; 147:473–88.
Kwon-Chung KJ, Bennett JE. Medical mycology. Philadelphia: Lea & Febiger, 1992.
Liu Z, Pagani M, Zinniker D, et al. Global cooling during the eocene-oligocene climate transition.
Science 2009; 323:1187–90.
Martinez LR, Garcia-Rivera J, Casadevall A. Cryptococcus neoformans var. neoformans (serotype
D) strains are more susceptible to heat than C. neoformans var. grubii (serotype A) strains. J
Clin Microbiol 2001; 39:3365–7.
McNab BK. Body weight and the energetics of temperature regulation. J Exp Biol 1970; 53:329–48.
Obendorf DL, Peel BF, Munday BL. Mucor amphibiorum infection in platypus (Ornithorhynchus
anatinus) from Tasmania. J Wildl Dis 1993; 29:485–7.
Perfect JR, Lang SDR, Durack DT. Chronic cryptococcal meningitis. Am J Path 1980; 101:177–93.
188 FUNGAL DISEASES
Ruben J. The evolution of endothermy in mammals and birds: from physiology to fossils. Annu Rev
Physiol 1995; 57:69–95.
Shapiro RS, Uppuluri P, Zaas AK, et al. Hsp90 orchestrates temperature-dependent Candida albicans
morphogenesis via Ras1-PKA signaling. Curr Biol 2009; 19:621–9.
Thomas MB, Blanford S. Thermal biology in insect-pathogen interactions. Trends Ecol Evol 2003;
18:344–50.
A7
SURVEILLANCE FOR EMERGING DISEASES IN WILDLIFE

Peter Daszak, Carlos Zambrana-Torrelio, and Tiffany Bogich26
Impact of Fungal Diseases on Wildlife

At this meeting, a number of presenters discussed the role of specific fungal
diseases in rapid declines and even extinctions of wildlife. Getting an accurate
measure of the impact of a pathogen or group of pathogens on wildlife is notori-
ously difficult. First, wildlife populations undergo often dramatic shifts that are
difficult to distinguish from the effects of an outbreak. These may be seasonal or
interannual, and occur in response to long-term climatic fluctuations, variation
in predator–prey cycles, or a range of other difficult-to-measure factors (Daszak
et al., 2005; Pechmann et al., 1991). Second, outbreaks of disease in wildlife may
cause significant mortality, but these events may be difficult to detect due to rapid
scavenging or decay of carcasses. Even when carcasses are found, they may be
too decayed to conduct proper pathological investigations. Third, despite a range
of infectious agents linked to recent declines, these are relatively new to ecolo-
gists and wildlife managers, so that die-offs are often attributed to other factors,
and diseases may not be examined. Despite these and other issues, emerging dis-
eases—indeed, emerging fungal diseases—have been shown to cause significant
population declines and even extinctions in wildlife (Daszak and Cunningham,
1999; Frick et al., 2010; Schloegel et al., 2006).
The emerging fungal disease chytridiomycosis is a good example of this
trend. As discussed elsewhere in this report, it is caused by the fungal pathogen
Batrachochytrium dendrobatidis, which infects the keratin-rich cells on the skin
of adult amphibians (Kilpatrick et al., 2010). This disease was discovered in the
1990s and associated with significant population declines in Australia and Cen-
tral America (Berger et al., 1998). Substantial support from other studies shows
it is the major cause of global amphibian declines (Crawford et al., 2010; Lips
et al., 2006; Skerratt et al., 2007). However, when this disease was first reported,
a debate took place in the literature over whether amphibians were undergoing
26â•›â•›EcoHealth Alliance, 340 West 34th Street, New York, NY 10001.

APPENDIX A 189
population declines, or simple fluctuations (Blaustein, 1994). Debate continued

on the importance of chytridiomycosis and other factors. The ecological com-
munity took about a decade to accept the role of disease in these wild animals as
important enough to call for large-scale global action to prevent further spread
(Mendelson et al., 2006).
Despite these issues, reports of emerging diseases affecting wildlife popula-
tions have grown rapidly (Aguirre and Tabor, 2008; Cunningham, 2005; Daszak
et al., 2000; Deem et al., 2001; Nettles, 1996; Wildlife and emerging disease,
2009; Williams et al., 2002). These include diseases that have caused a number
of high-profile declines, such as mycoplasmal conjunctivitis of house finches in
the United States (Fischer et al., 1997); trichomonosis in declining birds in the
United Kingdom (Robinson et al., 2010); chronic wasting disease of cervids in
the United States (Miller and Williams, 2004; Sigurdson, 2008); and white-nose
syndrome (Blehert et al., 2009) or geomycocis (Chaturvedi and Chaturvedi, 2011)
of bats. Among these are a surprising number of fungal diseases, which often
have a high impact. In humans, fungal pathogens do not represent a major cause
of emerging diseases.
Analysis of a global database of emerging pathogens (Jones et al., 2008)
suggests that fungi are responsible for only 5.9 percent of the emerging infectious
diseases of people in the past four decades (Figure A7-1). Yet in wildlife, fungi
have been implicated in global declines of amphibians, leading to extinction of
species (Schloegel et al., 2006), multistate declines of bat populations (Frick
et al., 2010), the near extinction of the Florida Torreya tree (Schwartz et al., 1995,
2000), and the collapse of eel grass beds, leading to global extinction of the eel
grass limpet Lottia albicans (Carlton, 1993; Carlton et al., 1991).
Challenges in the Surveillance of Wildlife for Fungal and Other Pathogens

The emergence of so many high-impact diseases in wildlife and the role of
wildlife as reservoirs for human emerging infectious diseases, or EIDs (Mahy
and Brown, 2000; Taylor et al., 2001), have led to expanding efforts in surveil-
lance of wildlife populations, both for new pathogens of human significance and
for potential EIDs affecting wildlife. However, a number of important factors
hinder this strategy. Here, we highlight the two most important challenges to
effective global surveillance in wildlife, as well as review some approaches to
address these challenges; and put them into context for emerging pathogens of
humans and wildlife, and for fungal diseases in particular.
Jurisdictional Problems in the Surveillance of Wildlife

The agencies, funding bodies, non-governmental organizations (NGOs),
and researchers that normally work with wildlife populations are usually distinct
from those involved in public health and agricultural diseases. For example, in
190 FUNGAL DISEASES
Number/
Percentage
173/48.3%
21/5.9%
13/3.6%
1/0.3%
42/11.7%
21/5.9%
87/24.3%
FIGURE A7-1â•‡ Proportion of emerging infectious diseases caused by different taxonomic

Figurefor
groups of pathogens. Fungi are responsible A7-1.eps
only 5.9 percent of emerging human dis-
ease, yet have a disproportionate impact on wildlife, with responsibility for a series of
bitmap
major population declines and w some
extinctions. The datavector
are takentype
from the database published
in a recent global analysis of emerging infectious diseases of humans over the last four
decades.
SOURCE: Adapted from Jones et al. (2008).
the United States, the U.S. Fish and Wildlife Service is the agency responsible for
protecting wildlife, but if the threat is an emerging pathogen, this agency has little
capacity for outbreak investigation and control. Similarly, the national agency
responsible for funding ecological research in the United States is the National
Science Foundation (NSF). The National Institutes of Health (NIH) oversees a
broad range of issues, from infections to organ dysfunction to mental health and
other areas. The U.S. Department of Agriculture oversees agricultural health. At
the international scale, the intergovernmental agency to protect wildlife is the
International Union for the Conservation of Nature (IUCN), whereas the global
health agenda falls under the World Health Organization (WHO), agricultural
health under the Food and Agriculture Organization (FAO), and trade-related
disease issues under the World Organisation for Animal Health (OIE). This siloed
approach is followed by many countries globally; they tend to have separate
ministries for health, agriculture, trade, and environment/forestry/wildlife. These
approaches work well until the threats to human health cross these jurisdictional
boundaries. With emerging diseases, they have done so repeatedly. For example,
the emergence of severe acute respiratory syndrome involved wildlife reservoir
APPENDIX A 191
species (Li et al., 2005), the national and international trade in hunted and farmed
wildlife and livestock (Xu et al., 2004), and international travel and migration
(Anderson et al., 2004). Likewise, the global emergence of amphibian chytrid-
iomycosis has been linked to trade and climate change (Lips et al., 2008), and
involves the medical industry (Weldon et al., 2004), the production of amphibians
for food (Schloegel et al., 2009), and introduced or invasive species (Kilpatrick
et al., 2010).
One simple approach to overcoming these challenges is to encourage cross-
disciplinary, cross-agency collaboration. This approach to research and policy
has been led by the fields of “conservation medicine” (Daszak et al., 2004), One
Health (Karesh and Cook, 2005), and EcoHealth (Daszak, 2009; Wilcox and
Daszak, 2006). In the United States, some efforts have successfully bridged the
funding gap between NIH and NSF, notably the Ecology of Infectious Diseases
program launched jointly by the NSF and the NIH John E. Fogarty International
Center in 2000 (Scheiner and Rosenthal, 2006). Likewise, there is a unique
U.S. federal agency with a specific remit to address with wildlife diseases, the
National Wildlife Health Center (NWHC) (Fleischli et al., 2004; Skerratt et al.,
2005). The NWHC has been conducting surveillance, monitoring, investigation,
research, and response on wildlife diseases for 35 years, and is registered with
the Centers for Disease Control and Prevention Select Agent Program, marking
it as a laboratory of significant relevance to human and livestock as well as wild-
life health. It has a sophisticated network of laboratories, including Biosecurity
Level 3 biocontainment labs, necropsy suites, and isolation rooms. In addition,
it publishes quarterly reports of mortality investigations, and acts as a national
focal point for similar activities in universities and NGOs. At the intergovern-
mental scale, there has been a recent flurry of activity to bring together agencies
around the One Health agenda, including formal links among the OIE, FAO, and
WHO, which originated from their collaborative efforts to tackle avian influenza
(Anderson et al., 2010). Additionally, wildlife health has two significant nuclei
within the United Nations system.
First, in the IUCN, there is the Species Survival Commission Wildlife Health
Specialist Group (http://www.iucn.org/about/work/programmes/species/about_
ssc/specialist_groups/specialist_group_pprofiles/veterinary_sg_profile/), which
has a network of more than 400 wildlife veterinarians and researchers globally.
Second, the OIE has a Working Group on Wildlife Diseases (http://web.oie.int/
wildlife/eng/en_wildlife.htm), which has operated for more than 15 years and
advises the OIE on wildlife health issues.
These initiatives have begun to bring diverse disciplines together to under-
stand the drivers and impacts of wildlife EIDs, and to conduct effective surveil-
lance and control of wildlife as reservoirs for human EIDs. However, they could
be improved significantly with some simple approaches. First, within the United
States, the government has the capacity to form interagency task forces for spe-
cific issues that cross agency mandates. In a previous administration, the complex
issue of amphibian declines was addressed with the formation of the Interagency
192 FUNGAL DISEASES
Taskforce on Amphibian Declines and Deformities. Similar task forces are likely
to be useful to address the need for better surveillance of the wildlife trade for
pathogens (Smith et al., 2009a), or the threat of white-nose syndrome in bats.
Second, at a global scale, strengthening of laboratory capacity and personnel for
wildlife diseases, and support for One Health approaches from the development
community, could be extremely useful in fostering linkages among disparate
ministries, universities, NGOs, and others. Recently, the U.S. Agency for Inter-
national Development launched an Emerging Pandemic Threats program that
specifically adopted a One Health approach to build capacity in the regions where
emerging zoonoses most commonly originate (http://www.usaid.gov/our_work/
global_health/home/News/ai_docs/ept_brochure.pdf) (Daszak, 2009). This in-
cludes specific collaboration among human and veterinary medical scientists;
ministries of health, agriculture, and environment; and OIE, FAO, and WHO.
Limited Resources for Surveillance and Lack of Predictive Capacity

During the past decade, significant economic investment has been made
in global surveillance for EIDs. Funding was provided by intergovernmental
agencies, and national governments in particular, to counter the specific threat
of H5N1 avian influenza. Although much of the surveillance and control efforts
focused on Southeast Asia, the recent emergence of an H1N1 triple reassortant
(Neumann et al., 2009; Smith et al., 2009b; WHO gets mixed reviews for H1N1
response, 2011) from a different region may have changed the public’s perception
of pandemic risk, and a shift of funding priorities. At the same time, the promi-
nence of H5N1 in the media over the past decade and its inability so far to mount
a pandemic heightens the perception of emerging diseases as difficult to predict
or forecast. With limited global resources for pandemic prevention or control,
predictive efforts that can help target resources are greatly needed.
Recent work has shown that analyzing past trends in disease emergence and
correcting data for surveillance biases allows us a strategy to identify the regions
most likely to propagate a new emerging zoonosis (Jones et al., 2008; King et al.,
2006; Taylor et al., 2001; Woolhouse and Gowtage-Sequeria, 2005). These ap-
proaches may provide a way to geographically target surveillance efforts. They
suggest that surveillance programs should target regions with high biodiversity
and dense human populations, essentially in tropical and subtropical regions
(Jones et al., 2008). This approach essentially analyzes the underlying drivers of
emerging diseases, which are usually socioeconomic factors (e.g., travel, trade,
agricultural changes) or environmental factors (e.g., climate, land use change).
If we adapt this to fungal pathogens, we see that two drivers in particular are
important—climate and trade. For human EIDs, analysis of the database in Jones
et al. (2008) suggests that the majority of emerging fungal diseases were associ-
ated with HIV/AIDS or antimicrobial use. For plant EIDs, analyses suggest that
agricultural trade and climate are the most important drivers of new emerging
diseases (Anderson et al., 2004). With the limited work that has been conducted
APPENDIX A 193
on emerging fungal pathogens of wildlife, trade appears to be an important driver

of emergence. For example, significant evidence points to the global amphibian
trade for food as a major driver of the spread of the amphibian fungal disease
chytridiomycosis (Schloegel et al., 2010). The similarity of U.S. isolates of Geo-
myces destructans (the causative agent of bat white-nose syndrome) with those
from Europe and its continued spread as if from a focal point at a tourist cave in
New York state strongly suggest anthropogenic introduction.
Two important recommendations emerge: First, targeting of wildlife trade
routes, wet markets,27 and international ports of entry for surveillance of wildlife
may be particularly fruitful in identifying novel EIDs (Karesh et al., 2005; Pavlin
et al., 2009; Smith et al., 2009a). Second, use of novel modeling approaches to
geographically targeting wildlife for surveillance may be particularly useful for
identifying novel pathogens. This “smart surveillance” approach (see Daszak,
2009) would help identify a pool of microbes harbored by a reservoir, some of
which ultimately may become zoonotic under the right circumstances. Clearly,
issues remain in that many microbes will be unable make the species jump to
humans, so that distinguishing the truly potential zoonoses from these is not a
simple task. However, conducting PCR-based surveillance in wildlife using con-
served (or “degenerate”) primers for known pathogen groups would allow the
targeting of novel agents related to known pathogens. This would enable a greater
understanding of the risk of new zoonoses from each wildlife species examined,
and would be a simple, cost-effective way to better evaluate the diversity of likely
zoonoses. Diagnostic assays could then be developed for these pathogens, and
used to screen people who live in close contact with the wildlife reservoirs.
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A8
GEOGRAPHY, CLIMATE, DUST, AND DISEASE:

EPIDEMIOLOGY OF VALLEY FEVER (COCCIDIOIDOMYCOSIS)
AND WAYS IT MIGHT BE CONTROLLED
John N. Galgiani28,29
Introduction
Human disease resulting from infection by Coccidioides spp. was first rec-
ognized late in the 19th century. Since then, with more information and chang-
ing demographics, our understanding of this problem and our perception of its
importance has evolved in many ways (Galgiani, 2007). First thought of as a rare
28â•›â•›Valley
Fever Center for Excellence, University of Arizona College of Medicine.
29â•›â•›Correspondence
and current address: John N. Galgiani, M.D.; Professor, University of Arizona
College of Medicine; Director, Valley Fever Center for Excellence; P.O. Box 245215, Tucson, AZ
85724; Tel.: 520-626-4968; Fax: 520-626-4971; e-mail: spherule@u.arizona.edu.
APPENDIX A 197
and always fatal illness, coccidioidomycosis later was appreciated as a common

and frequently self-limited illness known as valley fever, in which only a small
percentage of those affected suffered serious complications (Smith, 1940).
With U.S. military training within the endemic regions of California and
Arizona during World War II, the potential of coccidioidomycosis as a significant
problem affecting military readiness quickly became apparent, a problem that
persists for the military into the present (Crum-Cianflone, 2007; Smith, 1958).
With the rapid and extensive population expansion within south-central Arizona
over the past decades, the impact of coccidioidomycosis has emerged from a
rural to a much larger public health problem. With continued population growth
within the central valley of California, in Mexico along the U.S. border, and in
other endemic regions throughout the Western Hemisphere, it is expected that the
numbers of infected persons will continue to increase. In addition to the medical
problem for humans, a variety of other species, especially dogs, are susceptible
to coccidioidomycosis and suffer considerable morbidity and mortality as a result
(Shubitz, 2007).
A comprehensive assessment of coccidioidomycosis should address the fun-
gus as it exists in the environment as well as how it creates medical problems. In
both arenas, there are opportunities for better understanding which in turn could
lead to improvements in public health. Hopefully, this article will be useful to
call attention to where our knowledge is limited and how advances in those areas
could benefit prevention and management of coccidioidal disease.
Coccidioides spp. in the Environment

Our primary source for our understanding of the relative endemnicity for
regions within the United States is derived from studies conducted in the 1950s
of coccidioidin skin-test prevalence of naval recruits from across the country
(Palmer et al., 1957). These primarily include extensive portions of southern
California, the lower deserts of Arizona, west Texas, and smaller areas of Utah,
Nevada, and New Mexico. More recent information from California and Arizona
(Hector et al., 2011), where coccidioidomycosis is a reportable infectious dis-
ease, is consistent with the earlier estimate. Coccidioidal infections are found in
numerous other countries within the Western Hemisphere. What is known about
these areas also suggests that the endemic distribution has been stable over the
past several decades (Laniado-Laborin, 2007). On the other hand, the endemic
regions in a longer time scale may not be constant. Recent archeological findings
in Nebraska identified a Holocene bison dating back 8,500 years with coccidioi-
domycosis in a bone (Morrow, 2006). Also, models of wind pattern trends with
global warming would predict increased westerly currents from the Coccidioides-
endemic regions of west Texas toward the more populated portions of the state
(Reheis and Rademaekers, 1997). Thus, with climate change we might see the
endemic region expand.
198 FUNGAL DISEASES
Recent population studies of fungal isolates have identified genetic differ-

ences in isolates from patients in California (now classified as Coccidioides
immitis) as compared to isolates from elsewhere (now classified as Coccidioides
posadasii) (Burt et al., 1997; Fisher et al., 2000, 2001). This geographic sepa-
ration is surprising, given the likelihood that coccidioidal spores should travel
freely in and out of California, either by wind currents or as contaminants of
trains, planes, or road vehicles. One possibility is that C. immitis is much better
suited to its California niche than is C. posadasii and vice versa. Another expla-
nation might relate to the biology of how soil becomes infected and endemic
regions become established. For example, simply inoculating new soil may
not be sufficient to create a new coccidioidal colony. Instead, a more complex
interaction with rodents, plants, or other factors in the environment may be
needed. Reinforcing this speculation is the sparse distribution of Coccidioides
spp. within the environment. For example, in one systematic study in the central
valley of California, only four genetically distinct isolates were found from 720
soil samples (Greene et al., 2000). Soil sampling in southern Arizona has also
found isolates in a very small proportion of samples tested (Barker et al., 2010).
Researchers have known for some time that Coccidioides spp. are more
prevalent in specimens from rodent burrows than from random subsurface soil
(Elconin et al., 1957), although the reasons for this association remain unclear
(Barker et al., 2010). In contrast, repeated sampling from a site known to be posi-
tive often yields positive results. One site identified as containing Coccidioides
spp. in the 1960s (Converse and Reed, 1966) has again yielded C. posadasii half a
century later (Personal communication, January 2011, M. J. Orbach). The sparse
and stable nature of coccidioidal residence within the endemic regions affords
an implication as to what factors are more likely to be associated with risk of
exposure. Although there have been well-documented outbreaks associated with
archeological and other dirt-disrupting sites (Pappagianis, 1983; Werner et al.,
1972), in general occupational exposure does not appear to be a major risk fac-
tor (Kim et al., 2009). What might appear to be a paradox is actually consistent
with the sparse prevalence of the fungus within the soil of even its most endemic
regions, in which case much of the soil-disrupting activities occur at sites where
the fungus is not present. In contrast, climate and especially wind patterns have
been shown to affect seasonal incidence of infection (Comrie and Glueck, 2007;
Hugenholtz, 1957). Taken together, the evidence would suggest that simply
length of endemic exposure rather than a specific activity constitutes the more
dominant effect for risk of infection.
With our current understanding of Coccidioides spp. residing in the environ-
ment, it is not possible to predict whether it exists in a specific location with any
degree of precision from a physical or chemical analysis of the soil. Furthermore,
high-throughput methods do not exist for microbial detection of Coccidioides
spp. in soil samples. These missing tools prevent us from identifying specific
locations which, if disrupted, would likely create a release of fungal spores. If
APPENDIX A 199
methods were available to do this, methods for treating the soil exist that would
minimize or prevent this exposure from happening. Advances in this area would
have practical public health benefits.
Coccidioidomycosis: The Scope of the Problem

Statistics about newly diagnosed coccidioidomycosis from California and
Arizona were expected to total more than 16,000 new infections in 2010 (Fig-
ure A8-1). The sharp increase in reported cases in Arizona in 2009 is the result
of an administrative change by a single large clinical laboratory to report a more
sensitive coccidioidal serologic test as indicative of a new infection when posi-
tive. However, for both states there has been increased disease activity in the last
half of 2010 that is unexplained.
In Arizona, the Department of Health Services conducted a telephone ques-
tionnaire of 10 percent of newly identified patients in 2007. It provides a better
understanding of the impact of this disease (Tsang et al., 2010). According to
the survey, illness lasted for an average of 6 months; three quarters of employed
FIGURE A8-1â•‡ Annual cases of coccidioidomycosis.

Figure A8-1.eps
SOURCE: Figure courtesy of John N. Galgiani (data from the California Department of
bitmap
Health and Arizona Department of Health Services).
200 FUNGAL DISEASES
patients lost more than a month of work; a quarter of patients needed 10 or more
physician visits; and 40 percent of patients required hospitalization for their
illness (Tsang et al., 2010). The same report shows Arizona hospital costs for
coccidioidomycosis were more than $86 million. Extrapolating from these costs,
estimates that include all outpatient medical care, often lasting for years if not
entire lives, could easily reach a quarter of a billion dollars.
As significant as these findings are, other projections suggest that the actual
number of persons seeking medical attention for coccidioidal infection is several
times greater than those diagnosed and included in state public health statistics.
One study found that only 3–13 percent of patients with pneumonia in Phoenix
were tested for coccidioidomycosis (Chang et al., 2008). By contrast, a prospec-
tive study in Tucson in which patients with community-acquired pneumonia were
tested for coccidioidomycosis demonstrated that nearly a third of these subjects
had a coccidioidal infection (Valdivia et al., 2006).
State statistics show case rates for college-age persons in Pima County to be
from 34 to 48 cases per 100,000 annually. However, recent surveillance of schol-
arship athletes at the University of Arizona, which is in Pima County, indicated
374 cases per 100,000 (Stern and Galgiani, 2010). Further analysis suggested that
the most important reason for this much higher case rate was that the athletes
received many more serologic tests for coccidioidomycosis. Evidently, more pa-
tients would be accurately identified as to the true cause of their illness if patients
with endemic exposure to Coccidioides spp. were tested more routinely for this
possibility. This is now the recommendation of the Arizona Department of Health
Services and a growing number of Arizona state medical specialty societies and
other professional organizations (Tsang et al., 2010).
With the commercial and recreational growth of the southwestern United
States, coccidioidomycosis has become an increasing problem for the rest of the
country as well. For example, persons who develop a respiratory illness within
a month after returning from vacation or business conferences in south-central
Arizona would have the same risk (approximately 30 percent) that their illness is
due to Coccidioides spp. as would residents of the endemic regions. Using Ari-
zona Department of Tourism statistics for 2008, the chance of an individual visi-
tor developing any clinical illness would be expected to be small (approximately
1 in 17,000). However, because more than 22 million persons visit Arizona for
an average of 4 to 5 days, the total number of illnesses occurring after leaving
Arizona would add up to more than 1,300 per year. Evidence suggests that most
of these illnesses would be diagnosed incorrectly in the course of routine medical
care (Standaert et al., 1995).
Even if physicians obtain appropriate testing, establishing a diagnosis of
early coccidioidal infection is often difficult. Coccidioidal serology is very spe-
cific when results are positive. Moreover, in progressive forms of infection, serol-
ogy is very likely to be diagnostic (Fish et al., 1990; Pappagianis and Zimmer,
1990). However, these tests are not nearly as sensitive early in the course of the
APPENDIX A 201
acute pneumonia syndrome, the most common manifestation of a coccidioidal in-

fection. In one study, depending on the method of analysis, false-negative results
were estimated to occur from one third to two thirds of the time on first testing
(Wieden et al., 1996). Although improved methods for detecting Coccidioides-
specific antibodies may improve the sensitivity, approaches such as detection of
coccidioidal DNA by polymerase chain reaction (Clark and McAllister, 1996)
or detection of coccidioidal antigens by enzyme-linked immunosorbent assay
(ELISA) (Durkin et al., 2008; Helfrich et al., 2011) offer theoretical advantages
to earlier detection. Improvements in early diagnosis would be very useful to
clinicians trying to manage their patients.
Therapy for Coccidioidomycosis and Its Current Limitations

The most serious complications of coccidioidomycosis include progressive
chronic pneumonia and hematogenous spread of infection to parts of the body
outside of the chest. Patients with these problems have benefited greatly from
the advent of orally absorbed azole antifungal agents (Galgiani et al., 2005). Flu-
conazole and itraconazole are commonly used for these complications (Galgiani
et al., 1993, 2000; Tucker et al., 1990). The more recently available azoles,
voriconazole and posaconazole, offer additional options (Catanzaro et al., 2007;
Prabhu et al., 2004; Proia and Tenorio, 2004; Stevens et al., 2007). This class of
antifungal drugs has been found to be relatively safe and generally well toler-
ated for extended courses of administration. However, approximately a quarter
of patients with such infections do not respond adequately to these drugs. Even
in patients who appear to respond to azole treatment, relapses occur in approxi-
mately a third of those when treatment is discontinued. Thus, for some patients,
including all patients with coccidioidal meningitis, treatment is recommended
to be lifelong (Dewsnup et al., 1996). The most currently available drugs offer a
suppressive effect; they do not eradicate infections or cure patients.
Surprisingly, there is virtually no published experience on the use of any
antifungal drug for the most common manifestations of coccidioidal infection,
that of the early respiratory syndrome. In a recent prospective observational study
(Ampel et al., 2009), patients treated with oral azoles (usually fluconazole) ap-
peared neither to improve at a faster rate nor subsequently to avoid progressive
complication than did patients who did not receive antifungal treatment. Although
not a randomized controlled trial, this study presents the only information avail-
able and provides little encouragement for the value of early treatment.
Prospects for new drugs to treat coccidioidomycosis are limited (Ostrosky-
Zeichner et al., 2010). This is due in part to the general contraction in anti-
infective drug development in general (Talbot et al., 2006), but is especially
problematic for coccidioidomycosis because of its status as an orphan disease,
defined by the Food and Drug Administration (FDA) as having a U.S. prevalence
of less than 200,000. Even for new antifungals, such as voriconazole, posacon-
202 FUNGAL DISEASES
azole, and caspofungin (Gonzalez et al., 2001, 2007), which already have FDA
approval for other fungal diseases, there are very limited or no controlled clinical
trials conducted in patients with any form of coccidioidomycosis.
One exception to this pattern has been the persistent interest in bringing
nikkomycin Z into clinical trials. Nikkomycin Z is a competitive inhibitor of
chitin synthase, first discovered by German scientists in the 1970s. It was part of
a fungicide discovery program at the Bayer Company (Fiedler, 1988). Its poten-
tial as a therapeutic for coccidioidomycosis was identified in the 1980s (Hector
et al., 1990). In mice, nikkomycin Z treatment produced sterile lungs under
conditions in which the lungs of untreated mice yielded several million viable
fungal colonies. This observation raises the possibility that nikkomycin Z might
offer a curative treatment for coccidioidomycosis. If so, this would provide even
more incentive to diagnose coccidioidomycosis early in order to eradicate it and
thereby prevent later and serious complications. Clinical development of nikko-
mycin Z was begun in the 1990s by Shaman Pharmaceuticals (Galgiani, 2007).
However, the program became inactive when Shaman ceased to exist in 2000, and
for several years nikkomycin Z development remained dormant.
In 2005, the University of Arizona acquired the program along with several
kilograms of bulk nikkomycin Z that remained from Shaman’s program. Since
then faculty at the University of Arizona and a small start-up company, Valley
Fever Solutions, have successfully competed for research awards and small busi-
ness grants from the National Institutes of Health (NIH) and the FDA Office of
Orphan Products Development. With these funds as well as philanthropic support,
clinical trials with nikkomycin Z were resumed with a 2-week multidose safety
trial that was completed in 2009. This support is also being used to develop a
more efficient manufacturing process. Supplies of nikkomycin Z made by this
new process are planned to be available to begin a Phase II clinical trial in 2011 or
2012. It is hoped that this progress will advance the program sufficiently to attract
pharmaceutical or investment interest to complete the commercialization process.
Prospects for a Preventative Vaccine

A large majority of the estimated 150,000 U.S. coccidioidal infections oc-
curring annually resolve with or without symptoms and whether or not specific
antifungal treatment is instituted (Galgiani et al., 2005). Healing occurs as a result
of the patient’s cellular immunity, which is remarkably durable, usually lasting a
lifetime (Galgiani et al., 2010). The fact that natural infection so often produces
resistance to second infections has attracted a long-time interest in engendering
this immunity through active immunization, leading to a clinical trial of a whole-
cell killed vaccine (Pappagianis and Valley Fever Vaccine Study Group, 1993).
This preparation did not produce protection. In retrospect it was surmised that
the irritation at the injection site of the fungal cell wall polysaccharides prevented
sufficient doses of protective immunogens to be delivered during vaccination. For
APPENDIX A 203
the past 15 years, a collaboration of several research groups has yielded a number
of immunogenic coccidioidal proteins and vaccines prepared from recombinant
proteins with adjuvants, some of which have shown excellent protection in mice
and efficacy in primates (Cole et al., 2004; Cox and Magee, 2004; Herr et al.,
2007; Johnson et al., 2007; Shubitz et al., 2006; Tarcha et al., 2006).
The next step for existing recombinant vaccine candidates would be for them
to be moved into clinical trials. However, these candidates have met with major
challenges including developing a suitable manufacturing process; identifying a
suitable and available adjuvant; and compounding a suitable formulation appro-
priate for human experimentation (Galgiani, 2008). None of these challenges are
insurmountable, but all require significant development investment. The overall
impact of coccidioidomycosis within the endemic region is not so dissimilar to
that caused by polio in the United States before a polio vaccine was available
(approximately 10 per 100,000 population). However, the impact of coccidioido-
mycosis involves a much smaller population at risk as compared to the worldwide
distribution of polio. This difference in market size makes it unlikely that a com-
mercial vaccine manufacturer will invest in developing a coccidioidal vaccine
even though such a vaccine, once developed, could arguably be profitable to
manufacture and distribute (Barnato et al., 2001). Moving a coccidioidal vaccine
into clinical trials probably requires the discovery of new, more easily formulated
protective antigens; a breakthrough in vaccine technology that greatly reduces
the cost of development; or a growing public health imperative to underwrite the
costs needed for vaccine development.
Summary
Coccidioidomycosis is a major public health problem for a major, growing
segment of the U.S. population as well as other endemic regions throughout the
Western Hemisphere. A more complete understanding of its biology and ecology
where it exists in the endemic environment could lead to risk abatement strate-
gies not currently available. Improved recognition by healthcare professionals of
coccidioidomycosis as a cause of community-acquired pneumonia when it oc-
curs in their patients could improve management. This could be assisted further
by developing more sensitive and clinically available diagnostic tests based on
biosignatures such as DNA or proteins from the fungus itself. Curative therapies
are also needed, but none exist today. Finally, eliminating problems caused by
Coccidioides spp. might be possible if a preventive vaccine were developed. Even
though coccidioidomycosis is an orphan disease, pursuit of these objectives is
more than justified by the potential public health benefit and the reduced medical
costs to society that their achievement would provide.
204 FUNGAL DISEASES
Acknowledgments
This presentation was supported in part by Award Number U54AI065359
from the National Institute of Allergy and Infectious Diseases (NIAID). The
content is the sole responsibility of the authors and does not necessarily represent
the official views of the NIAID or NIH.
Disclosure
Dr. Galgiani is chief medical officer, chair of the board, and a significant
stock holder in Valley Fever Solutions, Inc.
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A9
CRYPTOCOCCUS GATTII: AN EMERGING

PATHOGEN IN THE UNITED STATES
Julie R. Harris30
The genus Cryptococcus comprises 37 different species, of which only 2

are relevant for clinical infection (C. gattii and C. neoformans). Cryptococcus
spores are inhaled from the environment, causing a primary lung infection
that may or may not be symptomatic. Disseminated disease may result in
meningitis and death (Li and Mody, 2010). Intricately linked with severe im-
munosuppression, C. neoformans was rarely reported before the 1950s, when
cancer treatments and organ transplants—conditions that often require immu-
nosuppressive treatments—began occurring with increasing frequency (Perfect
and Casadevall, 2011). The era of AIDS led to an exponential increase in the
30â•›â•›Centers for Disease Control and Prevention.

208 FUNGAL DISEASES
numbers of immunosuppressed persons, and a corresponding massive increase

in C. neoformans infections worldwide (Mitchell and Perfect, 1995; Perfect
and Casadevall, 2011). Today, cryptococcal infections due to C. neoformans
are among the most common AIDS-defining infections (Park et al., 2009;
Warkentien and Crum-Cianflone, 2010). In contrast, infections caused by C.
gattii are reported much less frequently. Gatti and Eeckels produced the first
report of C. gattii infection in 1970, from a 7-year-old boy in Congo in 1966
(Gatti and Eeckels, 1970). The boy was found to have a cryptococcal infection
clinically similar to C. neoformans, but with a different pathogen morphology
(Gatti and Eeckels, 1970). The newly observed pathogen was deemed to be a
variant of C. neoformans called C. neoformans var. gattii.
Today, C. gattii is considered its own species (Kwong-Chung et al., 2002).
During the mid-1980s, studies of geographic sources of C. gattii and C. neo-
formans isolates demonstrated that although C. neoformans was found from all
areas of the world, C. gattii was found only in tropical and subtropical climatic
zones (Kwon-Chung and Bennett, 1984a,b). The authors concluded that C. gattii
was likely to be restricted to locations where the minimum winter temperatures
typically remained above freezing (Kwon-Chung and Bennett, 1984a). During
the next decade, limited information became available about the epidemiology
of C. gattii, much of it from papers describing infections in endemic Australia
and Papua New Guinea (Ellis, 1987; Mitchell et al., 1995; Seaton et al., 1996b,
1997; Speed and Dunt, 1995). These studies demonstrated that C. gattii, unlike
C. neoformans, was found almost exclusively in immunocompetent persons.
In agreement with the findings of Kwon-Chung and Bennett (1984a), C. gattii
infections were still seen only in patients living in tropical and subtropical cli-
mates (Chen et al., 2000; Lalloo et al., 1994; Laurenson et al., 1993, 1996, 1997;
Mitchell et al., 1995; Seaton et al., 1996a, 1997; Slobodniuk and Naraqi, 1980;
Speed and Dunt, 1995).
Beginning in 1999, the rate of cryptococcal infections among HIV-uninfected
persons living on temperate Vancouver Island, British Columbia (B.C.), Canada,
began increasing rapidly (Hoang et al., 2004; Fyfe et al., 2002). Early investiga-
tions demonstrated that most of these infections were caused by C. gattii, rarely
reported before from Canada (Kwon-Chung and Bennett, 1984a). Veterinarians,
too, noted that a wide range of animals were becoming infected with C. gattii
where they had not previously been found with non-neoformans cryptococcosis
(Duncan et al., 2006). During the following years, the disease continued spread-
ing in B.C., infecting humans and animals on the nearby mainland who had no
history of travel to Vancouver Island (MacDougall et al., 2007), and by 2007, 218
human infections had been reported from B.C. Although four genetic groups of
C. gattii have been identified (VGI, VGII, VGIII, and VGIV), most infections
in British Columbia were caused by the relatively uncommon VGII genotype.
Due to the high numbers of isolates available from the outbreak, further genetic
subdivision of outbreak-associated isolates was performed, demonstrating that
APPENDIX A 209
approximately 90 percent of isolates in B.C. were of a “major strain” genetic

subtype VGIIa, with a smaller number of “minor strain” subtype VGIIb isolates
(Galanis and MacDougall, 2010).
Clinicians began noting C. gattii infections among patients in the Pacific
Northwestern (PNW) states of Washington and Oregon in 2004 and 2005, respec-
tively (MacDougall et al., 2007). In addition to the VGIIa and VGIIb infections, a
new genotype of infection was noted in the United States, called VGIIc (Byrnes
et al., 2010a). Beginning in October 2009, the Pacific Northwest Cryptococcus
gattii Public Health Working Group, comprising the U.S. Centers for Disease
Control and Prevention (CDC) and state health departments and laboratories
in the Pacific Northwest, began retrospectively and prospectively collecting
standardized information on C. gattii infections in the United States. As national
awareness about the outbreak began to grow, other states also reported rare infec-
tions or submitted isolates for speciation and genotyping at the CDC.
Outbreak of C. gattii in the United States

To date, nearly 80 laboratory-confirmed human cases have been reported in
the United States (Harris et al., 2010), most from Washington and Oregon (Fig-
ure A9-1). As awareness of the outbreak in the PNW has spread throughout the
United States, infections from other states have also been reported. Case counts
have increased each year, from a single case reported during 2004 to 24 cases
reported during 2010 (Figure A9-2). The increase in reported cases is likely to be
a result of both improved surveillance, as indicated by the recent increase in cases
being reported from areas outside the PNW (Figure A9-2), and actual increases
in case occurrences.
Among human patients in the United States identified through surveillance
as having laboratory-confirmed C. gattii infection, approximately half are male,
with a median age of 56 (range, 15–95). Patients aged 30 and older comprise
the majority of cases (Table A9-1). The most commonly reported symptoms are
headache, nausea, and cough, affecting more than half of all patients; more than
half present with pneumonia and approximately half have meningitis. Most (73
percent) patients have an underlying immunosuppressive condition, including
(but not limited to) a recent history of oral steroid use; lung, heart, kidney, or
liver disease; a history of cancer; or a solid organ transplant (Table A9-2). HIV
infection was more frequent among C. gattii patients than is found in the general
U.S. population (5.9 percent vs. 0.6 percent) (CIA World Factbook, 2010), but
was still the least commonly reported underlying condition. Of 59 patients with
data, 17 (29 percent) had no detectable underlying immunocompromising condi-
tion. Cryptococcomas, or fungal masses, were found in the lungs and/or brains
of substantial proportions of patients who received the corresponding scans or
x-rays. Nearly all patients were hospitalized, and nearly one third with follow-up
information died with or from their infections (Table A9-1).
210 FUNGAL DISEASES
17
51 *
FIGURE A9-1â•‡ Human infections with C. gattii, United States, December 2004–January
2011 (n = 79).
NOTE: * indicates patients that reported extensive travel to Washington and/or Oregon
A9-1 new
during the year before their illness onsets.
Outbreak-Strain vs. Non-Outbreak-Strain C. gattii Infections in the United

States
Currently, 77 of 79 reported U.S. infections have been genotyped. Of these
77, 38 (49 percent) were VGIIa; 19 (25 percent) were VGIIc; 6 (8 percent) were
VGIIb; and 14 (18 percent) were other genotypes (VGI, VGIII, and unrelated
VGII).
Clear delineations exist between C. gattii infections in Oregon and Wash-
ington (“PNW-associated infections”) and those occurring in other parts of the
United States (Figure A9-2). In particular, most PNW-associated infections are
genotype VGIIa, VGIIb, or VGIIc (“outbreak-strain” genotypes). Outbreak-strain
infections have also been reported from humans living in states outside of Wash-
ington and Oregon; those that have been reported were linked to these states
by residential or travel history (Harris et al., 2010). One case in Idaho (subtype
VGIIc) (CDC, 2010; Iqbal et al., 2010) and one case from Alaska (subtype
VGIIa) (Harris et al., 2010) are considered linked to the outbreak, with both
patients reporting extensive travel throughout Oregon, Washington, and/or B.C.
during the year before their illness onsets (CDC, 2010).
APPENDIX A 211
FIGURE A9-2â•‡ U.S. human cases of C. gattii, by year of illness onset (n = 71*).
Figure
* NOTE: Onset year is reported for A9-2.eps
62 patients and is estimated by initial report year for 9
bitmap2010 data are current as of January 2011;
patients for whom onset date was not available.
complete case data typically lag illness onset by several months.
PNW = Pacific Northwest.
While a small number of non-outbreak-strain infections have occurred in

the PNW, most have been reported from states outside the PNW. A patient from
North Carolina was diagnosed with C. gattii (genotype VGI) in 2007 following
travel to San Francisco (Byrnes et al., 2009), and two cases of C. gattii (genotypes
VGI and VGIII) were reported in 2010 from patients in New Mexico who did not
have any recent history of travel outside of the state (Harris et al., 2010). In 2010,
a patient was reported from Hawaii with a novel subtype of infection belonging
to the genotype VGII (Harris et al., 2010). Since 2004, two patients have been re-
ported from California and one from Michigan with VGIII-type infections. Thus,
at least one focus of infection involving genotypes novel in the United States ap-
pears to be ongoing in the PNW; it also appears that sporadic C. gattii infection
is occurring elsewhere, with genotypes distinct from those found in the PNW.
The collection of standardized data on U.S. outbreak-strain and non-
outbreak-strain infections has provided an opportunity to examine clinical
212 FUNGAL DISEASES
TABLE A9-1â•‡ Characteristics of C. gattii Patients in the United States,

2004–2010
Characteristic n N %
Age (mean, median, range), years 52, 55 (15–95)
≤ 18 3 64 5
19–29 3 64 5
30–49 23 64 36
50–69 27 64 42
70+ 8 64 13
Symptoms
Headache 32 55 58
Nausea 26 50 52
Cough 29 57 51
Dyspnea 24 51 47
Fever 25 57 44
Vomiting 22 50 44
Fatigue 17 40 43
Weight loss 21 50 42
Loss of appetite 15 46 33
Chills 16 52 31
Muscle pain 15 52 29
Chest pain 14 52 27
Neck stiffness 11 49 22
Night sweats 6 44 14
Photophobia 5 49 10
Seizure 4 45 9
Blurry vision 2 45 4
Any respiratory 39 55 71
Any central nervous system 19 44 43
Clinical findings
Pneumonia 27 49 55
Meningitis 26 51 51
Crypto lung 18 50 36
Crypto head 6 22 27
Outcomes of infection
Hospitalized 52 57 91
Died 16 52 31
differences by genotype and patient type. Although a relatively small number

of non-outbreak-strain infections have been reported, significant differences be-
tween these and outbreak-strain infections have been noted in the proportion of
infected patients who were immunocompromised and the propensity for respira-
tory and CNS symptoms (Table A9-2). It seems likely that different genotypes
of C. gattii might infect different patient types; in addition, clinical manifestation
of C. gattii infection might differ either by infecting strain genotype, patient im-
mune status, or both.
APPENDIX A 213
TABLE A9-2â•‡ Comparison Between Outbreak-Strain (VGIIa/b/c) and Other

Genotypes of Infection with C. gattii, United States, 2004–2010
Symptoms, Patient Characteristics, Outcomes VGIIa/b/c Other genotypes RR p
Respiratory symptoms
Cough 58% 11% 5.3 0.01
Dyspnea 53% 12% 4.3 0.05
Any respiratory symptom 76% 44% 1.7 0.10
Central nervous system symptoms
Seizure 3% 50% 0.05 0.01
Blurry vision 8% 43% 0.19 0.04
Any central nervous system symptom 34% 100% 0.04 0.00
Preexisting medical condition 78% 33% 7.1 0.01
Hospitalized 90% 100% 0.9 1.00
Died from or with C. gattii 32% 16% 2 0.65
NOTES: p: Fisher’s exact p-value; RR: Relative risk
Historical Isolates of C. gattii in the United States

In spite of extensive press coverage in the United States during 2010 about
the “new deadly fungus” in the United States and British Columbia (Discover
Magazine, 2010; Hutchison, 2010; Park, 2010), C. gattii infections are not en-
tirely novel in the United States: in particular, the infection repeatedly has been
found in California in the past. In a 1984 study, C. gattii was found in 46 of 315
clinical isolates of Cryptococcus from the United States; 30 (65 percent) of the
46 C. gattii isolates were from patients residing in Southern California. Among
all 71 Cryptococcus isolates from Southern California, C. gattii represented 42
percent, compared with 6 percent in the rest of the continental United States
(Kwon-Chung and Bennett, 1984b). The same group reported in a different paper
that both of the two clinical Cryptococcus isolates from Hawaii were C. gattii
(Kwon-Chung and Bennett, 1984a). Another study, which examined cryptococcal
isolates from HIV-infected patients from Los Angeles in the early 1990s, found
C. gattii in 12 percent of these isolates (Chaturvedi et al., 2005). Genotyping of
the isolates showed that 28 of 30 isolates were of subtype VGIII; 1 was VGI,
and 1 was of subtype VGII (Byrnes et al., 2010b). 31 C. gattii was also found in
three of 358 genotyped isolates collected from around the United States during
surveillance from 1992 to 1994; two isolates were from San Francisco (VGI and
VGIII) and one was from Atlanta (VGI) (Brandt et al., 1996). Finally, two isolates
of genotype VGIIa, indistinguishable by multilocus sequence typing (MLST)
from the major strain in Vancouver Island, were collected from the sputum of a
Seattle man in the 1970s (Diaz et al., 2000) and from a eucalyptus tree in San
31â•›â•›Also see contributed manuscript by Heitman in Appendix A (pages 226–248).

214 FUNGAL DISEASES
Francisco in 1992 (Fraser et al., 2005). Taken together, these data suggest that
Southern California, but probably not the rest of the country, has long been an
endemic area for C. gattii. In addition, sporadic infections appear to be occurring
from other states, although the travel history of these patients and their potential
exposure site is unknown. Recently, environmental isolates of C. gattii have also
been found in Puerto Rico from a variety of cacti and tree material (Loperena-
Alvarez et al., 2010).
C. gattii in the PNW vs. C. gattii in Other Areas of the World

Globally, the most common infecting strains of C. gattii and the immune
status of patients infected appear to vary. In endemic Australia and Papua New
Guinea, C. gattii (usually type VGI) appears to occur primarily in apparently im-
munocompetent patients and cause CNS disease (Seaton et al., 1996a,b; Speed
and Dunt, 1995). However, in several African countries, VGIV-type infections
are most frequent, and are reported exclusively from HIV-infected patients
(Litvintseva et al., 2005; Steele et al., 2010). By contrast, in Venezuela (Villanueva
et al., 1989), Brazil (Santos et al., 2008), Paraguay, Argentina (Castanon-Olivares
et al., 2000; Kwon-Chung and Bennett, 1984a,b), Peru (Bustamante Rufino and
Swinne, 1998; Kwon-Chung and Bennett, 1984a,b), and Colombia (Escandon
et al., 2006), infections (most commonly genotype VGII, although different from
the PNW outbreak strains) occur in both HIV-uninfected and HIV-infected pa-
tients. In Mexico, all four C. gattii genotypes have been reported (Olivares et al.,
2009), primarily from HIV-uninfected persons (Castanon-Olivares et al., 2000;
Lopez-Martinez et al., 1996). In Asia, C. gattii (primarily VGI and VGII) has
been reported from Vietnam, Cambodia, Thailand, Korea, Japan, Malaysia, India,
China, and Nepal (Chen et al., 2008; Choi et al., 2010; Kwon-Chung and Bennett,
1984a,b; Lui et al., 2006; Okamoto et al., 2010), in both immunocompetent and
immunocompromised persons (Chau et al., 2010; Ngamskulrungroj et al., 2010).
The outbreak of C. gattii in B.C. and the PNW is qualitatively different in
genotype, presentation, disease course, and outcome than has been seen with
C. gattii in other locations. In contrast to findings from areas outside of North
America, C. gattii patients in both B.C. and the PNW most commonly present
with respiratory rather than CNS symptoms (Galanis and MacDougall, 2010;
Harris et al., 2010). In addition, a high proportion of infections in both the PNW
and B.C. occur in immunocompromised (but usually HIV-uninfected) patients. In
B.C., 38 percent of patients are immunocompromised (Galanis and MacDougall,
2010); using the same case definition for immunocompromised state, in the
United States, approximately 59 percent of patients are immunocompromised
(CDC, unpublished data).
Interestingly, differences also might exist between C. gattii patients in the
United States and in B.C. U.S. patients were younger, more likely to be hospital-
APPENDIX A 215
ized, and more likely to die from or with their infection than were B.C. patients
(Harris et al., 2010). The reasons for these differences are unclear, but might
include differences in case ascertainment in the PNW compared with B.C., which
has led to capture of a lower proportion of non-hospitalized C. gattii patients in
the PNW. Alternately, they could relate to the different genotypes seen in the
PNW compared with B.C. A comprehensive review of patient medical charts is
under way. It might help elucidate the differences and provide some insight into
whether these differences are real, and if so, why they exist.
The differences in U.S. C. gattii infections in the PNW, U.S. infections
outside the PNW, and infections occurring in other areas of the world might be a
function of C. gattii subtype, tropism, environmental distribution, or surveillance
bias. That is, C. gattii infections might not be reported completely from other
areas because they have not been looked for systematically elsewhere. Regardless
of the reasons for the outbreak in North America, reports of the outbreaks do not
appear to be exclusively due to temporal changes in surveillance. Retrospective
speciation studies of isolates from B.C. before 1999 (Fyfe et al., 2008) and from
the Seattle area before 2004 (Upton et al., 2007) suggested that the increase in
reported cases represents a true increase in infections in the region. It remains to
be seen whether the U.S. C. gattii infections outside of the PNW are sporadic,
travel associated, or linked to part of a larger emerging health issue in other areas
of the United States. Below is a discussion about efforts to conduct surveillance
for C. gattii infections outside of the PNW.
Surveillance for Human C. gattii Infections Outside the PNW

To address the question of how frequently C. gattii infections are occur-
ring outside of the PNW, in October 2010 the CDC issued an alert through
ClinMicroNet (Dwyer, 2003), a listserv sent to directors of U.S. clinical mi-
crobiology laboratories. The alert described the ongoing outbreak in the Pacific
Northwest, and requested that any unspeciated Cryptococcus isolates (or those
already speciated as C. gattii) be sent to the Mycotic Diseases Branch labora-
tory at the CDC, with isolation date, source city, and source state. Isolates were
speciated at CDC and C. gattii isolates were genotyped. As of January 2011, 32
isolates, isolated between 2006 and 2010, from patients in seven states had been
submitted to the CDC. Of these, 10 were C. gattii; half were from California
(Table A9-3). Collection of isolates is ongoing and cases continue to be reported
to the CDC, albeit rarely, from states outside of Washington and Oregon. The
recent commercial availability of specialized culture medium (CGB medium) that
enables rapid discrimination between C. neoformans and C. gattii will hopefully
facilitate this process (Butler-Wu and Limaye, 2011).
216 FUNGAL DISEASES
TABLE A9-3â•‡ Sources and Species of Isolates of Cryptococcus Submitted

Following a Request Through ClinMicroNet, United States, October 2010–
February 2011
C. neoformans
State isolates C. gattii isolates Total isolates
AL 3 0 3
CA 1 5 6
GA 1 2 3
HI 0 1 1
IL 14 0 14
OR 0 1 1
TX 3 0 3
UT 0 1 1
Total 22 10 32
Clinical Aspects of C. gattii Infection and Differences from C. neoformans

Several attempts have been made to carry out head-to-head comparisons of
clinical disease caused by C. gattii and C. neoformans. In 1995, Speed and Dunt
(1995) published a paper comparing clinical symptoms among hospitalized C.
gattii and C. neoformans patients. In addition to noting that fewer than 10 percent
of C. neoformans infections in Australia occurred in otherwise healthy patients
while 100 percent of C. gattii infections occurred in healthy patients, the authors
also reported that C. gattii more frequently involved cerebral and meningeal sites,
had neurologic sequelae, required CNS or thoracic surgery to resect cryptococ-
comas, and required longer periods of treatment (Speed and Dunt, 1995). In ad-
dition, C. gattii infections more frequently resulted in relapse than C. neoformans
infections. The authors commented that the nearly three-fold longer therapy times
required for patients with C. gattii compared with patients with C. neoformans
infections stemmed from the difficulty in reducing the size of the cryptococcomas
and the inability to rapidly control infection in patients with C. gattii. However,
they also noted that both bloodstream infections and mortality were exclusively
limited to patients with C. neoformans (Speed and Dunt, 1995).
The same year, Mitchell et al. (1995) published a report directly comparing
the two cryptococcal species in immunocompetent patients with cerebral disease
(Mitchell et al., 1995). Similar to the findings by Speed and Dunt (1995) and later
by Chen et al. (2000), the authors reported that both lung and brain cryptococ-
comas were more common among patients with C. gattii than C. neoformans
infections. In addition, they reported that immunocompetent patients with C.
gattii infection were significantly more likely to have a “poor outcome”—defined
as moderate to major sequelae or death—than immunocompetent patients with
APPENDIX A 217
C. neoformans infections. When they compared outcomes among patients with

meningitis but normal brain imaging at initial presentation, they found no differ-
ences by infecting species; however, they did find that C. gattii patients present-
ing with mass lesions on initial intracranial scan were more likely to have poor
outcomes than those who did not (Mitchell et al., 1995). Taken together, these
data suggested that the epidemiology of C. neoformans and C. gattii in Australia
was different even when controlling for patient immune status.
However, a similar paper, published in 2010, compared infection with C.
gattii and C. neoformans in immunocompetent patients in Vietnam (Chau et al.,
2010) and failed to demonstrate differences in clinical phenotype by infecting
species. The authors of this report, in contrast to Mitchell et al. (1995), suggested
that host immune status was more influential on clinical course and outcome than
was infecting species. This was also a conclusion drawn by Chen et al. (2000),
who compared cryptococcal infections among both immunocompetent and im-
munosuppressed patients in Australia and New Zealand and demonstrated that
immunocompetent hosts were more likely to present with lung infections, species
type notwithstanding, than were immunocompromised hosts. Chen et al. (2000)
also noted that C. gattii infections were more likely to occur in the brain than C.
neoformans infections, but that cryptococcomas were associated both with infec-
tion with C. gattii and with immunocompetent status.
Lui et al. (2006) also compared cryptococcal infection in immunocompetent
and immunosuppressed patients in China, noting elevated proportions of immu-
nocompetent patients presenting with meningitis compared with immunocompro-
mised patients, more intense inflammatory responses, and a lower risk of death.
Although the paper also indicated that immune status might influence clinical
course more than infecting species, the small numbers of C. gattii infections made
evaluation of the effect of infecting species difficult.
Only two studies have directly compared C. gattii infections to C. neofor-
mans infections exclusively in AIDS patients. In Botswana, Steele et al. (2010)
compared C. gattii and C. neoformans infections in AIDS patients with crypto-
coccal meningitis, and found few differences in terms of clinical presentation
or in-hospital mortality (Steele et al., 2010). Morgan et al. (2006) found similar
results among South African patients with cryptococcal meningitis. It is pos-
sible that, among severely immunosuppressed patients who have a low capacity
to respond immunologically to infection, such as late-stage AIDS patients, the
infecting cryptococcal species is irrelevant to outcomes, while infecting species
have a stronger influence when patients are mildly to moderately immunosup-
pressed or not immunosuppressed. These studies did not carry out extensive brain
or thoracic imaging to evaluate the presence of cryptococcomas.
The poor prognosis of immunocompetent patients infected with C. neofor-
mans has been studied previously, and has been suggested to be due to the delay
in diagnosis, inappropriate treatment, and potentially the presence of an intact
immune system that might provoke an immune reconstitution inflammatory
218 FUNGAL DISEASES
syndrome (IRIS) (Ecevit et al., 2006; Lui et al., 2006). IRIS is a paradoxical
clinical deterioration that is well documented during treatment of cryptococcosis
following initiation of antiretroviral therapy in AIDS patients (Woods et al., 1998)
and is thought to be due to an overzealous “rebound” immune response in the
presence of significant amounts of infecting pathogen. An IRIS-like syndrome
has also been documented in patients infected with C. gattii, where the syndrome
was suggested to be due to concomitant immune rebound and decreases in IL-10
(Einsiedel et al., 2004). These same factors may contribute to the severity of C.
gattii infection in immunocompetent hosts. At least one report exists of a patient
whose condition improved with steroid treatment, suggesting that an overly
functional immune system could confound treatment efforts in some patients
(Lane et al., 2004).
Discussion
The described outbreaks of C. gattii infection in the temperate climates of
B.C. and the PNW demonstrate a much less restrictive geographic range for C.
gattii than previously thought, and a broader range of persons who are suscep-
tible to infection. In particular, a compromised immune status now appears to be
a significant risk factor for at least some subtypes of C. gattii infection (CDC,
2010; Galanis and MacDougall, 2010). In addition, data from patients associated
with these outbreaks suggest that different C. gattii genotypes might infect dif-
ferent types of patients, and/or demonstrate different clinical courses resulting
from infection. The mere existence of an outbreak associated with C. gattii, never
previously reported, suggests that genetic components might be important for
pathogen spread in ways that are still poorly understood. More than ever, collect-
ing data is important that disease recognition and optimal treatment of C. gatiii
infections can be investigated. Several existing challenges now face the field.
Existing Challenges
One existing challenge is diagnosis of infection. Although several methods
exist to identify cryptococcal infections, including culture, India Ink stains of
cerebrospinal fluid or sputum (Cohen, 1984), and commercially available cryp-
tococcal antigen (CrAg) test kits (Saha et al., 2008), these methods cannot dis-
tinguish between C. neoformans and C. gattii. A simple way to confirm whether
or not a cryptococcal isolate is species gattii is to plate the isolate on canavanine-
glycine bromothymol blue (CGB) agar (Klein et al., 2009; Kwon-Chung et al.,
1982), where C. neoformans will leave the medium unaffected in color (yellow
to green) due to a failure to grow, and C. gattii will turn the medium blue due
to use of glycine as a carbon source. This medium is currently available from
at least one commercial supplier, but is not widely used in U.S. clinical micro-
biology labs. Thus, many C. gattii infections likely are being misdiagnosed as
APPENDIX A 219
C. neoformans. Ensuring that clinicians are aware of C. gattii infection and the
possible need for clinical differentiation from C. neoformans, and that their refer-
ence laboratories are able to speciate Cryptococcus isolates (and have an interest
in doing so), is critical to evaluate fully the geographic spread of disease and the
clinical spectrum of infections.
Investigating whether or not the most recent findings warrant modified treat-
ment guidelines is an additional challenge. The Infectious Diseases Society of
America published guidelines in 2010 (Perfect et al., 2010) that refer to differ-
ences in the treatment of C. gattii infections, compared with C. neoformans infec-
tions: specifically, C. gattii infections might require lengthier, more aggressive
treatment when compared with C. neoformans infections. The increased pro-
pensity for C. gattii to form cryptococcomas is also noted (Perfect et al., 2010).
However, the guidelines were largely based on data from C. gattii infections
occurring in Australia and Papua New Guinea. Increasingly, our data suggest
that even among C. gattii infections, not all cryptococcal infections are alike.
However, it is unclear which factors—infecting species, infecting subtype, host
immune status, or perhaps even host genetics—are most influential on patient
presentation and infection. Data from rigorous clinical studies are of utmost
importance in ensuring that clinician guidelines provide sufficient guidance to
optimize patient care. To this end, a large-scale, longitudinal chart review of
C. gattii infections is ongoing as a collaborative effort among Australia, B.C.,
and the United States, designed to address some of these questions. Results are
expected sometime in 2012.
Finally, the development of prevention messages is a challenge. Unlike C.
neoformans, which grows in pigeon feces, C. gattii appears to live in associa-
tion with trees and soil surrounding them (Springer and Chaturvedi, 2010). The
tree type appears to be less important than the presence of a wood substrate for
growth, and C. gattii has to date been associated with more than 50 tree species
(Randhawa et al., 2001; Springer and Chaturvedi, 2010). It has also been found
in air and water samples. These findings notwithstanding, C. gattii has not been
found ubiquitously around the globe in a distribution similar to C. neoformans,
and thus we can postulate that at least some environmental restrictions remain
in place for this organism. Environmental organisms present a specific challenge
for public health prevention because infections are usually relatively rare, and
difficult to avoid without draconian measures (e.g., staying indoors and purifying
air). This represents a quandary for public health officials. It remains to be seen
whether “hot spots” of infection exist in the PNW for which generalized recom-
mendations can be made that would benefit patient health, perhaps for subgroups
of higher risk patients. The benefits of outdoor activity would need to be weighed
against any risk calculated for these patients, and such recommendations are
bound to be controversial.
220 FUNGAL DISEASES
Where Did C. gattii Come From, and Where Is It Going?

How did C. gattii arrive in the temperate areas of North America? Kidd et al.
(2007) demonstrated, during an environmental sampling study for C. gattii in
B.C., that anthropogenic activities could spread the pathogen, showing the pres-
ence of C. gattii on the shoes of human samplers and wheel wells of sampling
vehicles as they traveled from one sampling site to the next. In addition, they
showed that the pathogen was present in highly trafficked areas of Vancouver
Island; that it could be found in the air, in freshwater, and in seawater around the
area; and that the spores could survive for more than a year in many of these me-
dia. Thus, it is not difficult to hypothesize several methods by which the pathogen
could have found itself in temperate B.C., and easier still to imagine mechanisms
by which it could be transported into the United States. However, the question of
whether new, cold-tolerant strains of C. gattii were brought to B.C. or whether
they were formed there, through recombination of two or more preexisting or
“seeded” strains, is still unresolved. It is also possible that the pathogen was
always tolerant of temperate-weather climates, but existed in caches too small
in these regions to sustain human infection or to establish permanent habitats;
repeated seeding of these regions through global materials trafficking (e.g., wood
or trees) could have created a sustainable niche for the pathogen. Alternately,
changes in the global climate could be facilitating the optimal habitat develop-
ment and spread of C. gattii, providing minimum conditions under which the
pathogen can successfully propagate. Whole-genome sequencing is currently
being carried out with C. gattii isolates obtained from patients associated with
this outbreak, as well as more historical isolates (Lockhart et al., 2010), which
could shed some light on the origin of the current outbreak and provide ideas for
where it might move next.
Conclusion
The increase in the number of reports during the past decade related to the
occurrence of C. gattii infections outside of traditional endemic tropical and
subtropical regions has provided excellent opportunities to learn more about this
important pathogen. The differences between individual cryptococcal infections
appear to be linked not only to patient immune status and infecting species, but
also to genetic subtypes within a species. It is unclear if the species and subtypes
have preferences for infection among certain patient types, possibly due to a need
for host immune support (or lack thereof) for replication, or if differences in
environmental colonization patterns might influence the type of patient infected.
For example, a pathogen with a ubiquitous distribution and a preference for im-
munocompromised patients will have a much higher infection rate and a much
higher immunocompromised to immunocompetent patient ratio than would a
pathogen with “hot spot distribution,” which would infect fewer patients overall,
but be limited to patients living in its area of environmental distribution (most of
APPENDIX A 221
whom are immunocompetent). Thus, the immunocompromised to immunocom-

petent ratio might be nothing more than a function of the degree of distribution
of a Cryptococcus species in the environment and the types of patients living in
its area of distribution. Determining what governs the range of distribution of C.
gattii—and understanding when it reaches a stable environmental equilibrium in
new areas of emergence—is critical for understanding this relationship.
In spite of the recent flood of reports about C. gattii, much remains unknown.
The epidemiologic curve has not yet stabilized in the United States, and the tra-
jectory of future infections is unknown. The lack of comprehensive surveillance,
both within North America and without, and the genetic variety inherent in C.
gattii, has limited our current understanding of pathogen spread and pathogenesis.
The conditions that favor pathogen colonization and propagation are not known.
Evidence shows that infections in the United States and particularly the PNW are
qualitatively different from those occurring elsewhere, but it is unclear whether
or not these differences warrant modifications to existing treatment guidelines.
Continued collection of robust surveillance data will assist in answering some
of these questions. The coming years should see increasing amounts of informa-
tion on C. gattii infections globally, which should shed light on genotype- and
subtype-specific differences among C. gattii infections.
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226 FUNGAL DISEASES
A10
SEXUAL REPRODUCTION, EVOLUTION, AND

ADAPTATION OF CRYPTOCOCCUS GATTII IN
THE PACIFIC NORTHWEST OUTBREAK
Joseph Heitman,32,33Edmond J. Byrnes III32,34 and John R. Perfect32,33
Abstract
How microbial pathogens emerge to cause outbreaks and become established
as agents of disease in humans involves genetic exchange, zoonotic transmission,
and perturbations of ecosystems and habitats. The threat of emerging infec-
tious diseases is particularly poignant for eukaryotic pathogens, the fungi, and
parasites, given that these microbes are more difficult to treat and have complex
genomes and lifecycles. A sobering recent development has been the emergence
and reemergence of several fungal pathogens in both humans and other animals,
including Geomyces destructans in bats, Batrachochytrium dendrobatidis in
amphibians, Nosema ceranae in bees (colony collapse disorder), and Cryptococ-
cus gattii in humans and other animals in the Pacific Northwest. Here we review
issues surrounding the C. gattii outbreak that began on Vancouver Island in 1999
and has expanded into the United States in Washington, Oregon, and California
and has the potential to expand further. The focus will be on the emergence of C.
gattii in the United States, including the appearance of a novel, highly virulent
genotype and the potential role of sexual reproduction in the emergence of novel
pathogens and their dispersal via airborne spores.
Introduction
The early history of cryptococcosis was documented in single or small se-
ries of cases. From an initial case of tibial osteomyelitis with the encapsulated
Cryptococcus neoformans yeast in 1895 until a seminal monograph on this
disease by Littman and Zimmer in 1956, the entire repertoire of reports in the
medical literature numbered less than 300 cases (Littman and Zimmer, 1956).
This was a humble beginning for this cosmopolitan, encapsulated basidiomycete
that has now emerged into an outbreak mode in the new millennium. In the first
half-century of its known existence, many of the clinical features of cryptococ-
cosis were well described, including its propensity to invade the central nervous
32â•›â•›Department of Molecular Genetics and Microbiology, Duke University Medical Center.

33â•›â•›Division of Infectious Diseases, Department of Medicine, Duke University Medical Center.
34â•›â•›Current address: Division of Infectious Diseases, Johns Hopkins University School of Medicine,
Baltimore, MD, USA 21287-0005.

APPENDIX A 227
system. The occurrence of outbreaks of mastitis (persistent inflammation of the

udders) in dairy herds in which hundreds of animals were infected brought the
realization that cryptococcal infection outbreaks can occur in mammals (Pounden
et al., 1952; Simon et al., 1953). These outbreaks in animals and the link to the
environment again were emphasized with reports of goats developing Cryptococ-
cus gattii infection in Spain temporally linked to the importation of Eucalyptus
trees (Baro et al., 1998). These two outbreaks in animals vividly demonstrated
that cryptococcal infections could change from sporadic to outbreak as the en-
demic status changes. However, the first prescient report that recognized the
future emergence of human cryptococcosis was from Kaufman and Blumer at the
Centers for Disease Control and Prevention (CDC) when they called cryptococ-
cosis “an awakening giant” mycoses (Kaufman and Blumer, 1978). As clinical
mycologists in a major reference laboratory, they observed increasing numbers of
cases as the immunosuppressed population dramatically increased due to the use
of immunosuppressive therapies in modern medicine. In 1983 to 1984, reports of
opportunistic cryptococcal infections coinciding with the early natural history of
HIV infection provided insights into the emerging association of HIV infection
and cryptococcosis (Lerner and Tapper, 1984; Vieira et al., 1983). In a landmark
epidemiologic work in 2009, Park and colleagues from the CDC estimated that,
in association with the AIDS pandemic, there were approximately 1 million cases
of cryptococcosis per year worldwide, with at least 600,000 deaths per year in the
past 5 years due to cryptococcosis (Park et al., 2009). While this massive outbreak
continues, on another front, an outbreak of C. gattii infections on Vancouver
Island has been observed over the past decade and has now migrated down into
the Northwest United States, infecting humans and other mammals (Byrnes and
Heitman, 2009; Byrnes et al., 2009, 2010; DeBess et al., 2010; Fraser et al., 2005;
Kidd et al., 2004; MacDougall et al., 2007; Upton et al., 2007).35 Cryptococcosis
started as a medical curiosity in a few patients, but because of medical interven-
tions with immunosuppression, an immunosuppressive viral pandemic (HIV/
AIDS), and a change in local climates, this yeast has become more prevalent in
clinical medicine. Its present impact supports its title as a major emerging fungal
disease or, to be more direct, “the giant is fully awake.”
Vancouver Island C. gattii Outbreak and Expansion into the United States
Our focus in this chapter will be the C. gattii outbreak that began on Van-
couver Island in 1999 and has now expanded into the Canadian mainland in
British Columbia and into the United States. A considerable body of knowledge
is available with respect to the life and virulence cycles for this pathogenic
yeast (Heitman, 2011). C. neoformans and C. gattii are closely aligned species.
C. neoformans is prevalent in the environment globally, and C. gattii has been
35â•›â•›See also the contributed manuscript by Harris in Appendix A (pages 207–225).

228 FUNGAL DISEASES
thought to be geographically restricted to tropical and subtropical regions until

its recent emergence in the relatively temperate climate on Vancouver Island. We
are exposed to both organisms from the environment; C. neoformans is typically
associated with pigeon guano and less commonly with trees, whereas C. gattii
is commonly isolated from trees and soil, and also present in the air on Vancou-
ver Island. We are exposed by inhaling spores and desiccated yeast cells, both
of which can cause an initial pulmonary infection. This can be cleared, recede
into a dormant latent form, cause fulminant pneumonia, or even make its way
to the central nervous system via the bloodstream to infect both the covering of
the brain (meninges) and the brain itself (meningoencephalitis). For reviews of
the virulence and lifecycles, see Idnurm et al. (2005) and Kronstad et al. (2011).
Of particular importance are recent studies from the CDC’s Park and col-
leagues (2009) that reveal that Cryptococcus has reached pandemic proportions.
More than a million cases occur globally each year. This is largely in the context
of the AIDS pandemic and results in more than 600,000 attributable mortalities,
approximately a third of all AIDS-associated deaths. This is thought to be largely
attributable to C. neoformans, but there is also likely to be a more substantial bur-
den of C. gattii infection occurring globally than is currently appreciated given
that few clinical microbiology laboratories routinely assign Cryptococcus species
status. It is in this global context that we consider the expanding and ongoing
outbreak of C. gattii in the Pacific Northwest (Figure A10-1).
Cryptococcus is a species complex, and it is important to know which patho-
gen you are dealing with in the context of infection. For example, this is particu-
larly important with E. coli, in which various strains are causing infections such as
EPEC, EHEC, UPEC, and VTEC (enteropathogenic E. coli, enterohaemorrhagic
E. coli, uropathogenic E. coli, and verotoxin-producing E. coli). This is also true
with fungal pathogens. Two species are currently recognized: C. neoformans and
C. gattii (Figure A10-2). The two can be readily distinguished in clinical micro-
biology labs on L-canavanine glycine bromothymol blue (CGB) agar, exploiting
the ability of C. gattii to cause a pH change on this media, resulting in a blue
chromogenic reaction (Figures A10-2A and B). We now appreciate that the group
of isolates currently recognized as C. gattii spans four cryptic species groups.
Currently, these are recognized as the VGI, VGII, VGIII, and VGIV molecular
types (Figure A10-2C) based on molecular phylogenetic analyses that show
each as a distinct, well-defined group (Bovers et al., 2008; Fraser et al., 2005;
Ngamskulrungroj et al., 2009a). We also know from the whole-genome analysis
for the representative VGI (WM276) and VGII (R265) isolates, which has just
been completed at the Broad Institute and the University of British Columbia in
Vancouver by Jim Kronstad and colleagues, that at a whole-genome level the dif-
ferent molecular types are not exchanging genetic information (D’Souza et al.,
2011). Similarly, the molecular types are four genetically isolated cryptic species
based on robust analysis of “molecular barcodes” throughout their genomes us-
APPENDIX A 229
FIGURE A10-1â•‡The C. gattii outbreak expanded into, and emerged within, the United
Figure A10-1.eps
States (n = 56). BC = British Columbia.
bitmap
ing multilocus sequence typing (MLST) (Bovers et al., 2008; Fraser et al., 2005).
This distinction of cryptic species is important because the VGII cryptic species
is causing the outbreak in the Pacific Northwest, whereas the VGI lineage is more
prevalent and more commonly causing disease in Australia (see Heitman, 2011).
The outbreak of C. gattii in the Pacific Northwest began in 1999 and prior
to this, no C. gattii infections were reported as causing disease in this region of
the world. The first cases came to the attention of astute clinicians and veterinar-
ians on the southeastern shores in Naniamo and Parksville on Vancouver Island
(Duncan et al., 2005; Hoang et al., 2004; Stephen et al., 2002). Over the past
decade, there have been approximately 260 cases with an approximately 10 per-
cent attributable mortality rate, and many infections in animals. Interested read-
230 FUNGAL DISEASES
FIGURE A10-2â•‡ Cryptococcus pathogenic species complex. Panel A shows growth on

Figure A10-2.eps
niger seed media that detects production of the pigment melanin (+) or its absence (–),
bitmap
production of urease (+) as a pink color on Christensen’s agar, and growth and a blue color
change (+) on CGB agar. Panel B shows a summary of the phenotypic data shown in panel
A. Panel C is a phylogenetic tree of the relationships between the molecular types in the
species complex. C. neoformans: opportunistic, AIDS, global, pigeon guano. C. gattii:
primary, non-AIDS, tropical, arboreal. CGB = L-canavanine-glycine-bromothymol blue.
SOURCE: Panel A modified from Byrnes et al., PLoS Pathogens, 2010 (figure S2). Panel
B courtesy of Heitman. Panel C modified from Chapter 22, Figure 2 of Carter et al. (2011).
APPENDIX A 231
ers are referred to a reprint36 included in this volume from Karen Bartlett, who
played a critical role in identifying the environmental source of the organism on
Vancouver Island (Bartlett et al., 2008), and to the manuscript from Julie Harris
from the CDC Cryptococcus working group.37 Our charge in this article was to
consider two aspects of the outbreak: first, its expansion into the United States,
and second, the possible roles of sexual reproduction in the origin of the outbreak
isolates and the ongoing production of airborne infectious spores.
The first cases associated with the Vancouver Island outbreak in patients
from mainland British Columbia with no travel history to the island appeared
in 2003–2004 (Bartlett et al., 2008). Environmental sampling studies provided
evidence that C. gattii expanded across the water to a broader niche, including
the Canadian mainland (MacDougall et al., 2007). The southern tip of Vancouver
Island is very close to the U.S. border, and therefore a key question was if and
when the outbreak might spread into the United States. The San Juan Archipelago
is a part of Washington state, located as near as 5 km from the gulf islands off
the coast of Vancouver Island. The first C. gattii index case in the United States
was an elderly patient with leukemia on Orcas Island, Washington, who presented
with a pulmonary C. gattii nodule in January 2006 (Upton et al., 2007). Since
then, the outbreak has expanded into the United States from Canada, and we
summarize here the evidence and key findings.
From 2006 to 2010, approximately 70 cases in patients have been reported,
and the most complete records are attributable to the efforts of the CDC Cryp-
tococcus working group. The cases in humans and animals are shown in Figure
A10-1, and the icons represent cases for which we have isolates in the laboratory
that have been molecularly analyzed. Two types of isolates are circulating on
Vancouver Island: the VGIIa/major genotype, which causes approximately 95
percent of the infections, and the VGIIb/minor genotype, which represents the
remaining 5 percent of isolates (Fraser et al., 2005). Both of these genotypes have
been found in the environment on Vancouver Island and cause infection in both
patients and animals, and both have now emerged within the United States. Of
particular interest is a completely novel genotype that has emerged in Oregon:
VGIIc, or the novel genotype (Byrnes et al., 2009, 2010). VGIIc has not yet been
found in Washington state, Vancouver Island, or anywhere else in the world; thus,
it is a completely new emergence from 2005 to 2010 in Oregon.
We can identify genotypes by applying MLST of genetic barcodes through-
out the genome. In this technique, coding and non-coding regions of genes are
PCR amplified, sequenced, and assigned a unique allele number. The alleles are
then color coded and organized in a tabular format in which each line represents
a different isolate associated with the outbreak or a global isolate in the strain
collection. This allows one to appreciate that there has been what appears to be a
large clonal expansion of the VGIIa/major genotype in the region that dominates
36â•›â•›See contributed manuscript by Bartlett in Appendix A (pages 101–116).
37â•›â•›See contributed manuscript by Harris in Appendix A (pages 207–225).
232 FUNGAL DISEASES
the outbreak on Vancouver Island and its expansion into Puget Sound and beyond
in Washington state. There are fewer isolates of the VGIIb/minor genotype, but
these have been found on Vancouver Island, in Oregon, and most recently in
Washington state. The VGIIc/novel genotype has thus far been found only in
Oregon and has several, unique alleles not found to date in any other isolates
examined from global sources. The CDC has identified one isolate from a patient
in Idaho that appears to be closely related to the VGIIc/novel genotype, but it
may be the result of travel exposure (DeBess et al., 2010).
Where did these VGII outbreak genotypes originate? The VGIIa/major geno-
type is indistinguishable across 30 MLST loci and several variable number of
tandem repeat loci from the NIH444 strain, which was isolated in the early 1970s
from a sputum sample from a patient in Seattle (Fraser et al., 2005). This isolate is
the type strain for C. gattii and is molecularly indistinguishable from the VGIIa/
major outbreak strain at all loci examined thus far. Therefore, the major outbreak
strain appears to have been in this geographic region for at least 40 years and
possibly even longer. The VGIIb/minor genotype isolates are indistinguishable
from isolates from a fully recombining sexual population in Australia (Campbell
et al., 2005a,b; Fraser et al., 2005). The VGIIc/novel genotype has thus far only
been identified in isolates collected in Oregon, and we have not observed it in a
large collection of more than 200 C. gattii isolates collected globally. It appears
as if the VGIIc/novel genotype either emerged locally in Oregon or, alternatively,
it may be present in an undersampled environmental niche that remains to be
discovered. Further evidence that this new genotype in Oregon is novel involves
haplotype network mapping. In this approach, the MLST alleles are compared
and we apply a computer algorithm to predict the ancestral allele. The alleles are
then organized into a diagram rooted with the ancestral allele, and alleles derived
from it by mutations and genetic drift are indicated with lines and circles. In this
analysis alleles that are arisen from the ancestral allele long ago lie closest to the
ancestral allele, whereas alleles that arose recently lie distal. The MLST alleles
that are private to the VGIIc genotype are distal in these haplotype networks,
suggesting that they arose recently. The alleles that the VGIIc alleles are derived
from come from isolates that originated from either Australia or South America
(Byrnes et al., 2010). This analysis then suggests that those alleles might repre-
sent possible sites of origin of at least some of the genetic material in the Oregon
VGIIc/novel outbreak isolate.
We have conducted mammalian virulence studies in a mouse inhalation
model, both at the Wadsworth Center in Albany with our collaborators Ping Ren,
Sudha Chaturvedi, and Vishnu Chaturvedi and also at Duke University (Byrnes
et al., 2010; Fraser et al., 2005). The VGIIa/major genotype and the VGIIc/novel
genotype isolates are both highly virulent in the mouse model used, whereas the
minor VGIIb genotype is considerably attenuated by direct comparison (Byrnes
et al., 2010). Isolates collected globally that share many but not all markers with
the VGIIa/major genotype are considerably less virulent than VGIIa (Byrnes
APPENDIX A 233
et al., 2010). This includes, for example, isolates from both California and South
America. Of note, the NIH444 type strain (a VGIIa/major genotype isolate) is
also somewhat less virulent than contemporary outbreak VGIIa/major genotype
strains, which may reflect either attenuation as a result of long-term storage and
passage; variation in virulence even among the VGIIa/major isolates; or unknown
genetic differences between NIH444 and the VGIIa/major outbreak genotypes
in regions of the genome not yet analyzed. Therefore, it appears as if virulence
has increased with the emergence of the VGIIa/major outbreak genotype and the
VGIIc/novel genotype from their original source strains.
Several investigators have been addressing why outbreak isolates are more
virulent than other genotypes. There have been two prescient studies. Jim Kro-
nstad and colleagues at the University of British Columbia in Vancouver have
shown that there is less protective inflammation in the lungs of mice infected
with the outbreak isolates (Cheng et al., 2009). This may then lead to increased
virulence if the host fails to mount a sufficient, protective immune response. In
addition, the work from Robin May’s lab at the University of Birmingham has
described an increased intracellular proliferation rate of the VGIIa and VGIIc
outbreak isolates in macrophages and linked mitochondrial function to the robust-
ness of their interaction with host immune cells (Byrnes et al., 2010; Ma et al.,
2009). Given that Cryptococcus is a pathogen able to grow either outside or
inside of host immune cells, an enhanced proliferation rate in the context of the
macrophage intracellular milieu may lead to enhanced virulence. These studies
are excellent starting points to use to begin to dissect the virulence mechanisms
at the interface with host immune cells in the lung.
To summarize, this outbreak was originally restricted geographically to
Vancouver Island and then spread to the mainland of British Columbia. Starting
in 2006 (and maybe earlier in one case in Oregon in 2005), it emerged within the
United States. Thus, there clearly appears to have been a geographic expansion
from Vancouver Island across Puget Sound to reach Washington, mostly involv-
ing the VGIIa/major genotype. However, there is more diversity in strains from
Oregon, involving both the VGIIa/major and VGIIb/minor genotypes observed
on Vancouver Island, but also including the novel VGIIc genotype that has not
been identified on Vancouver Island. In essence, this looks like an outbreak
within an outbreak, which may be of independent origins. It is as though two
pebbles have been dropped into a pond at different times, one earlier than the
other, and they have generated concentric waves that are now expanding outward
and intersecting. There is considerable concern that this outbreak will continue
to expand geographically given that there are cases and isolates now from Idaho
and California (Personal communication, Shawn Lockhart, CDC Mycotic Dis-
eases Branch, May 2011). There is also a well-documented risk to travelers to the
endemic region, who then return to locations around the world with an infection
of the VGIIa/major isolate and present with a very unusual fungal infection that
is not commonly described in those regions and that might confuse clinicians
234 FUNGAL DISEASES
(Chambers et al., 2008; Georgi et al., 2009; Hagen et al., 2010; Lindberg et al.,
2007).
The Role of Sexual Reproduction in Pathogen Emergence and Outbreaks

The second charge of this article was to consider the role of sexual reproduc-
tion in the emergence of pathogens and how it might contribute to adapting to a
novel niche. We might start with just a very general question of, why sex? For
those investigators who focus on bacteria or other prokaryotes, their mechanisms
for genetic transfer typically involve horizontal gene transfer. But for the eukary-
otic pathogens, including fungi, parasites, and the oomycete plant pathogens such
as Phytophthora infestans (the cause of potato blight), their genetic exchange
mechanisms involve sexual reproduction. The general theme that emerges for
all of these groups of eukaryotic pathogens is that sexual reproduction plays a
critical role in their diversity and, in many cases, also in their infection cycles
(Heitman, 2006, 2010).
What benefits are conferred by sexual reproduction? This process enables the
exchange of genetic information and generates diversity, and it can also purge
the genome of deleterious mutations. It also allows these organisms to stay one
step ahead of their transposable elements, which might otherwise accumulate to
litter the genome.
We know a great deal about the sexual reproduction of Cryptococcus because
of the pioneering work of June Kwon-Chung at the National Institutes of Health
some 30 years ago (Kwon-Chung, 1975, 1976a,b). There are two mating types,
called a and a (Hull and Heitman, 2002; McClelland et al., 2004). A panoply of
signals regulates the interactions of cells of opposite mating type, allowing fu-
sion and completion of their sexual cycle, thereby generating infectious spores.
We know from both historic and recent experimental studies that these spores are
indeed infectious propagules (Botts and Hull, 2010; Botts et al., 2009; Giles et al.,
2009; Sukroongreung et al., 1998; Velagapudi et al., 2009; Zimmer et al., 1984).
Two of the many signals that regulate the C. gattii sexual cycle are interac-
tions with plants and extreme desiccation (Xue et al., 2007). As noted by Karen
Bartlett, there is more Cryptococcus found in the air in Vancouver Island in
July when it is very hot and dry (Bartlett, 2010). Indeed, these are also the ideal
conditions for stimulating the sexual cycle, at least under laboratory conditions.
Thus, Cryptococcus may mate more in the hot and dry conditions of July than in
other times of the year.
Sex in Cryptococcus produces spores, which are infectious. There is also a
link of the mating type to virulence that has focused interest on its sexual cycle
(Kwon-Chung et al., 1992; Nielsen et al., 2003, 2005a,b; Okagaki et al., 2010).
One of the most curious features of this species is that most of the Cryptococcus
population is just one mating type (a), even though the sexual cycle that was
originally defined in the laboratory requires both opposite mating types (Hull
APPENDIX A 235
and Heitman, 2002). This fact raised a central conundrum for the entire field of
cryptococcal pathogenesis: If there is a well-maintained a-a opposite-sex sexual
cycle, but the a mating type is extremely rare, if not completely absent in many
populations, how then can a sexual cycle be an important step of the infectious
cycle for this fungus? It might be that it is not important, such that these unisexual
populations were just clonally reproducing mitotically as yeasts. However, we
discovered that C. neoformans can undergo an unusual sexual cycle involving
only one of the two mating types (Lin et al., 2005).
The opposite-sex cycle that we have known about for 30 years is depicted in
the lower panel of Figure A10-3. Then in 2005, an alternative sexual cycle was
reported called unisexual reproduction or same-sex mating, and this is depicted
in the upper panel of Figure A10-3 (Lin et al., 2005; Wang and Lin, 2011). This
alternative sexual cycle only involves a cells. They can fuse with another a cell
from the population, or they can, in extreme cases, fuse with themselves, undergo
meiosis, and produce spores.
You might wonder, what would be the point of undergoing sexual reproduc-
tion with yourself? There is no genetic diversity to admix in this circumstance.
We always think about sexual reproduction as involving two parents with very
FIGURE A10-3â•‡ Cryptococcus neoformans can reproduce unisexually and bisexually.

Figure A10-3.eps
bitmap
236 FUNGAL DISEASES
different genetic compositions. It turns out that mating with yourself can also
lead to the generation of genetic diversity. Sex itself can serve as something of
a mutagen to generate genotypic and phenotypic diversity. This strategy turns
completely on its head what we traditionally think of as the primary role of sexual
reproduction. An analogy might be the appearance of mismatch repair muta-
tor mutations in bacterial pathogens, which arise to result in the generation of
genome-wide mutations and are then swept from the population as a consequence
of their concomitant deleterious effects.
As an example, in preliminary studies we isolated 100 progeny produced
by this same-sex mating cycle and looked for phenotypic diversity. We have
found within this set of just 100 isolates clear examples of azole resistance,
temperature-sensitive growth, hyperfilamentous growth, and increased melanin
production. By comparison, analysis of 100 progeny produced by mitotic clonal
growth yielded no such examples. Many of the phenotypically distinct isolates
turn out to be aneuploid in one way or another. This is based on comparative ge-
nome hybridization analysis showing that one of these morphologically distinct
isolates now has an extra copy of chromosome 10. The presence of an extra copy
of a chromosome is termed aneuploidy. We often associate aneuploidy with very
deleterious consequences. We do not have to go further than Down syndrome
as an example to think about what might be bad about aneuploidy. On the other
hand, in the microbial kingdom, aneuploidy can be a rich source of phenotypic
diversity. There are well-documented studies in Candida albicans that an a spe-
cial type of chromosome formed by duplicated arms of a chromosome (termed
an isochromosome) can form and drive fluconazole resistance (Selmecki et al.,
2006, 2008, 2009). Similarly, we also know that azole heteroresistance in Crypto-
coccus is driven by aneuploidy for chromosome 1 (Hu et al., 2008; Sionov et al.,
2009, 2010). A variety of other experimental evolution studies have brought to
the forefront the idea that aneuploidy might be a driving force for genotypic and
phenotypic plasticity (Pavelka et al., 2010; Rancati et al., 2008; Torres et al.,
2007, 2010). So this may be one mechanism by which a same-sex mating cycle
could engender phenotypic and genotypic diversity in a population without need-
ing a partner that is genetically divergent.
Another way to view same-sex mating is that it is a mechanism for selfing.
Selfing/inbreeding is common in fungi and the paradigm is mating type switching
in S. cerevisiae, which allows mother–daughter cell mating. Same-sex mating is
another route to self, but not via mating type switching. It is a question at some
level of outcrossing vs. inbreeding. Same-sex mating superimposes much more
limited genetic diversity on a well-adapted fungal genome that has run the gaunt-
let of natural selection. As such, unisexual reproduction may confer one of the
benefits of sex (generation of diversity), but at the same time avoid one of its costs
(breaking apart well-adapted genomic/genetic configurations). For organisms that
are well adapted to a niche, this more limited genetic diversity may better answer
subtle changes in selection pressures.
APPENDIX A 237
We have alluded to the fact that there are restricted geographic areas where
fungal sex is extant. Why is that? Why would you have these little areas and
pockets in Africa and that is where they have sex, and in the rest of the world they
don’t have sex except for the unusual unisexual cycle? At least for Cryptococcus,
what it looks like is, where they have opposite-sex mating seems to be where
there are restricted populations where both mating types still exist. We know the
most about this from C. neoformans, where there is an extant population that is
on a very specific indigenous tree in South Africa, in Botswana (Litvintseva et al.,
2011). But everywhere else in the world, you find just the alpha mating type (Hull
and Heitman, 2002). So there is a niche in nature where opposite-sex mating is
occurring. Until 5 years ago everyone presumed everywhere else it was asexual
and mitotic. But what we are coming to appreciate is that there is just a different
version of the sexual cycle occurring in these other populations where there is
just one mating type.
This is interesting as another classic example is Phytophthora infestans. In
Mexico and in Peru and other areas of South America, that is where the sexual
cycle occurs, new versions arise, and then they are often exported on potato
crops. That was the source of the Irish potato famine. But why there is a sexual
cycle in such a restricted place is not really clear. Toxoplasma gondii might be
another interesting pathogen to consider. It only has its sexual cycle in cats and
other felids, not in other animals of any sort (Heitman, 2006). So what is it about
the gastrointestinal tract of a cat that is so different from a mouse or a human that
allows this parasite to undergo its sexual cycle there? The parasite sexual cycles
are even more bizarre than some of the fungal ones, and restricted to extreme
niches (Heitman, 2006, 2010).
What about the specific example of C. gattii sexual reproduction? June
Kwon-Chung elucidated the C. gattii sexual cycle more than 30 years ago
(Kwon-Chung, 1976a,b). Spores were produced in her classic studies via an a-a
opposite-sex mating cycle that she defined. We were approached by several of
the Australian groups working on the VGI genotypes in the late 1990s, including
Wieland Meyer and Dee Carter, and we spent more than 5 years recapitulating
this sexual cycle under laboratory conditions. This required a great deal of heroic
effort on the part of several individuals in various laboratories, in large part be-
cause fecundity can be reduced with long-term passage and storage and because
the VGI molecular type that is predominant in Australia is more recalcitrant in
mating assays compared to the VGII and VGIII C. gattii molecular types. We
were finally able to recapitulate a sexual cycle for C. gattii involving the for-
mation of dikaryotic hyphae with special cells linking the hyphal cells (fused
clamp cells) culminating in the production of basidia and basidiospores, all of
the morphological features of the sexual cycle, including meiotic recombination
(Campbell et al., 2005a,b; Fraser et al., 2003). Based on this advance, we were
able to show that the vast majority of the outbreak isolates from Vancouver Is-
land are fertile in laboratory crosses (Byrnes et al., 2010; Campbell et al., 2005a;
238 FUNGAL DISEASES
Fraser et al., 2003). These are a-a matings that were conducted, but we stress
the point that every single isolate that is associated with the outbreak is of the a
mating type. No one has found any a isolates occurring anywhere on Vancouver
Island or in Washington or Oregon.
How might mating occur on Vancouver Island and in the Pacific Northwest in
this unisexual population? In previous studies we observed that an association of
Cryptococcus with plants can stimulate the fungal sexual cycle (Xue et al., 2007).
Part of the idea in testing for this phenomenon in the first place was that plants,
and more specifically trees, are the environmental niche for C. gattii (Heitman
et al., 2011). For the plant pathogenic fungus Ustilago maydis, which infects corn,
the infectious form is the filamentous dikaryotic hyphae produced by the sexual
cycle, which is stimulated by interaction with the plant. In fact, U. maydis has to
be in its sexual form to infect the plant host. We found something very similar
with Cryptococcus. We were unable to infect a plant efficiently with the haploid
yeast, but the filamentous dikaryotic state will infect plants and stimulate a plant
defense response (Xue et al., 2007). In turn, if we put seedlings distant from a
fungal mating mixture on a plate, the plants secrete small molecules, such as ino-
sitol, that stimulate completion of the sexual cycle and production of spores (Xue
et al., 2007, 2010). We observed this with seedlings of Arabidopsis or Eucalyptus
and are now exploring this with Douglas fir because of the link to this indigenous
tree species in the Pacific Northwest outbreak. This line of investigation suggests
that this may be one niche in nature where the sexual cycle is occurring to pro-
duce spores as small airborne infectious propagules.
Another critical line of investigation involves population genetic studies con-
ducted by Dee Carter from the University of Sydney, Australia. She has focused
on the sexual cycle that may be occurring in Eucalyptus tree hollows. She discov-
ered that in populations with both mating types, they can engage in opposite-sex
mating, but in other tree hollows where only a isolates are found, they are also
sexually recombining (Saul et al., 2008). So it seems as if the organism has two
extant sexual cycles, one opposite sex and one same sex, and whom you mate
with depends on who your neighbors are in the population. All of her studies
have focused on the VGI cryptic species, which has very limited genetic diversity
and an extremely high level of apparent inbreeding (Campbell and Carter, 2006;
Campbell et al., 2005a,b; Carter et al., 2007, 2011). All of the isolates on Vancou-
ver Island associated with the clonal outbreak are of a different cryptic species,
VGII. However, based on Dee Carter’s studies of VGI sexual reproduction, the
presumption is that there may be both forms of sexual reproduction occurring also
for the VGII isolates in nature, associated with the environmental niche in trees
or plants for the VGII lineage and involving both opposite and same-sex mating
depending on where a isolates are found in nature.
We have advanced the population genetic analysis in two ways with our
global isolates to look for measures of sexual reproduction and recombination
occurring in the population. One of these is a simple test called an allele com-
APPENDIX A 239
patibility test. The basic premise is that if you cross two strains that differ at
two markers, you obtain four types of progeny produced by sexual processes by
reassorting two unlinked loci (i.e., AB by ab yields AB, ab, Ab, and aB) (Carter
et al., 2011), which we all know from studying Punnett squares. So the multilo-
cus sequence loci can be analyzed as two unlinked alleles in the population. If
you find all four possible combinations, this is indicative of sexual reproduction
and recombination occurring in the population. By looking at the multilocus
sequence markers, we find that these measures of recombination are rampant
throughout the VGII global population (Byrnes et al., 2010). In addition, if we
just examine the sequences of the multilocus sequence alleles themselves, which
represent approximately 1 kilobase of sequence each, we can find examples of
hybrid recombinant alleles that implicate isolates as potential parents or potential
offspring (Byrnes et al., 2010). A lot of mitotic recombination must be occur-
ring to see evidence of recombination within just a random 1-kilobase sequence
in a 20-megabase genome. The potential parents that are identified by this type
of analysis originate from South America, Africa, and Australia (Byrnes et al.,
2010). These findings suggest that sexual reproduction is occurring globally in
multiple environments and locations.
We went on to examine these global isolates for fertility in the lab and
documented, by scanning with electron microscopy, the production of meiotic
spores, which are the products of sex that may be infectious propagules found in
the air on Vancouver Island. The majority of the a strains we analyzed are fertile.
We have found a very limited number of a strains in the global population. For
example, we found 6 out of 200 (~3 percent) are this minor mating type. Five
out of six of these a strains are fertile in the laboratory, and thus they may be
undergoing opposite-sex mating in nature. Because five of these six a isolates are
from South America, it may be a site in which opposite-sex mating is still extant
in the population (Escandon et al., 2007; Fraser et al., 2005; Ngamskulrungroj
et al., 2008). We know that C. neoformans has also retained extant opposite-sex
mating, but that it is geographically restricted to Botswana and South Africa and
occurring in the VNB C. neoformans lineage (Litvintseva et al., 2003, 2007).
When we return to consider the VGIIb minor genotype and its relationship
to global isolates, this provides critical insight. For instance, we looked at 30
MLST alleles, the VGIIb outbreak isolates are completely indistinguishable from
isolates from a fertile, unisexual, sexually recombining population in Australia
that was identified by Dee Carter and colleagues (Campbell et al., 2005a,b). It
suggests that this population may be the ancestral source for the VGIIb/minor
outbreak genotype isolates now circulating in the Pacific Northwest. While one
might posit just the opposite (transfer from Vancouver Island to Australia), the
diversity of the population in Australia supports this as the ancestral rather than
the derived population. Thus, the most parsimonious model is that the isolates
from the outbreak originated from Australia (Figure A10-4).
Based on a broad comparison of the isolates on Vancouver Island, the VGIIa/
240 FUNGAL DISEASES
Ongoing same sex mating?

Avirulent parent,
hypervirulent progeny
αxα
αxα
Eucalyptus trees,
original same-sex
mating a x α?
αxα
Avirulent parent
in a fertile recombining population
=VGIIa/major, highly virulent
=VGIIb/minor, less virulent
=VGIIc/novel, highly virulent
FIGURE A10-4â•‡ Sexual reproduction and the origin of an outbreak.
major and the VGIIb/minor genotypes A 10-4share

newhalf of their genetic markers and
Bitmapped map
differ at the other half. So in a very simple model, they might be progeny from a
genetic cross (i.e., siblings), or one might be a parent and the other an offspring.
In this simple conceptual framework, one could imagine an isolate undergo-
ing mating with an unknown isolate in nature to produce the outbreak isolates.
However, the VGIIa/major and the VGIIb/minor genotypes on Vancouver Island
are all of the same mating type, a. When we look in detail at the mating type
locus, we can see that they are molecularly distinct from each other (Fraser et al.,
2005) and are not identical by descent. So, in essence, they have two different
ancestries for this region of their genome that dictates their mating type. A more
parsimonious model then is that genetic crosses that led to the production of these
isolates would have involved two parents with different a mating alleles in a
same-sex mating cycle. It is also possible that both types of crosses have occurred
(opposite-sex and same-sex mating) and that there has been more than one cross
involved here in the genesis of these isolates that are responsible for the outbreak
and also for the global isolates to which they are related.
APPENDIX A 241
Conclusions and Perspective

To summarize, three molecular genotypes are found circulating in the Pacific
Northwest. The most prominent is the VGIIa/major genotype (Figure A10-1 in
yellow), and we can date its origin to at least the early 1970s to an isolate from
a patient in Seattle. The VGIIb/minor genotype is shown in red and it is mo-
lecularly indistinguishable from isolates from a fertile recombining population
in Australia. In green is a completely novel VGIIc genotype that has emerged
in Oregon. Two of these genotypes (VGIIa, VGIIc) are highly virulent in both
macrophage and mouse models (Byrnes et al., 2010).
From where do these VGII isolates originate? We find VGII isolates glob-
ally: in Africa, Australia, and South America. But based on available evidence
to date, the most parsimonious model is that at least one of these genotypes
(VGIIb/minor) came from a population in Australia, given that isolates from the
two are indistinguishable (Figure A10-4). In terms of the crosses hypothesized to
have been involved in the origin of the outbreak, all of the populations that have
been identified are all a. There are no a mating types of this lineage that have
been found on Vancouver Island, in the Pacific Northwest, or in Australia. The
only places where VGII mating type a isolates have been found so far are South
America and Greece. So it may be that in Australia and the Pacific Northwest that
same-sex mating drives diversity in the population, but opposite-sex mating is a
possibility in South America. In the context of the outbreak, we also envision that
same-sex mating is contributing to the production of infectious spores, which are
the aerosolized small particles detected with Anderson air samplers. It is entirely
possible that those propagules are desiccated yeast cells, spores, or a mixture of
both. Experiments to test if these are actually spores in nature are being planned,
and if spores are present in the air on Vancouver Island, it seems likely that they
are being produced by same-sex mating occurring in the environment, given that
the population is a unisexual.
Disease caused by C. gattii vs. C. neoformans has been the subject of several
reviews, and despite the impression that C. gattii frequently causes infection in
apparently immunocompetent hosts and commonly presents with focal crypto-
coccomas in the lung or brain (Chen et al., 2000; Mitchell et al., 1995; Speed and
Dunt, 1995), it has been difficult to clinically distinguish between C. neoformans
and C. gattii disease. In fact, the presentation of C. gattii infections in Vancouver
and the Pacific Northwest outbreak appear similar to C. neoformans infections,
and disease is not limited to the immunocompetent host. C. gattii infections such
as those caused by the VGIII molecular type/cryptic species can be even found in
HIV-infected individuals in endemic areas (Byrnes et al., 2011; Chaturvedi et al.,
2005). Another aspect that has been reported to distinguish these two species is
their response to therapy. It has been suggested that C. gattii infections require
longer therapy (West et al., 2008). However, in vitro susceptibility testing has pro-
duced inconsistent results; some investigators have found similar minimum inhib-
itory concentrations (MICs) whereas others have found higher MICs to azoles for
242 FUNGAL DISEASES
C. gattii compared to C. neoformans (Thompson et al., 2009; Torres-Rodriguez

et al., 2008). It is always difficult to be precise in determining the response to
therapy when in a normal host; a component of immune reconstitution inflam-
matory syndrome may influence clinical judgment of treatment failure. A recent
study primarily in HIV-infected patients demonstrated that there was no differ-
ence in the outcome of C. gattii vs. C. neoformans infections in a single medical
center (Steele et al., 2010). This is interesting and may be due to small numbers,
but early results comparing C. gattii infections in the Vancouver Island vs. the
Pacific Northwest outbreaks suggested a higher mortality in the United States
(Kluger et al., 2006). After review of present data on outcomes in the literature,
in vitro drug susceptibility studies, and clinical experience, the 2010 Infectious
Diseases Society of America Cryptococcal Guidelines support the treatment of
C. gattii infections in a manner similar to those of C. neoformans (Perfect et al.,
2010). However, C. gattii infections may produce more cryptococcomas in the
brain or lung, and chronic conditions such as hydrocephalus and a slow response
to antifungal therapy may be prominent features of disease with this species.
Despite the lack of precise differences in C. gattii vs. C. neoformans disease
clinically, which would require immediate identification of the species, there
are differences in epidemiology regarding the range of infections. It is also sug-
gested on a pathobiological basis that C. gattii may present more with acute
disease rather than reactivation disease. This hypothesis is based on (1) anti-
body studies in children where C. neoformans, but not C. gattii, antibodies are
frequently detected, and (2) the higher percentage of disease that is observed in
immunocompetent patients with C. gattii (Goldman et al., 2001). It has also been
shown that C. gattii may induce a different host immune response compared to
C. neoformans (Cheng et al., 2009). Furthermore, the Vancouver Island outbreak
represents an ideal arena to check for genetic susceptibility to C. gattii infection
because a vast number of individuals were exposed to high numbers of infectious
propagules, but only a minority of those exposed was identified with clinical
disease. However, it is clear through both animal studies and transplant cases
that C. gattii infections can reactivate (Dromer et al., 1992; Kluger et al., 2006).
Finally, although major virulence factors (melanin, capsule, high-temperature
growth) are similar, some of the networks such as the trehalose pathway connect-
ing these virulence factors are different in these two species (Ngamskulrungroj
et al., 2009b; Petzold et al., 2006). The possibility that differences in both host
and microbe genetics may influence the course and management of infections
will drive studies on this unique example of a fungal outbreak in humans as we
continue its analysis.
Acknowledgments
We thank Wenjun Li and Yonathan Lewit for their contributions to the analy-
sis of the outbreak; Dee Carter, Vishnu Chaturvedi, Jim Kronstad, Kieren Marr,
APPENDIX A 243
and Robin May for collaboration; Rory Duncan, Chris Lambros, and Dennis
Dixon from the National Institute of Allergy and Infectious Diseases (NIAID)
and Victoria McGovern from the Burroughs Wellcome Fund for their support;
and Stephen Johnston for his prescient questions. We also explicitly thank all of
the Forum members and the Institute of Medicine for shining a very bright light
on the fungal kingdom, and for the invitation to participate. Our research is sup-
ported by NIH/NIAID R37 grant AI39115.
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A11
YEAST INFECTIONS—HUMAN GENETICS ON THE RISE38

Steven M. Holland39 and Donald C. Vinh39
We lead inextricably mycotic lives: yeasts leaven our bread, ferment our
wine and beer, and inhabit our skins, mouths, and gastrointestinal tracts; however,
not all is harmony. Hippocrates reported aphthous ulcers consistent with thrush in
patients with severe debilitation, but it was not until the 1840s that in the newly
emerging field of clinical experimental medicine that thrush—as well as other
mycotic conditions, including ringworm—was recognized as being caused by
transmissible fungi. In 1923 Christine Marie Berkhout named what we now call
Candida albicans, for the white robe, toga candida, worn by Roman senators
and senatorial candidates (Emmons et al., 1977). Fungal infections in general,
and candida infections in particular, are important markers of innate or acquired
immune dysfunction. However, despite the estimated 1.5 million species of
fungi in the world, precious few cause human disease, and those that do (in the
38â•›â•›Originally published as Holland, Steven M; Vinh, Donald C.2009. Yeast Genetics on the Rise.
New England Journal of Medicine. Article DOI: 10.1056/NEJMe0907186. Copyright © 2009 Mas-
sachusetts Medical Society. Available at: http://www.nejm.org/doi/full/10.1056/NEJMe0907186.
39â•›â•›Steven M. Holland, M.D., and Donald C. Vinh, M.D. From the Laboratory of Clinical Infec-
tious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD.
APPENDIX A 249
absence of iatrogenic factors) usually cause inapparent or mucocutaneous infec-

tion. Therefore, genetic factors in both the pathogen and the host must be key to
an understanding of who gets disease and why.
Despite the ubiquity and importance of fungi, little is known about specific
human genetic predispositions to them. There are three distinct categories of
fungi: filamentous molds (e.g., aspergillus, mucor, and trichophyton), dimor-
phic fungi (e.g., the endemic mycoses, histoplasma, blastomyces, coccidioides,
and sporothrix, a non-endemic pathogen), and yeasts (e.g., candida, crypto-
coccus, and trichosporon). In the absence of iatrogenic factors, visceral infec-
tions of filamentous mold occur almost exclusively in patients with chronic
granulomatous disease, whose capacity to generate phagocyte superoxide is
defective. Severe invasive aspergillosis develops in these patients (who have
normal lungs), as do, on occasion, infections with invasive candida; interest-
ingly, mucocutaneous candidiasis is not encountered in chronic granulomatous
disease. In contrast, development of most other cases of pulmonary aspergillo-
sis that are unrelated to immunosuppression requires previous airway damage,
such as bronchiectasis or bullous disease. Dimorphic fungi cause inapparent or
relatively mild disease in the vast number of people who are otherwise normal.
Severe pneumonia and disseminated disease occur infrequently, as in cases of
advanced human immunoÂ�deficiency virus (HIV) and in patients with defects of
the interferon-γ–interleukin-12 axis, indicating that intracellular killing plays
a crucial role in the body’s defense against these infections. Mucocutaneous
candidiasis, vaginal candidiasis, thrush, and onychomycosis occur in a vari-
ety of disparate conditions, ranging from diabetes mellitus to the autoimmune
polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) syndrome,
to HIV, to Job’s (hyper-IgE) syndrome. Our recent identification of mutations in
signal transducer and activator of the transcription 3 gene (STAT3) as the cause
of Job’s syndrome unexpectedly provides a link to the important articles by Fer-
werda et al. (2009) and Glocker et al. (2009) in this issue of the Journal.
Whereas mucocutaneous candidiasis has long been recognized as a con-
sequence of profound lymphocyte dysfunction, or lymphopenia, it occurs in
Job’s syndrome in patients with a relatively normal number of functioning lym-
phocytes. Mutations in the gene encoding transcription factor STAT3 impair
numerous pathways, including the generation of interleukin-17–committed T
lymphocytes (Th17 cells). Th17 cells fill a gap in previous knowledge about
lymphocyte-mediated killing: If Th1 (interferon-γ–producing) lymphocytes are
controlling intracellular infection and Th2 (interleukin-4–producing) lympho-
cytes are directing antibody-mediated protection against extracellular infection,
which cells protect the exposed mucosa and epithelium? Among the products of
Th17 cells is interleukin-22, which synergizes with interleukin-17 in the epithe-
lial synthesis of cationic antimicrobial peptides, such as defensins. STAT3 is also
central to the induction and signaling of itself, interleukin-17, and interleukin-22
(Fig. A11-1) (Minegishi et al., 2009).
We encounter most microbes first at epithelial surfaces through cell-surface
250 FUNGAL DISEASES
Figure A11-1.eps
bitmap
APPENDIX A 251
FIGURE A11-1â•‡ (facing page). Mechanisms of Fungal Sensing and Control. The β-glucan
on the budding yeast forms of candida bind to dectin-1 on epithelial cells and phagocytes,
leading to activation of the CARD9 signaling complex (along with Bcl-10–MALT1)
(Panel A). This in turn produces cytokines that help drive CD4+ T lymphocytes toward the
Th17 phenotype, a STAT3-dependent process. Th17 lymphocytes elaborate interleukin-17
(augmenting neutrophil production and recruitment) and interleukin-22, which are syn-
ergic in the STAT3-dependent production of antimicrobial peptides by epithelial cells.
The generation of superoxide by the phagocytic NADPH oxidase system is crucial for
the killing of filamentous molds such as aspergillus (Panel B). Interactions between mac-
rophages and lymphocytes are required for the control of intracellular infections such as
the dimorphic fungi. In response to fungal infection, macrophages release interleukin-12,
which acts on its cognate receptor on T lymphocytes and natural killer (NK) lymphocytes.
Interleukin-12–stimulated lymphocytes release interferon-γ, which acts on macrophages
through STAT1 to kill intracellular fungi. Interferon-γ also augments tumor necrosis fac-
tor α (TNF-α), which acts in part through the nuclear factor κB (NF-κB) to kill fungi
and drive inflammatory responses. Molecules with identified mutations relevant to fungal
susceptibility are shown in red.
receptors. These receptors couple the binding of specific microbial components

to signal transduction pathways, which results in local and systemic immune
responses, including activation of the central activator of inflammation, nuclear
factor κB. Specific immunodeficiencies involving receptor molecules and their
signaling have been characterized for herpes simplex virus encephalitis, Neisseria
meningitidis, and Streptococcus pneumoniae (Beutler, 2009). As expected, the
closer the defect is to the initial contact with the pathogen, the more likely it is
that the susceptibility will be microbiologically narrow. In contrast, defects that
occur at the convergence of several pathways tend to be broader and more severe.
Dectin-1 is the cell-surface receptor for β-glucan, a major component of the
budding yeast-cell wall, and its signaling travels through a series of molecules,
including caspase-recruitment–domain (CARD) protein 9, leading to activation
of nuclear factor κB.
Ferwerda et al. identified a Dutch family with impaired in vitro responses
to β-glucan. The spectrum of disease was limited to nails and mucosa. Interest-
ingly, the ages at clinical disease presentation were 10 to 12 years for the homo-
zygous daughters but 40 and 55 years for the heterozygous mother and father,
respectively, suggesting both hormonal and gene-dose effects. Generation of
interleukin-6 and interleukin-17 was impaired only in response to the dectin-1
ligand, β-glucan. The specific stop codon the authors identified in dectin-1 is
remarkably common in some parts of Africa and Europe (allele frequency, 3 to
7%), suggesting that unrecognized forces maintain it in populations. Glocker
et al. identified an extended Iranian family with predominantly mucocutaneous
but also fatal candidiasis of the central nervous system caused by mutations in the
critical dectin-1 signal transduction molecule, CARD9, impairing both dectin-1
signaling and Th17 production. CARD9 also funnels a variety of other cell-
252 FUNGAL DISEASES
surface and intra-cellular signals, including the p38 mitogen-activated protein

kinase (MAPK) and Jun N-terminal kinase (JNK) pathways, possibly accounting
for its greater clinical severity as compared with isolated dectin-1 deficiency.
The CARD9 mutation appears to be rare, and its rarity is commensurate with its
severity.
Important caveats concerning both reports are that the key manifestations
are mucocutaneous, whereas the functional studies were performed on leuko-
cytes. It remains to be proven whether the key mechanisms in these cases of
severe candidiasis consist of impaired dectin-1 signaling at the epithelial level
or impaired leukocyte activation of epithelium, mediated through cytokines such
as interleukin-17.
The long and largely happy coexistence of humans and fungi has necessitated
the existence of ways to detect and control fungi, keeping them in their place.
Apparently, these pathways are different for candida, aspergillus, and mucor.
Although we do not yet understand the entire conversation between leukocytes
and epithelium, with these two reports we have now overheard some of the key
words that will enable us to listen more thoughtfully.
No potential conflict of interest relevant to this article was reported.
References
Beutler BA. TLRs and innate immunity. Blood 2009;113:1399–407.
Emmons CW, Binford CH, Utz JP, Kwon-Chung KJ. Medical mycology. 3rd ed. Philadelphia: Lea
& Febiger, 1977.
Ferwerda B, Ferwerda G, Plantinga TS, et al. Human dectin-1 deficiency and mucocutaneous fungal
infections. N Engl J Med 2009;361:1760–7.
Glocker E-O, Hennigs A, Nabavi M, et al. A homozygous CARD9 mutation in a family with suscep-
tibility to fungal infections. N Engl J Med 2009;361:1727–35.
Minegishi Y, Saito M, Nagasawa M, et al. Molecular explanation for the contradiction between
systemic Th17 defect and localized bacterial infection in hyper-IgE syndrome. J Exp Med
2009;206:1291–301.
A12
THE INCREASED RISK OF GLOBAL WHEAT RUST PANDEMICS:

PUTTING YELLOW RUST INTO PERSPECTIVE
Mogens Støvring Hovmøller40
Infectious diseases of humans are receiving great attention due to their di-
rect and immediate impact on human mortality (King et al., 2006; WHO, 2008),
40â•›â•›Aarhus University, Faculty of Agricultural Sciences, Department of Integrated Pest Management,
Flakkebjerg, 4200 Slagelse, Denmark. Mogens.hovmoller@agrsci.dk.

APPENDIX A 253
whereas the diseases of crop plants are subject to much less publicity although
they are threatening crop productivity, food security, and thereby the livelihood
of billions of people around the world. Currently, wheat rust fungi are among
the top 10 constraints for food production in many parts of the world, such as
sub-Saharan Africa, East Asia, and South Asia (FAO, 2010; Waddington et al.,
2010), mainly due to their epidemic potential and transboundary spread by wind
(Brown and Hovmøller, 2002).
According to U.S. Department of Agriculture statistics, global wheat produc-
tion was generally lower in 2010 compared to the previous year (USDA, 2011).
The reduced productivity was caused by several factors, such as unfavorable
weather conditions, resulting in large-scale flooding, droughts, and bushfires
in different parts of the world, as well as severe yellow rust epidemics (USDA,
2010).
Rust diseases have been recognized as being harmful to wheat since ancient
Greece. Theophrastus of Eressus (371–286 BC), the founder of botany and a
pupil of Aristoteles, noted in his book ΠΕΡΙ ΦΥΤΩΝ ΑΙΤΙΩΝ (The Causes of
Plants) that some plants of barley were more susceptible to the rusts than others,
the most susceptible denoted “Achilles Barley,” and he recognized the importance
of sowing in due time to reduce rust diseases on different crop plants such as
wheat, barley, peas, and beans (Theophrastus, 1990). Wheat rust may be caused
by three plant pathogenic fungi, Puccinia graminis (Leonard and Szabo, 2005),
Puccinia triticina (Bolton et al., 2008), and Puccinia striiformis (Hovmøller
et al., 2011). The corresponding diseases are termed black (stem) rust, brown
(leaf) rust, and yellow (stripe) rust, respectively (synonymous terms in brackets)
(Figure A12-1).
The three wheat rust fungi are biotrophic and heteroaecious, meaning they
generally require a primary wheat host for asexual reproduction and an alternate
host to complete sexual reproduction. Berberis spp. have been known to serve as
an alternate host for stem rust for more than two centuries. For instance, Schøler
(1818), who was a school teacher in Denmark, demonstrated a link between
stem rust on barberry and some cereals via repeated infection experiments. His
results started a so-called “barberry quarrel,” where the usefulness of barberry
eradication programs was discussed. Later, de Bary (1865, 1866) demonstrated
the complete life cycle of the stem rust fungus. However, the problems related to
barberry adjacent to cereal fields were observed by farmers in Europe much ear-
lier, with the first local laws against barberry being implemented in France in the
17th century and in the Americas in the 18th century (Roelfs, 1982). Surprisingly,
the discovery of barberry as an alternate host for yellow rust infecting wheat and
Poa grass, respectively, was not made until very recently (Jin et al., 2010). The
alternate host of P. triticina depend on the primary host: Isolates from cultivated
wheat and wild emmer have Thalictrum speciosissimum (in the Ranunculaceae)
as alternate host, whereas several species in the Boraginaceae, such as Anchusa
aggregata, Anchusa italica, Echium glomeratum, and Lycopsis arvensis, may
254 FUNGAL DISEASES
FIGURE A12-1â•‡ Typical macroscopic symptoms of rust infections on adult wheat plants.
Puccinia striiformis Westend (yellow/stripe rust) [left photo], Puccinia triticina (brown/
Figure
leaf rust) [center photo], and Puccinia A12-1.eps
graminis tritici (black/stem rust) [right photo].
bitmap
serve as alternate host for P. triticina from wild wheat and rye (Anikster et al.,
1997). Although this paper deals with all three wheat rusts, the primary focus is
yellow rust, which has spread at unprecedented scales in recent years and caused
severe epidemics even in areas where the disease was previously non-significant
or absent (Hovmøller et al., 2010).
Yellow Rust Epidemiology

Wheat rusts are highly epidemic on susceptible cultivars in a rust-favor-
able environment. In the past, yellow rust was considered most harmful in cool
and wet climates, whereas leaf rust and stem rust epidemics were favored by
warmer temperatures. They are all characterized by passive spreading of airborne
urediniospores carried by the wind, potentially across hundreds of kilometers
(Hovmøller et al., 2002; Kolmer, 2005; Leonard and Szabo, 2005; Zadoks, 1961).
Since 2000, epidemics of yellow rust in particular have accelerated in many areas
(Hovmøller et al., 2010). In the United States, annual losses due to wheat yellow
APPENDIX A 255
rust exceeded 1 million metric tons over several years, and in each of the years
2003 and 2010 they mounted to 2.4 million tons (Long, 2000–2010), despite
increased and widespread use of agrochemicals already in 2003 (Chen, 2005). In
China, yellow rust is considered the most damaging disease on wheat, which is
grown on more than 20 million hectares (Wan et al., 2004). In three yellow rust
epidemic years, annual losses varied between 1.8 and 6 million tons; in 2002,
losses of 1.3 million tons were recorded and 1.9 million tons were saved by
treating at least 6 million hectares with fungicides (Wan et al., 2004). Yellow rust
epidemics reached record levels in northern Africa in 2009 (Ezzahiri et al., 2009)
and also in central and western Asia (Mboup et al., 2009), where more than 90
percent of important wheat varieties were susceptible to the disease (Sharma et
al., 2009). Areas particularly affected by yellow rust epidemics in 2009 and 2010
are illustrated in Figure A12-2.
New genetic variability of the pathogen, resulting from natural population
dynamic forces such as mutation, recombination, and migration, may be fol-
lowed by host-induced selection, which is a major driver of changes in gene and
genotypic frequencies of biotrophic plant pathogens (Hovmøller et al., 1997).
2009 2010
FIGURE A12-2â•‡ Map indicating the distribution of global wheat production and regions
of recent yellow rust epidemics.Figure
Each red A12-2.eps
dot is equivalent to 20,000 tons of wheat grain
production. The blue and orange circlesbitmap
indicate some of the areas in which severe yel-
low rust epidemics on wheat were reported in 2009 and 2010. Yellow rust severity varied
greatly from field to field within the highlighted areas, depending on susceptibility of
actual host cultivar, local environmental conditions, pathogen race distribution, and disease
management practices. The epidemics in Northwest Europe in 2009 (dotted circle) were
restricted to relatively few cultivars of wheat and triticale.
SOURCE: Adapted from Trethowan et al. (2002). Dr. Dave Hodson, Food and Agricul-
ture Organization of the United Nations, Rome, is acknowledged for kind permission to
reprint the map.
256 FUNGAL DISEASES
In agricultural systems, where crop cultivars with similar or identical resistance

specificities are cultivated across large areas, the role of selection is greatly en-
hanced (Bayles et al., 2000; Hovmøller et al., 1997; McDonald and Linde, 2002).
Therefore, crop varieties that are resistant when first deployed in agriculture may
become susceptible after some years in cultivation. This phenomenon, which is
often referred to as the boom-and-bust cycle, has been reported in numerous cases
for the wheat rusts (e.g., Bayles et al., 2000; Chen, 2005; Enjalbert et al., 2005;
Hovmøller, 2001; Wellings, 2007). The boom-and-bust cycle is closely related to
resistance genes in the host and two categories of pathogen traits, virulence and
aggressiveness.
Virulence and Aggressiveness

The yellow rust fungus is specialized on the cereal host at both species and
cultivar levels (Hovmøller et al., 2011). The former refers to variation in the in-
fection and reproduction capacity of the pathogen on different host genera (e.g.,
wheat, barley, rye, tricicale), whereas the second level of specialization occurs
within the host genus, that is, at the cultivar level. The latter is typically based on
the interaction between host resistance gene products and pathogen avirulence
gene products in a gene-for-gene relationship (Flor, 1956). Mutation in the aviru-
lence gene may result in virulence, which can be defined as the qualitative ability
of the pathogen to cause disease by compromising a matching resistance gene in
the host. In case the host cultivar possesses more than a single resistance gene, the
virulence phenotype, representing a specific combination of different virulences,
becomes critical in determining the fate of host susceptibility (e.g., Hovmøller
and Henriksen, 2008; Johnson, 1992; Line and Qayoum, 1992). The virulence
phenotype can be resolved from the outcome of a race analysis, which is car-
ried out by inoculating a rust isolate onto a set of host differential lines carrying
known resistance genes, for details (see, e.g., Hovmøller and Justesen, 2007a).
The study of race dynamics in P. striiformis populations has often been an
important part of early warning and yellow rust strategies in Europe (Hovmøller
and Henriksen, 2008; Johnson, 1992), North America (Chen and Penman, 2005;
Line and Qayoum, 1992), China (Wan et al., 2007), and Australia (Wellings,
2007). Since 1967, where a national race survey was established in the United
Kingdom (Johnson, 1992), similar programs were established in Germany (Flath
and Bartels, 2002), France (De Vallavieille-Pope et al., 1990), and Denmark
(Hovmøller, 2001). In recent years, European data on P. striiformis virulence
dynamics have been made publicly available via the Eurowheat database at www.
eurowheat.org, which also describes options for disease control, such as cultural
practices and fungicide efficacies (Jørgensen et al., 2010). Systematic race sur-
veys were initiated in the United States in the late 1960s after a series of severe
yellow rust epidemics on wheat (Line and Qayoum, 1992). Systematic race sur-
veys have also been implemented in China (Chen et al., 2009; Wan et al., 2004),
APPENDIX A 257
India (Prashar et al., 2007), Australia (Wellings, 2007; Wellings and McIntosh,
1990), and South Africa (Boshoff et al., 2002).
In addition to the virulence phenotype, pathogen aggressiveness, which is
a quantitative measure of the ability of a virulent isolate to cause disease on a
susceptible host plant, is a key determinant for the rate of pathogen spread and
evolution. Some of the recent epidemics since 2000 have been ascribed to the
emergence of yellow rust strains with increased aggressiveness and tolerance to
warm temperatures (Hovmøller et al., 2008). In this context, strain was defined
as a group of P. striiformis isolates sharing both molecular marker phenotype
and virulence phenotype. For instance, Milus et al. (2009) demonstrated a sig-
nificantly increased aggressiveness at both high and low temperatures, compared
with isolates of pre-2000 races from North America and Europe (Milus et al.,
2009). At the low-temperature regime, gradually changing from 10°C (night) to
18°C (day), the aggressive strains produced more than 70 percent more spores
per infected leaf area compared to reference isolates from before 2000. At the
high-temperature regime (12°C at night to 28°C during the day), isolates of the
aggressive strains produced approximately 150 percent more spores per infected
leaf area. Milus and colleagues (2009) concluded that the aggressive strain had
most likely enhanced the yellow rust epidemics in North America and contrib-
uted to the spread of yellow rust into areas that were previously considered too
warm for yellow rust epidemics. The aggressive strain detected in North America
was indistinguishable from a strain detected as exotic incursion in Australia in
2002 (Hovmøller et al., 2008; Wellings et al., 2003). In Europe, a nearly identi-
cal strain was first detected in 2000, whereas first appearance of this strain in
the Red Sea area and in western and central Asia is unknown (Hovmøller et al.,
2008). According to time and area, these observations may be comparable to the
famous panglobal spread of the Irish potato famine fungus Phytophthora infes-
tans (Goodwin et al., 1994).
The distinction between virulence and aggressiveness may be subject to con-
troversy because virulence toward a resistance gene with minor effects may be
manifest by increased disease progress and spore production. These are the same
variables used to measure aggressiveness. However, in case of virulence, there
should be evidence for a significant host–pathogen interaction, whereas in the
case of aggressiveness, the host–pathogen interaction is ideally non-significant.
This implies that for a range of host cultivars with varying levels of rust suscep-
tibility, they should rank the same for aggressive and non-aggressive isolates.
The Need for Coordinated Global Action

The number of foreign incursions of wheat rust races has increased signifi-
cantly in recent years. Park et al. (2010) reported nine foreign incursions of wheat
stem rust and wheat leaf rust into Australia between 1925 and 2005. Of these
seven had occurred since 1969. Hovmøller et al. (2011) documented at least six
258 FUNGAL DISEASES
exotic incursions of yellow rust at continental scales since 1979, often resulting
in the spread of yellow rust epidemics to new regions or continents. Scherm and
Coakley (2003) noted that the rate of exotic pathogen invasion in the United
States had increased from about five instances per decade from 1940 to 1970
to more than three times that number during the 1990s. This increasing rate of
incursions of the yellow rust fungus may partly be ascribed to the emergence of
new, highly aggressive rust strains combined with increasing human travel and
commerce. However, regardless of the reasons for the increasing spread, the situ-
ation escalates, emphasizing the relevance of pathogen surveys covering larger
areas and a need for global coordination. The use of crop cultivars with similar
or identical resistance specificities across large wheat-growing areas supports
this conclusion.
The accelerating wheat yellow rust epidemics requires coordinated multina-
tional action to complement the ongoing initiatives of the Borlaug Global Rust
Initiative (www.globalrust.org) to fight the multivirulent strain of wheat stem
rust, known as Ug99 (Northoff, 2007, 2008). The speed by which threatening
new strains can be diagnosed and reported widely is essential, and the precision
by which traits such as virulence and aggressiveness are diagnosed is another
bottleneck (Hovmøller et al., 2011). Precise diagnosis of both traits require pure
and correct identification of the seed stocks of standard differential wheat variet-
ies used for the race assays, genetically pure pathogen isolates, and well-defined
experimental conditions for temperature, humidity, light, and plant nutrition, as
well as trained staff who can assess and interpret the virulence phenotype and
aggressiveness. The acknowledgment of these challenges led to the establish-
ment of a Global Rust Reference Center (GRRC) in 2008 targeting yellow rust,
supported by the International Center for Agricultural Research in the Dry Areas
(ICARDA), the International Maize and Wheat Improvement Center (CIMMYT),
and Aarhus University (Denmark) (Hovmøller et al., 2010). GRRC is accessible
for receiving yellow rust samples on a year-round basis, which is a major advance
complementing the capacities of existing regional and national rust diagnostic
laboratories. At the Borlaug Global Rust Initiative (BGRI) Coordination Meet-
ing in Syria in September 2009 (Dold, 2009), a proposal was made to extend the
activities of GRRC to all wheat rust fungi, that is, yellow rust, brown rust, and
stem rust.
The long-term aims of a Global Rust Reference Center would be the track-
ing of new wheat rust incursions and assessment of ongoing pathogen evolution
at global scales, supplying data to online early-warning systems for farmers,
breeders, and policy makers, like the current “Rust Spore” monitoring system
run by the Food and Agriculture Organization of the United Nations (FAO,
2010). Another main activity would be to assist in training of students and junior
scientists in rust pathology, and maintenance and extension of a unique world
wheat rust collection to facilitate resistance breeding efforts. The activities should
complement ongoing regional and national survey activities and research efforts
APPENDIX A 259
by BGRI partners, national counterparts, and advanced research institutions,

resulting in stronger global efforts to mitigate the consequences of the inherent
pathogen dynamics. At present, the activities at GRRC are limited to race analysis
of relatively few pathogen samples per year in addition to training activities. To
become sustainable in the long term, GRRC must contain a considerable portfolio
of activities to reduce vulnerability due to potential changes in staff, management,
and political awareness.
The identification of sources of resistance from cultivated and wild crop rela-
tives to wheat is probably the most urgent task to reduce the threats posed by the
wheat rusts, including yellow rust. Plants have an immunity system with several
layers. Race-specific resistance provides protection only during one or few steps
of the infection process and is therefore considered vulnerable to pathogen evolu-
tion whereas partial resistance, based on several or many sources of resistance, is
considered more durable. Nevertheless, breeding for increased partial and non-
host resistance protecting wheat against a broad spectrum of pathogen species and
races can be done by large-scale field screening of thousands of breeding lines by
applying targeted pathogen species and strains.
Prospects for New Research

The serious impacts and characteristics of wheat yellow rust epidemics
should remind society about the vulnerability of global food production and the
need to understand the interactions between agricultural crops, their pathogens,
and the environment. Until recently, investigations of the basic biological and mo-
lecular mechanisms of yellow rust evolution have been hampered by the biotro-
phic lifestyle of yellow rust, precluding the use of molecular tools used for fungi
that can be cultured on artificial media, and by the lack of an experimental system
for genetic studies. These limitations may soon be overcome by the significant
advances of molecular technologies, new insights into pathogen biology, and the
cloning of host resistance genes, which were achieved in recent years. Molecular
markers are probably the best established tools for resolving recent as well as
past dispersal and evolutionary events in P. striiformis (Hovmøller and Justesen,
2007b; Hovmøller et al., 2008; Mboup et al., 2009). Next generation sequenc-
ing is currently the most promising tool for the identification of genetic changes
related to virulence in fungal pathogens (Stergiopoulos and de Wit, 2009). A
Danish–British genome sequencing initiative was started in 2009 (Walter et al.,
2009), followed by a more comprehensive U.S.-led initiative (Broad Institute,
2009) aiming to resolve the whole genome sequence of P. striiformis. Such
knowledge will most certainly speed up the discovery of genes involved in
pathogenicity. The recent discovery of barberry as sexual host for P. striiformis
(Jin et al., 2010), which has solved a century-old mystery about the yellow rust
lifecycle, represents another major breakthrough that can serve as the basis for
the development of an experimental system allowing classical genetic studies.
260 FUNGAL DISEASES
Functional characterization of isolated rust genes is still a great challenge

because of the biotrophic lifestyle, which is hampering genetic transformation
of P. striiformis. The identification of sources of resistance in wheat, especially
for resource-poor areas in Africa and Asia, is probably the most urgent task to
reduce the threats posed by wheat rust pathogens. However, this will require a
long-term commitment and combined efforts of breeders, pathologists, and biolo-
gists to keep pace with the wheat rusts, which can evolve rapidly to compromise
genetic resistance of the wheat host (Hovmøller and Justesen, 2007b; Wellings
and McIntosh, 1990).
Concluding Remarks
The fight against infectious crop diseases has become a collective responsi-
bility and requires a collective investment with a global, long-term political and
scientific commitment. Such a global network will need to establish the physical
and human resources to support the progress of wheat rust management tech-
nologies, that is, pathogen monitoring and resistance breeding, training programs
for farmers and pathologists, and scientific progress. Hence a major priority of
a global network should be to link researchers and plant breeders (1) with each
other and with appropriate partners in resource-poor countries that are affected
most by sudden wheat rust epidemics, and (2) with political authorities to allow
for rapid actions. The establishment of the BGRI in 2006, inspired by Dr. Nor-
man Borlaug, the pioneer of the Green Revolution and the front leader of cereal
rust resistance breeding, represents a major milestone in this respect. The real
challenge is to ensure sustained activities beyond ongoing, short-term projects.
In the long term, surveillance and prevention measures of wheat rust epidemics
will only be successful if performed on a coordinated, global scale. Borlaug used
to say: “Rust never sleeps”—and events of recent years have shown how right
he was.
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264 FUNGAL DISEASES
A13
FUNGAL PATHOGENESIS IN PLANTS AND ANIMALS:

SIMILARITIES AND DIFFERENCES
Barbara Howlett41
Introduction
Although the vast majority of fungal species do not cause disease, ones that
affect plants are responsible for significant economic losses (Skamnioti and Gurr,
2009). In the past couple of decades, fungal diseases of humans have become an
increasing threat, especially for people who are immunologically compromised
(Sexton and Howlett, 2006). Consequently there has been a rapid explosion in
knowledge about fungal pathogenesis of animals and this has been taken up by
people studying plant pathogenic fungi. Pathogenesis involves the interaction of
two partners with input from the environment, a concept described as the “dis-
ease triangle” in plant pathology. The “damage-response” concept developed for
animal pathogens emphasizes that the outcome of an interaction is determined
by the amount of damage incurred on the host (Casadevall and Pirofski, 2003).
These concepts are useful reminders of the complexity of the interaction and the
interdependence of host and pathogen.
Tools to Study Fungal Pathogenesis

The large number of fungi with sequenced genomes and recent advances
in genetic manipulation of fungi are leading to an improved understanding of
mechanisms associated with disease. Comparative genomics is being used to
identify candidate genes involved in disease. Amplification of particular gene
families within a genome is consistent with lifestyles: For instance, many plant
pathogens have large gene families that encode enzymes to degrade the cuticle
and cell wall, which coats plant cells, while animal pathogens (e.g., Coccidi-
oides immitis, the cause of valley fever) have gene families encoding enzymes
that degrade proteins of the skin, which is usually the initial barrier to invasion
(Sharpton et al., 2009). Transcriptomics can show which genes are turned on;
microarrays have been used extensively and many datasets are publicly available
(Cairns et al., 2010). Proteomic analysis of culture filtrates provides information
about secreted proteins (secrotome) that are accessible to the host and thus may
play a role in the interaction; this approach has been applied to identify such
proteins of Leptosphaeria maculans, the blackleg pathogen of canola (Vincent
et al., 2009).
41â•›â•›The University of Melbourne.

APPENDIX A 265
The generation of fungal mutants via tagged random insertional mutagen-

esis allows identification of novel genes involved in disease, while targeted gene
knockout or gene silencing allows functional analysis of candidate pathogenic-
ity genes. Analyses of human pathogenic fungi generally rely on cell lines and
animal models with different immunosuppression regimes. By contrast, plant
pathogens can be studied directly on their hosts. Many more fungal species
infect plants compared to animals and thus more plant fungal systems than
animal fungal systems are studied, but with a shallower focus. Plants obviously
can be manipulated with fewer ethical issues than those associated with animal
experimentation. Findings from model systems can often be applied to other
host–pathogen interactions. The best studied plant fungal system for ascomy-
cetes is rice and Magnaporthe oryzae (cause of rice blast) (Ebbole, 2007) and
for basidiomycetes is maize and Ustilago maydis (cause of corn smut) (Brefort
et al., 2009). Model animal fungal systems include immune-suppressed mice
and Aspergillus fumigatus, Candida albicans, or Cryptococcus spp; recently
amoeboid and non-vertebrate animal model systems, particularly the insect Gal-
leria mellonella, have been used (Mylonakis et al., 2007). General similarities
and differences between fungal pathogens of plants and animals are described in
Table A13-1 and are discussed in more detail later in this chapter.
The Invasion and Disease Process

The disease process can be considered as a series of consecutive steps begin-
ning with recognition between host and fungus, penetration of the host, coloni-
zation and avoidance of host defenses, development of disease symptoms, and
finally fungal reproduction and dispersal. These steps are developmentally regu-
lated, and fungal mutants can be generated that are arrested at particular stages
(for review see Sexton and Howlett, 2006). The mutated genes are often referred
to as pathogenicity genes and are broadly categorized as to where they appear
to act in the disease pathway. This is an arbitrary classification for convenience.
Clearly there are pathogenicity genes with pleiotropic effects at many steps in
the pathway, as some processes (e.g., recognition, pH regulation, oxidative burst,
signaling) occur several times. Pathogenicity genes of fungi that attack plants
have been reviewed recently (Van de Wouw and Howlett, 2011). Interestingly
the steps related to symbiotic relationships between plant and fungi are similar
to those involved in pathogenesis; thus the classification of plant–fungus interac-
tions as pathogenic or symbiotic is indistinct.
Fungal inoculum of plants is usually spores (sexual or asexual) that then
germinate on the surface of the plant. Animal pathogenic fungi can exist in
forms such as yeasts, conidia, or hyphal fragments. Some such fungi undergo
dimorphic switching. In some cases, the yeast form is more pathogenic than the
hyphal phase, while in others the converse is true. A key class of hydrophobic
proteins, hydrophobins coat the fungal cell wall, which is composed generally of
266 FUNGAL DISEASES
TABLE A13-1â•‡ General Similarities and Differences Between Fungal

Pathogens of Plants and Animals
Feature Plants Animals
Number of fungal pathogens Many Few
Importance Cause 25–30% crop losses Usually affect
immunocompromised animals
Lifestyle and environmental Often biotroph; necrotroph; Often soil saprophytes; not
niche also saprophytes obligates
Experimental systems Many; often shallow focus Few; different host cell and
immunosuppression regimes of
non-humans
Host specificity Often host species or cultivar Sometimes species specific, not
specificity genotype specific
Mode of entry into hosts Via stomata, or breaching Via inhalation, ingestion, or
surface barriers via enzymes or wounds in skin
pressure created by infection
structures
Dispersal Between hosts Not usually between hosts
Reproduction Sexual and asexual Usually asexual (ascomycetes)
Overall pathogen Often complex set of physical Inocula small enough to enter
requirements and molecular barriers to host, survive at 37°C and avoid
overcome immune responses
chitin, a polymer of b 1,4 N-acetyl glucosamine, b 1,3 glucans, as well as other

polysaccharides and proteins.
Plants have more complex physical barriers to invasion than animals do. Epi-
dermal cells on the plant surface have a cuticle comprising epoxy fatty acids, and
a cell wall that is composed of a matrix of proteins and interacting carbohydrates,
including b 1,3 and b 1,4 glucans such as cellulose (Carpita and Gibeaut, 1993).
By contrast the animal surface, the stratum corneum, is much thinner and the cells
are highly keratinized (Proksch et al., 2008). Fungi enter animals by inhalation,
ingestion, or wounds. Basically, for a fungus to be an animal pathogen, its major
features include being small enough to enter tissue, able to survive at 37°C, and
able to evade host immune responses (Sexton and Howlett, 2006). Indeed, the
temperature optimum for many fungi is significantly lower than 37°C, which
has been proposed as an explanation for why relatively few fungi are important
pathogens of mammals (Robert and Casadevall, 2009). The mammalian charac-
teristics of maintenance and close regulation of body temperature are proposed
to have a selective advantage in conferring resistance to many fungal pathogens
(Bergman and Casadevall, 2010).
APPENDIX A 267
Fungi enter plants via the stomatal apertures, where air exchange occurs;
by digesting the cuticle and cell wall with hydrolytic enzymes; or by develop-
ing infection structures, appressoria, which accumulate high concentrations of
glycerol and puncture the surface due to high turgor pressure (Van de Wouw and
Howlett, 2011). As mentioned above, fungi enter animals through the skin or via
inhalation or wounds; an exception is Histoplasma capsulatus, which enters the
host via receptor-mediated endocytosis (Woods, 2003).
The first basal or innate layer of defense is very similar in plants and ani-
mals. It involves binding of pathogen associated molecular patterns (PAMPs) to
pattern recognition receptors on the host membrane surface. This triggers signal-
ing pathways that induce a range of defense responses, including production of
reactive oxygen species and sometimes programmed cell death. This response
is termed “pathogen triggered immunity.” PAMPs common to plant and animal
pathogenic fungi include wall carbohydrate fragments, such as chitin oligosac-
charides or b 1,3 glucans. As well as conservation among PAMPs, there is also
conservation in structural domains of Pattern Recognition Receptors (for review
see Zipfel, 2009).
In plants often the responses triggered by innate basal immunity are not
strong enough to stop pathogen invasion. Consequently a second round of rec-
ognition occurs involving effector-triggered immunity (Jones and Dangl, 2006).
Often there is a “gene for gene” interaction between avirulence (effector) genes
in the pathogen and resistance genes in the plant, such that the pathogen is unable
to attack host genotypes with the corresponding resistance genes. Thus there is
usually a high degree of host specificity with plant diseases, with only particular
varieties (genotypes) of a single species being susceptible. In contrast to plant
diseases, most animal diseases do not display host genotype specificity, although
some fungi only cause disease in certain animal species. Apart from innate basal
immunity, the immune system of mammals is very different from that of plants.
The responses that come into play if the pathogen is not stopped by the innate
basal immunity response include the innate complement system, circulating cells
such as phagocytes that can internalize and destroy pathogen cells, and adaptive
antibody-mediated defenses (Speth et al., 2004).
Fungal plant pathogens have a range of lifestyles and nutritional require-
ments. Many have a saprophytic phase, and some are obligate, surviving only on
their hosts. Some (biotrophs) require hosts to be living, while others (necrotrophs)
kill plant tissues. Some fungi are both biotrophs and necrotrophs at different
stages during growth in planta. Some fungi such as mycorrhizae and endophytes
colonize plants in a symbiotic relationship deriving carbon from photosynthesis
by the plant and often conferring drought tolerance on the plant. Fungi that infect
animals often have a saprophytic lifestage in the soil, and few if any are obligate.
After invasion, fungi then need to colonize the host, derive nutrition, and
avoid or subvert host defense responses. In many plant–pathogen interactions, de-
fense responses include the hypersensitive response, whereby an oxidative burst
268 FUNGAL DISEASES
by the plant generates reactive oxygen species associated with the programmed
cell death of host cells. This can arrest pathogen growth, particularly that of bio-
trophs. Necrotrophic fungi can subvert this defense process to derive nutrition
from the dead host tissue. Toxins produced by necrotrophic fungi also kill plant
tissue. However, they are generally not often important disease determinants.
This is also often the case for secondary metabolite toxins produced by fungi
that attack animals. Their role may be to protect fungi against predators such as
insects, nematodes, and amoebae during their saprophytic growth phase in the
soil (Kempken and Rohlfs, 2010).
To complete its lifecycle, a fungus must reproduce and exit the host to find
another host. Many animal pathogenic ascomycetes do not have a sexual phase.
However, the sexual cycle of basidiomycetes such as Cryptococcus spp. is ex-
tremely important in virulence, as discussed by Heitman. 42 Both animal- and
plant-pathogenic fungi reproduce mitotically within the host. Although plant-to-
plant transmission of fungal disease is very common, direct transmission of fun-
gal pathogens between mammalian hosts is unusual. In plant pathogens, conidia
(vegetative spore) production usually occurs after infection has been established
and lesions have developed. Conidia are spread from plant to plant by wind or
in water droplets. However, many pathogens of mammals are transmitted by
inhalation as hyphae or as arthroconidia, in dust and wind (e.g., C. immitis) or
soil. Increasingly, biofilms, whereby a community of microorganisms attaches
to a solid surface, mediate dispersal of fungi in hospital environments (Ramage
et al., 2009).
A Fungal Pathogen That Infects Plants and Animals

Given some of the similarities in strategies that fungi use to cause disease in
plants and animals, it is perhaps surprising that few organisms have been reported
to infect both plants and animals. A single isolate of Fusarium oxysporum f. sp.
lycopersici can infect both plants and animals. The animal experimental system
involves injection of the tail vein of immunodepressed mice and the plant system
involves incubation on intact tomato roots (Ortoneda et al., 2004). Mutants in sev-
eral genes of F. oxysporum f. sp. lycopersici have been tested for their virulence
in the animal and plant models. A mitogen activated protein kinase gene (fmk1)
and an Rho1 GTPase, which are both involved in signaling, are not required for
virulence in mice, but are essential for virulence in tomatoes. In contrast, the
transcription factor PacC, which mediates the environmental pH signal, and the
photosensor WC-1, which interacts with a light-sensitive transcription factor,
WC-2, are necessary for full virulence in mice, but are not essential for virulence
in tomatoes (Martínez-Rocha et al., 2008; Ortoneda et al., 2004; Ruiz-Roldan
et al., 2008). These findings highlight redundancy in some signaling pathways
42â•›â•›See contributed manuscript by Heitman in Appendix A (pages 226–248).

APPENDIX A 269
TABLE A13-2â•‡ Fungicides Used to Control Plant and Animal Diseases

Fungicide Group Target Plants Animals
Azoles Ergosterol biosynthesis Widely used Widely used
in fungal membrane
Echinocandins b 1,3 glucan synthesis Not used New drug
Polyenes: Ergosterol biosynthesis Not used Yes
Amphotericin B
Nikkomycin Z Chitin synthesis CHS I Not used Trialed against C.
immitis
Strobulurins Mitochondrial Used but resistance can Not used; toxic
cytochrome bc develop: G143A mutations
complex in cytochrome bc
Bion Salicylic acid analog: Used in horticulture; Not effective as
Mimics systemic expensive dependent on plant
acquired resistance in defense responses
plants
and the complexity of these interactions. It will be interesting to compare global

transcriptional analyses of tomato and mouse tissue infected with this fungus
to further identify networks and signaling components regulated in response to
interaction with two extremely different host types.
Control of Fungal Diseases

Animal diseases are controlled mainly by a well-functioning immune system
of a potential host. There are no approved vaccines against fungi, and fungicides
often are not efficacious (Ostrosky-Zeichner et al., 2010). Plant diseases are con-
trolled by a combination of approaches. These include management of inocula;
for instance, removing infected stubble (crop trash) at the end of the growing
season; using fungicides; and introgressing (breeding) resistance genes, often
from related species, into varieties. For diseases of both types of hosts, a range
of fungicides is employed; choices depend on toxicity and cost/benefit ratio
(Table A13-2). Azoles target ergosterol biosynthesis in the fungal membrane
and are the most common group of fungicides used for diseases on both types of
hosts. Echinocandins, which target synthesis of b 1,3 glucans in the fungal cell
wall, are now being used to combat fungal diseases of humans, but would not be
effective against fungal plant pathogens given that plant cell walls also have b
1,3 glucans. Nikkomycin Z, which targets chitin synthesis, is being used to con-
trol C. immitis infections (Ostrosky-Zeichner et al., 2010),43 while strobilurins,
43â•›â•›See article by Galgiani in Appendix A (pages 196–207).

270 FUNGAL DISEASES
which target mitochondrial cytochrome bc complex, are deployed against plant

diseases, but resistance to them can develop and these molecules have a degree
of toxicity toward animals (Bartlett et al., 2002). Bion, which is a salicyclic acid
analogue, can induce systemic acquired resistance in some plants (Beckers and
Conrath, 2007). This molecule is used more in horticulture than broad-acre grain
crops due to its cost.
Deploying resistance genes is an important strategy in controlling plant dis-
ease. When corresponding avirulence genes are mutated or deleted, the fungus is
not recognized by the plant, and infection ensues. Thus resistance is overcome,
often resulting in severe yield losses. Some fungi can more readily overcome re-
sistance than others; such fungi are deemed to have “high evolutionary potential”
and they usually outcross prolifically, producing large numbers of wind-borne
sexual spores as inocula (McDonald and Linde, 2002). These properties enable
such fungi to adapt to selection pressures imposed by extensive sowing of crop
varieties with resistance conferred by single genes. Thus the frequency of virulent
isolates will increase and resistance in the plant can break down. An example of
such a breakdown of resistance occurred in 2003 when resistance in canola to
blackleg disease caused by L. maculans broke down (Sprague et al., 2006). This
resulted in up to 90 percent yield losses in regions of Australia, costing farmers up
to $20 million (B. J. Howlett, unpublished). The resistance had been conferred by
a canola gene named Rlm1. The corresponding avirulence effector gene, AvrLm1,
has been cloned and is located in a very “plastic” part of the L. maculans genome
(Gout et al., 2006).
Recently the genome sequence of this fungus has been acquired (Rouxel
et al., 2011). The fungus has a unique structure. More than one third comprises
repetitive elements composed of degenerated transposons. Gene-rich regions
with high GC content alternate with gene-poor regions with high AT content
with sharp boundaries between them. Disease-related genes such as effectors are
interspersed in the gene-poor regions among repetitive DNA. This organization
contributes to the readily changeable nature of the fungal genome and enables
effector genes to be readily gained, lost, or mutated (Rouxel et al., 2011; Van de
Wouw et al., 2010). Markers are now available for several other avirulence ef-
fector genes (Fudal et al., 2007; Parlange et al., 2007), and thus the frequency of
virulent isolates within fungal populations in the field that are virulent toward a
particular resistance gene can be monitored. This information allows farmers to
rotate the disease resistance genes in the canola varieties that they sow from year
to year. This strategy, which can be applied to other plant diseases, can help pre-
vent outbreaks and maximizes the duration of effectiveness of resistance genes.
Closing Remarks
The development of fungal diseases of plants and animals has many parallels.
A fungus must overcome many hurdles to successfully invade a plant and cause
APPENDIX A 271
disease. There is a continuum from disease to damage to symbiosis with plant–

fungal interactions. The most important properties of a human pathogen are to be
able to survive at 37°C and to avoid the immune responses. The use of fungicides
to control disease is made on the basis of specificity and after cost/benefit analy-
ses. Such decision making about a broad acreage crop is very different from that
about a human patient. An important strategy to control plant disease is to incor-
porate resistance genes. However, virulence toward this resistance can be selected
for, due to the high evolutionary potential of some fungi. Thus resistance genes
have to be managed (rotated) by farmers to maximize longevity of resistance.
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A14
CLIMATE, GLOBALIZATION, AND TRADE: IMPACTS ON

DISPERSAL AND INVASION OF FUNGAL PLANT PATHOGENS
Michael Jeger,44 Marco Pautasso,44 and James Stack45
Climate change is likely to become a major issue in plant pathology over the
coming years. In this overview, we provide recent evidence for this statement.
Moreover, we point out the importance for future plant disease management of
climate change interactions with other global change drivers, such as increased
long-distance trade. Recent advances in aerobiology, together with new molecu-
lar tools used in landscape and geographical genetics, can help in addressing the
challenges posed by an increasing number of emerging plant diseases worldwide.
There is increasing evidence that climate change will be a key issue in how plant
pathogens will affect food security and ecosystem health.
Less knowledge is available on the potential impacts of climate change on
biological control of exotic fungal plant pathogens. Network theory is a promis-
ing tool to improve biosecurity in the face of the increased volumes of traded
plants coupled with climate warming. Although there are now many reviews of
the literature on the topic of climate change and plant diseases, there is a need to
keep up with the rapid development of the subject.
Introduction
The emergence of fungal plant pathogens can occur as the result of at least
three processes: (1) the evolution of new genotypes within a pathogen population
that is endemic to a habitat or location (e.g., through genetic recombination); (2)
the introduction of an exotic pathogen genotype into a new, receptive habitat or
location; and/or (3) the natural selection of new pathogen genotypes from an
endemic population as a result of changes in host genotype, host species, or ex-
ternal pressures (e.g., environmental conditions, fungicide applications, changes
in plant cultural practices). Emergence is a function of introduction (via dispersal
or evolution), establishment (i.e., adaptation to the habitat or location), and spread
44â•›â•›Division
of Biology, Imperial College London, Silwood Park, Ascot, SL5 7PY, U.K.
45â•›â•›Department
of Plant Pathology, Kansas State University, 4024 Throckmorton Plant Sciences
Center, Manhattan, KS 66506Â�Â�Â�-5502.
274 FUNGAL DISEASES
(i.e., dispersal via natural or human-mediated mechanisms). Weather variables,

including wind patterns and storms, and the global trade in plants and plant prod-
ucts facilitate the dispersal of plant pathogens and thus provide opportunities for
the emergence of fungal plant pathogens. Further, weather is a primary regulator
of invasion into new habitats and locations by plant pathogens. Consequently,
climate change may provide unprecedented opportunities for the emergence of
fungal plant pathogens through the dispersal of fungal pathogens to new locations
and/or through habitat modifications for both pathogens and plants.
Climate, globalization, and trade are major drivers of the dispersal and in-
vasion of fungal plant pathogens, affecting agricultural and horticultural crops,
plantation and forest trees, and plants in natural/semi-natural environments. Pat-
terns of weather, notably large-scale air movements, have been implicated in
long-range dispersal of several economically important pathogens between and
within continents. In some cases, continental dispersal of plant pathogens is
tracking and catching up with previous movements of crop plants, in turn accom-
panying human population migrations that occurred over centuries, sometimes
millennia. The rate of movement of humans, crop plants, and plant pathogens
has intensified markedly in recent decades with the increased globalization of
the world economy. Expansion of trade pathways and technological innovations
make possible the production, harvesting, storage, marketing, shipping, and air
freight of food crops, ornamental plants, and plant products to an extent that
previously was not possible (Figure A14-1).
From a broad perspective, climate and global change are drivers of large-
scale ecological perturbations that facilitate novel “biomixing” and “ecological
fitting” (Agosta et al., 2010). These phenomena can lead to rapid host switching,
the emergence of hybrid pathogens, and invasion of new infectious diseases and
pests (Brasier, 2008; Palm, 1999). Changes in plant phenology occur in a variety
of ways, depending on species and geography. Such changes impact on plant
interactions with fungi in general (Gange et al., 2011; Kauserud et al., 2010) and
with fungal pathogens in particular (Grulke, 2011; Marçais et al., 2009). The
consequence is that multiple species at many sites need to be studied in order
to understand and predict regional change and impact (Ibanez et al., 2010). An
alternative strategy has been to focus on multiple drivers of global change on a
single species (Baeten et al., 2010; Matesanz et al., 2009; Paajanen et al., 2011).
Global environmental change arises from CO2 enrichment, increased nitrogen
deposition, climate shifts, biotic interactions, and land use change (Tylianakis
et al., 2008). These factors have pervasive effects on antagonistic and mutualistic
interactions between plants and fungi and in some cases increase the severity of
pathogen infection while weakening mutualisms. Interactions must be expected
between tree genetic diversity, variation in phenology, resistance to defoliators
and fungal pathogens, increased CO2, and ozone concentration affecting tree
growth and mortality; some of these interactions have been reviewed by Pautasso
(2009).
APPENDIX A 275
FIGURE A14-1â•‡ The increase in goods (109 tons × km) moved in the United Kingdom
from the 1930s to the 1990s. Figure A14-1.eps
SOURCE: Schulz (2004).
bitmap
In this overview we consider the impact of climate, globalization, and trade

on dispersal and invasion of fungal plant pathogens in the broader ecological
context described above. We make a distinction between dispersal that is medi-
ated by natural means, mostly atmospheric, and that which is mediated by human
intervention of one form or another. This is partly a matter of convenience: we
recognize that at some scales of dispersal both means can be important. With
respect to pathogen emergence, dispersal is ineffective without establishment,
spread, and persistence—the elements of invasion.
Natural Dispersal of Fungal Plant Pathogens

Fungal spores that escape the boundary layer of crop canopies can be trans-
ported over long distances subject to their biophysical characteristics and meteo-
rological conditions. The study of such transport belongs firmly in the domain
of aerobiology, a discipline that owes much of its development to the effect of
aero-allergens on human health (Dallafior and Sesartic, 2010). An account of an
early pioneer of aerobiology, Philip Gregory, and the link with fungi of agricul-
tural and human health concern, can be found in Lacey et al. (1997). Weather has
276 FUNGAL DISEASES
significant effects on the incidence of aero-allergens, including the abundance and

biodiversity of spores of the fungal plant pathogen Alternaria alternata (Magyar
et al., 2009). Under conditions predicted by climate change, changes in plant-
ing practices and modified crop management may be required to keep allergen
concentrations under control (Beggs, 2010).
Even if climate change is becoming important in allergen aerobiology
(D’Amato and Cecchi, 2008), vegetation normally will be the main source of
fungal spores in the atmosphere. Thirty-two genera of fungi arising from vegeta-
tion sources were found across both cultivated and urban areas in three regions
in Egypt, with a clear association with weather conditions and many implications
for the spread of human and plant diseases (Awad, 2005). Fungal concentration
in the atmosphere may not be the best indicator of health risk, which may be
more associated with the predominant aero-allergen present (Awad, 2005). The
effects of meteorological factors on atmospheric dispersal, in biophysical terms
relevant for plant pathogens (including viruses and bacteria), was provided in a
recent review (Jones and Harrison, 2004).
In some cases, the dispersal of fungal plant pathogens has been modeled
explicitly using biophysical principles. A recent example has been the dispersal
of Phakopsora pachyrhizi, causing soybean rust (Andrade et al., 2009). The
American continent was free of P. pachyrhizi until 2001. After its introduction in
Paraguay, the pathogen rapidly became established throughout Bolivia, Brazil,
and Argentina, possibly due to a combination of large-scale cultivation of the
plant host, international movement of infected material, and long-distance natural
dispersal. Urediniospores of this fungus have been shown to remain viable long
enough to be able to travel hundreds of kilometers (Savage et al., 2010). In 2004,
Asian soybean rust was reported in the continental United States (Goellner et al.,
2010). The fungus now overwinters in warm southern U.S. locations. Spore es-
cape was modeled and combined with a standard large-scale transport model to
forecast spore deposition over U.S. soybean production areas. Canopy turbulence
and canopy porosity were found to be key determinants of spore escape.
Some effort has been made to test the validity of transport models (Skelsey
et al., 2009). Spijkerboer et al. (2002) evaluated the Gaussian plume model
(GPM) for predicting and describing spore dispersal over a potato crop. The main
purpose was prediction of Phytophthora infestans, but for experimental purposes
they used a commonly used fern spore. They concluded that the GPM was not
applicable in risk assessments, unless combined with site-specific information at
the source, such as spore escape in relation to wind speed.
Other more empirical approaches can also be used for specific purposes.
High-speed imaging showed that, by synchronizing the ejection of thousands of
spores, ascomycete fungi such as the pathogen Sclerotinia sclerotiorum form an
air flow that carries spores around intervening obstacles to atmospheric currents
and new infection sites (Roper et al., 2010). Mundt et al. (2009) used simple
empirical relationships based on the inverse power law to describe the spread of
APPENDIX A 277
plant diseases such as wheat stripe rust, wheat stem rust, potato late blight, and
Southern corn leaf blight. Much of the earlier literature on this approach is cited
by the authors. They found that the estimated power law parameter varied little
over five orders of magnitude on a distance scale. Evidence was found to support
the hypothesis that disease advances through accelerating, rather than constant,
waves. Integration of (unmanned) aerial measurements of spore concentration at
various distances from the source with simulation of spore flight trajectories is
important to develop reliable decision support systems to predict risk of disease
spread, as shown for Phytophthora infestans in potato fields in Virginia (Aylor
et al., 2011; Techy et al., 2010).
A feature of recent work on dispersal has been the integration with popula-
tion genetics aspects of pathogen diversity (Hovmøller et al., 2008; Montarry
et al., 2010). This can operate at the level of host specialization and biotrophy and
the ways in which wind dispersal acts as a survival mechanism—what has been
termed “Oases in the desert” (Brown et al., 2002). Equally, the strongly stochastic
nature of long-distance dispersal can lead to founder effects in the pathogen popu-
lation (Brown and Hovmøller, 2002). In such cases, pathogen genotypes that suc-
cessfully establish in new regions and/or on new cultivars may be different from
those at the source—the so-called founder effect. Such effects were found in the
migration of the fungus causing black Sigatoka of bananas, Mycosphaerella fi-
jiensis, from Southeast Asia to Africa and Latin America (Brown and Hovmøller,
2002; Rivas et al., 2004). In both of the new regions founder effects were present
in the new invasions. Unresolved for this pathogen is the relative importance of
air-dispersed ascospores (probably limited) and the movement of infected plant
material (largely unrecorded). The fungus Corynospora cassiicola has a wide
geographical range in the tropics and subtropics and many plant hosts. Common
fungal lineages were widely distributed geographically, indicating long-distance
dispersal of clonal lineages, but also previously unrecognized genetic diversity
involving some degree of host specialization on some hosts (Dixon et al., 2009).
The advent of modern molecular tools in epidemiology provides a step change
in both the tracking of dispersal of novel fungal genotypes and in risk assess-
ment of emerging fungal diseases in plants and in animals (Gladieux et al., 2011;
Moslonka-Lefebvre et al., 2011).
Climate and Plant Diseases

Weather and climate generally have major impacts on diseases caused by
fungal plant pathogens. This topic has had extensive historical coverage that it is
not possible to cover in this overview. What is more significant and of immediate
concern is how climate change will impact the distribution and severity of dis-
eases caused by known pathogens and the emergence of new invasive pathogens.
This topic has also had its fair share of literature reviews: recently, e.g., in the
context of structural change in the international horticultural industry (Dehnen-
278 FUNGAL DISEASES
Schmutz et al., 2010); the evolution of the phytosanitary regulatory framework

(MacLeod et al., 2010); forest health and adaptive management (Parks and
Bernier, 2010); the impacts on plant health and carbon sequestration in Australia
(Singh et al., 2010); cool season grain legume crops and their diseases (Thomas,
2010); urban trees and their pathogens (Tubby and Webber, 2010); diseases in
tropical plantation crops (Ghini et al., 2011); the disease triangle and changes
in plant phenology (Grulke, 2011); rice diseases and pests (Haq et al., 2011);
diseases of food crops (Luck et al., 2011); the geographical distribution of plant
pathogens (Shaw and Osborne, 2011); and plant pathogens in Sweden (Roos
et al., 2011).
Here, we refer to climate change to include trends in air composition as well
as trends in global warming. Air composition in terms of SO2 concentration has
been shown to be associated with the relative prevalence of two important fungal
pathogens of wheat (Fitt et al., 2011), a finding made possible by the application
of molecular analytical tools to archived plant material from the long-term Broad-
balk experiments at Rothamsted Research in the United Kingdom. Elevated atmo-
spheric CO2 and ozone concentrations decreased the incidence of downy mildew
disease in soybean, but increased the severity of disease caused by Fusarium
virguliforme (Eastburn et al., 2009). Changes in precipitation and temperature
resulted in increased disease severity for both diseases, and there were indirect
effects due to treatment effects on canopy structure and leaf age. Similar kinds
of results were obtained in studies of four pepper diseases (Shin and Yun, 2010).
Elevated CO2 and temperature treatments increased the rate of progress for two
bacterial diseases, but not for a stramenopile disease (Phytophthora capsici) or a
fungal disease (Colletotrichum acutatum).
The relationship between climate change, plant diseases, and food security
(Chakraborty and Newton, 2011) considers international cooperation and inte-
grated solutions, including disease management issues, to be essential to meet the
food demands of the growing world population. Plant breeding for climate-related
traits such as drought avoidance or tolerance (Khan et al., 2010) must also take
into account disease resistance. The arable sector will be critical in this respect,
with mitigation and adaptation strategies with respect to plant disease control
likely to become a key area (Fitt et al., 2010). Because of the variation in crop
growth and pathogen environmental requirements, geographical divides in crop
yield and productivity may become more pronounced (Butterworth et al., 2010).
Climate change will also impact on the incidence of mycotoxins in food (Russell
et al., 2009), a much-neglected topic in relation to food security and human and
animal health.
Forest health is one area where the impact of climate change and biologi-
cal invasions is providing a clear signal (Kliejunas et al., 2008; Sturrock et al.,
2011; Woods et al., 2010). In turn, emerging forest diseases under climate change
can lead to a positive feedback by reducing the carbon stocks of affected forests
(Peltzer et al., 2010), as seen in British Columbia with the developing Dothis-
APPENDIX A 279
troma needle blight epidemic following the devastating mountain pine beetle
outbreaks. The resilience of northern boreal forests to rapid climate change can
be questioned (Chapin et al., 2010), as can forest establishment under condi-
tions of permafrost thaw where fungal pathogens may affect seedling survival
(Camill et al., 2010). In forest tree nurseries in Finland, rust, powdery mildew,
and other fungal leaf diseases are already causing more problems because of
climate warming (Lilja et al., 2010). A more optimistic outlook due to improved
adaptive management practices is presented with respect to white pine blister
rust (Hunt et al., 2010), where white pines have broad ecological ranges and are
less likely to be maladapted thus succumbing to the disease, and hence may be
more resilient in the long term. Similar issues will need to be faced with regard
to urban trees (Tubby and Webber, 2010) and Mediterranean forests (Attorre
et al., 2011; La Porta et al., 2008), where the impact of non-native insect pests
and fungal pathogens introduced through trade pathways (see later sections) is
already being observed.
The above discussion concerns general issues regarding climate change and
plant diseases. There have been many accounts of global/climate change impacts
on diseases caused by specific fungal plant pathogens in recent years. A selection
of these is cited with summary comments in Table A14-1.
Climate and Beneficial/Biocontrol Fungi

Compared with studies on plant pathogenic fungi, there have been relatively
few studies on the effects of climate change on beneficial plant-associated fungi.
There has been some consideration of mycorrhizal associations and endophytic
fungi, but little on tritrophic mycoparasitic interactions (Singh et al., 2009). In
a review of the results of 135 studies, Compant et al. (2010) found that elevated
CO2 had a positive influence on the incidence of arbuscular and ectomycorrhi-
zal fungi, whereas the effects on endophytic fungi were more variable. Effects
of temperature were idiosyncratic, with positive, neutral, and negative effects
equally common. Plant growth-promoting fungi (as with bacteria) positively af-
fected plants subject to a degree of drought stress. Considerable research has been
done with tritrophic interactions among arthropod pests and their natural enemies
(Thomson et al., 2009), including fungal entomopathogens; similar research is
lacking for biocontrol of plant pathogens (Ghini et al., 2008). The expectation
must be that prediction will be difficult because of the indirect and direct effects
on biocontrol fungi, unless there is a good understanding of tritrophic interac-
tions (Thomson et al., 2009). Biological control of weeds using fungi is simply
a case of a plant pathogenic fungus being used for a beneficial purpose. Cirsium
arvense is a troublesome weed with world-wide distribution. Climate change will
exacerbate invasion and persistence of the weed and make biological control us-
ing a variety of pathogenic fungi more difficult (Tiley, 2010).
280 FUNGAL DISEASES
TABLE A14-1â•‡ Selected Papers Illustrating the Effects of Climate and Global
Change Factors on Specific Pathogen–Host Systems
Pathogen Host Reference Comments
Valsa Alder Worrall Previous oscillation period in damage of ~21 years
melandiscus et al. likely to dampen with warmer summers and with
(2010) no period of recovery
Erisiphe Grapevine Pugliese Increase in CO2 concentration did not affect
necatrix et al. incidence possibly due to increased photosynthesis
(2010) with higher CO2 and temperature
Phytophthora Beech Fleishmann Elevated CO2 and low N supply enhanced
citricola et al. susceptibility, but host compensation followed
(2010)
Cercospora Red bud and McElrone Incidence/severity under elevated CO2 greater
spp. Sweet bud et al. with above average rainfall and temperature (one
(2010) species), but mitigated by higher photosynthetic
efficiency
Leptosphaeria Oilseed rape Stonard Geographic variation in species may be exacerbated
maculans and et al. by climate change with more damaging species
L. biglobosa (2010) expanding in range
Didymella Chickpea Frenkel Isolates infecting crop and wild Cicer show
rabiei et al. different adaptation to high temperature, with the
(2010) potential for hybrids infecting both hosts over a
broader temperature range
Phakopsora Soybean Del Ponte Integrating epidemiological and meteorological
pachyrhizi and Esker factors suggest no restricted overwintering areas in
(2008) Brazil
Seiridium Mediterranean Zocca et al. Pathogen is associated with insect vectors, which
cardinale cypress (2008) are able to reach the range margin and thus the
continuous threat of arrival in the expanding range
Cronartium White pine Frank et al. Pathogen arrived in New Mexico in ~1970 with
ribicola (2008) upper flow synoptic classification indicating early
June 1969 most favourable
Fusarium Wheat Xu et al. Environmental conditions differentially affect
spp. (2008) infection and colonization process and the
comparative abundance of six toxigenic species in
the head blight disease
Fusarium Maize Horvath Damage by beetles provides conducive growing
spp. (2003) conditions for the toxigenic fungi, with feeding
behavior changing under drought conditions
APPENDIX A 281
Human-Mediated Dispersal of Fungal Plant Pathogens

Thus far we have considered natural dispersal and invasion of fungal plant
pathogens, where the main drivers are weather variables, or more generally cli-
mate. Although pathogens have always accompanied humans and their crops over
the centuries, most notably with the historical migrations of human populations
(Guillemaud et al., 2011; Money, 2007), there have been instances where crops
moved to new areas have escaped, at least temporarily, the pathogens endemic in
the original distributional range of the crops (Mitchell and Power, 2003). Equally,
there are cases where crops grown in a new environment have been exposed to
novel pathogens. These movements have taken place over centuries with periods
of time for adaptation (assisted through plant breeding) or for the pathogen to
reencounter the host in a new environment. With the globally connected world
that now exists, these time scales are much shorter—decades or, in some cases,
much less. The consequence has been the emergence of new fungal plant patho-
gen species or levels of subspecific variants that were previously unrecorded.
For example, since the publication of Erwin and Ribeiro’s 1996 Handbook on
the genus, 39 new species of Phytophthora and two species of hybrids have been
formally described (Ersek and Ribeiro, 2010). It is unlikely that this increase is
due solely to the advent of modern molecular taxonomic techniques. More likely
it is due to the ability of this genus to adapt to new hosts in new environments,
through encounters made possible by new pathways.
Less spectacular has been the historical accumulation of non-indigenous for-
est pests in the United States, where some 450 insect and pathogen species have
colonized since European settlement (Aukema et al., 2010). Some 16 pathogenic
species have caused substantial damage to trees. This finding is more in keeping
with analyses over 10 taxonomic groups of alien species (including fungi), which
suggested a historical legacy going back at least a century (Essl et al., 2011). This
result would imply that Britain is more at risk for invasion of new exotic species
than other European countries (as has happened, for instance, for Phytophthora
ramorum), given its historical links to its Commonwealth (Figure A14-2). How-
ever, even where that is the case, the corollary is that the impact of current global
activity will be even more manifest in the decades to come (Crooks, 2005).
Dispersal Through Trade Pathways

Even in a country with an efficient and visible plant quarantine service, such
as Australia, there are problems in defining what is present and what is absent
(Hyde et al., 2010). These authors make a case for a reinventory of Australia’s
plant pathogens and consider five fungal groups in which what were thought to
be species were in fact species complexes. Without this level of discrimination
concerning what is in the country, it will not be possible to operate an effective
quarantine and plant protection service. The often tortuous relationship between
plant quarantine and trade barriers can be a problematic political issue (MacLeod
282 FUNGAL DISEASES
FIGURE A14-2â•‡ The world in 1897, with British possessions marked in red.
Figure A14-2.eps
SOURCE: Cambridge University Library available at Wikimedia Commons.
bitmap
et al., 2010; Perrings et al., 2010). Emerging plant diseases can be seen as nega-
tive externalities deriving from the international trade in plants (Lansink, 2011).
The removal of trade barriers, however, can be beneficial and in some cases
desirable. For example, seed trade legislation was designed primarily to protect
trade and return royalties to contemporary plant breeders. Increasingly, the im-
portance of exploiting the genetic diversity present in cereal land races has been
recognized, but to exploit their use fully, changes in legislation will be required
(Newton et al., 2010). Also the genetic diversity of target plant pathogens should
be used in building comprehensive collections to allow efficient, reliable, and
specific diagnostic and detection tools in the national and international trade
(Barba et al., 2010).
The Sanitary and Phytosanitary Agreement of the World Trade Organisation
specifies that countries cannot regulate against unknown pests, yet many invasive
alien forest insects and pathogens were new to science when first recorded in a
new environment (Britton et al., 2010). To counter this, effective surveillance
systems are required to facilitate early detection; these are lacking in many na-
tions. Britton et al. (2010) propose a global network of sentinel plantings based
in historical gardens and arboreta to enable early detection and rapid response
APPENDIX A 283
to such incursions. To support early detection, diagnostic capability and capacity

are needed to provide rapid and accurate identification of emerging or introduced
pathogens. Progress has been made in establishing diagnostic capability in rural
areas of some developing nations and improving diagnostic capability in some
developed nations (Boa, 2007; Miller et al., 2009; Stack, 2010).
Mitigating the nursery stock pathway (for forest trees and ornamentals)
for undescribed pathogens will be extremely difficult. In general, analysis has
been lacking on how the structure of trade pathways affects the spread of plant
pathogens in the nursery trade. Pautasso et al. (2010c) analyzed the hierarchical
structure in terms of the proportions of producers, wholesalers, and retailers in-
volved in the trade. Despite the many uncertainties associated with commercially
sensitive trade information, it was concluded that disease management options
should concentrate on the middle tier of the nursery hierarchy, particularly in the
absence of hubs (superconnected nodes). If hubs are present, then control is bet-
ter targeted at them because (ceteris paribus) the higher the correlation between
links in and out of nodes, the lower the epidemic threshold (Moslonka-Lefebvre
et al., 2009). In addition, the number of outgoing links from the starting node
of simulated epidemics in small-size directed networks (a realistic assumption
for plant trade among countries and within regions) is strongly correlated with
epidemic final size at equilibrium (Pautasso et al., 2010b). These analyses were
motivated by the outbreaks of the emerging plant pathogen Phytophthora ramo-
rum but have broader generality for systems where susceptibility and infection
are not too incompatible. Rather, the states form two poles of a continuum (a
realistic assumption for the plant trade, where premises and plant shipments can
have a varying proportion of infected plants).
In 2009, the United States imported 1.2 billion live plants, 2.6 million
pounds of seed for planting, and countless shipments of horticultural plants that
pass through inspection and enter the retail distribution system in less time than
the latent period for most diseases. The tremendous volume of trade of plants
and plant products crossing borders and ecological areas and the speed of that
trade precludes inspection and interception as a primary strategy for preventing
the emergence of plant pathogens (Stack, 2010; Stack et al., 2010). On average,
1–2 percent of U.S. imports are inspected. Even with effective surveillance and
diagnostic systems, the emergence of fungal plant pathogens will continue. Ex-
isting natural dispersal pathways at the site of introduction, in addition to local
and regional trade channels, provide opportunities for spread from the sites of
introduction.
The first comprehensive inventory of alien fungi and oomycetes recorded
in France since 1800 was recently reported (Desprez-Loustau et al., 2010), with
some 65 percent being plant pathogens. Using this dataset, the factors influencing
invasion success were investigated, with an emphasis on forest tree pathogens.
There was an influence of climatic factors, but human population size and its
relationship with imports of plants in trade was a major explanatory variable.
284 FUNGAL DISEASES
In some cases, it is not simply the occurrence of a plant health problem in

trade that is the issue, but that of human or animal health, notably with regard to
mycotoxins. For example, the occurrence of tracheomycosis associated with Gib-
berella xylarioides on Robusta coffee in East Africa (Hindorf, 1998) has added
to the long-known mycotoxin problem in raw coffee, limiting imports, especially
to the European Union. Similarly, the increasing trade volumes of fresh produce
due to consumer appreciation of the health benefits brought by consumption of
fresh fruit and vegetables are associated with a greater occurrence of outbreaks of
foodborne human pathogens, such as Salmonellae, Escherichia coli, noroviruses,
and hepatitis A (Berger et al., 2010).
Dispersal of Plant Pathogens as a Cause and Consequence of Warfare and

Social Unrest
Perhaps it is no coincidence that sometimes new pathogen problems emerge
during periods of warfare and social upheaval. The Bengal famine in the mid
1940s due to Cochliobolus miyabeanus was associated with severe weather, but
it also occurred at the end of World War II, when many military, political, and
social issues constrained the ability to deal with the problem: enough food was
there, but it was not distributed to those who needed it (Padmanabhan, 1973). In
some cases, famines due to fungal pathogens can lead to, rather than be caused
by, periods of social change, as shown by the Irish migrations following the
famine caused by Phytophthora infestans in the middle of the 19th century (Fry
and Goodwin, 1997). In central Italy, the invasion of stone pine stands by North
American isolates of Heterobasidion annosum was linked to the movement of
U.S. troops during World War II (Gonthier et al., 2007). Since that time, there has
been a rate of advance of 1.3 km/year along invasion corridors, with the North
American taxon dominant, but also some hybridization with the European taxon.
The two pathogens are active in the Circeo National Park, where they cause ex-
tensive (up to 30 m in radius) gaps in Pinus pinea plantations (Scirè et al., 2011).
Subsequent studies showed that invasion success was due to the reproductive
potential of the invader not being reduced during the dry seasons, when compared
with the resident (Garbelotto et al., 2010). Unusually, this example of a fungal
plant pathogen has been commented on (“Trees become casualties of war”) in a
respected medical journal (Dixon, 2005). Although there have been cases of plant
pathogen outbreaks during wars and revolutions, today there is a trend away from
looking at climate change with a disaster-focused perspective to an emphasis on
long-term livelihood security and adaptation (Conway and Schipper, 2011). For
example, the shift from today’s widespread reliance on chemical fungicides to
the adoption of alternative and less toxic products such as inorganic salts may
happen following democratic regulations and in agreement with consumers’
perceptions, rather than as a result of, for example, mass protests or social unrest
(Deliopoulos et al., 2010).
APPENDIX A 285
The intentional introduction of plant pathogens as a strategy of warfare

has long been considered (Madden and Wheelis, 2003). Concern regarding the
creation and deployment of novel pathogen genotypes with high virulence and/
or expanded host range could certainly result in the emergence of novel fungal
plant pathogens. Although there are clear examples of state-sponsored programs
from the past, there is considerable speculation about the relative importance of
intentional introductions in the face of introductions resulting from trade and
natural events (Stack et al., 2010; Young et al., 2008).
Dispersal Associated with the Introduction of Novel Crops and Plant Species
Farmers world-wide tend to maximize the potential of the crops they grow,
in ways that are appropriate to their socioeconomic circumstances, but often
only sensible in the short term or not considering side effects such as enhanced
opportunities for plant pathogens. For example, modern agriculture relies heav-
ily on increasing the size of fields to enable a more efficient mechanization and
economies of scale. But this leads to more uniform landscapes, with potential
repercussions on epidemic thresholds of plant pathogens (Moslonka-Lefebvre
et al., 2011). Not just in natural ecosystems (Scherrer and Körner, 2011), but
probably also in agriculture, small-scale topographic variability will be important
for plant species to be able to cope with climate change. In some cases, there
may be opportunities to grow a novel crop because the changing climate makes
production possible in areas not previously suitable. This may lead to hosts for
plant pathogens that were previously unavailable.
In other cases, the cultivation of novel crops arises from other, often politi-
cal inducements: for example, the requirement that each country in the Euro-
pean Union must derive a certain percentage of its energy use from renewable
sources. Hence, there has been a diversion of food crop (cereal) production to
bioenergy use, and the planting of non-food crops such as willow, and other
short-rotation coppiced trees, and grasses such as Miscanthus for such use. As
pointed out by Stewart and Cromey (2010), new disease threats, often from
fungal plant pathogens, are likely to emerge among the existing ones. This is
likely to occur because the crop is new or because the cultivation method (e.g.,
high-density monoculture) is different from previous practice. In conservation
biology, there is a hefty discussion on the issue of assisted migration (managed
relocation), which is thought to be going to be necessary for the many species
not able to track the predicted rapid variations in climate, although it will result
in artificial shifts in species distributions and ecosystems (Loss et al., 2011).
In such debates, little attention has been paid to the likelihood that assisted
migration will result in unwanted introductions of, for example, exotic plant
pathogens.
286 FUNGAL DISEASES
Invasion Biology and Biodiversity

Dispersal and invasion of pathogenic fungi are significant not only for crop
and other managed plant populations such as commercial forests and grasslands,
but also for native plant communities (Alexander, 2010; Mordecai, 2011). In
such plant communities, invasive alien plants have long been considered a threat
to native biodiversity. Conversely, we now admit that native fungal diseases are
important components of healthy forests (Ostry and Laflamme, 2009). How-
ever, invasive plant pathogens (either introduced with alien plant invaders or
through transfer from plants in trade) can pose a threat to ecosystems (Busby
and Canham, 2011; Evans and Finkral, 2010; Holzmüller et al., 2010; Loo, 2009;
Newcombe and Dugan, 2010; Orwig, 2002).
Phytophthora ramorum is one such example. On the West Coast of the
United States, the pathogen affects a range of forest tree and shrub species, from
bay laurel to redwoods and tanoaks. In the United Kingdom (U.K.), the main
host in woodlands that supports sporulation of Phytophthora ramorum is Rho-
dodendrum ponticum, itself an invasive plant against which eradication efforts
have been targeted, whereas the main native tree species that suffers mortality
is beech. The heathland environment and its biodiversity, notably Vaccinium and
heather species, are also at risk (Harwood et al., 2009). More worryingly, the
pathogen has now been discovered affecting large areas of commercially planted
Japanese larch (Larix kaempferi), introduced into the United Kingdom in the
19th century. Japanese larch also supports sporulation of Phytophthora ramorum.
Consequently, this poses a threat not only to commercial production, but also to
neighboring native trees not previously thought to be susceptible, or never tested
(Brasier and Webber, 2010). As has happened in California, infection, tree mortal-
ity, and disease spread affect, and in turn are impacted by, forest composition and
other aspects such as landscape connectivity, proximity to infected plant nurser-
ies, and woodland history (Davis et al., 2010; Xu et al., 2009).
This example supports the hypothesis mentioned in the Introduction that
emerging fungal diseases often result from host shift speciation within a particu-
lar ecological context (Giraud et al., 2010). The impact on native biodiversity can
usefully be seen from this viewpoint. In return, reduced biodiversity frequently
increases disease transmission, including in plants, despite the observation that
areas of high endemic biodiversity may harbor a greater source pool of pathogens
(Keesing et al., 2010). In general, conserving diverse plant communities reduces
the incidence of disease (Pautasso et al., 2005; Turnbull et al., 2010). Of course
there are two-way interactions between wild and crop plants, as noted earlier in
the case of chickpea and native Cicer. Macrophomina phaseolina affects a broad
range of native plant communities and crops in Kansas. Saleh et al. (2010) found
that isolates from crops grown in the vicinity of plants in tallgrass prairie could
interchange with the more diverse wild isolates with potential dangers arising
from novel recombinations.
Irrespective of how an initial introduction occurred—through natural or
APPENDIX A 287
human-mediated means—invasion into endemic communities depends very much

on ecological circumstances. For example, the origins of fungi in New Zealand
are diverse, a few ancient with many more recent arrivals. Some more recent
arrivals have evolved into local endemic species, whereas others are maintained
through regular transoceanic arrivals (Johnston, 2010). Since 1980 the number
of fungal species (saprobic, mycorrhizal, and pathogenic) has doubled. The kinds
of fungi present in New Zealand are constantly changing as a consequence of
historical changes in plant and animal biota as well as those exotic fungal spe-
cies that have become naturalized. The fungal biodiversity of New Zealand may
appear today as a rather special case in its exotic nature, but could well become
the norm for many other countries if current trends in global trade and carbon
emissions will carry on unrelented.
The rates of spread and ecological impact of an introduced plant pathogen
through a natural plant community depend on the phylogenetic structure of
the plant community (Gilbert and Webb, 2007). Although many of the tropical
fungal pathogens investigated by Gilbert and Webb (2007) are polyphagous, the
likelihood of any one infecting two species decreases with phylogenetic distance.
However, using arbitrary cut-offs, such as plant genus or family, may underesti-
mate the risks of local spread. It would be interesting to integrate this phyloge-
netic approach with the framework provided by the geographic mosaic theory of
coevolution, which was tested for mining moths and leaf rust (Melampsora) of
Populus tremula along a latitudinal gradient in Sweden (Albrectsen et al., 2010).
Climate change can be expected to lead to twists in geographic clines of parasite
damage, as well as to shifts in the phylogenetic signal of plant host susceptibility,
but it is likely that phylogenetic and spatial structure will still be present even in
novel communities and climates.
Much of this overview has been concerned with dispersal and invasion of
fungal plant pathogens into crop plants or forest trees (Liebhold et al., 1995).
Grassland constitutes a large proportion of the Earth’s terrestrial ecosystems, with
varying levels of human exploitation (Ellis et al., 2010). Grassland ecosystems
have been less studied than crops or forests in the context of climate change and
plant health. Reductions in grassland plant richness, a measure of plant biodiver-
sity, appear to increase vulnerability to the spread of fungal plant diseases (Knops
et al., 1999), with aspects of global change, especially elevated CO 2 and nitrogen
addition, increasing the pathogen load, suggesting this is an important mechanism
by which global change affects grassland ecosystems.
Concluding Comments
In cropland, forests, and grasslands, experimentation is needed at the land-
scape level to investigate plant health adaptation approaches to climate change
(Holdenrieder et al., 2004). Similar experimentation, if accompanied by subse-
quent data analysis and dissemination, is also likely to be beneficial for pathway
288 FUNGAL DISEASES
control and diagnostic systems (Albers et al., 2010; Norton and Taylor, 2010).
Monitoring of plant pathosystems and associated organisms under changing
conditions is key to refine models, so as to successfully predict further changes
(Luedeling et al., 2011). Similarly, review of the burgeoning literature on the
subjects of climate change, land use, and plant health is essential and should be
funded on a long-term basis (Jeger and Pautasso, 2008; Pautasso et al., 2010a).
Analyses of past and current trends and simulations of future plant health sce-
narios are likely to be more influential and effective if accompanied by stake-
holder involvement and interactions with policy experts (Dwyer, 2011; Mills
et al., 2011). Climate change and other global change drivers are not the only
factors involved in plant disease epidemiology, so it is still important to consider
the important role of, for example, improvements in agronomic and silvicultural
practices in reducing current disease potential, whether or not the climate is
likely to become more conducive to a certain chronology of diseases in the com-
ing decades (Savary et al., 2011). Nonetheless, it is time to start adapting to the
challenges to plant health posed by the future climate change scenarios (Juroszek
and von Tiedemann, 2011; Legrève and Duveiller, 2010; Olesen et al., 2011;
Planinšek et al., 2011).
The emergence of fungal plant pathogens, facilitated by climate change
and globalization, will challenge our ability to meet future food demands for
a growing population and to achieve sustainable environments that provide the
ecosystem services on which societies depend. Policies informed by science and
an infrastructure that supports plant health are prerequisites to that future.
Acknowledgments
Many thanks to C. Brasier, B. D. L. Fitt, M. Garbelotto, K. Garrett, O.
Holdenrieder, M. W. Shaw, and J. Webber for insights and discussions. This
overview was partly funded by the Rural Economy and Land Use Programme
(RELU), U.K.
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A15
EMERGING FUNGAL DISEASES OF WILD ANIMAL SPECIES

Luis R. Padilla46
Summary
Several fungal diseases of non-domestic animal species have been described
as agents of epizootic proportions in wild animals in recent years. The emergence
of these diseases has reshaped the understanding of the role of fungi as contagious
diseases having an impact on wild animal populations. The recent description of
fungal epizootics caused by Batrachochytrium dendrobatidis (Bd) in amphibians
worldwide and Geomyces destructans in North American bats has called attention
to the factors driving the emergence of these diseases. Two other fungal diseases
of wild animals, lobomycosis in dolphins and penicilliosis in wild bamboo rats,
have been recognized for their zoonotic potential and highlight the need for
comprehensive, multidisciplinary approaches to understanding the ecology and
epidemiology of these diseases in wild animal populations. Continued efforts
aimed at mitigating the effects of fungal epizootics on wildlife populations and on
46â•›â•›Department of Animal Health, Smithsonian Conservation Biology Institute.

APPENDIX A 297
public health will only be successful through the identification of factors driving
the emergence of these diseases across different taxa, and embracing the concept
that wildlife may serve as sentinels of ecosystem health.
Introduction
Anthropogenic activities have been the likely driving factors behind the
emergence of some diseases in wildlife (Daszak et al., 2000). In some cases,
animal species whose status may have been threatened by other factors may now
be faced with extinction as the emergent disease spreads through a diminished
population. The recent spread of two major fungal epizootic agents, namely
Batrachochytrium dendrobatidis and Geomyces destructans in amphibians and
bats, respectively, could result in the largest changes to vertebrate populations
in recorded history (Frick et al., 2010; Skerrat et al., 2007). The discovery of
these fungal agents as major causes of wildlife epizootics in the past decade has
revolutionized the way that biologists approach the detection and diagnosis of
fungal diseases, challenging the prior misconception that fungal infections only
occurred “sporadically or in small outbreaks” and were more important to captive
wildlife, where captivity was thought to “increase susceptibility to these diseases”
(Burek, 2001).
As novel pathogens have been discovered, or known ones recognized in
novel hosts, in novel geographic areas, or with increased incidence, wildlife
conservationists have been faced with the emergence of fungal diseases that
threaten the status of wild animal populations. The factors driving disease in
free-ranging wild animal species can no longer be easily differentiated from
those affecting captive wildlife. The different factors have been blurred into a
continuum of factors through modern globalization, inadvertent movement of
disease, disease vectors, or animals themselves, through the trade of animals
across geographical barriers and the mixing of potential hosts of disease that
may not have been exposed to each other in their native habitats. Many of these
anthropogenic actions have expanded the geographic range of some diseases
or removed the natural barriers that had prevented their spread, exposing naïve
hosts to pathogens to which they were not previously exposed. Concurrently,
many of the environmental factors thought to predispose their captive counter-
parts to infectious diseases have been identified and minimized through modern
captive animal science aimed at reducing stress levels, providing better environ-
mental conditions, and through better quarantine, improving disease screening
and recognition procedures.
Institutions dedicated to the captive care of animals for conservation pur-
poses often encounter infectious diseases that would otherwise go unreported
in those species. These provide a useful baseline of understanding the host–
pathogen relationship systems. Captive animal populations can serve as viable
models for recognizing or refining the understanding of disease mechanisms
in individual hosts, which may not be possible in free-ranging animals. For in-
298 FUNGAL DISEASES
stance, in the 1990s, investigations by pathologists at the Smithsonian’s National

Zoo of an apparently novel fungus affecting captive frogs led to the isolation of
Batrachochytrium dendrobatidis (Pessier et al., 1999) and fulfillment of Koch’s
postulates (Nichols et al., 2001) in what would subsequently be recognized as
the most important infectious disease affecting wild amphibian populations on
a global scale.
The widespread recognition of other fungal pathogens and their prevalence
in wildlife populations has also changed the understanding of their role as agents
of contagious potential and concern to human public health. Concern to human
health from fungal diseases carried or propagated by wild animals has brought
attention to the role of wildlife and interactions with humans in changing envi-
ronments as indicators of global health and ecosystem stability. Direct zoonotic
potential or a shared susceptibility to disease where wildlife have a high preva-
lence of infection is worthy of direct public concern, but fungal pathogens where
the infection potential is limited to wild animals still carry an inherent cost to the
health of an ecosystem and carry indirect impacts to human society that may be
difficult to quantify.
This review article summarizes some of the fungal diseases currently known
to affect wild animal species that could be encountered by veterinarians, conser-
vationists, or animal professionals. This review is not intended as a comprehen-
sive reference on the subject. Instead, it is meant to be a common point for calling
attention to the impact of the recent emergence of specific fungal pathogens in
wildlife species within the context of wildlife as sentinel species for recognizing
changes to the health of an ecosystem.
Emergent Fungal Pathogens of Wild Animal Species
Batrachochytrium dendrobatidis
Batrachochytrium dendrobatidis is a member of a basal group of fungi, the
Chytridiomycota, and the only member known to affect vertebrates. Bd infects
amphibian species, with frogs most often reported. This emerging pathogen was
discovered in the late 1990s (Berger et al., 1998; Longcore et al., 1999; Pessier
et al., 1999) and has been recognized as a significant pathogen partially impli-
cated in the global decline of amphibian populations (Schloegel et al., 2006;
Skerrat et al., 2007). It has been retrospectively identified in North American
amphibians as early as 1961 (Ouellet et al., 2005). Bd may be the most signifi-
cant fungal infectious disease agent of vertebrate species, based on the global
distribution, wide species host range, pathogenic potential, and ability to cause
large-scale mortality.
Molecular genetic evidence from isolates collected from different locations
worldwide suggests that recent Bd outbreaks were caused by a pathogen recently
disseminated (Morehouse et al., 2003), and anthropogenic spread is suspected
APPENDIX A 299
in at least some introductions (Weldon et al., 2004), but the origin is still not
conclusively known. Virulence and pathogenic potential varies among isolates,
and some may show little or no pathogenicity to their hosts (Berger et al., 2005;
Goka et al., 2009), but these host-adapted strains may still serve as reservoirs
of strains that are pathogenic to other species. The naturally occurring bacterial
flora of the host may produce antifungal proteins that decrease the ability of the
fungi to colonize amphibian skin, and these bacteria may improve host survival
(Harris et al., 2006). Mortality in affected amphibians is likely associated with
physiologic abnormalities (electrolyte loss and osmotic imbalances) caused by
damage to the permeable amphibian skin.
Clinical signs in affected frogs can be subtle and not detectable before sud-
den death (Pessier et al., 1999). Affected frogs show excessive shedding of skin,
usually on the legs, feet, and ventrum (Nichols et al., 2001). Molecular techniques
have been established as a cost-effective and rapid method to detect the organism
in amphibian samples (Boyle et al., 2004; Kriger et al., 2006). Histopathology,
showing intralesional organisms in keratinized layers of skin, and cytology of
shed skin or imprints showing chytrid organisms have been used for diagnosing
infections (Nichols et al., 2001). Bd has no known zoonotic potential.
Chrysosporium Anamorph of Nanniziopsis vriesii (CANV)

Nanniziopsis vriesii is an ascomycetous fungus recognized as a pathogen
from skin lesions of reptiles, specifically lizards, snakes, and crocodilians. When
cultured, Nanniziopsis vriesii produces an anamorph form that is described in
the genus Chrysosporium and is undistinguishable from reptilian isolates (Paré
and Sigler, 2006). Mycotic infections may be morphologically misidentified as
other species of Chrysosporium, Geotrichum, or even Trichophyton. Historical
reports in the literature of reptilian mycoses attributed to unspeciated members of
these genera should be interpreted with caution, as CANV infections may have
been previously underdiagnosed. CANV deserves special attention as an emerg-
ing fungal pathogen of reptiles because clinical disease has been recognized in
a multitude of species with increasing frequency (Abarca et al., 2008; Bertelsen
et al., 2005; Bowman et al., 2007; Nichols et al., 1999; Paré and Sigler, 2006;
Paré et al., 2006; Thomas et al., 2002), but most reports involve captive or farmed
reptiles. Unlike opportunistic fungal pathogens, which are often ubiquitous in the
host’s environment, the CANV is extremely rare on healthy reptile integument
(Paré et al., 2003), suggesting that this organism is a primary fungal pathogen
in reptiles.
Preliminary molecular genetic evidence has suggested that the CANV repre-
sents a species complex with distinct host affinities (Paré and Sigler, 2006). Until
further molecular work is completed, the magnitude of the CANV as a pathogen
of free-ranging and captive reptile populations cannot be properly described,
300 FUNGAL DISEASES
but its potential as an emerging primary fungal pathogen of reptiles cannot be

ignored.
Clinical signs vary by species, but when seen in inland bearded dragons
(Pogona vitticeps), it causes a yellow skin discoloration dubbed “yellow skin
disease” or “yellow fungus disease.” The disease can have a protracted, chronic
course involving deep tissues (including muscles and bones) and cause disfigure-
ment in addition to the necrotic skin lesions seen. The corresponding lesions can
be diagnosed as CANV through histopathology of lesions and fungal cultures.
Histopathology typically shows severe granulomatous, necrotic dermatomycosis
with the characteristic intralesional fungal organisms. The CANV forms solitary,
single-celled conidia that can be confused with the microconidia of dermato-
phytes (e.g., Trichophyton), and arthroconidia caused by fragmented hyphae,
which may resemble the arthroconidia of Geotrichum or other Chrysosporium
(Paré and Sigler, 2006).
A single case report of a mycotic brain abscess attributed to the Chrysospo-
rium anamorph of Nanniziopsis vriesii in an HIV-seropositive Nigerian man has
suggested that this reptile pathogen can be a zoonotic agent, although the route or
source of exposure or infection in this patient could not be identified (Steininger
et al., 2005).
Geomyces destructans
Geomyces destructans is a newly described psychrophilic fungus that affects
North American bats during hibernation (Gargas et al., 2009), causing the char-
acteristic fungal white growth after which the syndrome was originally named.
White-nose syndrome (WNS) could more accurately be dubbed bat geo-
mycosis (per Chaturvedi and Chaturvedi, 2011), but the denomination of WNS
has been used since its recognition (Blehert et al., 2009). Its impact on wild bat
populations has earned it the distinction of being the second most significant
vertebrate pathogen in recorded history (after Bd), if only because Bd has been
documented in more species, with a wider global impact.
Bat geomycosis is a true emerging disease of epizootic proportion, appearing
in upstate New York in 2006 (Blehert et al., 2009), but quickly spreading south
and westward and devastating bat populations (Blehert et al., 2009; Frick et al.,
2010). Host susceptibility to infection seems to vary with species. The fungus has
also been identified in Europe (Puechmaille et al., 2010; Wibbelt et al., 2010),
but to date, the severe, widespread mortality seen in North American bats has not
been documented. Among North American bat species, either pathologic lesions
or G. destructans DNA has been detected in the gray bat (Myotis grisescens),
the Indiana bat (M. sodalis), the little brown bat (Myotis lucifugus), the northern
long-eared bat (M. septentrionalis), the eastern small-footed bat (M. leibii), the
southeastern bat (M. austroriparius), the cave bat (M. velifer), the tricolored bat
APPENDIX A 301
(Perimyotis subflavus), and the big brown bat (Eptesicus fuscus) (Foley et al.,
2011).
Geomyces destructans infections in bats result in premature arousal from
hibernation and abnormal behavior. Although the mechanisms by which death
occurs are still under investigation, the inability to forage in the winter after pre-
mature arousal (due to lack of prey availability) and direct damage to the wing
membranes, resulting in irreversible physiologic and homeostatic deficits from
which bats cannot recover (Cryan et al., 2010), are significant factors.
Limiting human access to caves of concern has been a management tool
implemented to limit the spread of the Geomyces fungus. One of the first theories
to explain the rapid appearance and spread of this pathogen in North American
bat populations suggested that the fungus may have been endemically established
in European bat populations and that a recent anthropogenic introduction into
North America may have led to disease in naïve populations, but this theory
has not been tested. Histopathology of rostral muzzle and wing membranes is
deemed important to confirm infections and establish true prevalence of this dis-
ease (Meteyer et al., 2009), although advanced diagnostic tools are likely being
developed and refined.
As bats are significant providers of ecological services, such as insect control
and pollination, the extinction or even reduction of bat populations is likely to
have economic impacts on society beyond the immediate loss of biodiversity.
Penicillium marneffei
Penicillium marneffei is the only fungus of this genus known to be a primary
pathogen of mammals. Penicilliosis marneffei is a systemic fungal disease of wild
rodents and humans recognized in northeast India and Southeast Asia (Thailand,
the Guangxi region of China, Vietnam, Taiwan, and Hong Kong). Penicillium
marneffei was first identified from hepatic lesions in a bamboo rat (Rhizomys
sinensis) from Dalat, South Vietnam (Caponi et al., 1956), and was subsequently
identified as a human pathogen following an accidental exposure by a researcher
(Vanittanakom et al., 2006). It is an emerging human disease, a primary pathogen
to bamboo rats, and a threat to public health. Penicilliosis marneffei is third only
to tuberculosis and cryptococcosis as the most common opportunistic infections
in patients with AIDS in northern Thailand (Vanittanakom et al., 2006), and the
source of more than 100 cases per year in the Guanxi region of China (Cao et al.,
2011).
Affected rodents show ascites and enlargement of the liver, spleen, and
lymph nodes. The fungal pathogens can be identified in multiple organs and
ascitic fluid, but is most commonly cultured from the lung of affected rodents
(Ajello et al., 1995).
Wild bamboo rats may be good sentinels of this sapronotic disease, and are
significant in the ecology of this disease, although their exact role is not com-
302 FUNGAL DISEASES
pletely understood. Penicillium marneffei has been isolated from the internal
organs of four species of bamboo rats (Rhizomys sinensis, Rhizomys pruinosus,
Rhizomys sumatrensis, and Cannomys badius) and from the soil associated with
their burrows (Vanittanakom et al., 2006). A high prevalence of infection and le-
sions among wild bamboo rats of the genera Rhizomys and Cannomys suggested
that these wild animals may serve as enzootic reservoirs, but the prevalence var-
ies across regions, and a wildlife reservoir has never been conclusively identified
(Cao et al., 2011; Deng et al., 1986; Gugnani et al., 2004; Vanittanakom et al.,
2006). When sympatric rodents have been sampled, only bamboo rats appear to
harbor the organism (Gugnani et al., 2004), suggesting distinct host differences.
Possibly, bamboo rats are exposed from either an unidentified wildlife vector
or an environmental source (Vanittanakom et al., 2006), and are not the wildlife
hosts of this disease, although they can transmit the disease to humans. The
role of wild bamboo rats as amplifiers or dispersers of infectious stages has not
been eliminated. The initial case of human infection in 1959 in a researcher who
became infected after injection from a needle used in laboratory inoculations
(Vanittanakom et al., 2006) underscores the zoonotic risk posed by wildlife.
Although most human cases involve immunosuppressed individuals, infec-
tions in humans with normal immunity—and the lack of evidence for immu-
nosuppression in affected bamboo rats—suggests that P. marneffei could be a
primary mammalian fungal pathogen (Duong, 1996). A complete understanding
of the role of wildlife in this disease and its dynamics in rodent populations is
essential to better managing the threat to human health in endemic regions and
mitigating the possible anthropogenic spread of this disease through travel, move-
ment of commercial products (some of which may be environmental reservoirs),
and the primary trade of wild animals. Penicilliosis marneffei remains one of the
most enigmatic emerging fungal diseases of wildlife, and one whose primary
zoonotic potential is not fully understood.
Lacazia loboi
Lobomycosis is a zoonotic disease of dolphins caused by Lacazia loboi, a
cutaneous fungus in the order Onygenales that has never been cultured (Herr
et al., 2001). The disease affects humans and dolphins in tropical and transitional
tropical climates. Lobomycosis is endemic in certain human populations in Cen-
tral and South America, and is likely endemic in regional dolphin populations
(Murdoch et al., 2008), although the overall prevalence and many factors of its
ecology are still unclear. The disease has been confirmed in two dolphin species,
Guiana dolphins (Sotalia guianensis) (Van Bressem et al., 2009) and Atlantic
bottlenose dolphins (Tursiops truncatus) (Caldwell et al., 1975), and has been
suspected in Indo-Pacific bottlenose dolphins (T. aduncus) (Kiszka et al., 2009).
Transmission between dolphins is likely by direct contact, and affected
dolphins show chronic white to pink verrucous, raised lesions that may coalesce
APPENDIX A 303
into large plaques or nodules predominantly on dorsal and pectoral fins, the head,
fluke, and caudal peduncle (Murdoch et al., 2010). Lesions may be associated
with sites of prior trauma, and many affected dolphins have shown impaired
adaptive immunity, suggesting that the disease may represent an opportunistic
infection in an immunocompromised host (Reif et al., 2008).
Some have suggested that the disease in dolphins is being more frequently
reported (Van Bressem et al., 2009) and the reported geographic range may be
expanding (Rotstein et al., 2009), making it a true emerging fungal disease of
wildlife. In the Indian River lagoon of Florida, measured prevalence was 30 per-
cent in Atlantic bottlenose dolphins (Tursiops truncatus) from the southern part of
the lagoon in a 2006 survey (Reif et al., 2006). It was not detected in the northern
portion (near Charleston, South Carolina), suggesting that localized geographical
factors or environmental stressors may play a significant role in the incidence and
distribution of the disease. A subsequent study (Murdoch et al., 2008) suggested
that the disease is endemic and not an emerging disease in the Indian River dol-
phin population. A survey of Guiana dolphins (Van Bressem et al., 2009) in the
Paranaguá estuary in Brazil suggested an increasing detection of a lobomycosis-
like disease (missing histological confirmation) and suggested that the change
was an indication of the health of the marine environment.
Coccidioides immitis and Coccidioides posadasii

Coccidioidomycosis is a systemic fungal disease known to affect a large
number of domestic and non-domestic animal species, both in captivity and in
the wild. It is a primary pathogen and can affect otherwise healthy individuals,
although clinical disease is often seen in animals with recognized limited immune
function or underlying disease. Coccidioides immitis is considered endemic to the
Western Hemisphere, and is commonly recognized in the southwestern United
States in the Sonoran life zone—areas characterized by alkalinic soils and an arid
climate. Coccidioidomycosis is also reported in Mexico and Central and South
America, although C. posadasii is often implicated in infections outside of the
Southwestern United States. Anthropogenic disturbance of soil (e.g., construc-
tion), dust storms, and cycles of drought may precede outbreaks, but wind-blown
spores have been hypothesized to play a role in cases of affected marine mam-
mals far from endemic areas. Fungal spores may be concentrated around rodent
burrows, but wild animals are not likely reservoirs of this disease and contagion
is unlikely to occur between animals. Human exposure may occur during nec-
ropsies of affected carcasses by aerosolization of spores, but wild animals are
likely insignificant in the epidemiology of the disease in wild animal populations.
Coccidioides has affected some captive endangered species disproportion-
ately more than other species, and can be a threat to species with limited immune
function. Specifically, the Przewalski’s horse (Equus przewalskii) has been dis-
proportionately affected in areas where the disease may be endemic (Terio et al.,
304 FUNGAL DISEASES
2003). In one area of southern California, coccidioidomycosis was associated

with high incidence of infection and was the leading cause of death in a popula-
tion of this endangered species, posing a threat to the captive propagation of this
species. The disease showed a predilection toward younger male horses, and it
has been suggested that the immune system of some of these horses may not
respond appropriately to infection with coccidioides (Terio et al., 2003). Limited
immune function can be a limitation of propagation of endangered species with
a limited genetic pool, and infectious diseases can have disproportionate effects
on these populations, even when the epizootic potential is limited.
Actions for Managing and Mitigating the Effects

of Fungal Diseases on Wild Animals
A basic understanding of the role of fungal diseases in populations is the
most important, fundamental need to manage the spread of these diseases. Public
expectation for action in the face of spreading epizootics is high, and basic knowl-
edge gaps must be addressed to develop and implement effective management
plans. Although the goal is to correct the product of detrimental anthropogenic
influences on the occurrence or emergence of diseases, intentions and action plans
should be tempered by the recognition that naturally occurring diseases are and
have been a driving evolutionary and ecological force responsible for shaping
and adapting community structures in wild populations. Adaptive management
strategies should be scientifically sound.
In the absence of proven action plans for the control of specific fungal patho-
gens, general action plans for the adaptive management of fungal epizootics can
be modeled following the template considered for the control of WNS in bats
(Foley et al., 2011). These strategies can generally include: continuing disease
and population surveillance; limiting further anthropogenic spread through bios-
ecurity and quarantine measures; providing individual animal treatments to in-
crease survival; creating protected populations or rescue populations with the goal
of preserving genetic diversity; increasing population resistance to the disease;
modifying the environment or environmental sources of exposure; selective cull-
ing of populations to limit transmission; and disseminating factual information
to address both public perception and foster scientific collaboration and research.
Prospective monitoring and surveillance of disease and populations allows
wildlife managers to establish objective criteria that can be used to document their
impact following the emergence of a pathogen. In the case of a novel pathogen,
such as the Geomyces destructans in North American bats, historical, basic data
on the population status of native bat populations has allowed the documentation
of changes to the population after the emergence of this disease. By contrast,
the lack of historical specimens collected at the time of initial decline of am-
phibian populations, whose decline has been attributed to chytridiomycosis, has
raised unanswerable questions about the cofactors that may have precipitated the
APPENDIX A 305
emergence of this fungal pathogen or its true impact on populations (McCallum,

2005). Active surveillance should rely on standardized case definitions, sampling
methodologies, and data analysis and centralized sharing of results. Molecular
techniques, some of which were in their infancy a mere 10 years ago, have al-
lowed the differentiation of pathogenic strains for different fungal organisms and
their relationship to the host and their immune defenses, and a better understand-
ing of the ecology of fungal diseases. For instance, the advancement of molecular
techniques as an adjunct to field surveillance techniques has proven valuable in
understanding the dynamics of amphibian chytridiomycosis. The characterization
and dissemination of reports of fungal diseases in novel hosts or new presenta-
tions in known hosts is an implied responsibility of veterinarians and animal bi-
ologists working with wildlife species, both in captive or free-ranging situations.
In the face of spreading epizootics, movement and entry restrictions or
quarantine regulations are often imposed to halt or slow down the spread of an
infectious disease. These restrictions can be as simple as prohibiting (human)
visitor access to caves where bat geomycosis has been documented, or as com-
plex as trade regulations and sanctions against the commercial movement of spe-
cies. Instituting proper quarantine procedures when entering endemic areas and
educating visitors and researchers about their own potential as vectors of disease
can be effective methods to avoid irresponsible spread of pathogens. Responsible
disinfection protocols can be established by scientists repeatedly monitoring af-
fected populations.
Individual animal treatments may not be feasible or effective in halting
the large-scale spread of epizootics, but are valuable if the animals treated are
being used for captive propagation programs or if treatment is only needed for
extremely small populations. Treatment of infected individuals may include
antifungal agents, although the efficacy and safety of these for individual fungal
pathogens has not always been established, and a specific discussion of antifun-
gal agents and their efficacy in the treatment of specific pathogens is beyond
the scope of this manuscript. The biggest limitations in delivering treatments to
free-ranging wild animals is lack of access to treat individual animals, and lack
of knowledge on the proportion of a population that would need to be effectively
treated to reduce the continued spread of a fungal disease. Large-scale aerosoliza-
tion (“fogging”) treatments have been suggested for specific cave environments
affected by bat geomycosis, but the environmental effects of “untargeted” envi-
ronmental treatments are likely to disrupt microbial communities within delicate
cave ecosystems, and the efficacy for the treatment is unknown at this time.
The captive breeding of endangered species vulnerable to emerging infec-
tious disease pandemics has been advocated as a temporary “rescue” measure
until the threat of infectious disease can be mitigated. Captive breeding is par-
ticularly appealing as a putative solution when the infectious disease has had an
anthropogenic component, or when human activities have compounded a threat
to vulnerable populations. In principle, captive stock can be subsequently used to
306 FUNGAL DISEASES
supplement wildlife populations or reintroduce them, after the infectious disease

threat is mitigated, to areas where the species underwent local extinction. Indeed,
many species have been successfully bred in captivity for purposes of reintroduc-
tion after the initial threat of an infectious disease has threatened their survival.
A successful captive breeding and propagation program undertaken by the
U.S. Fish and Wildlife Service (FWS) in the 1980s was aimed at the recovery
of the black-footed ferret (Mustela nigripes). Although the species was consid-
ered extinct in 1979, the rediscovery of a small population in Wyoming in 1981
launched a recovery program for the species. When this extremely small popula-
tion was further threatened by an outbreak of canine distemper virus after a syl-
vatic plague outbreak (Forrest et al., 1988; Williams et al., 1988), the FWS started
a rescue effort to propagate the species in captivity from those wild founders. This
program has now resulted in the reintroduction of the species into the wild at
multiple sites, and has resulted in adequate early success in a relatively short time
frame for the recovery of an entire species. Between 1987 and 2010, more than
6,500 ferret kits were produced, and over 2,300 animals have been released into
the wild through 19 projects across 8 states, Canada, and Mexico (FWS, 2011).
Over the years, this program has benefited from advances in veterinary
medicine to mitigate the threats posed by infectious diseases in the wild and in
captivity; advances in captive husbandry and nutrition; establishment of bios-
ecurity measures; application of modern principles of genetic management; and
the use of assisted reproductive techniques to enhance genetic diversity. Similar
multidisciplinary programs could be applicable to supplement endangered popu-
lations at risk of extinction from a fungal disease.
The unprecedented emergence of amphibian chitridiomycosis and the global
scale of this threat have triggered the launch of concerted recovery efforts for
amphibians under the Amphibian Ark (AARK). The AARK is a joint initiative
by international partners (the World Association of Zoos and Aquariums, the
International Union for Conservation of Nature [IUCN] Conservation Breeding
Specialist Group, and the IUCN Amphibian Specialist Group) dedicated to “en-
suring the global survival of amphibians, focusing on those that cannot currently
be safeguarded in nature” (AARK, 2011). The AARK prioritizes and rescues
amphibian species at risk of extinction while developing in-country capacity
for the continued research and propagation of those species. Although aimed at
the overall decline of amphibian populations, the prominent role of the chytrid
fungus in the decline has prompted the AARK to develop and refine biosecurity
procedures and management techniques to control and mitigate the effects of
this disease. This active network of professionals is a model for the recovery of
species through ex situ propagation and investigation, with the goal of returning
and recovering threatened populations of amphibians to their natural habitats.
However, captive propagation may not always be the most viable solution in
the face of a wildlife epizootic, and should never be seen as a substitute to directly
addressing the anthropogenic factors driving the emergence of diseases threaten-
APPENDIX A 307
ing wildlife populations. The financial cost of captive propagation programs is

variable and species-dependent, but inevitably significant. In addition, the life his-
tory and biological needs of some species may make them unsuitable candidates
for captive recovery programs. Captive breeding should be seen as a short-term
strategy for the recovery of endangered or threatened species, and should only be
considered as a last resort, as in the previous examples, with defined goals and
a target time line. Captive breeding should only be undertaken in conjunction
with comprehensive efforts to create in situ capacity for disease and population
monitoring, continued basic disease research, and efforts to contain or mitigate
the spread of disease in the wild. The limitations of captive breeding programs
have been previously discussed (Lynch and O’Hely, 2001; Snyder et al., 1996)
and should always be weighed as part of the risk assessment on the viability of
captive breeding strategies. Limitations include problems with establishing self-
sufficient captive populations; species-specific and possibly limited success in
reintroductions; relatively high costs; possible domestication and the selection
and propagation of traits that may not be ideal for wild species survival; and
possible exposure and establishment of unrecognized diseases in captive stock
that could be carried to their wild counterparts.
Techniques that increase the population resistance to infection could be
applicable to the management of fungal epizootics, specifically through vaccina-
tions or bioaugmentation of biological defenses. The acquisition of immunity
against fungal pathogens is not well known for all pathogens, and the complex-
ity of pathogen–host relationships further exacerbates our gaps in knowledge.
Although not widely used, fungal vaccines have been developed, even if their
efficacy has varied widely. Different strategies in vaccine development, from
targeting fungal cell wall characteristics (Cassone and Torosantucci, 2006) to
developing recombinant vaccines that induce immunity to specific fungal pro-
teins, could be employed. However, these techniques take time to develop and
effectively implement on a large scale, and are potentially costly.
A novel technique being used to convey immunity to amphibians at risk
of chytridiomycosis involves the use of skin bacteria that produce antifungal
compounds. The addition of Janthobacterium lividum to the skin of mountain
yellow-legged frogs (Rana muscosa) has been demonstrated to convey protection
against challenge with B. dermatitis (Harris et al., 2009). Surveillance of wild
populations has suggested that the presence of symbiotic skin bacteria capable of
producing anti-Bd compounds is beneficial to populations in the wild (Lam et al.,
2010). The bioaugmentation of anti-Bd bacterial flora has the potential to be a
significant advance in increasing the survival of amphibians in areas where the
fungus has become endemic. The recognition of similar symbionts in the skin of
other species could help direct action plans toward increasing resistance to fungi
in natural populations.
Environmental manipulations could be effective in captive efforts, or dur-
ing short-term recovery of individual infected animals, but are not necessarily
308 FUNGAL DISEASES
feasible to apply in wild environments. Manipulating the pH of soil or water,

and changing the temperature, humidity, or other microclimatic conditions, are
possible ways to discourage the growth of certain life stages of fungal organ-
isms. However, basic biological research is necessary before these manipulations
could even be considered or applied in the wild. The detrimental effects of these
manipulations to the environment would need to be understood and minimized,
particularly when applied at scales large enough to make a difference to the health
of a population of wild animals.
The mass culling of populations known to be affected by an emerging infec-
tious disease has been advocated by domestic animal regulatory agencies and
has been an effective tool in the eradication of diseases of agricultural interest
and zoonotic concern. The efficacy of culling wildlife populations has not been
established and efforts have been met with mixed results. Success would depend
on a multitude of factors, including the pathogen, transmission dynamics, popu-
lation biology of hosts and the reservoir species, prevalence and incidence of
the disease, and culling techniques. Wildlife culling has been effective at meet-
ing the immediate management goals in some wildlife health scenarios, but the
overall impacts to ecosystems are often unknown, typically complex, and not
easily predicted. For instance, long-term efforts to cull wild European badgers
(Meles meles) by the British government to control tuberculosis (TB) in cattle
have been successful at reducing incidence of TB in cattle in the immediate area
where badgers are culled, but actually increased the incidence in adjoining areas
(Donnelly et al., 2007). A disease model to examine the efficacy of culling bats
in hibernacula as a way to slow the spread of WNS suggested that this strategy
would be ineffective, in part due to high contact rates among colonial bat species
(Hallam and McCracken, 2011). Culling is not likely to be a large-scale effective
strategy for managing fungal infectious diseases.
Conclusion
Several emerging fungal diseases have recently been shown to affect wild
animal species. Some of these diseases carry a zoonotic risk potential, but even
those that do not directly affect human health are likely to carry a societal cost in
terms of ecosystem health. Wild animal species can be sentinels of emerging dis-
eases and are early indicators of overall ecosystem health. Advances in veterinary
medicine have aided in the recognition of fungal pathogens, and new techniques
are being developed to mitigate their impact on wild populations. However, the
inherent responsibility falls to veterinarians, physicians, conservation biologists,
and public health professionals to properly document and disseminate the find-
ings of fungal diseases in novel hosts, geographic locations, or areas of increased
frequency. Continued multidisciplinary surveillance of fungal diseases is es-
sential to understand the impact of these emerging pathogens on wild animal
populations, humans, and ecosystems.
APPENDIX A 309
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A16
THE EMERGENCE OF PHYTOPHTHORA RAMORUM

IN NORTH AMERICA AND EUROPE
David M. Rizzo,47 Ross K. Meentemeyer,48 and Matteo Garbelotto49
Introduction
The emergence of fungal and fungal-like plant pathogens in agricultural and
natural ecosystems can be triggered by a number of key changes in the host–
pathogen–environment interaction (Anderson et al., 2004; Desprez-Loustau and
Rizzo, 2011; Desprez-Loustau et al., 2007). The evolution of pathogens owing
to selection pressure due to fungicides, deployment of resistant plant varieties, or
changes in the environment (global to local) can allow previously known patho-
gens to increase in incidence locally or across wide geographic areas (Anderson
et al., 2004). Movement of fungi from one part of the world to another may
also lead to disease emergence and large-scale epidemics of plant pathogens
(Desprez-Loustau et al., 2007; Rizzo, 2005). Because of the well-known impacts
of exotic plant pathogens, much effort has been made at regional, national, and
47â•›â•›Department of Plant Pathology, University of California-Davis.

48â•›â•›Department of Geography and Earth Science, University of North Carolina-Charlotte.
49â•›â•›Department of Environmental Science, Policy and Management, University of California-Berkeley.
APPENDIX A 313
international levels to restrict movement of plant pathogens in order to pro-

tect agricultural crops. However, some of the greatest impacts of exotic plant
pathogens have occurred in natural ecosystems. Well-known, high-impact fungal
diseases that are considered to be caused by exotic pathogens include chestnut
blight (caused by Cryphonectria parasitica), white pine blister rust (caused by
Cronartium ribicola), Dutch elm disease (caused by Ophiostoma ulmi and O.
novo-ulmi), and jarrah dieback (caused by Phytophthora cinnamomi) (Aukema
et al., 2010; Desprez-Loustau et al., 2007; Rizzo, 2005).
Recently, Phytophthora ramorum has emerged as a presumed exotic causal
agent of the forest disease “sudden oak death” that has had important impacts
on coastal oak forests in California and Oregon (Rizzo and Garbelotto, 2003)
and more recently in woodlands in the United Kingdom (Brasier and Webber,
2010). There are several reviews available that cover disease symptoms, biology,
ecology, management, and history of P. ramorum (e.g., Davidson et al., 2003;
Garbelotto and Rizzo, 2005; Grünwald et al., 2008; Kliejunas, 2010; Rizzo and
Garbelotto, 2003; Rizzo et al., 2005). Information on the pathogen and the dis-
eases it causes is updated regularly on the website of the California Oak Mortal-
ity Task Force (http//:www.suddenoakdeath.org). In this brief review, we give an
overview of some of the factors responsible for the emergence of P. ramorum
followed by current work on management of the pathogen.
The Pathogen
Phytophthora ramorum was unknown before it was observed to cause dis-
eases of a number of host species in the mid-1990s in Europe and California.
The pathogen was officially named P. ramorum in 2001 (Werres et al., 2001).
Population studies indicate that P. ramorum in both North America and Europe
has a genetic structure consistent with that expected of an introduced species and
that it reproduces exclusively clonally (Ivors et al., 2004, 2006). P. ramorum has
two mating types (A1, A2); to date, all North American isolates of P. ramorum
have been found to be mating type A2, while all isolates in Europe (with rare
exceptions) are A1 (Grünwald et al., 2008). Sexual reproduction outside of the
laboratory has not been documented (Grünwald et al., 2008), although laboratory
attempts at crossing the P. ramorum mating types have produced viable progeny
with similar virulence to the parent types (Boutet et al., 2010). Three genetically
distinct lineages (NA1, NA2, and EU1) have been identified within P. ramorum
(Grünwald et al., 2008; Ivors et al., 2006). Only NA1 is present in California and
Oregon forests, all three lineages are found in North American nursery popula-
tions, and only EU1 has been found in European nurseries and woodlands (Goss
et al., 2009, 2011; Grünwald et al., 2008; Ivors et al., 2006).
The putative exotic nature of P. ramorum in North America is also supported
by the findings that it is present in nurseries (Yakabe et al., 2009) yet is absent in
historical herbarium collections (Monahan et al., 2008). However, the geographic
origin of P. ramorum remains unknown. From a phylogenetic perspective, its
314 FUNGAL DISEASES
nearest relative is P. lateralis, another presumed exotic pathogen in California

and Oregon that is the causal agent of Port-Orford cedar root disease (Rizzo
et al., 2002). The geographic origin of P. lateralis was also unknown until it was
recently found in the mountains of Taiwan (Brasier et al., 2010). This suggests
a potential origin for P. ramorum in Asia (Brasier et al., 2010), but there is no
direct evidence at this time.
P. ramorum is a generalist plant pathogen and has been found to infect more
than 125 plant species including ferns, gymnosperms, monocots, and dicots
(Grünwald et al., 2008; Kliejunas, 2010). The U.S. Department of Agriculture
Animal and Plant Health Inspection Service Plant Protection and Quarantine
maintains an updated list of regulated and associated hosts (http://www.aphis.
usda.gov/). The diseases that P. ramorum causes on this wide range of hosts are
expressed in two ways: canker infections that may cause tree mortality and non-
lethal foliar and twig infections (known as ramorum blight) (Rizzo et al., 2005).
Canker symptoms have been primarily described from oaks and other members
of Fagaceae (known as sudden oak death). Recently, canker symptoms have
been reported on trunks of Japanese larch in plantations in the United Kingdom
(Brasier and Webber, 2010). No host-specific population genetic structure has
been detected in P. ramorum. While there is variation in virulence within P. ramo-
rum populations, isolates of the pathogen from one host show similar virulence
on unrelated hosts (Hüberli and Garbelotto, 2011).
Transmission of P. ramorum in forests is primarily driven by spore produc-
tion from foliar infections rather than from lethal cankers on the main stem of
trees (Davidson et al., 2005, 2008, 2011). P. ramorum spreads naturally via spores
over both short and long distances. Spread is primarily by rain splash, with the
majority of propagules appearing to travel less than 10 m (Davidson et al., 2005;
Mascheretti et al., 2008). Genetic information has been used to infer inoculum
dilution curves and spread potential of the pathogen (Mascheretti et al., 2008,
2009). The curve describing such a relationship is bimodal and reminiscent of
dispersal curves for relatively large particles, with a steep gradient indicating lim-
ited dispersal ability (most dispersal within 10 m, and decreasing to a minimum
at about 500 m) and a secondary peak at 1–3 km, indicating medium-distance
dispersal (Mascheretti et al., 2008). These values match observations based on
the onset of new infestation foci in Oregon (Hansen et al., 2008). Long-distance
spread of the pathogen in both North America and Europe appears to be primarily
associated with movement of nursery plants (Goss et al., 2009; Ivors et al., 2006;
Prospero et al., 2009).
Emergence of P. ramorum in North America

A new disease, described as sudden oak death (SOD), was first associated
with mortality of tanoak (Notholithocarpus densiflorus) and coast live oak (Quer-
cus agrifolia) in the San Francisco Bay area during the mid-1990s (Garbelotto
et al., 2001). A Phytophthora species was identified and confirmed as the causal
APPENDIX A 315
agent of the disease in 2000 (Rizzo et al., 2002). In the United States, P. ramo-
rum’s current geographic range in native forests extends from the Big Sur area in
central California to southern Mendocino County, with two disjunct populations
in Humboldt County, California, and one small population in Curry County, Or-
egon (Figure A16-1). P. ramorum has not become established in forests outside
of this area. Potentially millions of tanoak and oak trees have been lost to the
disease over the past 10 years (Meentemeyer et al., 2008c, 2011).
The pathogen has been associated with the horticultural industry in both
the United States and Canada and has been consistently found in a number of
nurseries. While dozens of plant species have been found infected in nurseries,
the majority of infections have been associated with the genera Rhododendron,
Camellia, Pieris, Kalmia, and Viburnum (Osterbauer et al., 2004; Parke et al.,
2010a). Although P. ramorum has not been found in forests outside of California
and Oregon, the pathogen has become established in streams in several states,
including Washington, Alabama, Mississippi, Georgia, and South Carolina (Oak
et al., 2010). These streams have been associated with infestations in nearby nurs-
eries; in several rare instances vegetation along the banks has become infected by
P. ramorum, but there is no evidence of terrestrial colonization of forests at this
time outside of California and Oregon (Oak et al., 2010).
The emergence of P. ramorum in California and Oregon forests can be linked
to several key factors: movement of ornamental plants (both into and within
California) (Ivors et al., 2006; Prospero et al., 2009), the susceptibility and dis-
tribution of host plants in coastal forests (Anacker et al., 2008; Dodd et al., 2004,
2008; Hayden et al., 2011; Hüberli and Garbelotto, 2011; Meentemeyer et al.,
2008a,b,c; Rizzo et al., 2005), and a suitable climate (Meentemeyer et al., 2004,
2011). P. ramorum appears to have originally spread from two focal points in
California. Two P. ramorum populations (Marin and Santa Cruz) were identified
using population genetic analysis as the two oldest sources of the pathogen in
the state (Mascheretti et al., 2008, 2009). This genetic analysis corroborated an-
ecdotal evidence from field observations and nursery records. By reconstructing
the epidemic from this point, other locations were determined to be of intermedi-
ate age. For example, the Big Sur population was shown to be derived from the
Santa Cruz population originally associated with nursery stock (Mascheretti et al.,
2008). This analysis suggests at least eight separate introductions of P. ramorum
within California from the two focal populations. Most of these introductions
were likely human related via movement of nursery plants, but some introduc-
tions were likely caused by natural spread of the pathogen from forests (Cushman
and Meentemeyer, 2008; Mascheretti et al., 2008, 2009).
P. ramorum can be found in a number of forest types along California and
Oregon coasts; these range from drier oak mixed evergreen forests dominated by
coast live oak to wetter forest types dominated by coast redwood or Douglas fir
(Rizzo and Garbelotto, 2003). In these conifer-dominated forests, tanoak is the
316 FUNGAL DISEASES
FIGURE A16-1â•‡ Current distribution of Phytophthora ramorum in California and Oregon

forests. Red circles represent areas where P. ramorum and sudden oak death have been
confirmed. The blue arrows indicate the general
Figure locations (Marin and Santa Cruz counties)
A16-1.eps
where P. ramorum was initially introduced into California.
bitmap University of North Carolina-Charlotte.
SOURCE: Figure courtesy of Ross K. Meentemeyer,
APPENDIX A 317
host most affected by sudden oak death. As forest types vary across a topographi-
cal landscape, there are associated changes in microclimate and, consequently, a
likelihood of variation in the host–pathogen interaction and pathogen transmis-
sion (Condeso and Meentemeyer, 2007; Meentemeyer et al., 2008a, 2011). These
changes in microclimate may be due to variation in edaphic factors such as as-
pect, soil strata, and hydrology that underlie the growth of a particular vegetation
type. Differences in the ensuing physical structure of the vegetation itself also
affect light availability and, consequently, temperature and moisture. These are
crucial factors that determine survival and sporulation of most pathogens, includ-
ing Phytophthora species. On the level of fine-scale host-pathogen interactions,
these environmental variables may affect the timing and production of inoculum,
the length of the infectious period, the degree of host susceptibility or pathogen
virulence, pathogen survival through adverse conditions (dormancy), the timing
and incidence of new infections, and the overall magnitude and pattern of disease
epidemics.
Although P. ramorum infects more than 25 host species in these woodlands,
nonlethal foliar lesions on bay laurel (Umbellularia californica) are the most
important host tissue for sporulation by P. ramorum in the oak woodlands of
California (Davidson et al., 2005, 2008, 2011). Levels of inoculum in through-fall
rain are up to 20 times higher under bay laurel as opposed to other forest trees.
In addition, at the landscape level, infection on bay laurel is known to precede
infection on oak and tanoak trees, and the presence of this host is associated with
higher levels of oak mortality (Cobb et al., 2010; Maloney et al., 2005). Conse-
quently, infections on bay laurel leaves drive the spread of P. ramorum and the
onset of lethal infections on oak and tanoak. Sporulation does occur on tanoak
leaves and this species does appear capable of driving epidemics in the absence
of bay laurel (Davidson et al., 2008). This situation is occurring in Oregon forest,
where bay laurel is a relatively minor component of the forests (Hansen et al.,
2008).
Various modeling efforts have been made to predict the spread and estab-
lishment of P. ramorum in California, Oregon, and other locations within North
America (e.g., Kelly et al., 2007; Meentemeyer et al., 2004, 2008a,b, 2011;
Vaclavik et al., 2010; Venette and Cohen, 2006). For example, a recent epidemio-
logical model was used in combination with geographical modeling to predict
the spread of P. ramorum through host populations in wildland forests, subject
to fluctuating weather conditions (Meentemeyer et al., 2011). Application of the
model to Californian landscapes over a 40-year period (1990–2030), since the ap-
proximate time of pathogen introduction, suggests that in the absence of extensive
control, a 10-fold increase in disease spread will occur between 2010 and 2030
with most infection concentrated along the northern coast of California between
San Francisco and Oregon (Meentemeyer et al., 2011). Long-range dispersal
of inoculum to susceptible host communities in the Sierra Nevada foothills and
coastal southern California leads to little secondary infection due to lower host
318 FUNGAL DISEASES
availability and less suitable weather conditions. However, a shift to wetter and
milder conditions in future years would double the amount of disease spread
in California through 2030 (Meentemeyer et al., 2011). In other areas of North
America, the forests of the southern Appalachian Mountains are considered to
be at the highest risk (Kelly et al., 2007; Rizzo et al., 2005; Venette and Cohen,
2006). The combination of susceptible oaks (e.g., Q. rubra), potential sporulating
hosts found in the understory (Rhododendron, Kalmia), and a moist, relatively
mild climate has the potential to support the emergence of P. ramorum if it is
introduced into these areas (Spaulding and Rieske, 2011).
Emergence of P. ramorum in Europe

Around the same time that SOD was noted in California, a new Phytophthora
species was observed to infect rhododendrons in nurseries and gardens in Ger-
many and the Netherlands (Werres et al., 2001). A connection was made between
the European and California Phytophthora species in December 2000 (Rizzo
et al., 2002). Although initially described from Germany and the Netherlands,
P. ramorum has now been identified in nurseries or gardens of most countries
in Western Europe (Kliejunas, 2010). To date, P. ramorum has not caused the
extensive damage in native European woodlands that has been seen in California
forests; stem cankers caused by P. ramorum have only been found in the United
Kingdom and the Netherlands. The pathogen is most widespread in the United
Kingdom (including Ireland) in mixed-species woodlands, planted woodland
gardens, heritage gardens, and national plant collections (Fichtner et al., 2011).
Surveys for P. ramorum in Great Britain were initiated in 2001 and the pathogen
has since been found at hundreds of sites, both in nursery systems and in wood-
lands (Kliejunas, 2010). P. ramorum causes extensive foliar necrosis and shoot
dieback on Rhododendron ponticum (considered an invasive plant species) and
bleeding cankers on European beech (Fagus sylvatica) as well as other tree spe-
cies. Early surveys of disease incidence found that the majority of infected trees
are located within 2 m of infected R. ponticum, suggesting a role of R. ponticum
in disease transmission.
P. ramorum has recently emerged as a serious pathogen in Japanese larch
(Larix kaempferi) plantations in the United Kingdom (Brasier and Webber, 2010).
Japanese larch is an important timber tree in the United Kingdom and is grown in
large plantations. While other conifers had been reported as hosts (e.g., redwood,
Douglas fir), this was the first observation of extensive damage caused by P.
ramorum to conifers. The key finding was that larch can serve both as a canker
host, leading to death of large trees (as on oaks), as well as a foliar host that sup-
ports sporulation of P. ramorum (Brasier and Webber, 2010). The identification of
larch as both a foliar and a canker host was unexpected and points to many gaps
in the knowledge about the host range and biology of P. ramorum.
APPENDIX A 319
Disease Management
Management of P. ramorum–associated diseases in forests, woodlands, and
urban areas has taken a multiscale approach ranging from individual trees to
landscapes to international quarantines (Alexander and Lee, 2010; Frankel, 2008;
Rizzo et al., 2005). Disease prevention and mitigation at the individual plant level
or urban–wildland interface in California has been focused on chemical control or
other programs designed to maintain health of plants. Some fungicides have been
developed that act as protectants (e.g., phosphonates) against infection, but few
chemicals have been developed that work once the plant is infected (Garbelotto
and Schmidt, 2009; Garbelotto et al., 2009). Removal of inoculum-producing
plants, such as bay laurel or rhododendron, has also been important at smaller
scales to protect high-value oaks (Swiecki and Bernhardt, 2008). Education and
involvement of local communities has been critical at the urban–wildland inter-
face to the implementation of management programs (Alexander and Lee, 2010).
At larger landscape scales in wildland forest communities, management
strategies for P. ramorum have included prevention, eradication, treatment, and
restoration (Rizzo et al., 2005; Valachovic et al., 2008, 2010). Eradication has
been attempted in some cases, most notably with tanoak forests in Oregon
(Kanaskie et al., 2010) and larch plantations in the United Kingdom (Brasier
and Webber, 2010), but has met with mixed success. Important successes have
been balanced by continuing tree mortality in many areas (Kanaskie et al., 2010).
Difficulties have been encountered in detecting the pathogen at an early enough
stage for eradication to be completely effective at a landscape scale. Cryptic
infection (i.e., with minimal or no symptoms) of foliage during the initial inva-
sion of a site by P. ramorum has allowed the pathogen to stay one step ahead
of detection efforts in many cases. The development of management strategies,
beyond eradication, for forest lands following invasion by P. ramorum is still
in the early stages (Rizzo et al., 2005; Valachovic et al., 2008, 2010). Decision
making requires the ability to fit disease management into the context of other
management goals (e.g., fire prevention, wildlife) within the broader forest land-
scape (Rizzo et al., 2005). Examples of approaches that are being tested include
forest stand thinning to remove inoculum-producing hosts and use of prescribed
fire (Valachovic et al., 2008, 2010).
The broadest scale for disease management, regional to international, is
driven by regulations and management practices designed to prevent further
spread of Phytophthora (Brasier, 2008; Frankel, 2008; Rizzo et al., 2005). In
recent years, broadening of national and international quarantines designed to
prevent pathogen movement has led to an increased effort to manage all Phytoph-
thora diseases in nursery settings (Osterbauer et al., 2004; Parke et al., 2010a,b).
While dozens of plant species have been found infected in nurseries, the major-
ity of infections have been associated with the genera Rhododendron, Camellia,
Pieris, Kalmia, and Viburnum (Osterbauer et al., 2004; Parke et al., 2010a). These
plant species have become the focal point for development of best management
practices and pathogen detection strategies (Parke et al., 2010a). The need for
320 FUNGAL DISEASES
pathogen detection in nursery plants as part of quarantine inspections has resulted

in the development of a number of PCR-based molecular tests (e.g., Hayden et al.,
2004; Vettraino et al., 2010).
One consequence of increased detection ability and monitoring for P. ramo-
rum has been the discovery of many additional species of Phytophthora in
nurseries and wildlands (e.g., Brasier, 2008; Hansen et al., 2003; Yakabe et al.,
2009). Since 2000, the number of described species of Phytophthora has nearly
doubled (Brasier, 2009). The ecology and potential long-term impacts of these
many “new” species is unknown at this time. Limited knowledge of the baseline
biodiversity of Phytophthora (and other fungal plant pathogens) in many areas
makes it very difficult to determine if an organism is a newly arrived “exotic”
species. Therefore, it becomes very difficult to predict if any of these recently
described species will have the large ecological impacts of P. ramorum.
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A17
CLIMATE CHANGE, EXTREME WEATHER EVENTS,

AND FUNGAL DISEASE EMERGENCE AND SPREAD
Compton J. Tucker,50 Karina Yager,50,51 Assaf Anyamba,50,52 and
Kenneth J. Linthicum53
Abstract
Empirical evidence from multiple sources shows the Earth has been warm-
ing since the late 19th century. More recently, evidence for this warming trend
is strongly supported by satellite data since the late 1970s from the cryosphere,
atmosphere, oceans, and land. Those data confirm increasing temperature trends
and their consequences (e.g., reduced Arctic sea ice, rising sea level, ice sheet
mass loss, etc.). At the same time, satellite observations of the Sun show remark-
ably stable solar cycles since the late 1970s, when direct observations of the
Sun’s total solar irradiance began. Numerical simulation models, driven in part
by assimilated satellite data, suggest that future warming trends will lead to not
only a warmer planet, but also a wetter and drier climate, depending on location,
in a fashion consistent with large-scale atmospheric processes. Continued global
warming poses new opportunities for the emergence and spread of fungal disease,
as climate systems change at regional and global scales, and as animal and plant
species move into new niches.
Our contribution to this proceedings is organized as follows: First, we re-
view empirical evidence for a warming Earth. Second, we show the Sun is not
responsible for the observed warming. Third, we review numerical simulation
50â•›â•›Laboratory for Hydrospheric and Biospheric Science, NASA/Goddard Space Flight Center,
Greenbelt, MD 20771.
51â•›â•›Oak Ridge Associated Universities (ORAU).
52â•›â•›GESTAR–Universities Space Research Association (USRA), Columbia, MD 21044.
53â•›â•›USDA/ARS Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL.
APPENDIX A 325
modeling results that project these trends into the future, describing the pro-
jected abiotic environment of our planet in the next 40 to 50 years. Fourth, we
illustrate how Rift Valley fever outbreaks have been linked to climate, enabling
a better understanding of the dynamics of these diseases, and how this has led
to the development of an operational predictive outbreak model for this disease
in Africa. Fifth, we project how this experience may be applicable to predicting
outbreaks of fungal pathogens in a warming world. Last, we describe an example
of changing species ranges due to climate change, resulting from recent warming
in the Andes and associated glacier melt that has enabled amphibians to colonize
higher elevation lakes, only to be followed shortly by the emergence of fungal
disease in the new habitats.
Introduction: Observational Evidence for Global Warming

Among many non-scientists, there appears to be controversy over climate
change and its causes. This is paradoxical because there is little debate over
human-caused climate change within academic communities, except for some
peripheral issues, as noted by Lockwood (2009) and others. Let us look to obser-
vational evidence from multiple and different sources to better understand climate
change and its implications in this persisting pseudo-controversy (Figure A17-1).
A number of methods are used to collect consistent and long-term datasets
to monitor Earth’s properties. Observations of temperatures by thermometers at
the surface and by satellite microwave “sounding” of atmospheric temperature
profiles are important components of our understanding of global temperature.
Since 2003, we have been able to measure ocean temperature profiles using
the ARGO global array of 3,000 free-drifting robotic probes that measure the
temperature and salinity of the upper 2,000 meters of the ocean. This provides
continuous monitoring of ocean temperature, salinity, and currents, with all data
made publicly available within hours after collection (Lyman et al., 2010; Wells
et al., 2009). The ARGO data are fundamental to climate studies because the
oceans absorb ~90 percent of the heat from global warming.
We can measure the extent of glaciers and their variation over time, fre-
quently drawing on historical paintings, photographs, maps, and satellite image
archives to determine if they are getting smaller or larger. We also measure the
extent of sea ice weekly and monthly using passive microwave radiometers. More
recently, we have been able to measure ice mass variations for the Greenland
and Antarctic ice sheets, using gravity data from the joint U.S.–German Gravity
Recovery and Climate Experiment (GRACE) satellite mission (Swenson and
Wahr, 2002).
We have measured sea level globally since 1993 using radar altimeters on-
board satellites. Sea level is an unequivocal proxy for global warming: As the
Earth warms, sea level rises; as it cools, sea level falls. Lastly, since 1979 we have
been able to measure the Sun’s energy output using satellites above the Earth’s
326 FUNGAL DISEASES
FIGURE A17-1â•‡ Summary of observations that show the Earth is warming (red arrows)
Figure A17-1.eps
while the Sun has been constant over the same period of time.
bitmap
surface. The convergence of observational evidence from all of these sources

makes a compelling case that the Earth is warming, and this warming is not due
to the Sun.
Convergence of Observations Showing Global Warming
Surface Thermometers
Four global surface temperature datasets are available from the following
locations: (1) National Aeronautics and Space Administration (NASA)/Goddard
Institute of Space Studies (Hansen et al., 2010), (2) National Oceanic and Atmo-
spheric Administration’s National Climatic Data Center (Smith et al., 2008), (3)
the University of East Anglia’s Climate Research Unit (Rayner et al.. 2006), and
(4) the Japanese Meteorological Agency.54 These four datasets all use the same
input data and differ only in interpolation techniques between sparse observa-
tions, how the polar regions are treated, and the reference period for which means
are calculated. Not surprisingly, they are very similar (Figure A17-2).
Atmospheric Temperature Profiles

Since late 1978, polar-orbiting satellites have provided global air tempera-
ture profiles with altitude or “soundings” using passive microwave radiometers
operating between 23 and 89 GHz frequencies. These measurements started with
54â•›â•›See http://www.jma.go.jp/jma/indexe.html.
APPENDIX A 327
FIGURE A17-2â•‡ A comparison of the existing four global surface temperature datasets
that are used in climate analyses.Figure A17-2.eps
These datasets are based on the same input data and dif-
fer by interpolation among stations, treatment of missing data, and the length of the record.
type is outlined
The data in this figure have been adjusted to a common baseline period.
SOURCE: Figure courtesy of Robert Simmon, NASA Earth Observatory, provided by
Compton J. Tucker NASA Goddard Space Flight Center.
the microwave sounding-unit instruments and were followed by the advanced

microwave sounding-unit instruments that began operation in 1988. As is not
uncommon in science, early work using atmospheric temperature soundings pro-
duced a range of temperature trends. Recent work on atmospheric temperatures
has shown no reasonable evidence of disagreement between these measurements
and surface observations (Thorne et al., 2011).
Arctic Sea Ice

Satellite observation of sea ice is accomplished with a very high degree of
accuracy. This results from spectral emissivity differences between open water
(~0.4–0.5) and sea ice (~0.9–1.0) at a wavelength of 1.5 cm (Kwok, 2002). Fur-
thermore, passive microwave radiometers are translucent to clouds, are weather
insensitive, and operate from a polar orbit that provides near-daily observations
of Arctic sea ice. According to the National Snow and Ice Data Center, all
months (i.e., all Januaries, all Februaries, all Marches, etc.) from the late 1970s
to 2010–2011 show declining Arctic sea ice with time.55
55â•›â•›See http://nsidc.org/arcticseaicenews/.
328 FUNGAL DISEASES
Sea Level
Sea level is of direct interest to climate science because it varies directly with
global mean temperature over short time scales. Temperature affects sea level
through two mechanisms: (1) sea level rises through the thermal expansion of
water as it warms, or it falls through thermal contraction of water as it cools; and
(2) warmer global temperatures melt ice stored on land in glaciers and ice sheets,
and the resulting ice loss raises sea level, while cooler global temperatures result
in more water being stored on land in glaciers and ice sheets and sea level falls.
Thus sea-level variations are an excellent, unambiguous indicator of planetary
cooling or warming. For example, at the last glacial maximum, occurring before
20,000 years ago, sea level was >100 m lower than it is now (Clark et al., 2009).
This huge quantity of water was stored on land in the form of glaciers and ice
sheets (Lambeck et al., 2010).
Although tide gauges provide centennial-scale sea-level records from nearly
10 locations around the world, these few locations are insufficient for a global
study of sea level. Researchers have also measured vertical accretion rates in
salt marshes as a sea-level proxy, using radiocarbon, pollen, foraminifera, and
other markers (reviewed in Mitchum et al., 2010). Since 1993, however, radar
altimeters have measured sea level globally and directly with a high precision
(Figure A17-3).
We have briefly reviewed global surface thermometer data, atmospheric
temperature profile data from satellites, variations in Arctic sea ice, and sea-
level data. All of these data unambiguously show the effects of increasing global
temperatures.
Earth’s Climate and the Sun

Since the late 1970s, the study of the Sun with instruments on satellites has
progressed with continuous observations being collected. Lockwood and Frohlich
(2007, 2008) have shown that the three mechanisms where the sun can influence
the Earth’s temperature (total solar irradiance, changes in the spectral distribu-
tion of solar irradiance, and the solar wind–magnetic field–cosmic ray–cloud
hypothesis) have all been opposite to the observed increases in temperatures (Fig-
ure A17-4). The Sun’s output is currently at record low values since the satellite
era began in the late 1970s (Lockwood, 2009). Thus the Sun is not to blame for
the observed global warming since the late 1970s to the present.
Projections of Warming Trends on Weather and Climate

Numerical simulation models of the Earth’s weather and climate are called
general circulation models because they simulate the circulation of the atmo-
sphere. They are representations of the ocean, land, sea ice, and atmosphere
where the Earth is a series of grid cells driven by energy, moisture, and pres-
APPENDIX A 329
FIGURE A17-3â•‡ Sea-level rise based on radar altimeters from TOPEX and Jason, with
seasonal variations removed. Figure A17-3.eps
SOURCE: Mitchum et al. (2010). bitmap
sure. Each grid cell interacts with adjacent cells horizontally and vertically to
simulate climate (Figure A17-5). Model interactions are governed by systems
of differential equations and incorporate climate-forcing factors such as land
cover change, volcanic aerosols, and increasing greenhouse gas concentrations.
Weather and climate models have been shown to be realistic at reproducing the
global temperature and precipitation patterns of the 20th century and are widely
used in weather and climate research (Delworth et al., 2006).
Climate model simulations, incorporating increasing greenhouse gas concen-
trations in the atmosphere, have been used to extrapolate precipitation patterns
into the 21st century as surface temperatures increase. Several of these climate
model simulation predictions can be described as “the wet getting wetter and the
dry getting drier” (Held and Soden, 2006). The displacement of arid and semi-
arid zones northward results from an expansion from the Hadley circulation cell
under global warming (Figure A17-6) (Lu et al., 2007). These changes in climate
have direct impacts on vegetation and biodiversity across the globe, including
species range shifts, changing phenology, new invasive species, and new disease
outbreaks (Parmesan and Yohe, 2003; Walther et al., 2002).
330 FUNGAL DISEASES
FIGURE A17-4â•‡ A comparison between the total solar irradiance (top) and the NASA/
GISS surface temperature data (bottom),
Figure both from 1979 to 2010. This shows the sun is
A17-4.eps
not responsible for the 1979 to 2010 increased surface temperatures.
bitmap
Linkages Between Vector and Non-Vector Diseases and Climate

A variety of infectious diseases have been linked to variations in climate
(reviewed in Patz et al., 2005). These include diarrheal diseases (Checkley
et al., 1997), cholera (Colwell, 1996; Pascual et al., 2000), salmonella (Kovats
et al., 2004), viral pneumonia (Ebi et al., 2001), hantavirus (Glass et al., 2002),
influenza (Viboud et al., 2004), flea-associated plague (Parmenter et al., 1999),
Culicoides biting midge-associated bluetongue (Baylis et al., 1999; Purse et al.,
2005), African horse sickness (Baylis et al., 1999), mosquito-associated Murray
Valley encephalitis (Nicholls, 1986), Ross River virus (Woodruff et al., 2002),
dengue (Hopp and Foley, 2003; Linthicum et al., 2008), chikungunya (Chretien
et al., 2007), malaria (Bouma and Dye, 1997; Bouma et al., 1996), and Rift Valley
fever (Linthicum et al., 1999). Of these, we use the example of Rift Valley fever
outbreaks to show the use of climate data to understand in what regions and at
what times these disease outbreaks will occur.
The link between epizootics of Rift Valley fever and rainfall was first docu-
mented by Davies et al. (1985). Through an analysis of time-series rainfall data
APPENDIX A 331
FIGURE A17-5â•‡ Representation of a general circulation model illustrating the grid cell
nature of the model on the right,Figure
while on A17-5.eps
the left, many of the different important com-
ponents of these models are shown. bitmap
SOURCE: Figure courtesy of the Center for Multiscale Modeling of Atmospheric Pro-
cesses (CMMAP), Colorado State University, http://www.cmmap.org/learn/modeling/
whatIs2.html.
FIGURE A17-6â•‡ Change in precipitation between the 1971–2000 average and the 2091–
2100 average in inches of liquidFigure
water/yearA17-6.eps
(Held and Soden, 2006).
SOURCE: Geophysical Fluid Dynamics Laboratory, National Oceanic and Atmospheric
Administration.
bitmap
332 FUNGAL DISEASES
records from numerous stations in Kenya between 1950 and 1982, it was deter-
mined that periods with extended positive surplus rainfall corresponded to periods
when Rift Valley fever epizootics occurred. Widespread, frequent, and persistent
rainfall was shown to be a prominent feature of all epizootic periods. Heavy rain-
fall raises the level of the water table in certain areas, flooding grassland depres-
sions that are the habitat of the immature stages of certain ground-pool–breeding
mosquitoes of the genus Aedes. These findings have been collaborated by find-
ings in southern Africa (Swanepoel, 1976) and West Africa (Bicout and Sabatier,
2004). Rift Valley fever virus is thought to be initially transmitted transovarially
in these species. Under prolonged flooded conditions, large numbers of Culex
species mosquitoes emerge and are an amplification vector for Rift Valley fever.
Following the development of these conditions, Rift Valley fever first occurs in
animals and subsequently in humans.
Linthicum et al. (1999) established that outbreaks of Rift Valley fever are
closely coupled with above normal rainfall that is associated with the occur-
rence of the warm phase of El Niño/Southern Oscillation (ENSO) (Cane, 1983;
Nicholson, 1986; Ropelewski and Halpert, 1987) and warm events in the equato-
rial western Indian Ocean (Anyamba et al., 2002; Birkett et al., 1999; Saji et al.,
1999). Such warm ocean events precede by 2 to 3 months above normal and
extended rainfall over East Africa, and are further enhanced when both the sea
surface anomalies in the western Indian Ocean and equatorial central-eastern
Pacific are synchronized. More than 90 percent of Rift Valley fever outbreak
events since 1950 have occurred during warm ENSO events (Linthicum et al.,
1999) (Figure A17-7). The interepizootic period is dominated by La Niña events
(the cold phase of ENSO), which results in drought in East Africa and wetter
than normal conditions in southern Africa (Anyamba et al., 2002; Nicholson
and Entekhabi, 1986). Recent evidence shows that Rift Valley fever outbreaks
in southern Africa are coupled with La Niña patterns (Anyamba et al., 2010).
Interannual variability, in part driven by ENSO events with differential impacts
on rainfall anomaly patterns in Eastern and Southern Africa, largely influences
the temporal outbreak patterns of Rift Valley fever.
Our work on Rift Valley fever prediction thus uses climate data to inform
us when and where regionally we should expect outbreaks. Subsequent detailed
daily satellite observations identify where outbreaks will occur with a high degree
of geographical specificity (~60 percent).
Prediction of Rift Valley Fever Outbreaks

Developed by Anyamba et al. (2002), prediction of Rift Valley fever out-
breaks includes several components: (1) mapping of potential epizootic/epidemic
regions through the combined use of satellite data, climate data, and historical
reports; (2) closely following sea surface temperature anomalies with reference
to phase and amplitude in the NINO 3.4 tropical Pacific and equatorial western
5
4 1951-3 1957 1961-3 1968-9 1977-9 1982-3 1989 1997-8 2006 2010
-1
-2
-3
Southern Oscillation Index Anomalies

RVF Activity
-4
-5
1951 1955 1959 1963 1967 1971 1975 1979 1983 1987 1991 1995 1999 2003 2007 2011
FIGURE A17-7â•‡ Rift Valley fever major outbreak events plotted against time and the Southern Oscillation Index, a measure of the phase of El
Niño/Southern Oscillation events. Most Rift Valley fever outbreak events have occurred during the warm phase of ENSO (negative Southern
Oscillation Index shown in blue).
SOURCE: Linthicum et al. (1999), and updated.
Figure A17-7.eps
333
landscape
type is 3.69 point--some type is outlined
334 FUNGAL DISEASES
Indian Ocean areas; (3) monitoring patterns of outgoing long-wave radiation

anomalies to infer and detect large-scale changes and shifts in the major atmo-
spheric centers of tropical convection as a result of ENSO; and (4) monitoring
patterns of normalized difference vegetation index anomalies over Africa as a
proxy for excessive rainfall.
The first successful prediction using this system was made in 2006 (Anyamba
et al., 2006, 2009) and provided a lead time of 3 to 4 months (Figure A17-8) to
respond, although response and mitigation activities only started a month before
the first reported outbreak. The predictions were subsequently confirmed by en-
tomological and epidemiological field investigations of virus activity in the areas
mapped to be at risk in Kenya, Somalia, and Tanzania with a geographic accuracy
of 60 percent (Anyamba et al., 2009). Following the outbreak in East Africa,
this system provided further predictions of outbreaks in Sudan in late 2007 and
January 2008, 2009, and 2010 in southern Africa. These predictions and outbreak
assessments are described in detail in Anyamba et al. (2010).
How Tools and Previous Approaches Could Have Relevance to

Anticipating Conditions for Fungal Disease Emergence
We (Linthicum, Anyamba, and Tucker) have been studying the use of satel-
lite data to predict Rift Valley fever outbreaks since the mid-1980s. Our study of
Rift Valley fever occurrence led us to the antecedent role of high sea-surface tem-
peratures in the tropical Pacific and western Indian Oceans that results in higher
than average rainfall in East Africa, which triggers Rift Valley fever outbreaks.
Alerted when the antecedent sea-surface temperature conditions are present, we
then step up our near-real-time satellite data surveillance in East Africa that pro-
vides very specific location information for control measures to be put in place.
We propose a similar approach for anticipating conditions for fungal disease
emergence: Use satellite data to map the abiotic conditions associated with fungal
disease outbreaks; evaluate historical fungal outbreaks with respect to anteced-
ent abiotic conditions; and use this knowledge to predict where and when future
fungal outbreaks would occur.
A recent example how our prediction model could be applied elsewhere
was the role that the very heavy 2011 summer rains have played in increased
transmission of Murray Valley encephalitis virus, Ross River virus, and Kunjin
virus in Australia (ProMed, 2011b). This same climate anomaly also produced
widespread moist soil conditions and increased the likelihood of fungal diseases
of cereal crops such as Puccinia graminis f. sp. tritici producing wheat stem rust
(Figure A17-9), resulting in the release of warnings for the occurrence of fungal
diseases in cereal crops in eastern and southern Australia (ProMed, 2011a).
APPENDIX A 335
A B
C D
RVF risk areas Identitified as Non-Risk

RVF potential epizootic areas Identitified as Risk
FIGURE A17-8â•‡ Summary Rift Valley fever (RVF) risk maps for (A) Eastern Africa:
September 2006–May 2007; (B)Figure A17-8.eps
Sudan: May 2007–December 2007; (C) Southern Africa:
4 bitmaps w vector
September 2007–May 2008; and (D) Madagascar: type & ruling
September 2007–May 2008. Areas
shown in green represent Rift Valley fever potential epizootic areas. Areas shown in red
represent pixels that were mapped by the prediction system to be at risk for RVF activity
during the respective time periods. Blue dots indicate human cases identified to be with
the Rift Valley fever risk areas, while yellow dots represent human cases in areas not
mapped to be at risk.
SOURCE: Adapted from Anyamba et al. (2010).
336 FUNGAL DISEASES
FIGURE A17-9â•‡ Stem rust symptoms on wheat.

SOURCE: Agricultural research service, U.S. Department of Agriculture (http://www.ars.
Figure A17-9.eps
usda.gov/images/docs/9910_10104/stemrust_inset.jpg).
bitmap
Tropical Glacier Recession, Amphibian Migration,

and Subsequent Fungal Migration
Our group at NASA/Goddard Space Flight Center has documented New
World tropical glacier variation from the mid-1980s to the present, including
glaciers in the Cordillera Vilcanota in Peru. This heavily glaciated range, with
multiple peaks over 6,000 m, is a key watershed for regional river systems, in-
cluding the Amazon. Rapid environmental changes are documented in the region,
including record tropospheric warming of 0.3°C per decade between 1974 and
1998 (Vuille et al., 2003), rise in freezing level (Diaz and Graham, 1996), and
deglaciation (Bradley et al., 2006; Thompson et al., 2003). According to our cur-
rent estimates, between the mid-1980s and mid-2000s, there has been approxi-
mately 30 percent glacial loss in this particular mountain range of southern Peru
(Slayback and Tucker, in preparation). We have found warmer temperatures were
largely responsible for tropical glacier recession in these areas (Figure A17-10).
No variations in cloud cover or precipitation have been found, indicating global
warming was the primary driver of glacial change.
Interdisciplinary research in the Cordillera Vilcanota, around Lake Sibina-
cocha, has been conducted for several years to investigate the impacts of climate
change on local ecosystems (Seimon et al., 2009). This work includes research
APPENDIX A 337
FIGURE A17-10â•‡ False-color Landsat satellite data (RGB 642) showing glaciers as the
Figure A17-10.eps
blue colors. The green colors represent green vegetation and the red colors represent areas
of rock, sand, and soil. bitmap
SOURCE: Figure provided by Karina Yager, NASA-Goddard Space Flight Center.
on glaciers, vegetation, colonization of microbes in newly deglaciated soils, fossil

plants, agropastoralism, species migration, and amphibian studies (Halloy et al.,
2006; Nemergut et al., 2007; Seimon et al., 2007; Yager et al., 2010). Glacier re-
treat at higher elevations in the watershed has been rapid, resulting in the creation
of new corridors and newly habitable areas for species migration and the upward
range extension of numerous species, including plants, animals, amphibians, and
pathogens.
Herpetologists on the team have documented the higher elevation coloniza-
tion of three species of anurans—Telmatobius marmoratus, Rhinella spinulosa,
and Pleurodema marmorata—that have expanded their ranges and moved to
unprecedented elevations for amphibians (5,200–5,400 m) into new lakes and
ponds created by recent deglaciation (Seimon et al., 2007). In the case of P. mar-
morata, climatic warming has resulted in an approximate 200 m vertical increase
in its range, corresponding to the amount of glacier retreat since 1880 (Seimon
et al., 2007).
338 FUNGAL DISEASES
These amphibian species are opportunistic in their adaptation to the warm-

ing climate by migrating to and spawning in ever-higher terrain. However, new
climate conditions are also proving advantageous for the spread of epidemic dis-
ease, and in particular Chytridiomycosis. This pathogenic chytrid (Bd) produces
aquatic zoospores on amphibian skin, and under certain conditions becomes a
highly lethal infection (Seimon et al., 2005). Chytrid fungus has been linked
to amphibian population declines and even species extinction across the globe
(Daszak et al., 1999; Pounds et al., 2006).
New challenges are being presented for the long-term survival of amphibian
species in this watershed with climate change. Since 2003, a year after Bd was
first detected in this region, all three species have been decreasing in number,
and T. marmoratus has not been documented in the Sibinacocha watershed since
2005 (Seimon et al., 2007; Yager et al., 2010). The current research indicates that
recent warming, and intense solar heating of glacier ponds during the day, may
be contributing to the ability of chytrid to expand and thrive at unprecedented
altitudes and terrain (Seimon et al., 2007). In addition, as the glaciers continue
to melt, ponds that were once inhabited by amphibians are experiencing a re-
duction in meltwater or are drying up altogether, leading to loss of habitat and
contributing to subsequent population declines. Amphibians are some of the most
sensitive species to environmental changes, and are becoming more susceptible
to life-threatening disease and possible extinction under current climate patterns.
Conclusions
The Earth’s climate is warming and our Sun is not responsible. Weather and
climate simulation models project even warmer temperatures by the middle of
this century, with some areas getting wetter and others drier. These changing pat-
terns of temperature and precipitation will alter endemic areas for various plant
and animal diseases, including fungal pathogens. We reviewed how knowledge
of climatic linkages is being used to predict the outbreak regions of Rift Valley
fever in Africa, complemented by detailed satellite observations to identify spe-
cific locales where control measures should be undertaken. We advocate a similar
approach to identify areas where fungal diseases may emerge: understand the
biology of specific fungal pathogens; use satellite data to establish temperature
and precipitation climatology in the areas of interest; associate this information
with documented fungal outbreaks; and use this knowledge in conjunction with
satellite data to predict the impacts of a changing and variable climate on fungal
pathogens.
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A18
HOST-PATHOGEN DYNAMICS OF AMPHIBIAN

CHYTRIDIOMYCOSIS: THE ROLE OF THE SKIN
MICROBIOME IN HEALTH AND DISEASE
Vance T. Vredenburg,56 Cheryl J. Briggs,57 and Reid Harris58
Abstract
Amphibian biodiversity is currently facing a severe crisis having recently
experienced declines in 42 percent of all species, and as many as 32 percent are
56â•›â•›Department of Biology, San Francisco State University, San Francisco, CA.
57â•›â•›Department of Ecology, Evolution and Marine Biology, University of California Santa Barbara,
Santa Barbara, CA.
58â•›â•›Department of Biology, James Madison University, Harrisonburg, VA.
APPENDIX A 343
threatened with imminent extinction. The most alarming extinctions and declines
have occurred enigmatically in protected, apparently pristine habitats. An emerg-
ing infectious disease, chytridiomycosis, is directly linked to the recent extinction
or serious decline of hundreds of amphibian species and is increasingly proposed
as a primary threat to amphibians. Chytridiomycosis, caused by the fungal patho-
gen Batrachochytrium dendrobatidis (Bd), infects the skin of amphibians and has
been described as causing the greatest loss of vertebrate biodiversity due to dis-
ease in recorded history. The severity of the current amphibian biodiversity crisis
suggests that Bd is a fundamentally new challenge to amphibians from previous
global and environmental changes. While many amphibian species are susceptible
to this fungal pathogen, others are silent carriers exhibiting no signs of disease.
How amphibian hosts survive with Bd infection is still unknown; however, host
immunity, differences in pathogen virulence, and environmental differences that
may limit growth and reproduction of the pathogen have all been proposed as pos-
sible mechanisms that could lead to Bd-infected host survival. Here we examine
amphibian declines in one of the best-documented systems, the Sierra Nevada of
California, and review the role that the amphibian skin microbiome may play in
host–pathogen dynamics of the chytridiomycosis-amphibian host system.
Introduction
The amphibians are long-term survivors (existing on earth for more than
350 million years) that endured four previous mass extinctions (e.g., 95 percent
of all living species were lost in the Permian-Triassic extinction) (Wake and
Vredenburg, 2008). Through these extinctions, not only did all three orders of
amphibians escape extinction, but most families and genera survived (Wake and
Vredenburg, 2008). Today, the amphibians, presently including more than 6,800
species (AmphibiaWeb, 2011), are the most threatened group of vertebrates with
over 40 percent of species in decline and over 30 percent threatened with extinc-
tion (Stuart et al., 2004). There are many potential causes for the declines, but
an emerging infectious disease, chytridiomycosis (Longcore et al., 1999), caused
by the infectious fungal pathogen Batrachochytrium dendrobatidis (Bd) is the
most alarming (Daszak et al., 2000). This fungal pathogen is associated with
the recent decline or extinction of more than 200 species of amphibians (Fisher
et al., 2009; Skerratt et al., 2007). Epizootic waves of chytridiomycosis have
been identified in Australia, Central and South America, and the western United
States (Fisher et al., 2009). In each case the epizootic caused mass mortality and
collapse of amphibian faunas (Berger et al., 1998, Lips et al., 2006, Vredenburg
et al., 2010a). Once Bd invades naive amphibian host populations, they can col-
lapse very quickly. In Panamanian sites, 50 percent of species were extirpated 4
to 6 months after Bd invaded (Lips et al., 2006). The surviving species declined
in population size by 80 percent (Lips et al., 2006). In California, a moving epi-
zootic wave of Bd is causing the collapse of entire metapopulations of ranid frogs
344 FUNGAL DISEASES
in some of the most protected and pristine areas in the United States (Vredenburg
et al., 2010a). In Australia, most of the damage was done before the pathogen
was identified (Laurance et al., 1996), but post hoc studies estimate that Bd was
responsible for the decline and disappearance of a majority of amphibian species
along the diverse eastern tropical montane areas (Fisher et al., 2009).
The Decline of the Mountain Yellow-legged Frog

in the Sierra Nevada of California
The dramatic decline of California’s mountain yellow-legged frog (a species
complex consisting of Rana muscosa and Rana sierrae; Vredenburg et al., 2007)
is emblematic of global amphibian declines (Stuart et al., 2004). Historically,
lakes in California’s Sierra Nevada were often inhabited by hundreds of frogs
and thousands of tadpoles (Grinnell and Storer, 1924) of this diurnal and highly
aquatic taxon (Vredenburg et al., 2005). By 1997, despite the fact that the major-
ity of their habitat is fully protected, R. sierrae and R. muscosa had disappeared
from 93 percent and 96 percent, respectively, of their historic range (Vredenburg
et al., 2007; Figure A18-1). As a consequence of this decline the two species of
mountain yellow-legged frog have gone from being the most common vertebrates
in the Sierra Nevada (Grinnell and Storer, 1924) to species classified as “criti-
cally endangered” (Stuart et al., 2004). These two species are not the only ones
in trouble in the protected landscape of the Sierra Nevada. In fact, five of the
seven species of amphibians that occur in the high-elevation areas of the Sierra
Nevada (>2,000 m above sea level) are threatened in the range (R. muscosa, R.
sierrae, Bufo canorus, B. boreas, and Ambystoma macrodactylum), while only
two species (Hydromantes platycephalus and Pseudacris regilla) are thought to
have stable populations (Stebbins and Cohen, 1995).
The declines of amphibians that occur in high-elevation protected habitats
around the world have been described as enigmatic (Stuart et al., 2004). Decline
hypotheses in these areas include the negative effects of increasing UV radia-
tion (Blaustein et al., 1994), pesticide drift (Davidson, 2004), introduced species
(Vredenburg, 2004), climate change (Pounds et al., 1999), and disease (Longcore
et al., 1999). In the Sierra Nevada, most studies have focused on the decline of the
two species of yellow-legged frog. To date, studies have not directly addressed
climate change and have shown no evidence for UV radiation effects (Vredenburg
et al., 2010b) or pesticide drift (Bradford et al., 2011), but they have shown
large negative effects from introduced species (Knapp and Matthews, 2000) and
disease (Vredenburg et al., 2010a). Like many montane areas worldwide, the
vast majority of the high-elevation portion of the Sierra Nevada, consisting of
more than 15,000 glacial lakes, was historically fishless (Knapp and Matthews,
2000). Despite the fact that most of the habitat is protected from habitat destruc-
tion, humans changed the landscape dramatically by planting non-native fishes.
By 1997 most of the lakes and streams in the area contained self-reproducing
APPENDIX A 345
Exnct
Extant (1997) B
FIGURE A18-1â•‡ Decline of (A) Sierra Nevada mountain yellow-legged frog, Rana sier-
rae, and (B) southern mountain yellow-legged frog, Rana muscosa, in California, USA.
Yellow points indicate extant populations in 1997 and red points indicate extinct popula-
tions compared to historically documented sites.
SOURCE: Adapted from Vredenburg et al. (2007).
A18-1
non-native trout, which have contributed significantly to the decline of the two
species of yellow-legged frog (Knapp and Matthews, 2000; Vredenburg, 2004).
In 2004 a whole-lake experimental study determined that predation on tadpoles
by introduced trout was causing frog population extirpations (Vredenburg, 2004).
The same study also demonstrated that threatened frog populations would recover
quickly when habitat was restored by removing the non-native fish (Vredenburg,
2004). Additional research showed that habitat restoration (to the fishless con-
dition) worked across the mountain range for both yellow-legged frog species
(Knapp et al., 2007). Unfortunately, further research ultimately revealed that
amphibian declines in the Sierra Nevada, perhaps like other areas around the
world, are complex in nature and can be caused by multiple factors that may act
346 FUNGAL DISEASES
synergistically. Although it was not understood until 2010, an infectious fungal

disease (chytridiomycosis) was also causing population collapse of the yellow-
legged frog (Vredenburg et al., 2010a), but it just happened that fish-removal
studies occurred ahead of the epizootic wave.
Differential Outcomes for Frog Host Populations After Pathogen Invasion

Chytridiomycosis is caused by the waterborne fungal pathogen Batrachochy-
trium dendrobatidis, whose only known hosts are larval and adult amphibians.
This pathogen was first described in the late 1990s (Berger et al., 1998; Longcore
et al., 1999) and is now known to inhabit six continents (Skerratt et al., 2007). The
infective stage is a free-living flagellated zoospore that encysts in the skin of an
amphibian and develops into a zoosporangium. Zoosporangia produce zoospores
via asexual reproduction (sexual reproduction may also occur; Morgan et al.,
2007) that are released into the water through a discharge tube (Berger et al.,
2005). Tadpoles of most species are minimally affected by chytridiomycosis
and the effects on frogs are highly variable, with some species succumbing to
the disease within weeks and others experiencing few negative effects (Fisher
et al., 2009; Skerratt et al., 2007). Chytridiomycosis likely causes frog mortality
by severely disrupting epidermal functions and leading to osmotic imbalance
(Voyles et al., 2009).
Despite a growing literature on chytridiomycosis (Fisher et al., 2009; Rosen-
blum et al., 2010), we have little predictive power in assessing why Bd presence
in some areas causes epizootic die-offs while in other areas amphibian hosts sur-
vive Bd infections in an apparently enzootic state for years with little or no effect
on host population survival (Briggs et al., 2010). Our studies of yellow-legged
frogs in California offer one of the most complete insights into the host–pathogen
dynamics of chytridiomycosis (Briggs et al., 2010; Vredenburg et al., 2010a).
Along with an important study from Central America (Lips et al., 2006), we show
that Bd epizootics occur when Bd invades completely naive host populations. We
describe the dynamics of Bd and its spread into three naive metapopulations of
yellow-legged frogs, each consisting of collections of connected subpopulations
(n = 80) within widely separated drainage basins in Sequoia and Kings Canyon
National Park (SEKI). Our study was the first to capture the spread of Bd on a
small scale (more than hundreds of kilometers) and provided insights into how
the pathogen spreads between host populations. The Bd epizootic moved linearly
across the landscape at approximately 700 m per year. In one basin (Sixty Lake
Basin, Figure A18-2, panels F–J), Bd took 4 years to invade all of the subpopula-
tions (Figure A18-2) and we proposed that Bd was probably invading new host
populations through movement of frog hosts as opposed to movement by water
(movement was upstream) or by birds or other flying organisms that might carry
Bd. (Bd always infected the nearest uninfected host population.) With the data
we collected from 1996 to 2008, we demonstrate that Bd invaded naive host
FIGURE A18-2â•‡ Maps of the three study metapopulations showing the spread of Bd and frog population status (adults only) during a 4-year
period following the initial detection of Bd. Depicted are (A–E) Milestone Basin, (F–J) Sixty Lake Basin, and (K–O) Barrett Lakes Basin.
Lake color (green, yellow, and black) shows the Bd infection and frog population status, and the light gray shaded region surrounds the area
in which frog populations were Bd-positive in each year. Lakes shown with a thick black outline are fishless, and a thin gray outline indicates
that non-native fish were present. The infection status of frog populations depicted in panels A and K is based on mouthpart surveys of 459
tadpoles. The infection status of frog populations in panels B–J and L–O is based on 4,591 skin swabs analyzed using a real-time PCR assay.
347
SOURCE: Vredenburg et al. (2010a).

348 FUNGAL DISEASES
populations of frogs and that infection prevalence quickly reached 100 percent
(Figure A18-3, panel B), but mass mortality and host population collapse did not
occur until the average Bd infection load (or intensity of infection on individu-
als) for a population reached more than 104 zoospore equivalents (Figure A18-3,
panels A and C; Vredenburg et al., 2010a). This condition was later named the
“Vredenburg 10,000 Zoospore Rule” (Kinney et al., 2011) and was shown to also
predict mortality in neotropical salamanders (Cheng et al., 2011).
In the Sierra Nevada, infection by Bd did not always lead to epizootic events
and host population collapse. About 100 km north of our SEKI study area in
and around Yosemite National Park, we describe the Bd–frog dynamics under
stable enzootic conditions (Briggs et al., 2010), which contrast sharply with
conditions we describe above in the same species of yellow-legged frog in SEKI
(Vredenburg et al., 2010a). We discovered that in the Yosemite area, frog host
population dynamics are strikingly different and that host populations survive, at
least for a decade, despite infection by Bd (Briggs et al., 2010). We determined
that while infection prevalence (proportion of infected hosts in a population)
could be high (more than 60 percent), the infection intensity of hosts remained
low (Briggs et al., 2010), well below the Vredenburg 10,000 Zoospore Rule
(Kinney et al., 2011). In fact, several other studies have found that host amphib-
ians do not die when Bd infection intensities remain low (Cheng et al., 2011;
Kinney et al., 2011; Kriger et al., 2007; Retallick et al., 2004). Why the dynamics
of the host–pathogen interaction is so different in different sites and among differ-
ent species remains a mystery, but we believe the key to host survival is the low
infection intensities on individual host frogs (Briggs et al., 2010; Kinney et al.,
2011; Vredenburg et al., 2010a) and salamanders (Cheng et al., 2011).
Mutualistic Bacteria Play a Role in Amphibian

Resistance to Fungal Disease
There are several factors that may prevent fungal infection intensities from
reaching a lethal threshold in amphibian hosts, such as exposure to a thermal
environment that limits Bd growth (Piotrowski et al., 2001), immune response by
the host (Ramsey et al., 2010; Rollins-Smith et al., 2000), or density-dependent
host-pathogen dynamics (Briggs et al., 2010). Another possibility is that a host’s
microbiome may disrupt fungal invasion and growth; in particular, species of mu-
tualistic bacteria may be able to produce compounds that deter fungal coloniza-
tion and growth. The idea that microbial symbionts may protect amphibian hosts
from fungal pathogens was discovered, in a sense, by accident. Until relatively
recently, it was assumed that amphibians had few microbes living on their skin
since it is generally accepted that amphibians wear their defenses, like armor,
on the outsides of their bodies (Duellman and Trueb, 1986). Amphibians are
distinct among vertebrates in that they contain specialized skin secretion glands
(granular glands) that produce toxins, presumably to deter predators (Duellman
APPENDIX A 349
Figure A18-3.eps
FIGURE A18-3â•‡Frog–Bd dynamics in eight intensively sampled populations in Mile-
bitmap
stone and Sixty Lake basins before and after detection of Bd: (A) frog counts (adults +
subadults) from visual encounter surveys; (B) infection prevalence, defined as the fraction
of skin swabs collected from each population on each date positive for Bd; and (C) infec-
tion intensity, defined as the average zoospore equivalents on swabs collected from each
population on each date. Data are from frog populations that were sampled more than
once per year, that experienced more than 80 percent declines by the end of 2006, and for
which the decline in the number of frogs was greater than 10. This last criterion excluded
populations that were very small before Bd arrival. Populations are aligned along the x axis
such that “0” represents the date on which each frog population began to decline. This was
calculated for each population by determining the date at which the number of postmeta-
morphic frogs dropped below 20 percent of the average population count before that point.
SOURCE: Vredenburg et al. (2010a).
350 FUNGAL DISEASES
and Trueb, 1986). Many species are well known for this characteristic, such as the
poison dart frogs of the neotropics (genus Dendrobates) that produce neurotoxins
strong enough to incapacitate monkeys hit by blow-darts from hunters who rub
the darts on the skin of the frogs, or the toxic newts (genus Taricha) that produce
the same chemical defense compounds, known as tetrodotoxins, found in deadly
puffer fish.
Amphibians, although generally tied to water, have colonized many diverse
habitats, including many with no standing or running water (Duellman, 1999). In
fact, many diverse species have developed reproductive modes that free them in
some capacity or completely from water (e.g., direct developers). The four-toed
salamander (Hemidactylium scutatum), although not a direct developer, is of
interest for this chapter because the female will lay her eggs outside water along
steep banks or edges of ponds and she will brood them or guard them until they
hatch as larvae. Originally it was presumed that the female was guarding her
eggs against egg predators, but behavioral experiments showed that unattended
eggs were not eaten by egg predators; in fact, the eggs were unpalatable to many
predators (Hess and Harris, 2000), and died instead from fungal infections.
Egg-brooding females protect their eggs by inoculating them with colonies of
beneficial bacteria (Harris et al., 2006). A laboratory study later showed that the
bacterium, Janthinobacteria lividum, cultured from female four-toed salamanders
inhibited the growth of Bd (Harris et al., 2006).
Could the information from egg-brooding salamanders in the eastern United
States provide insight into why some populations of yellow-legged frogs in Cali-
fornia survived Bd infection while others did not? In 2005 we collected bacterial
cultures from susceptible and nonsusceptible populations of Rana muscosa and
R. sierrae. We found that individual frogs from nonsusceptible populations were
more likely to contain culturable populations of the symbiotic bacterium Jan-
thinobacteria lividum, the same species of bacteria discovered on the four-toed
salamander (Woodhams et al., 2007). A laboratory study to test whether inocula-
tions of J. lividum could protect susceptible R. muscosa from Bd infections was
then initiated. Adult R. muscosa were collected from the wild in SEKI in an area
ahead of the Bd epizootic wave and were transported to the Harris Laboratory at
James Madison University. The laboratory experiment consisted of three treat-
ments of frogs: one group received J. lividum only, another group received Bd
only, and a third was first inoculated with J. lividum and was then infected with
Bd. As expected, all of the frogs in the Bd-only treatment died from chytridiomy-
cosis within several weeks, but the frogs inoculated with J. lividum before being
infected survived, as did the frogs that only received inoculations of J. lividum,
providing evidence that bacterial symbionts could alter the outcome of Bd infec-
tions in susceptible host populations (Harris et al., 2009).
APPENDIX A 351
Could Bioaugmentation of the Skin Microbiome on

Host Frogs Make Bd Epizootics Less Deadly?
The successful laboratory experiment with yellow-legged frogs showed that
inoculations with Janthinobacterium lividum can effectively inhibit Bd growth
and colonization (Harris et al., 2009). The production of violacein, an antifungal
compound (Brucker et al., 2008), appears to be the mechanism used by J. lividum
to combat the colonization of Bd (Harris et al., 2009). Our field cultures from ad-
mittedly limited Rana muscosa and R. sierrae populations indicated that a small
proportion of individual frog hosts even in susceptible populations did naturally
include J. lividum in their skin microbiome (<10 percent compared to >80 per-
cent from susceptible and nonsusceptible populations of hosts, respectively; Lam
et al., 2010). Nevertheless, because the Bd epizootic was extirpating hundreds of
yellow-legged frog populations, we decided to initiate a field bioaugmentation
experiment of J. lividum on frogs at a Bd epizootic site in the Sierra Nevada. We
planned a replicated whole-lake experiment in the spring of 2010 at a site called
Dusy Basin in the northeastermost section of Kings Canyon National Park. This
site represented some of the very last susceptible populations of R. sierrae that
had yet experienced the Bd epizootic by 2009. By the time we arrived after the
spring snowmelt in 2010, there was only one population in Dusy Basin of R.
sierrae left containing living adults; all other frogs in the basin were dead, pre-
sumably killed by Bd.
In July 2010 we captured 40 adult R. muscosa at a single lake in Dusy Basin
and collected bacteria culture samples. Of the 40 frogs sampled, only one suc-
cessfully resulted in a bacterial culture of J. lividum. We used this single culture
to grow large volumes of J. lividum and returned 2 weeks later to Dusy Basin to
inoculate as many frogs as we could find with live cultures of J. lividum. We cap-
tured 100 adult frogs at the single remaining population in the basin and treated
80 with bacterial baths, leaving 20 as controls. All frogs were marked individually
with microchips (PIT tags) and were treated equally in terms of handling time.
Control frogs were soaked in pond water from the lake while treatment frogs were
soaked in provosoli solution containing live populations of J. lividum in broth.
Each animal was weighed, measured, swabbed (for Bd PCR assay; Boyle et al.,
2004; Hyatt et al., 2007), and released. We then revisited the site five times in
the remaining days of summer, capturing as many frogs as we could find. Each
frog was checked for a microchip, weighed, swabbed, and released. By the end
of summer, 6 control frogs had been recaptured and 62 bacterially treated frogs
had been recaptured. The highest Bd infection intensities were recovered on the
control frogs, several control frogs contained infections with >10,000 zoospore
equivalents, a lethal infection for the species (Vredenburg et al., 2010a). Due
to the high elevation of the site (~3,352 m) and the extremely large amount of
precipitation during the winter of 2010–2011, we are not able to return to the site
until mid-July 2011. At this time we have no way of knowing what the fate of the
population may be. If the microbiome bioaugmentation technique is successful,
352 FUNGAL DISEASES
it holds great hope for the conservation of susceptible populations of amphib-

ians globally. If the technique does not work, then we must continue to try to
understand the factors that may keep Bd infection intensities on individual host
amphibians low, because we currently have no way of controlling the spread of
this highly virulent pathogen.
Acknowledgments
We thank Harold Werner, David Graber, and Danny Boiano of the National
Park Service for their support of our project and to SEKI for providing the proper
research permits. We especially thank Lilia Torres, without whom we would not
have been able to culture Janthinobacterium lividum, and David Daversa, who
collected most of the field data. We thank Anand Varma for taking wonderful
photographs; Celeste Dodge, Cory Singer, Sam McNally, and Lauren Gillespie
for all the hard work; and Roland Knapp and his field crew for logistics, field
work, and planning. We also thank all of the Forum members and the Institute of
Medicine for the invitation to participate and LeighAnne Olsen for her patience
and kindness to the lead author. Our research is supported by National Science
Foundation Grant EF-0723563 as part of the joint National Science Founda-
tion–National Institutes of Health Ecology of Infectious Disease program and by
startup funds provided to V. Vredenburg by the College of Science and Engineer-
ing at San Francisco State University.
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APPENDIX A 355
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A19
THE EFFECT OF TRADE-MEDIATED SPREAD OF

AMPHIBIAN CHYTRID ON AMPHIBIAN CONSERVATION
Ché Weldon59 and Matthew C. Fisher60
Introduction
Accelerating losses of amphibian biodiversity were brought to the forefront
of conservation concerns in 1989 at the First World Congress of Herpetology,
and the National Research Council Workshop the following year (Blaustein and
Wake, 1995). Although obvious causes of amphibian declines were known (e.g.,
habitat destruction and alteration, introduction of exotic species, pollution), the
causes of some declines and disappearances were undetermined, such as enig-
matic declines in tropical Australia, Meso-America, and western United States.
Detailed retrospective studies of declines in California national parks (Drost and
Fellars, 1996) and Monteverde Cloud Forest Preserve in Costa Rica (Pounds
et al., 1997) gave conclusive evidence of community-wide declines. Amphibian
species have disappeared or declined at such an alarming rate over the past few
decades that the phenomenon is now considered to be an amphibian extinction
crisis (Mendelson et al., 2006; Wake and Vredenburg, 2008).
In 2004, the Global Amphibian Assessment (GAA) conducted by the Interna-
tional Union for Conservation of Nature (IUCN) revealed that 32.5 percent of the
world’s ca. 6,000 amphibian species are currently threatened with extinction, and
more than 120 have already disappeared (Stuart et al., 2004). Furthermore, half
of 435 rapidly declining species were threatened by enigmatic processes (e.g.,
disease, climate change) and disease was detected in a significant proportion of
families (15/36 families) with threatened species (Stuart et al., 2004). While long
suspected, little direct evidence for the emergence of infection existed until a new
pathogen to science, the fungal chytrid Batrachochytrium dendrobatidis (Bd),
was found in dead and dying frogs collected in the mid-1990s from declining
populations in Australia and Panama (Berger et al., 1998; Longcore et al., 1999).
59â•›â•›Unit for Environmental Research: Zoology, Private Bag X6001, North-West University, Potchef-
stroom 2520, South Africa.

60â•›â•›Department of Infectious Disease Epidemiology, Faculty of Medicine, Imperial College London,
London W2 1PG, U.K.

356 FUNGAL DISEASES
Many amphibian declines considered at the time to be the cause of introduced

species or anthropogenic activities are now known to have been partly due to
chytridiomycosis (Green et al., 2002; Muths et al., 2003). More recently, Bd has
been identified as a rapidly emerging amphibian pathogen, capable of acting as
both the proximate and ultimate cause of amphibian extinction (Fisher et al.,
2009a; Schloegel et al., 2006).
In this article, the contribution of the amphibian–chytridiomycosis spread
pathway to amphibian declines is investigated, and conventional as well as novel
approaches to amphibian conservation with regard to disease threat are evaluated.
The essay argues for greater collaboration among science, conservation, and
policy if the chytridiomycosis panzootic is to be warded off more efficiently, and
summarizes possible strategies to meet that goal.
The Chytridiomycosis–Amphibian Spread Pathway

Through trade and travel, humans introduce species both intentionally and
inadvertently. There is a lucrative global trade in amphibians for purposes ranging
from experimental animals to cuisine delicacies to pets. The GAA reported that
there are 278 amphibian species in pet trade, with the main centers for export
being the wet tropics (Stuart et al., 2008). The commercial collection of wild
amphibians has often been unsustainable, resulting in reductions in even highly
protected amphibian populations such as the Japanese Giant salamander and the
Mountain Chicken frog of the islands of Montserrat and Dominica (Anita et al.,
2007; Wang et al., 2004).
The conservation threat of traded species affects local and endemic species
when introduced exotic and non-native species become invasive and establish
feral populations. In addition, diseases and parasites are often carried along un-
intentionally with their hosts, allowing them to cross geographic boundaries that
historically contained these agents at their source of origin (Cunningham et al.,
2003). Not many pathogens become established with invasive species, but those
that do have the potential to seriously threaten native wildlife (Lyles and Dobson,
1993). Amphibians are known for their ability to become invasive, as demon-
strated by the Invasive Species Specialist Group (ISSG) list of 100 of the world’s
most invasive alien species, which includes three amphibians (ISSG, 2008). Also
listed is Bd, for which invasive amphibians are now known to be the primary
vectors of spread (Garner et al., 2006). In Australia, for example, Bd is found
among others in Cane toads, a recently introduced invasive alien species (Fisher
and Garner, 2007). This stands as a testimony to the complexity of biological
invasion issues where not only is Bd an invasive alien species, but its spread is
facilitated by an invasive alien toad. Thus, aspects of the disease impacts on am-
phibian populations around the world result from the unintentional introduction
of a pathogen as an invasive alien species. This leads to a paradox in conservation
management, namely that in order to contain the pathogen threatening the organ-
APPENDIX A 357
isms targeted for conservation (e.g., amphibians), the very group conservationists
are trying to protect need to be mitigated to prevent the pathogen from spreading.
The disease pathway is not always a single invasive host–pathogen system,
but may involve multiple host species that are either directly or indirectly related
to the source population. Several invasive frog species play a role in the exporta-
tion of Bd from South Africa and its ongoing spread and impact on amphibian
populations around the world. Trade in African clawed frogs (Xenopus laevis)
since the 1930s was identified as one of the first human-mediated pathways for
spreading Bd internationally (Weldon et al., 2004). Feral populations that be-
came established in countries where the species had been moved (Weldon et al.,
2007) allowed for potential transmission of Bd to naïve amphibian species. Such
a link was hypothesized to exist between X. laevis and the American bullfrog
Rana catesbeiana, an invasive species and major vector of the disease (Daszak
et al., 2004; Weldon et al., 2004). Various other strong links have been confirmed
between the international amphibian trade for use in the food, pet, and labora-
tory industry, and the global dispersal of Bd (Fisher and Garner, 2007; Garner
et al., 2006; Schloegel et al., 2009). A second species traded from South Africa,
Xenopus gilli, was identified as the source of a Bd infection transmitted to captive
Alytes muletensis in Spain (Walker et al., 2008). These infected toads were later
reintroduced into their native habitat on the island of Mallorca, thus introducing
the pathogen into this disease-naïve ecosystem. What makes this case even more
extraordinary is that Bd was transmitted from one critically endangered species
to another, dismissing the assumption that the more common, widely distributed
species are mostly responsible for disease spread. This case study demonstrates
the caution with which reintroduction programs should be approached when there
is a potential for disease organisms to be vectored alongside their hosts; it is now
clear that invasive frogs and toads have spontaneously exposed many native spe-
cies of amphibians around the world to Bd, often with catastrophic results.
Conventional Amphibian Conservation Strategy

Conventional approaches to amphibian conservation focus on mitigating fac-
tors that have been threatening amphibian populations for more than a century.
Primary threats that have traditionally received conservation attention include
habitat loss and fragmentation, overexploitation, and species invasion (Stuart
et al., 2008). Because disease is a relative newcomer on the scene of threats to
amphibian biodiversity, it has traditionally received little conservation attention
and proven mitigation measures are generally underdeveloped.
A common approach to conserving species is to make habitat or site the
conservation priority as some form of protected area. The species that require
protection are then viewed as important features that are present at that site and
that qualify it for a particular protected-area designation status (Tucker, 2005).
However, no amphibian species is protected from becoming infected with Bd by
358 FUNGAL DISEASES
the site conservation designations approach because the distribution of disease

agents is not restricted by political boundaries. This observation is underscored
by the fact that many of the population declines at the start of the chytridiomy-
cosis panzootic occurred at pristine and protected areas.
A close relationship exists between emerging infectious disease and alien inva-
sive species, as demonstrated by the pathways of spread for the chytridiomycosis-
amphibian system (Fisher et al., 2009a). So, how can we manage the spread of
such trade-associated pathogens? Invasive species can either be managed through
the control of introduced species (physical, chemical, or biological control) or
through the prevention of introduction (mainly through cross-boundary policies
and laws). However, the strategies used in the control or prevention of invasive
species do not necessarily account for the spread of infectious disease. Physical
control is more effective for relatively contained invasion of conspicuous spe-
cies and therefore not appropriate for pathogen control. Chemical (antifungal)
or biocontrol would be more suitable for a widespread invasion such as Bd, but
non-target mortality of amphibian hosts or other fungal species resulting from in
situ application remains a high risk. Ultimately, the effective control of introduced
species requires rapid response by designated authorities when an invasion is first
detected, given that a country’s border-level biosecurity has failed. However, un-
less detection is rapid, the costs of removing an invasive host species is high, and
there is no guarantee that cross-transmission will not have occurred to endemic
species (e.g., as was found with the transmission of Bd from North American
bullfrogs to European Bufo bufo) (Garner et al., 2006). Thus, it is essential that
invasion pathways are not only identified, but effectively regulated if invasion
is to be prevented. Because the spread of Bd is associated with the amphibian
trade, all intentional introductions of amphibians should be regarded as potential
intentional introductions of the pathogen. In general, control of intentional intro-
ductions fall under the jurisdiction of governmental agencies responsible for the
introductions. Unintentional introductions of Bd (e.g., infected frogs that escape
from captivity) occur at a smaller spatial and temporal scale and are more chal-
lenging to manage. Regardless of scale, effective prevention of invasion requires
a certain amount of legislation and policy development.
Another shortfall of conventional conservation is that the laws governing the
conservation of biodiversity do not sufficiently deal with wildlife disease issues.
International laws governing management and the conservation of biodiversity
consist of agreements by cooperating countries through treaties, conventions,
and voluntary participation. For instance, the Convention on Biological Diversity
(CBD) consists of members who pledge to pursue a comprehensive approach to
conserving biological diversity. The CBD states that all participating governments
should attempt to prevent the introduction of, eradicate, or control those alien
species that threaten ecosystems, habitats, or species. The conservation challenge,
however, is for the individual countries that are either the source or recipient of in-
vasive species to act on these general statements of good intent. A good example
APPENDIX A 359
is the Lacy Act of 1900 that identifies a priority list of invasive species requiring
conservation management. The federal agencies responsible for implementing
the law are the U.S. Fish and Wildlife Service (FWS) and the National Oceanic
and Atmospheric Association, but the FWS does not provide specific authority to
prohibit the importation of a pathogen.
International trade in species whose existence may be threatened as a result
of commerce and other trafficking is regulated by the Convention on International
Trade in Endangered Species of Wild Fauna and Flora (CITIES). However, com-
prehensive data covering all amphibians is hard to find because there is no global
database or monitoring system for the trade in non-CITIES species (Schlaepfer
et al., 2005). Consequently, control over the international spread of Bd is not
regulated under this process when the vectors are non-threatened species such as
X. laevis and R. catesbeiana. The World Organisation for Animal Health (OIE)
provides guidelines that can be used by member countries to protect themselves
from the introduction of diseases and pathogens. Only two amphibian pathogens
(Bd and Ranavirus) are currently listed as notifiable to the OIE, thus requiring
regulation of the amphibian trade aimed at the prevention of disease spread.
Because non-members are under no obligation to adhere to the guidelines set
by the OIE and because recommendations rely on voluntary participation and
lack any legally binding mechanisms, amphibians traded by both member and
non-member countries are often not accompanied by veterinary certification.
This shortfall in international legislation may cause ambiguity with respect to
biosecurity associated with trade, ultimately contributing to the unabated global
spread of pathogens.
Conservation Strategy for Amphibian Disease Threat

The contemporary approach to biological conservation is to begin with an
assessment of threatened taxa, which can be used by conservationists as a source
of reference for identifying species that should be prioritized for conservation
research. When species are threatened by a pathogen with no apparent host
specificity, however (as is the case for Bd), species evaluation should take on the
form of a risk assessment based on biological and environmental characteristics
that predispose species to disease susceptibility. Prediction models are best ap-
plied in a precautionary context, when information is used proactively to prevent
a catastrophe to biodiversity (e.g., introduction of a novel pathogen). Preventing
the importation of non-indigenous species in the first place is an important tool to
invasive species management, but a strategy is also needed to effectively contain
harmful non-indigenous species once they have become established (Schlaepfer
et al., 2005).
360 FUNGAL DISEASES
Predict Disease Susceptibility and Emergence

The GAA indicated disease as the third most significant threat to Anura glob-
ally, but only the seventh for salamanders, with no caecilians known to be threat-
ened by disease (Stuart et al., 2008). When finer analyses are performed (family
down to species level), the data become more appropriate as a conservation
management tool. Various studies at global and regional scales have produced
predictive Bd emergence maps and species priority lists for conservation based on
multiple characteristics associated with susceptibility to Bd (www.bd-maps.net;
Bielby et al., 2008; Rödder et al., 2009; Ron, 2005) (Figure A19-1). Some of the
more common denominators collectively used in Bd predictive modeling include
climate, altitude, conservation status, and reproductive mode of amphibian spe-
cies. These integrated models can be used to predict which specific groups in any
newly infected region would have a high probability of being impacted by the
disease. The question of chytridiomycosis emergence is complicated by evidence
that different strains of Bd can exhibit varying degrees of virulence (Berger et al.,
2005; Fisher et al., 2009b). Therefore, the cross-regional/continental introduction
of infected amphibians could introduce novel strains of Bd that are more virulent
than those already present in a region. Consequently, there is an urgent need to
abate the introduction of Bd into regions already affected by chytridiomycosis.
Population Management Strategies

Despite a growing wildlife disease phenomenon as human encroachment
into natural areas intensifies, making ecological systems more susceptible to
infection and its consequences, surprisingly little research has been done on the
management of wildlife diseases (Daszak et al., 2001; Fenichel and Horan, 2007).
Mendelson et al. (2006) stated that the only hope to save amphibian species from
extinction is management through a combination of coordinated in situ actions
with ex situ husbandry programs (e.g., survival-assurance and research colonies)
on an unprecedented scale. The mandate of the IUCN is that all critically endan-
gered and extinct in the wild taxa should be subjected to ex situ management to
ensure recovery of wild populations (IUCN, 2004).
Assurance-survival populationsâ•‡ A management strategy that results from using

predictive modeling is to collect endangered species for assurance-survival popu-
lations by moving ahead of the planned spread of disease. Thus, by collecting
wild animals before the arrival of disease and breeding them in captivity disease-
mediated decline is prevented. Assurance-survival populations have particular
application when species face threats that cannot be easily or swiftly mitigated
in the wild (Pavajeau et al., 2008). The 2005 IUCN Amphibian Conservation
Action Plan (ACAP) white papers state that assurance-survival populations are
mandatory for amphibian species that will not persist in the wild long enough
to recover naturally once environments are restored (e.g., disease is controlled)
APPENDIX A 361
A.
B.
FIGURE A19-1â•‡ Maps indicating (A) the global prevalence and (B) regional U.S. preva-
Figure asA19-1.eps
lence of Batrachochytrium dendrobatidis mapped by www.bd-maps.net.
SOURCE: The Global Bd-Mapping Project, www.bd-maps.net.
2 bitmaps w some vector type
(IUCN, 2005). This strategy was successfully harnessed in Panama ahead of an

encroaching Bd epidemic wave (Lips et al., 2006). Threatened, regional endemics
and other specifically selected amphibian species were rescued from El Copé and
El Valle and placed in the El Valle Amphibian Conservation Center, an in-country
ex situ facility with the objective of long-term maintenance of species until perti-
nent threats could be mitigated and the species could be reintroduced (Gagliardo
et al., 2008). A group of concerned conservation organizations, including the
IUCN/SSC (Species Survival Commission) Conservation Breeding Specialist
362 FUNGAL DISEASES
Group, the World Association of Zoos and Aquariums, and the IUCN/SSC Am-
phibian Specialist Group initiated the Amphibian Ark (AArk), an organization
that provides all levels of support to ex situ actions around the world (Pavajeau
et al., 2008). Ex situ conservation centers, in addition to maintaining captive as-
surance populations, provide a great opportunity for much-needed research on
the management of wildlife diseases.
Some concerns that have to be addressed when initiating captive assurance
populations is the need of substantial resources for the construction of biosecure
facilities, training of keepers, and support of amphibian husbandry requirements
(Gagliardo et al., 2008; Pavajeau et al., 2008). In addition, for animals salvaged
from areas with recognized disease-mediated die-offs, it is necessary to control
the population-limiting infection to help ensure the success of the captive colony
and to prevent introduction of disease to other collection animals (Pessier, 2008).
Disease eradicationâ•‡ Fortunately, a variety of commonly used disinfectants,

including 1 percent sodium hypochlorite (chlorine bleach) and quaternary am-
monium compounds, as well as heat (60°C for 5 min) and desiccation, are effec-
tive against Bd (Johnson et al., 2003; Webb et al., 2007). Infected and diseased
animals from zoological collections have been treated successfully with various
antifungal medicines, especially itraconazole baths (Nichols et al., 2000; Parker
et al., 2002; Pessier and Mendelson, 2010). Before any chemical treatment can
be widely applied, its efficacy must be tested because no treatment for chytrid-
iomycosis has proven to be consistently effective across different amphibian
species and life stages (Young et al., 2007). The use of elevated environmental
temperatures (37°C for 16 hours) may also clear Bd infection in heat-tolerant
species (Woodhams et al., 2003).
However, treatment of Bd in wild amphibian populations presents its own
challenges, and we are still far from being able to eliminate Bd from the environ-
ment or stop its dispersal. Although Bd can be killed by a range of antifungal
medications, it is both impractical and ecologically dangerous to attempt such
treatments in the wild due to unknown effects on ecosystems fungal components.
However, mathematical models predict that mass mortalities can be reduced if
infection loads in affected populations are reduced (Briggs et al., 2010). Fur-
thermore, it is possible to eradicate a disease if the aggregate wildlife popula-
tion is harvested at a specific level: below a threshold population density and
below which disease prevalence declines and above which a disease becomes
epidemic if a threshold exists (McCallum et al., 2001). Some of these alternative
approaches have been attempted by Bosch et al. (2010) at Peñalara Natural Park
in Madrid with the Common midwife toad (Alytes obstetricans), the Mallorcan
midwife toad (A. muletensis), and the Betic midwife toad (A. dickhilleni).
Methods employed on wild-caught tadpoles included (1) thermal treatment
(21°C) in captivity followed by release after metamorphosis, (2) chemical treat-
ment with itraconazole baths in captivity and subsequent release of tadpoles into
APPENDIX A 363
ponds that had been desiccated during treatment, and (3) itraconazole baths in
situ in artificial pools combined with habitat improvement, which included desic-
cation before releasing the tadpoles. The different methods resulted in varying
success, although generally, released tadpoles became reinfected and metamorphs
had a higher survival rate than what was determined for previous seasons. It
seems that Bd eradication is difficult to achieve and that attempts at keeping
infection levels controlled in the hope of achieving natural selection against the
disease may be the only current approach for in situ mitigation (Bosch et al.,
2010). In tandem with antifungal mitigations, the use of probiotic amphibian-
associated bacteria holds promise for managing levels of chytrid infection in
nature. Specifically, Janthinobacterium lividum has shown to clear infections
in captive populations and is currently being widely trialed in a variety of field
settings (Harris et al., 2009).
According to Schlaepfer et al. (2005), management practices that rely on
continuous intervention are not sustainable indefinitely due to human and mon-
etary resource limitations. Instead, Schlaepfer et al. (2005) suggest a novel ap-
proach where native species can respond to changes in their selective regime via
evolution or learning that allows coexistence of native and introduced species in
cases where the eradication of invasive species is not successful. Although this
approach is suggested for invasive species that threaten local species through
predation or competition, its application for a pathogen like Bd—capable of
rapid population extirpations and even extinction through disease—remains to
be explored. Some evidence exists that amphibian populations that have expe-
rienced chytridiomycosis-associated decline may partially recover and persist
with endemic Bd infection (Retallick et al., 2004; Woodhams et al., 2007). These
observations raise hope that some species can acquire resistance to chytridiomy-
cosis in the wild, lessening the reliance on the development of survival-assurance
colonies for species threatened by this disease (Mendelson et al., 2006; Young
et al., 2007).
Reintroductionâ•‡ Ultimately, the goal of population management that begins with

the capture of wild amphibians is to combine ex situ treatment with the selection
of resistant individuals that could form the basis of lineages for future reintroduc-
tion. Before amphibians can be released into the wild, a number of precautions
related to infectious disease need to be considered. Collections that comprise
several species increase the likelihood of infection with a variety of potentially
novel infectious agents (Cunningham, 1996), a situation that warrants prerelease
screening for chytridiomycosis and other infectious disease, both known and
unknown. Pessier (2008) demonstrated how the spectrum of infectious agents
affecting amphibians is more varied than realized previously and argued for
enhancement of the understanding of disease in conservation programs, which
includes permanent quarantine housing of species combined with comprehensive
disease surveillance and treatment. However, it is possible that hosts raised in
364 FUNGAL DISEASES
captivity, who are continually treated to eliminate infections, may suffer a disad-
vantage when released into the wild because of increased susceptibility to infec-
tion caused by relaxed selection for resistance, especially in traits that are costly
to maintain (Smith et al., 2009). Given this possibility, species management
programs, especially those that include captive breeding and reintroductions, may
need to focus on maintaining the levels of immunity or variation in resistance that
are present in natural populations (Altizer and Pedersen, 2008).
Summary
The diversity and apparent increase in wildlife diseases, including chytrid-
iomycosis, have raised concerns that pathogens may pose a substantial threat
to biodiversity. The amphibian extinction crisis has been held to represent the
greatest species conservation challenge in the history of humanity (Pavajeau
et al., 2008). In part, this crisis has been accelerated by the spread of Bd through
the global amphibian trade. The ability of some amphibians to become alien
invasive species while functioning as carriers of chytridiomycosis complicates
conservation management of the matter. Response to amphibian threats usually
involves introducing or rising protection measures of the species and/or their
habitats. However, dealing with chytridiomycosis in wildlife populations re-
quires novel strategies that conventional approaches to and policy for amphibian
conservation do not adequately provide. It requires greater collaboration among
wildlife ecologists, veterinarians, and conservation organizations to provide a
more comprehensive mitigation strategy that may significantly reduce the risk of
chytridiomycosis-mediated population declines.
The global conservation community responded to this need in the ACAP, of
which the AArk plays an integral part, to select species that would otherwise go
extinct for captive assurance populations until they can be secured in the wild
(Pavajeau et al., 2008). Such facilities are ideally positioned to combine the man-
agement of endangered species through assurance colonies with opportunities for
conservation research that may include the development of new approaches to
mitigate wildlife diseases. Future efforts to better manage and protect the planet’s
amphibian diversity will depend on innovation from both the sciences and the
world of policies and regulation. A wide spectrum of international and domestic
regulation regimes already play a significant role in shaping the conservation
status of amphibian biodiversity, but considerate input from the scientific com-
munity will make implementation more effective.
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368 FUNGAL DISEASES
A20
WHITE-NOSE SYNDROME FUNGUS (GEOMYCES

DESTRUCTANS) IN BATS, EUROPE61
Gudrun Wibbelt, Andreas Kurth, David Hellmann, Manfred Weishaar,
Alex Barlow, Michael Veith, Julia Prüger, Tamás Görföl,
Lena Grosche, Fabio Bontadina, Ulrich Zöphel, Hans-Peter Seidl,
Paul M. Cryan, and David S. Blehert62
White-nose syndrome is an emerging disease in North America that has

caused substantial declines in hibernating bats. A recently identified fungus
(Geomyces destructans) causes skin lesions that are characteristic of this disease.
Typical signs of this infection were not observed in bats in North America before
white-nose syndrome was detected. However, unconfirmed reports from Europe
indicated white fungal growth on hibernating bats without associated deaths. To
investigate these differences, hibernating bats were sampled in Germany, Swit-
zerland, and Hungary to determine whether G. destructans is present in Europe.
Microscopic observations, fungal culture, and genetic analyses of 43 samples
from 23 bats indicated that 21 bats of 5 species in 3 countries were colonized by
G. destructans. We hypothesize that G. destructans is present throughout Europe
and that bats in Europe may be more immunologically or behaviorally resistant
to G. destructans than their congeners in North America because they potentially
coevolved with the fungus.
White-nose syndrome (WNS) is a recently emerged wildlife disease in North
America, which in 4 years has resulted in unprecedented deaths of hibernating
bats in the northeastern United States (Blehert et al., 2009; Reichard and Kunz,
2009; Turner and Reeder, 2009), and is a widespread epizootic disease among
bats. Although we have searched the literature describing observations of hiber-
nating bats, we have been unable to find any similar historical accounts of white
fungus growing on live hibernating bats in North America before the recent
emergence of WNS.
61â•›â•›Emerging Infectious Diseases * www.cdc.gov/eid * Vol. 16, No. 8, August 2010.
62â•›â•›Author affiliations: Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (G.
Wibbelt); Robert Koch Institute, Berlin (A. Kurth); University of Oldenburg, Oldenburg, Germany
(D. Hellmann); Bat Conservation Working Group, Gusterath, Germany (M. Weishaar); Veterinary
Laboratory Agency, Somerset, UK (A. Barlow); Trier University, Trier, Germany (M. Veith); Coor-
dination Agency for Bat Protection in Thuringia, Erfurt, Germany (J. Prüger); Nature Conservation
Foundation of Tolna County, Szekszárd, Hungary (T. Görföl); Echolot GbR, Münster, Germany (L.
Grosche); SWILD–Urban Ecology and Wildlife Research, Zurich, Switzerland (F. Bontadina); Saxo-
nian State Office for Environment, Agriculture and Geology, Dresden-Pillnitz, Germany (U. Zöphel);
Technical University Munich, Munich, Germany (H.-P. Seidl); US Geological Survey, Fort Collins,
Colorado, USA (P. M. Cryan); and US Geological Survey, Madison, Wisconsin, USA (D. S. Blehert).
â•›â•›DOI: 10.3201/eid1608.100002
APPENDIX A 369
In North America, WNS is known to affect 6 species of bats that use hiber-
nation as their winter survival strategy: the big brown bat (Eptesicus fuscus),
the eastern small-footed bat (Myotis leibii), the little brown bat (M. lucifugus),
the northern long-eared bat (M. septentrionalis), the tricolored bat (Perimyotis
subflavus), and the Indiana bat (M. sodalis) (Blehert et al., 2009; Courtin et al.,
2010; Turner and Reeder, 2009). Since its detection in February 2006 in a popular
tourist cave near Albany, New York, USA, WNS has spread >1,300 km into Con-
necticut, Massachusetts, New Hampshire, New Jersey, Pennsylvania, Tennessee,
Vermont, Virginia, and West Virginia in the United States and the provinces of
Ontario and Quebec in Canada (Blehert et al., 2009, Turner and Reeder, 2009;
United States Geological Survey, 2010) in a pattern suggesting the spread of an
infectious agent.
A recently discovered psychrophilic (cold-loving) fungus, Geomyces de-
structans (Gargas et al., 2009), has consistently been isolated from bats that meet
the pathologic criteria for WNS, including colonization of skin by fungal hyphae
causing characteristic epidermal erosions and ulcers that can progress to invasion
of underlying connective tissue (Meteyer, et al.,2009; Reichard and Kunz, 2009).
G. destructans is identified by its distinctive asymmetrically curved conidia
and has a unique taxonomic position among other Geomyces spp. described to
date (Gargas et al., 2009) Its closest genetic relative is G. pannorum, a ubiqui-
tous psychrophilic, keratinolytic fungus that has been isolated from a variety of
sources and geographic regions, including soil and the fur of wild mammals in
France (Chabasse et al., 1987), floors of trains and ferryboats in Italy (Mercantini
et al., 1989), boreal forests in Canada (de Bellis et al., 2007), and environmental
samples from Arctic regions (Kochkina et al., 2007; Mercantini et al., 1989).
G. pannorum var. pannorum has been reported as an unusual dermatophyte
infecting fingernails and superficial skin of humans who have a history of close
contact with soil and dust (Gianni et al., 2003, Zelenková, 2006). However, G.
destructans differs from other common soil fungi of North America in its ability
to invade the living tissues of hibernating bats.
After WNS was described in North America (Blehert et al., 2009), reports
dating back to the early 1980s (Feldmann, 1984) described repeated observations
of white fungal growth on muzzles of hibernating bats in Germany. However,
these bats lacked the characteristics of WNS such as associated deaths. Moreover,
fungus was not identified. In response to WNS in North America, researchers in
Europe initiated a surveillance effort during the winter of 2008–09 for WNS-like
fungal infections among hibernating populations of bats in Europe. G. destruc-
tans in Europe was previously reported in 1 hibernating bat that was sampled in
France during March 2009 (Puechmaille et al., 2010).
In this report, we describe results of a more extensive effort by scientists
from 4 countries in Europe (Germany, United Kingdom, Hungary, and Switzer-
land) to obtain and analyze samples from hibernating bats with white patches
on their faces or wing membranes. Our objectives were to identify the fungus
370 FUNGAL DISEASES
colonizing such affected hibernating bats in Europe and to clarify its geographic
distribution over a broad area of Europe.
Materials and Methods

During ongoing annual population surveys of caves and mines conducted by
national nongovernmental organizations, hibernating bats with obvious fungal
growth on their bodies (Figure A20-1, panel A) were opportunistically sampled
in Germany, Switzerland, and Hungary; samples were also obtained from 2 dead
bats from the same hibernaculum in the United Kingdom. Approximately 366
hibernacula were visited during mid-February–mid-April 2009: 336 in Germany,
20 in Hungary, and 10 in Switzerland. Two to 214 hibernating animals were
observed at each site, with the exception of 2 sites in Germany, which harbored
2,000–7,000 animals at each site.
Samples were collected from live bats by using 2 methods. Touch imprints
were obtained by holding adhesive tape against affected areas of skin or fur, or
fur clippings were obtained from affected areas of bat muzzles. All species of bats
in Europe are strictly protected under the Flora, Fauna, Habitat Guidelines of the
European Union (92/43/ EEC) (http://ec.europa.eu/environment/nature/legisla-
tion/ habitatsdirective/index_en.htm) and The Agreement on the Conservation of
Populations of European Bats (www. eurobats.org). We did not have permission
to invasively sample or kill individual animals for histologic analysis to confirm
skin infection by G. destructans (Meteyer et al., 2009). Samples were shipped to
the Leibniz Institute of Zoo and Wildlife Research (IZW), Berlin, Germany, for
further investigations.
Twenty adhesive tape samples were first screened by using light microscopy,
and 21 hair samples were examined by using scanning electron microscopy for
conidia characteristic of G. destructans (Figure A20-1, panel B). Two of the sub-
mitted samples (2 greater horseshoe bats from the United Kingdom) consisted of
entire bat carcasses. Although the carcasses were examined externally for fungal
growth on muzzle skin and hair, specimens were too decomposed to conduct
internal pathologic examinations. Tape or hair samples from all bats were further
investigated by using direct PCR amplification of fungal rRNA gene internal
transcribed spacer (ITS) region DNA (ITS1, 5.8S, and ITS2). Total nucleic acids
were extracted from culture, tape, or hair samples by using PrepMan Ultra Re-
agent (Applied Biosystems, Darmstadt, Germany) following the manufacturer’s
instructions.
The rRNA gene ITS region DNA was amplified by using PCR with prim-
ers ITS4 and ITS5 (White et al., 1990) and GoTaq DNA polymerase (Promega,
Madison, WI, USA). Cycling parameters were an initial 2-min denaturation at
98°C; followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C
for 30 s, and extension at 72°C for 1 min; and a final extension at 72°C for 7 min.
For fungal isolates, rRNA gene small subunit (SSU) DNA was amplified by us-
APPENDIX A 371
FIGURE A20-1â•‡ (A) Greater mouse-eared bat (Myotis myotis) with white fungal growth
around its muzzle, ears, and wing membranes (photograph provided by Tamás Görföl).
Figure A20-1AandB.eps
(B) Scanning electron micrograph of a bat hair colonized by Geomyces destructans. Scale
bar = 10 Î¼m. bitmap
372 FUNGAL DISEASES
ing PCR with primers nu-SSU-0021–5′ (White et al., 1990) and nu-SSU-1750–3′
(Gargas and Taylor, 1992) as above, except the extension time was increased to
2 min. Sequencing primers were PCR primers; nu-SSU-0402–5′ (Gargas and
Taylor, 1992), nu-SSU-1150–5′ (White et al., 1990), nu-SSU-0497–3′ (Gargas
and Taylor, 1992), and nu-SSU-1184–3′ (Gargas et al., 1995) were added for
SSU. PCR products were sent to the Robert Koch Institute, Berlin, Germany, for
direct sequencing.
Culture analyses of samples were performed at Munich University Hospital
and IZW. After examining tape impressions by using light microscopy, we identi-
fied small areas with fungal conidia characteristic of G. destructans and excised
them with a sterile scalpel blade. Half of the excised material was used for PCR;
the remaining sample and samples of individual hairs with microscopic indication
of G. destructans were immediately placed onto Sabouraud dextrose agar plates
containing gentamicin and chloramphenicol and incubated at 4°C and 8°C. G.
destructans isolates obtained during this study are maintained at IZW.
Results
We obtained and analyzed samples from live bats with obvious fungal growth
on their bodies found between mid-February and the end of March 2009 at 11
sites (8 in Germany, 1 in Hungary, and 2 in Switzerland). Samples were also ob-
tained from an additional bat in Germany in February 2008 and from 2 dead bats
from a site in the United Kingdom in March 2009 (Tables A20-1, A20-2) All 12
hibernacula sampled contained 1–5 animals that exhibited obvious fungal growth.
Forty-three samples were obtained from these 12 hibernacula and represented 23
adult bats of 6 species: 1 Brandt bat (M. brandtii), 3 pond bats (M. dasycneme),
1 Daubenton bat (M. daubentonii), 1 lesser mouse-eared bat (M. oxygnathus), 15
greater mouse-eared bats (M. myotis), and 2 greater horseshoe bats (Rhinolophus
ferrumequinum).
After direct PCR amplification and DNA sequence analysis of fungal rRNA
gene ITS regions, genetic signatures 100% identical with those from G. destruc-
tans type isolate NWHC 20631–21 (GenBank accession no. EU884921) were
identified from 21 of 23 bats examined: 15/15 from Germany, 2/2 from Hungary,
and 4/4 from Switzerland. Both bats from the United Kingdom were colonized
by Penicillium sp. (Tables A20-1, A20-2). Fungi with conidia morphologically
identical to those of G. destructans (Figure A20-1, panel B) as described by
Gargas et al. (2009) were isolated in axenic cultures from 8 of 23 bats examined:
3/15 from Germany, 1/2 from Hungary, and 4/4 from Switzerland) (Tables A20-1,
A20-2; Figure A20-2).
Consistent with published descriptions for G. destructans (Gargas et al.,
2009), fungal colonies grew slowly and within 14 days attained diameters of 1.0
mm at 4°C and 4.0–5.0 mm at 8°C; no growth occurred at 25°C. The sensitiv-
ity of our method for isolating G. destructans from bat hair was comparable to
APPENDIX A 373
TABLE A20-1â•‡ Bats Tested for Geomyces destructans by Using Microscopy,

Fungal Culture, or PCR Analysis, by Country, Europe*
No. positive/no. tested
Species (common name) Germany Switzerland Hungary United Kingdom
Myotis myotis (greater mouse-eared bat) 10/10 4/4 1/1 -
M. dasycneme (pond bat) 3/3 - - -
M. daubentonii (Daubenton bat) 1/1 - - -
M. brandtii (Brandt bat) 1/1 - - -
M. oxygnathus (lesser mouse-eared bat) - - 1/1 -
Rhinolophus ferrumequinum (greater - - - 0/2
horseshoe bat)
*-, species not obtained in this country.
published diagnostic sensitivity for culturing G. destructans from bat skin (Lorch
et al., 2010). Subsequent PCR/DNA sequencing analyses of the 8 isolates indi-
cated that they all had rRNA gene ITS and SSU region DNA sequences identical
to those of G. destructans type isolate NWHC 20631–21 (GenBank accession
nos. EU884921 for ITS and FJ231098 for SSU).
Unlike other bats sampled in this study, the 2 greater horseshoe bats from the
United Kingdom were found dead, and their nostrils were colonized by Penicil-
lium sp. These bats did not fulfill the pathologic criteria for WNS (Meteyer, et al.,
2009) because fungal hyphae did not invade the epidermis but remained within
the superficial layer of the epidermal stratum corneum. A more complete descrip-
tion of the postmortem analysis of the greater horseshoe bats has been reported
(Barlow et al., 2009). G. destructans was not isolated in culture, and its genetic
signature was not identified by PCR and DNA sequencing of samples collected
from greater horseshoe bats.
Discussion
Laboratory analyses demonstrated that 5 species of the genus Myotis in
Europe harbored G. destructans; male and female bats were equally affected.
Despite laboratory confirmation that bats obtained in Germany, Switzerland, and
Hungary were colonized by G. destructans, deaths were not observed at collec-
tion sites. Puechmaille et al. (2010) reported a similar observation with a greater
mouse-eared bat in France. Additionally, a lesser mouse-eared bat from Hungary
with visible fungal infection during hibernation, from which G. destructans was
isolated, was recaptured 5 months later (August 2009) and showed no external
signs of fungal infection. On February 19, 2010, the same bat was again observed
in the same hibernaculum without any visible sign of fungal growth. However, 7
other bats within that group of 55 animals displayed obvious fungal growth but
were not sampled for this study.
TABLE A20-2â•‡ Fungal Culture and PCR Results for 23 Bats with Evidence of Fungal Colonization Tested by Light or
374
Electron Microscopy, Europe*

GenBank accession no.
Country/location Sample Collection No./ PCR Culture
no.† source Species date hibernacula result result ITS SSU rRNA
Germany/4 Hair 2 Myotis dasycneme 2008 Feb 25 10 + + GU350437 GU350442
Germany/8 Hair 7 M. myotis 2009 Mar 3 214 + + GU350436 GU350441
Germany/7 Tape 8 M. myotis 2009 Mar 7 57 + + GU999986 GU999983
Hungary/9 Hair 16 M. myotis 2009 Mar 29 64 + + GU350434 GU350439
Switzerland/10 Tape 10 M. myotis 2009 Apr 5 25 + + GU350433 GU350438
Germany/1 Hair 1 M. myotis 2009 Feb 21 65 + – HM222616 –
Germany/6 Hair 20 M. myotis 2009 Mar 13 100 + – HM222617 –
Germany/2 Tape 1 M. myotis 2009 Feb 26 ≈2,000 + – HM222618 –
Germany/2 Tape 2 M. myotis 2009 Feb 26 ≈2,000 + – HM222619 –
Germany/8 Tape 5 M. myotis 2009 Mar 3 214 + – HM222620 –
Germany/8 Hair 6 M. brandtii 2009 Mar 3 214 + – HM222624 –
Germany/5 Hair 3 M. dasycneme 2009 Feb 28 29 + – HM222625 –
Germany/6 Tape 17 M. dasycneme 2009 Mar 13 100 + – HM222626 –
Germany/3 Hair 17 M. daubentonii 2009 Mar 5 ≈7,000 + – HM222627 –
Hungary/9 Tape 13 M. oxygnathus 2009 Mar 29 64 + – HM222628 –
United Kingdom/12 Hair 10 Rhinolophus ferrumequinum 2009 Mar 11 558 –‡ –‡ HM222629 –
United Kingdom/12 Hair 11 R. ferrumequinum 2009 Mar 11 558 –‡ –‡ HM222630 –
*ITS, internal transcribed spacer; SSU, small subunit; tape, touch imprint with adhesive tape.
†Location number corresponds to a hibernation site in Figure A20-2.
‡Although samples were negative for G. destructans, they were positive for Penicillium sp. by PCR and culture.
APPENDIX A 375
FIGURE A20-2â•‡ Locations in Europe of bats positive for Geomyces destructans by PCR
alone (circles) or by PCR and culture (solid stars) and bats negative for G. destructans
Figure A20-2.eps
but positive for other fungi (square). Numbers for locations correspond to those in Table
bitmap
A20-2. Sites 7, 8, and 9 had additional bats that were positive for G. destructans only by
PCR. Location of a bat positive for G. destructans in France (Puechmaille et al., 2010)
is indicated by an open star. Some sites had >1 bat species with evidence of colonization
by G. destructans.
In contrast, decreases in hibernating bat colonies infected by G. destructans

in North America are often >90% (Reichard and Kunz, 2009; Turner and Reeder,
2009), and mortality rates similar in magnitude would be difficult to miss among
closely monitored winter populations of bats in Europe. Biologists in Germany
and Switzerland have conducted annual censuses of bat hibernacula since the
1930s and 1950s, respectively. In Hungary, the largest hibernacula have been
annually monitored since1990. Similar death rates to those caused by WNS in
hibernating bats in North America have never been documented in countries in
Europe in which G. destructans has now been identified.
Although distribution of G. destructans in bats across Europe has not been
exhaustively characterized, opportunistic sampling conducted as part of this
study during the winter of 2008–09 demonstrated that the fungus was present on
bats in 3 countries (Figure A20-2). The 2 most distant points from which bats
colonized with G. destructans have been identified were separated by >1,300 km.
Despite the observed distribution of G. destructans in Europe (Figure A20-2), the
5 bat species from which G. destructans was detected migrate average distances
<100 km between their summer and winter roosting sites (Hutterer et al., 2005),
376 FUNGAL DISEASES
indicating that the fungus is most likely spread as local bat populations emerge
from hibernacula, disperse, and interact with populations within their dispersal
range. Identification of bats colonized by G. destructans from such distant sites,
in addition to the relatively homogenous distribution of the fungus among sites
in Germany, suggests that G. destructans may be widespread in Europe.
Regardless of widespread occurrence of G. destructans among bat species
in Europe (Figure A20-2), deaths of bats in Europe caused by WNS, similar to
those caused by WNS in North America, have not been observed. Although no
bat species migrates between Europe and North America or is present on both
continents (Dietz et al., 2009; Nowak, 1999), many species of the genus Myotis
are infected by G. destructans on each continent. Although the mechanism(s) by
which hibernating bats died because of infection with G. destructans in North
America is not yet understood, bat species in Europe may exhibit greater resis-
tance or respond differently to infection by this fungus than their counterparts in
North America.
Before the emergence of WNS in North America, large aggregations of
hibernating bats ranging from 1,000 to 50,000 animals were common in caves
and mines of affected regions, and many hibernation sites in regions of North
America still unaffected by WNS contained tens of thousands of bats during
winter (some contain hundreds of thousands) (Barbour and Davis, 1969). In
contrast, aggregations of bats hibernating in caves and mines in Europe rarely
exceed 1,000 animals. However, larger hibernating groups have been observed
at a few natural sites, such as a cave in northern Germany with 13,000–18,000
bats (Petermann and Boye, 2006) and human-made structures (e.g., Daubenton
bats in bunkers and catacombs) (Dietz et al., 2009). If host density plays a role
in G. destructans transmission or deaths of bats, such as through increased dis-
turbance of clustered bats, the bats in Europe may experience lower mortality
rates because they form smaller hibernation groups composed of small clusters
or individual bats. Apparent continental differences in susceptibility of hibernat-
ing bats to deaths associated with skin infection by G. destructans may indicate
either circumstantial or evolved resistance in bats in Europe.
G. destructans has been detected in North America only in states and prov-
inces where WNS has also been observed and in contiguous states. Recent emer-
gence and spread of G. destructans with associated deaths of bats throughout
hibernacula in the northeastern United States (Turner and Reeder, 2009) may
suggest ecologic release of an exotic pathogen into an uninfected ecosystem.
Although this suggestion remains a hypothesis and how G. destructans may have
been introduced to the United States is not known, initial documentation of WNS
in a popular tourist cave near Albany, New York (Blehert et al., 2009), suggests
that a human vector could have been involved.
There are many examples of unintended introductions of fungal pathogens,
particularly of those affecting plants and ectothermic animals with tissue temper-
atures permissive to fungal infection (Casadevall, 2005; Desprez-Loustau et al.,
APPENDIX A 377
2007; Robert and Casadevall, 2009). One case with striking similarities is the
panzootic chytrid fungus (Batrachochytrium dendrobatidis), which has caused
global decreases among amphibian species (Fisher et al., 2009). As with skin in-
fection by B. dendrobatidis in amphibians, which can alter body electrolyte levels
and lead to cardiac arrest (Voyles et al., 2009), skin infection by G. destructans
in hibernating bats may also kill by causing irreversible homeostatic imbalance
because wing membranes play major roles in water balance, circulation, and
thermoregulation of hibernating bats during winter (Davis, 1970; Makanya and
Mortola, 2007).
Bat species in Europe may be immunologically or behaviorally resistant
to G. destructans because of having coevolved with the fungus. Additionally,
microbial flora of bat skin or other abiotic surfaces in bat hibernacula in Europe
may have also coevolved to incorporate G. destructans as a nonpathogenic com-
ponent of the microbial community. Conversely, possible recent introduction of
G. destructans into the United States, with subsequent infection of bat species in
North America and ecosystems not infected with the fungus, provides a potential
explanation for the devastating effects of WNS in North America. Although bats
are reservoirs of various pathogens (Calisher et al., 2006; Wibbelt et al., 2009),
research into the immune function of bats, particularly during hibernation, is just
beginning.
In conclusion, nondetrimental colonization of bat species in Europe by G.
destructans may be relatively common (Figure A20-2), and historical reports
(Feldmann, 1984) suggest that such colonization of hibernating bats in Europe
has occurred for several decades. In contrast to recent mass deaths associated
with G. destructans skin infection, which is the hallmark of WNS in North
America, bats in Europe appear to coexist with G. destructans. Studies to inves-
tigate mechanisms of pathogenesis, microbial ecology, and phylogeography of G.
destructans will be essential for developing a comprehensive understanding of
WNS. In particular, testing the hypotheses that bats in Europe are more resistant
to fungal skin infection by G. destructans, that G. destructans was introduced
from Europe to North America, and that environmental circumstances limit the
pathogenicity of G. destructans in Europe seem warranted. Divergent manifesta-
tions of infection by G. destructans in bats in Europe and North America provide
a unique opportunity to address these research objectives with the ultimate goals
of better understanding WNS and developing sound strategies to manage the
devastating effects of this emerging wildlife disease in North America.
Acknowledgments
We thank N. Jahn, D. Viertel, A. Kus, and C. Kohl for excellent technical
assistance; A. Beck, T. Filip, M. Franz, S. Gloor, R. Güttinger, A. Kiefer, V. Korn,
G. Mäscher, B. Máté, C.D. Morawitz, C. Morris, P. Paulovics, M. Piskorski, F.
Putzmann, W. Schorcht, and C. Tress for help surveying sites and retrieving sam-
378 FUNGAL DISEASES
ples; A. Gargas, K. Schuler, and 2 anonymous reviewers for providing thoughtful

comments on earlier drafts of the manuscript; and M. Riccucci for help with the
literature search for previous reports of fungi in bats in Europe.
This study was supported by Bat Conservation Switzerland.
Dr Wibbelt is a senior veterinary pathologist at the Leibniz Institute for Zoo
and Wildlife Research, Berlin, Germany. Her research interests include infectious
diseases in wild animals, particularly bats.
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380 FUNGAL DISEASES
A21
PAN-EUROPEAN DISTRIBUTION OF WHITE-NOSE

SYNDROME FUNGUS (GEOMYCES DESTRUCTANS)
NOT ASSOCIATED WITH MASS MORTALITY*
Sébastien J. Puechmaille,63,64,65,66 Gudrun Wibbelt,66,67 Vanessa Korn,68
Hubert Fuller,63 Frédéric Forget,69 Kristin Mühldorfer,67 Andreas Kurth,70
Wieslaw Bogdanowicz,71 Christophe Borel,72 Thijs Bosch,73 Thomas Cherezy,74
Mikhail Drebet,75 Támás Görföl,76 Anne-Jifke Haarsma,77 Frank Herhaus,78
Guénael Hallart,79 Matthias Hammer,80 Christian Jungmann,81 Yann Le Bris,82
Lauri Lutsar,83 Matti Masing,84 Bart Mulkens,85 Karsten Passior,86
Martin Starrach,87 Andrzej Wojtaszewski,88 Ulrich Zöphel,89 and
Emma C. Teeling63,64
63â•›â•›School of Biology and Environmental Science, University College Dublin, Dublin, Ireland
64â•›â•›Conway Institute of Biomolecular and Biomedical Research, Dublin, Ireland
65â•›â•›Email: s.puechmaille@gmail.com
66â•›â•›These authors contributed equally to this work.
67â•›â•›Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany
68â•›â•›Office for Faunistic and Landscape Ecology, Schöneberg, Germany
69â•›â•›Plecotus Working Group,Association Natagora, Brussels, Belgium
70â•›â•›Robert Koch Institute, Berlin, Germany
71â•›â•›Museum and Institute of Zoology, Polish Academy of Sciences, Warszawa, Poland
72â•›â•›Commission de Protection des Eaux, du Patrimoine, de l’Environnement, du Sous-sol et des
Chiroptères—Lorraine, Velaine-en-Haye, France

73â•›â•›Dutch Bat Workers Group,Nijmegen, The Netherlands
74â•›â•›Coordination Mammalogique du Nord de la France, Béthune, France
75â•›â•›Podilski Tovtry National Nature Park, Kamenets-Podilsky, Ukraine
76â•›â•›Nature Conservation Foundation of Tolna County, Szekszárd, Hungary
77â•›â•›Centre for Ecosystem Studies, Alterra and Wageningen University, Wageningen, The Netherlands
78â•›â•›Biology Station Oberberg, Nümbrecht, Germany
79â•›â•›Société d’Etude et de Protection de la Nature en Thiérache, Le Chaudron, Origny-en-Thiérache,
France
80â•›â•›Department of Biology, Center for Bat Conservation in Northern Bavaria, Erlangen University,
Erlangen, Germany
81â•›â•›Nature and Biodiversity Conservation Union Rhineland-Palatine, Birkenfeld, Germany
82â•›â•›Bretagne Vivante SEPNB, Roussimel, Glénac, France
83â•›â•›Estonian Fund for Nature, Tartu, Estonia
84â•›â•›Sicista Development Centre, Tartu, Estonia
85â•›â•›Bat Working Group, Natuurpunt VZW, Belgium
86â•›â•›Nature and Biodiversity Conservation Union Southern Lower-Saxony,Nordstemmen, Germany
87â•›â•›Biotope Mapping Cooperation, Herford, Germany
88â•›â•›Institute of Natural Fibres and Medicinal Plants, Poznan, Poland
89â•›â•›Saxonian State Office for Environment Agriculture and Geology, Dresden-Pillnitz, Germany
â•… *Originally printed as: Sébastien Peuchmaille, Gudrun Wibbelt, Vaness Korn, Hubert Fuller,
Frédéric Forget, et al. ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½å°“ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½ï¿½
(2011) Pan-European Distribution of White-Nose Syndrome Fungus (Geomy-
ces destructans) Not Associated with Mass Mortality. PLoS ONE. 6(4): e19167. doi:10.1371/journal.
pone.0019167.
APPENDIX A 381
Abstract
Background:
The dramatic mass mortalities amongst hibernating bats in Northeastern
America caused by ‘‘white nose syndrome’’(WNS) continue to threaten popula-
tions of different bat species. The cold-loving fungus, Geomyces destructans,
is the most likely causative agent leading to extensive destruction of the skin,
particularly the wing membranes. Recent investigations in Europe confirmed
the presence of the fungus G. destructans without associated mass mortality in
hibernating bats in six countries but its distribution remains poorly known.
Methodology/Principal Findings:
We collected data on the presence of bats with white fungal growth in 12
countries in Europe between 2003 and 2010 and conducted morphological and
genetic analysis to confirm the identity of the fungus as Geomyces destructans.
Our results demonstrate the presence of the fungus in eight countries spanning
over 2000 km from West to East and provide compelling photographic evidence
for its presence in another four countries including Romania, and Turkey. Further-
more, matching prevalence data of a hibernaculum monitored over two consecu-
tive years with data from across Europe show that the temporal occurrence of
the fungus, which first becomes visible around February, peaks in March but can
still be seen in some torpid bats in May or June, is strikingly similar throughout
Europe. Finally, we isolated and cultured G. destructans from a cave wall adja-
cent to a bat with fungal growth.
Conclusions/Significance:
G. destructans is widely found over large areas of the European continent
without associated mass mortalities in bats, suggesting that the fungus is native to
Europe. The characterisation of the temporal variation in G. destructans growth
on bats provides reference data for studying the spatio-temporal dynamic of the
fungus. Finally, the presence of G. destructans spores on cave walls suggests that
hibernacula could act as passive vectors and/or reservoirs for G. destructans and
therefore, might play an important role in the transmission process.
Introduction
White nose-syndrome (WNS) is a devastating disease causing mass mortali-
ties in hibernating bats in North-America. In May 2009, it was estimated that
over one million bats had died from the disease which was first documented
in February 2006 at Howe’s Cave, West of Albany, New York (Anonymous,
2009). A visually conspicuous white fungus grows on the face, ears, or wings of
382 FUNGAL DISEASES
stricken bats with hyphae penetrating deep into the connective tissue of glabrous
skin and snout (Meteyer et al., 2009) and causing severe damage (Reichard and
Kunz, 2009). The fungus associated with WNS is a newly described, psychro-
philic (cold-loving) species (Geomyces destructans) (Gargas et al., 2009), closely
related to other psychrophilic species of Geomyces (Rice and Currah, 2006;
Puechmaille et al., 2010). Although it is not yet conclusively proven whether G.
destructans is the causative agent of the disease or if other co-factors are neces-
sary for disease to occur, the fungus is always found on bats at WNS sites where
hibernating bats experience mass mortalities (Blehert et al., 2009). To date, bac-
teriological, virological, parasitological and pathological evaluations as well as
studies of toxic contaminants have not identified the consistent presence of any
other agents/cause of death. The lack of evidence for the involvement of other
agents or compounds reinforces the suspicion that G. destructans is the causative
agent of WNS mortality (Meteyer et al., 2009; Blehert et al., 2009; Kannan et al.,
2010; Courtin et al., 2010).
Geomyces destructans has been found in nine species of bats in North-
America, from the provinces of Ontario and Quebec in Canada south and west
to the states of Tennessee and Oklahoma in the USA (Anonymous, 2010). Three
recent studies investigating samples collected in 2008–2010 have shown that
G. destructans was also present in six European countries (France, Germany,
Switzerland, Czech Republic, Slovakia & Hungary) (Puechmaille et al., 2010;
Martínková et al., 2010; Wibbelt et al., 2010). Nevertheless, the geographic
coverage of these studies was limited and the extent of the distribution of G. de-
structans in Europe remains poorly known. In this paper, we combine previously
published data on the distribution of G. destructans in Europe (Puechmaille et al.,
2010; Martínková et al., 2010; Wibbelt et al., 2010) with new data from twelve
countries covering 2,400 km from West to East (France to Turkey) and 1,900 km
from North to South (Estonia to Turkey) to demonstrate the widespread presence
of G. destructans on multiple species of hibernating bats in Europe without as-
sociated mass mortality.
Results
Review of data on Geomyces destructans in European bats, 2008–2010

Although photographs of bats with fungal growth similar to G. destructans
were published in Germany in the 1980’s (Feldmann, 1825), and also taken in
the 1990’s in the Czech Republic (Martínková et al., 2010), there have been no
confirmed records of G. destructans in Europe prior to 2008 (Puechmaille et al.,
2010; Wibbelt et al., 2010). In 2010, G. destructans has been confirmed by
morphological and genetic analyses from samples collected during the winters
2007/2008, 2008/2009 and 2009/2010 in six European countries (Puechmaille
et al., 2010; Martínková et al., 2010; Wibbelt et al., 2010). In France, Hungary,
APPENDIX A 383
Switzerland and Slovakia, the fungus has been confirmed from 1–2 location(s) per
country, whereas it has been confirmed at 8 sites in Germany and 23 sites in the
Czech Republic (Puechmaille et al., 2010; Martínková et al., 2010; Wibbelt et al.,
2010). All confirmed detections of G. destructans in Europe have been made by
isolating and/or genetically identifying the fungus from hairs, swabs or touch im-
prints from bats (Puechmaille et al., 2010; Martínková et al., 2010; Wibbelt et al.,
2010). In Europe, eight species of Myotis have been observed being colonised by
G. destructans: M. Myotis, M. blythii (referred to as M. oxygnathus in [Wibbelt
et al., 2010]), M. mystacinus, M. daubentonii, M. dasycneme, M. nattereri, M.
bechsteinii and M. brandtii. Species from other bat families were present in the
caves with infected individuals (e.g., Miniopteridae: Miniopterus schreibersii;
Rhinolophidae: Rhinolophus hipposideros and R. ferrumequinum),but no G. de-
structans has been confirmed from these species. Previous extensive surveys
of cave fungi in Europe (i.e., Nováková, 2009; Mosca and Campanino, 1962;
Bastian et al., 2009) or fungi associated with insects hibernating in underground
sites (Kubátová and Dvořák, 2005) never reported G. destructans in their inven-
tory, although some other species of Geomyces were recovered (Nováková, 2009;
Mosca and Campanino, 1962; Bastian et al., 2009).
New data on G. destructans in Europe 2003–2010

During winter hibernation counts, a total of 107 bats from 56 sites in
twelve European countries were reported to have visible white fungal growth
(Tables A21-1, A21-2, A21-3 and Figure A21-1). This represents the first records
from nine countries (Austria, Belgium, Denmark, Estonia, The Netherlands, Po-
land, Romania, Turkey and Ukraine). One hundred and five bats were alive and
two of them were found dead in hibernacula. These 107 bats belonged to eight
different species of Myotis: M. Myotis (59), M. dasycneme (26), M. mystacinus
(9), M. daubentonii (4), M. Myotis/blythii (3), M. blythii (3), M. nattereri (1),
M. escalerai/sp. A (1) and M. brandtii (1). Of these, molecular and morphologi-
cal identifications of the colonising fungus were carried out in 23 cases (Table
A21-1), while only photographic evidence was obtained for a further 50 cases
(Table A21-2 and Figure A21-2). The remaining 34 cases were based on reports
of visual observations of a white fungal growth on bat snouts and/or ears (Table
A21-3), which was very similar to pictures presented in Figure A21-2. All 84 bats
reported in Tables A21-2 and A21-3 are considered as Gd-suspects (bats showing
fungal growth that is thought to be G. destructans).
Geomyces destructans identification

Out of a total of 107 bats with fungal growth, 22 were sampled, 16 with
touch imprints and 6 with cotton swabs. The 22 bats sampled (20 alive and 2
dead) belonged to the species of Myotis from which G. destructans had been pre-
384 FUNGAL DISEASES
TABLE A21-1â•‡ Confirmed Records of Geomyces destructans on Hibernating

Bats in Europe and Details of the Culture and Genetic Analyses
GenBank
Country Lat Lon Date Host species Culture PCR No.
France* 49.9 4.1 04/03/2010 Myotis mystacinus GuH-04032010 -‡ n/a
France* 50.6 2.5 22/02/2010 Myotis nattereri ThC-22022010 -‡ n/a
France† 47.7 –2.1 04/03/2010 Myotis myotis Mmyo-FR-1 + JF502415
Belgium 49.8 5.3 03/04/2010 Myotis myotis Mmyo-BE-1 + JF502414
Belgium† 50.8 5.6 18/03/2010 Myotis mystacinus Mmys-BE-1 + JF502407
Belgium† 50.8 5.6 18/03/2010 Myotis mystacinus n/a + n/a
Netherlands† 52.0 5.8 09/03/2010 Myotis daubentonii Mdau-NL-1 + JF502411
Netherlands 52.1 4.3 27/02/2010 Myotis dasycneme Mdas-NL-1 + JF502410
Germany† 49.7 7.4 10/03/2010 Myotis myotis Mmyo-DE-12 + JF502401
Germany 49.8 9.6 22/03/2010 Myotis myotis Mmyo-DE-14 + JF502403
Germany 51.2 8.1 21/03/2010 Myotis mystacinus Mmys-DE-2 + JF502409
Germany 51.2 8.1 21/03/2010 Myotis mystacinus Mmys-DE-3 + n/a
Germany 51.2 8.1 07/03/2010 Myotis myotis Mmyo-DE-16 + n/a
Germany† 52.3 9.5 23/03/2010 Myotis myotis Mmyo-DE-15 + JF502404
Germany† 52.3 9.4 23/03/2010 Myotis mystacinus Mmys-DE-1 + JF502408
Hungary 47.1 17.6 24/03/2010 Myotis myotis Mmyo-HU-2 + JF502405
Hungary 47.1 17.6 24/03/2010 Myotis myotis Mmyo-HU-3 + n/a
Poland 50.8 16.7 07/03/2010 Myotis myotis Mmyo-PL-1 + JF502413
Estonia#,† 59.3 24.6 01/06/2010 Myotis brandtii EsT-01062010 + JF502412
Ukraine 48.7 26.6 17/03/2010 Myotis myotis Mmyo-UA-1 + JF502406
*Dead bat.
#Environmental sample (individual observed 23/05/2010; see text for further explanations).
†Photograph of the bat shown in Figure A21-2.
‡Samples were negative for G. destructans but amplified another fungus. doi:10.1371/journal.
pone.0019167.t001
viously isolated (see list above). In some cases, we were not able to discriminate
between M. Myotis and M. blythii (referred to as M. Myotis/blythii) as well as
between the newly recognised M. escalerai (Ibañez et al., 2006; Cabrera, 1904)
and Myotis sp. A (Garcia-Mudarra et al., 2009), a yet undescribed cryptic species
from the M. nattereri species complex (Ibañez et al., 2006; Mayer et al., 2007).
Additionally, swab samples were collected from the tunnel wall of an Estonian
hibernaculum. On the 23rd May 2010, a M. brandtii was observed in this hiber-
naculum with white fungal growth on its snout (Figure A21-2A) but no sample
was collected at the time. When the site was revisited for sample collection on the
1st of June 2010, the bat had left the site so samples were collected by swabbing
the wall of the tunnel where the bat was seen nine days before. Four cotton swabs
were used to sample different areas a few centimetres around the location where
APPENDIX A 385
TABLE A21-2â•‡ Suspected Photographic Records of Geomyces destructans on

Hibernating Bats in Europe.
Country Lat. Lon. Date Host species No. Individual
France 44.8 1.6 25/04/2008 Myotis myotis 1
France† 42.6 2.2 26/06/2010 Myotis escalerai/sp.A 1
France† 45.0 2.0 13/02/2010 Myotis myotis 2
France 50.4 3.5 01/03/2008 Myotis mystacinus 1
Belgium 50.8 5.6 09/02/2008 Myotis dasycneme 1
Belgium 50.8 5.6 20/03/2008 Myotis daubentonii 1
Belgium† 50.3 5.9 07/03/2010 Myotis myotis 1
Netherlands 52.1 4.3 26/03/2008 Myotis dasycneme 1
Netherlands 52.0 5.7 04/03/2010 Myotis mystacinus 1
Denmark† 56.4 9.1 14/03/2010 Myotis dasycneme 2
Germany 51.8 10.8 02/02/2008 Myotis myotis 1
Germany† 52.3 9.5 23/03/2010 Myotis mystacinus 1
Germany 52.3 9.5 23/03/2010 Myotis dasycneme 1
Germany 52.1 8.2 21/03/2007 Myotis daubenonii 1
Germany 52.1 8.2 14/03/2007 Myotis dasycneme 1
Austria† 46.8 16.0 07/02/2007 Myotis myotis 1
Hungary 47.1 17.6 24/02/2007 Myotis myotis 1
Hungary 47.1 17.6 23/02/2009 Myotis myotis 1
Hungary 46.2 18.1 03/03/2009 Myotis myotis/blythii 1
Hungary 48.5 20.5 18/02/2010 Myotis blythii 1
Hungary 47.1 17.6 19/02/2010 Myotis blythii 1
Hungary† 47.1 17.6 19/02/2010 Myotis myotis 2
Poland† 50.8 16.7 07/03/2010 Myotis myotis 1
Ukraine† 48.8 26.6 13/02/2010 Myotis myotis 1
Ukraine 48.8 26.6 17/03/2010 Myotis myotis 8
Romania† 46.8 22.6 29/03/2008 Myotis blythii 1
Romania 45.4 25.2 14/03/2009 Myotis myotis/blythii 1
Turkey† 41.9 27.9 22/03/2009 Myotis myotis/blythii 1
†Photograph of the bat shown in Figure A21-2. doi:10.1371/journal.pone.0019167.t002
the bat was observed. The four swabs were then streaked onto four Sabouraud’s
agar plates each and monitored regularly to physically remove any fungal growth
that was not similar to G. destructans. Although the amount of fungi varied per
swab sample, G. destructans was recovered from all four swabs, henceforth con-
sidered as a single sample, bringing the total of samples analysed to 23. No mass
mortality was reported at any of the sites investigated.
386 FUNGAL DISEASES
TABLE A21-3â•‡ Suspected Visual Records of Geomyces destructans on

Hibernating Bats in Europe
Country Lat. Lon. Date Host species No. Individual
Germany 50.9 13.3 23/03/2010 Myotis daubentonii 1
Germany 49.9 7.4 14/03/2010 Myotis Myotis 1
Ukraine 48.8 26.6 17/03/2010 Myotis Myotis 4
Romania 47.0 22.4 08/04/2008 Myotis Myotis 1
doi:10.1371/journal.pone.0019167.t003
Out of 23 samples investigated in the laboratory, 14 of the 16 touch imprint

samples presented characteristic conidia when observed under a microscope and
two of them were doubtful; none of the cotton swabs were inspected under a
microscope prior to culture. Cultures from 22 of these samples were successful.
The two dead bats investigated did not reveal the presence of G. destructans but
other fungal species such as Mucor sp. and Cladosporium sp. were identified
(data not shown).
DNA was isolated from the 22 cultures of which 20 showed morphological
similarity to G. destructans (e.g., curved conidia) and from one touch imprint
with unsuccessful culture attempts. Amplification and sequencing of the internal
transcribed spacer ITS) region (ITS1, 5.8S, and ITS2) was preferred over the
small subunit (SSU) rDNA as it was shown to be more informative and was
comparable to both European (Puechmaille et al., 2010; Wibbelt et al., 2010) and
North American G. destructans (Blehert et al., 2009, Chaturvedi et al., 2010). All
sequences obtained were identical and showed 100% similarity with previously
published ITS sequences of G. destructans available on GenBank (retrieved on
APPENDIX A 387
FIGURE A21-1â•‡ Distribution of confirmed and suspected records of G. destructans on

hibernating bats in Europe. Data Figure A21-1.eps
are presented for genetically confirmed records of G.
bitmap
destructans in red (circles, this study; triangles, published records), photographic evidence
in yellow, visual reports in green. Dead bats from Northern France which culture and
genetic analysis did not reveal the presence of G. destructans are depicted as black dots.
Countries abbreviated names are as follows: AUT: Austria, BEL: Belgium, CHE: Switzer-
land, CZE: Czech Republic, DEU: Germany, DNK: Denmark, EST: Estonia, FRA: France,
HUN: Hungary, NLD: Netherlands, POL: Poland, ROM: Romania, SVK: Slovakia, TUR:
Turkey, UKR: Ukraine. doi:10.1371/journal.pone.0019167.g001.
October 13th) (Puechmaille et al., 2010; Blehert et al., 2009, Wibbelt et al., 2010;
Chaturvedi et al., 2010).
Seasonal distribution of G. destructans

The monitoring of one site over two consecutive winters showed an absence
of Gd-suspect bats from September until the end of January (Figure A21-S1).
The first Gd-suspect bats were reported in February each year (16/02/2007 and
07/02/2008) and their numbers peaked in March (Figure A21-3). In April, the
total number of bats and the number of Gd-suspect bats decreased as bats left
the hibernacula. However, as the number of Gd-suspect bats decreased more
slowly than the total numbers of bats, the highest prevalence was observed in
April (Figure A21-S1). Prevalence varied between years for the same period of
the year and reached values in the range of 18–25% in 2007 (14th–28th March)
388 FUNGAL DISEASES
Figure A21-2.eps
bitmap
APPENDIX A 389
FIGURE A21-2â•‡ (facing page). Photographic evidence showing bats with confirmed or
suspected growth of G. destructans. Photographs of cases confirmed by genetic analysis,
from (A) Estonia (M. brandtii, May 23rd 2010, © L. Lutsar), (B) Poland (M. myotis,
March 7th 2010, © A. Wojtaszewski), (C) Germany (M. myotis, March 10th 2010, © C.
Jungmann), (D) France (M. myotis, March 4th 2010, © Y. Le Bris), (E) Netherlands (M.
daubentonii, March 9th 2010, © T. Bosch), (F) Germany (M. myotis, March 23rd 2010,
© K. Passior) (G) Belgium (M. mystacinus, March 18th 2010, © B. Mulkens), (H) Ger-
many (M. mystacinus, March 23rd 2010, © K. Passior) or bats with white-fungal growth
suspected as G. destructans from (I) Denmark (M. dasycneme, March 14th 2010, © B.
Ohlendorf), (J) Austria (M. myotis, February 2nd 2007, © O. Gebhardt), (K) Hungary (M.
myotis, February 19th 2010, © T. Görföl), (L) Belgium (M. myotis, March 7th 2010, © F.
Forget), (M) France (M. myotis, February 13th 2010, © J. Vittier), (N) Ukraine (M. myotis,
February 13th 2010, © A.-T. Bashta), (O) France (M. escalerai/sp. A, June 25th 2010, © F.
Blanc), (P) Turkey (M. myotis/blythii, March 22nd 2009, © M. Doker), and (Q) Romania
(M. blythii, March 29th 2008, © B. Szilárd).
doi:10.1371/journal.pone.0019167.g002
or 28–55% in 2008 (13th–28th March) when the numbers of Gd-suspect bats

are at the highest. The distributions of reported cases were similar between the
two years, although more cases were reported in April in the winter 2007/2008
(Figure A21-3). In April 2008, the monitoring of three marked bats with white
fungal growth clearly showed that after a bat had changed its position within the
hibernaculum or when it was leaving the hibernaculum, the visible white fungal
growth disappeared (Figure A21-4), most likely as a result of self-grooming.
The temporal distribution of reported cases of live Gd-suspect bats from
throughout Europe (this study, n = 105) was combined with information avail-
able from previously reported cases of G. destructans (Puechmaille et al., 2009;
Wibbelt et al., 2010) (n = 22) to investigate the seasonal variation across multiple
sites in Europe. The temporal range of reported cases of Gd-suspect live bats and
bats confirmed with G. destructans (n = 127) was not evenly distributed through-
out the winter/ spring, with about 2/3rd of the cases reported in March (81/127;
Figure A21-3). The number of reported cases more than doubled between Febru-
ary (30 cases) and March (81 cases). The earliest case was reported on January
17th from Belgium and the three latest cases were observed on May 23rd in
Estonia, in June 24th in the Netherlands and June 25th in France (Tables A21-1,
A21-2, A21-3, Figures A21-2A and A21-2).
390 FUNGAL DISEASES
FIGURE A21-3â•‡ Seasonal changes of the number of live bats reported with white fungal
growth in Europe. The number of bats with visible white fungal growth at an hibernacu-
lum in Germany was monitoredFigureduring theA21-3.eps
winter 2006/2007 (blue line) and the winter
2007/ 2008 (green line). The vertical redbitmap
lines represent the number of Gd-suspect bats (or
confirmed) observed across twelve European countries (n = 127) from 2003 until 2010.
In the X-axis, the thick marks represent the start of each month. doi:10.1371/journal.
pone.0019167.g003
Discussion
Presence of G. destructans in Europe

G. destructans was first identified in Europe in 2008–2009 (Puechmaille et al.,
2009; Wibbelt et al., 2010) but increasing photographic evidence suggest that the
fungus was present in Europe well before this date (this study, [Martínková et al.,
2010; Feldmann 1984]). Most previous studies investigating fungi in European
caves, including bat guano (Nováková, 2009; Mosca and Campanino, 1962;
Groth et al., 1999; Nováková and Kolařik, 2010) reported Geomyces species, but
none had curved conidia so far typical of G. destructans. In the Czech Republic,
APPENDIX A 391
FIGURE A21-4â•‡ Indirect evidenceFigure

of batsA21-4.eps
grooming off G. destructans during hiberna-
tion. Photographic evidence showing three different M. dasycneme individuals (A–B, C–D
bitmap
and E–F) observed at two different dates, first with visible fungal growth (A, C, E) and
later without visible fungal growth (B, D, F). The bat in A–B changed its position within
the hibernaculum whereas the other two (C–D and E–F) were captured when leaving the
hibernaculum (© V. Korn). doi:10.1371/journal.pone.0019167.g004
392 FUNGAL DISEASES
Kubátová & Dvořák (2005) investigated fungi associated with insects hibernating
in underground sites but did not find Geomyces species. To our knowledge, only
one study in Europe has investigated fungi present in bats’ skin and hair samples
where, based on our current knowledge, G. destructans is most likely to be found.
During the winter 1999/2001, Larcher et al. (2003) collected 25 samples of hair
and skin swabs from six species, including three Myotis Myotis, but did not find
any Geomyces species. It is important to note that most fungal cultures have been
carried out at temperatures above 24–25°C, temperatures at which G. destructans
does not grow (Gargas et al., 2009; Chaturvedi et al., 2010), which could explain
why although present, this fungal species had never been reported in Europe
before the study of Puechmaille et al. (2010).
Combining previously published data from France, Germany, Switzerland,
Hungary, The Czech Republic and Slovakia (Puechmaille et al., 2010; Martínková
et al., 2010; Wibbelt et al., 2010), additional data collected from France, Germany
and Hungary (this study), and new data from Belgium, The Nether-lands, Poland,
Estonia and Ukraine (this study), we demonstrate here that G. destructans is
widespread in Europe. We consider the photographic evidence of bats with white
fungus matching the characteristic growth pattern (e.g., Figure A21-2; pictures
from Romania and Turkey) to most likely represent G. destructans, because so far
all tested live European bats with such white fungal growth on their nose, simi-
lar to Figure A21-2, have been confirmed to carry that species of fungus. These
findings further support the fact that G. destructans is widespread across Europe.
However, to confirm the presence of G. destructans in Europe prior to 2008, his-
torical collections of bat specimens (or cave soil samples), especially specimens
collected during the hibernation period, should be screened for the fungus.
As depicted in Figure A21-1, most cases of bats with G. destructans (con-
firmed and suspected) have been found from North-eastern France through Bel-
gium, The Netherlands, Germany and the Czech Republic. However, it is not
clear whether this pattern reflects an actual higher occurrence and/or prevalence
of the fungus in these regions or if it is at least partly due to sampling bias,
whereby the fungus is more likely to be detected in regions with a higher number
of underground sites visited every winter or in regions were the fungus is specifi-
cally sought. In our opinion, it is most likely that this large-scale pattern is due to
a sampling bias. For example, the largest number of sites with G. destructans in
any European country was reported from the Czech Republic (76 localities with
suspected or confirmed G. destructans) were most sites have been searched for
signs of the fungus (>800 hibernacula) (Martínková et al., 2010).
G. destructans growth on bats

The clear seasonal peak in the number of observations of bats with white
fungal growth indicates an increasing prevalence or detectability of G. destruc-
tans as winter passes. This suggests that bats might acquire G. destructans late
APPENDIX A 393
during the hibernation period or that the fungus is carried by bats at the onset of
hibernation but needs time to develop the visible white fungal growth due to the
phenology of the fungus. Therefore, the absence of visible white fungal growth
on bats when observed with the naked eye may not directly reflect the absence
of G. destructans, but rather just the absence of visible fungal colonies. Further
complicating matters, our ability to detect G. destructans growth on bats can
substantially differ with proximity to the bats (i.e., low ceiling versus high ceil-
ing) or the location of the bat (ceiling versus crevices).
Our results confirm the suggestion of Martínková et al. (2010) by showing
that during the hibernation period, bats can remove the fungus from their snout,
ears and wings to a point where the fungus is no longer visible to the naked eye,
although some spores might still be present on their skin. During hibernation,
bats arouse every two weeks on average (Brack and Twente, 1985; Twente et al.,
1985) and if bats consistently groom off the fungus on these occasions, our ability
to visually detect the fungus, if present, will be considerably reduced. We also
showed that towards the end of the hibernation period, bats were emerging from
the hibernaculum without visible signs of the fungus despite showing visible
white fungal growth from two weeks to five days before leaving the hibernacu-
lum. It would be important to investigate whether bats carry spores out of hiber-
nacula and as a result could possibly contaminate maternity roosts and maternity
mates as suggested by Hallam and McCracken (2011).
Factors affecting G. destructans prevalence

Although it is not possible to clearly identify the mechanism responsible
for the sudden increase in the prevalence of G. destructans in late February and
March, these data suggest that shorter winter periods should be associated with
lower prevalence. This prediction seems to hold as in the Mediterranean region,
where hibernation periods are shorter (Rodrigues, 2003), no bats with visually
conspicuous fungal growth have yet been reported during winter cave surveys.
The case reported from Southern France (June 25th 2010, Figure A21-2) was
found in the Pyrenees mountains at ca. 1700 m a.s.l. and hence, is not considered
typical of the Mediterranean climate. It is nevertheless too early to conclude on
this association between G. destructans prevalence and the hibernation duration,
as other factors would need to be considered such as for example, the higher
temperature observed in hibernacula in the Mediterranean region compared to
other regions in Europe (Rodrigues, 2003). Higher temperatures in hibernacula
have been associated with more frequent arousals in Rhinolophus ferrumequinum
(Ransome, 1971; Arlettaz et al., 2000; Park et al., 2000). Considering that this
association holds for other species, as a consequence of more frequent arousals,
bats are expected to groom more often and therefore, reduce the probability of a
visible fungal growth to develop. More surveys and strategic sampling efforts are
needed to uncover whether the length of the hibernation period and/or climatic
394 FUNGAL DISEASES
conditions have a direct or indirect effect on the growth rates, prevalence, and
detectability of G. destructans on bats.
It is crucial that the change in prevalence or detectability over the hiberna-
tion period is considered when comparing prevalence across sites and/or years.
Our results from monitoring one site throughout the hibernation period over
two consecutive years as well as reported cases from multiple sites in multiple
years show that bats with fungal growth are first seen in January, the number of
cases slowly increases into February and peaks in March, then in April when
bats emerge from hibernation it drops again. Our results are in agreement with
recent results from the Czech Republic where in the winter 2009/2010, the num-
ber of sites with bats with white fungal growth increased from 4.1% in January/
February (33/800 sites; regular bat monitoring) to 77.5% in late February/March
(76/98 sites; additional inspections) (Martínková et al., 2010) The Czech study
reported that this increase in G. destructans prevalence was ‘‘suggestive of an epi-
zootic spread of the fungus’’ (Martínková et al., 2010); we propose an alternative
explanation whereby the increase in prevalence of G. destructans in late winter
(March) might regularly (yearly) occur in Europe but has gone unnoticed. Nearly
all hibernation counts in previous years were carried out between December and
mid-February when prevalence/detectability of G. destructans is low, but not in
March (Battersby, 2010) when the prevalence/detectability of G. destructans is at
its highest (Figure A21-3). Although the total numbers of bats in the hibernacula
decreased through April as bats left for the maternity colonies, our results show
that there is a high probability of fungal growth developing on the remaining
individuals. This further supports our hypothesis proposed above and links the
duration of the hibernation period with the prevalence of G. destructans. By
increasing the sample size, some cases might be reported earlier in the hiberna-
tion season or later through the summer, but we expect that the general pattern
observed will not change. Despite these difficulties in assessing the occurrence of
the fungus on bats, our data are consistent with other studies (Puechmaille et al.,
2010; Martínková et al., 2010; Wibbelt et al., 2010), and also demonstrate that
the most commonly encountered bat species with G. destructans in Europe is the
largest species of Myotis on the continent, Myotis myotis. In countries/regions
(i.e., the Netherlands, Northwest Germany) where M. dasycneme is more com-
monly encountered in hibernacula, G. destructans prevalence can reach high lev-
els in that species. It is interesting to note that neither Pipistrellus pipistrellus nor
Miniopterus schreibersii have been observed with G. destructans (Puechmaille
et al., 2010; Martínková et al., 2010; Wibbelt et al., 2010), although these two
species are known to hibernate in aggregations of tens of thousands of individu-
als, especially the latter (Furman and Özgül, 2004; Nagy and Postwana, 2011;
Benda et al., 2003; Serra-Cobo et al., 1998). Although rare, hibernacula of a few
thousands and up to about 34,000 individuals are also known for species of Myo-
tis in Europe (Furman and Özgül, 2004; Nagy and Postwana, 2011; Kokurewicz,
2009; Arthur and Lemaire, 2009; Sachanowicz et al., 2006; Dietz et al., 2009).
APPENDIX A 395
G. destructans outside of the hibernation period

We observed three individual bats with white fungal growth around their
nose (one confirmed as G. destructans) from May and June, when they were still
torpid in cold underground sites. This represents the first mention of individu-
als with G. destructans colonisation outside of the hibernation period and raises
questions about the role of these individuals in the persistence of the fungus in bat
populations. Pipistrellus pipistrellus During the summer period, while females ag-
gregate in colonies to raise their young, it remains largely unknown where males
are roosting (e.g., Senior et al., 2005). Furthermore, during the swarming season
in late summer/autumn, large numbers of individuals aggregate in caves, mines
or tunnels and come in close contact with each other (chasing, mating) (Senior
et al., 2005; Parsons and Jones, 2003; Parsons et al., 2003a; Parsons et al., 2003b;
Rivers et al., 2006; Rivers et al., 2005), which could represent an opportunity for
G. destructans to be transmitted between individuals.
We isolated G. destructans from the environment surrounding hibernating
bats. The presence of viable spores of G. destructans on the surfaces of hiberna-
tion sites has huge implications for the understanding of disease transmission
mechanisms and disease modelling (Hallam and McCracken, 2011) It seems
likely that cave walls could serve as a passive vector and/or reservoir for G. de-
structans spores. It is not yet known how long these spores can remain viable but
fungal spores generally remain viable for extended periods. Bats entering these
sites in autumn (for swarming and/or hibernation) could become contaminated
with spores of G. destructans left from bats infected during the previous winter.
In North-America, Lindner et al. (2010) successfully amplified ITS sequences
identical to G. destructans DNA from soil samples collected during the winter
2008–2009 at three bat hibernacula and stressed the importance of considering
the environment as a reservoir for G. destructans and in the dynamics of WNS
transmission. Our results confirm this and further suggest that more work is
needed to understand the persistence of G. destructans on hibernacula walls
(reservoir or passive vector) where they are in physical contact with bats.
Insights into the origin of G. destructans and WNS

The wide distribution of G. destructans in Europe and the absence of associ-
ated mortality supports the hypothesis that G. destructans has co-evolved with
European bats and only recently arrived in North America where it is causing
unprecedented mass mortalities (Puechmaille et al., 2010; Blehert et al., 2009;
Martínková et al., 2010; Wibbelt et al., 2010). Alternatively, G. destructans could
have been present on both continents and a virulent strain could have evolved
in North-America. Until the relationships between G. destructans populations
across continents are clarified, precautions should be taken to minimise the
chances of transcontinental movement of viable G. destructans (Puechmaille et
al., 2011).
396 FUNGAL DISEASES
During the two years monitoring at one site in Germany where G. destructans
prevalence reached high levels in March-April, not a single dead bat was found.
This is in agreement with previous studies (Puechmaille et al., 2010; Martínková
et al., 2010; Wibbelt et al., 2010) reporting that the presence of G. destructans
in bats from Europe is not associated with mass mortality. This sharply contrasts
with mass mortalities reported in North America where hundreds or thousands
of dead bats are found in hibernacula towards the end of the hibernation period.
Recent pathological investigations of bats dying from WNS in North America led
Cryan et al. (2010) to propose that mortality was caused by important disruptions
of wing-dependant physiological functions due to infection by G. destructans. In
North America, the fungus deeply invades wings tissues (Meteyer et al., 2009)
and causes damages that are thought to alter homeostasis and water balance,
resulting in more frequent arousals than bats can afford with their fat reserves,
leading to death by starvation (Cryan et al., 2010). The pathology associated with
G. destructans colonisation in Europe is not yet known. We believe that the first
step in understanding mortality differences between bats from Europe and North
America rely on understanding pathological differences incurred by the fungus
on the bats’ wings. As a result, we urge the necessity to carry out pathological
investigation of live bats from Europe colonised by G. destructans. Despite the
absence of mortality associated with the presence of G. destructans in Europe,
it would be necessary to investigate whether chronic infections with the fungus
are compromising the health of individuals, especially in M. Myotis and M.
dasycneme, which show high prevalence of the fungus towards the end of the
hibernation period.
Phylogeographic studies of European bat species have shown that in the
last 100,000 years, some species colonised Europe from Western Asia (Flanders
et al., 2009), including Myotis blythii (Berthier et al., 2006; Currat et al., 2008)
which has been found with G. destructans (Wibbelt et al., 2010). Assuming that
G. destructans can be transported over long distances by bats, we speculate that
the distribution of G. destructans is probably not limited to Europe and possibly
extends eastwards into Russia, Western and Central Asia. Further surveys are
necessary to clarify the global distribution of G. destructans.
Conclusions
We have shown here that G. destructans, the most likely causative agent of
WNS in North America, is widespread in Europe, but is not associated with mass
mortality. The prevalence of visible fungal growth on bats increases in February/
March before sharply decreasing when bats emerge from hibernation. We also
isolated viable G. destructans from the walls of an underground site suggesting
that the hibernacula could act as passive vectors and/ or reservoirs for G. de-
structans and therefore, might play an important role in the transmission process.
Further research is needed to clarify the global prevalence of G. destructans and
APPENDIX A 397
identify variables (e.g., temperature, humidity and hibernation length) explaining

regional differences. Finally, further research is needed in different parts of the
globe, especially temperate region of the Northern and Southern hemispheres, to
precisely determine the global distribution of G. destructans.
Materials and Methods
Sample collection
During ongoing population censuses carried out at hibernacula in different
countries across Europe and during additional hibernacula surveys carried out for
the purpose of this study, information on bats with visible white fungal growth
on snouts and/or ears was recorded. Whenever possible, sterile dry cotton swabs
(Puechmaille et al., 2010) or adhesive tape touch imprints (Wibbelt et al., 2010)
were used to collect fungal material from the bats. In Estonia, samples were
collected from the wall of the tunnel where a bat with characteristic white fun-
gus was observed nine days prior to the sampling. Where no sample collection
was possible, a photograph was taken of the bat (photographic record). In cases
where neither sample collection nor photographic evidence was obtained, the
record was classified as visual observation. Live hibernating bats with powdery,
white fungal growth on their noses were considered suspects of infection by G.
destructans (Gd-suspects) but not suspected of having WNS. There is presently
no data supporting the occurrence of WNS in Europe and the co-occurrence of
the fungus with lesions characteristic of WNS (Meteyer et al., 2009) has not (yet)
been reported in Europe (Wibbelt et al., 2010; Barlow et al., 2005). Although,
prevalence of G. destructans can reach high levels in some European species
(i.e., Myotis myotis, M. dasycneme) in late winter (especially in March), it can
be expected that by chance alone some bats dying from causes unrelated to the
presence of G. destructans will also be carrying the fungus. Unless the criteria
for the diagnosis of WNS are met (confirmation by histo-pathology and PCR)
(Meteyer et al., 2009) WNS should not be assumed as a cause of mortality in dead
bats found in hibernacula of Europe. Various species of fungi have been identified
on dead bats (Wibbelt et al., 2010; Voyron et al., 2011), most of them likely being
saprophytes that colonise bat carcasses post-mortem.
Fungal cultures
In the laboratory, samples were treated as in Puechemaille et al. (2010) for
swabs and following Wibbelt et al. (2010) for touch imprints. Briefly, swabs
were streak-plated onto plates of Sabouraud’s agar, supplemented with 0.1%
mycological peptone. For touch imprints, small areas with fungal conidia char-
acteristic of G. destructans were identified by light microscopy and the tape was
disinfected and excised before being transferred for culture to Sabouraud’s agar.
398 FUNGAL DISEASES
The plates were sealed with parafilm and incubated inverted in the dark at 10°C.
A fungal growth developed within 14 days, from which single spore cultures
were established.
Molecular identification
Each culture was sequenced for one molecular marker, the rRNA gene
internal transcribed spacer (ITS, ca. 930 bp.) region (ITS1, 5.8S, and ITS2) to
further confirm species identity. The DNA extraction, PCR amplification and
DNA sequencing followed protocols described in Puechmaille et al. (2010).
Briefly, DNA was extracted using the Qiagen Blood and Tissue kit following the
manufacturer’s instructions with slight modifications (after step 3, we added an
incubation time of 10 minutes at 70°C). PCR reactions were carried out in 25
mL containing 1 mL of DNA extract (at 10–75 ng/mL), 1.5 mmol/L MgCl2, 0.4
mmol/L each primer (Forward: ITS4, 5′-TCCTCCGCTTATTGATATGC – 3′;
Reverse: ITS5, 5′-GGAAGTAAAAGTCGTAACAAGG – 3′; (White et al., 1990),
0.2 mmol/L dNTP, 1x PCR buffer and 1 U Platinum Taq DNA Polymerase High
Fidelity (Invitrogen). PCR cycling conditions were: initial step 15′ at 95°C, then
10 cycles of 30″ at 95°C, 1′45″ at 60°C (reduce of 2°C every 2 cycles), 1′ at
72°C, following by 30 cycles of 30″ at 95°C, 1′45″ at 50°C and 1′ at 72°C. PCR
products were purified and sequenced by Macrogen Inc. (Seoul, Korea) in both
directions using the PCR primers. Complementary sequences were assembled
and edited for accuracy using CodonCode Aligner 3.0.3 (www.codoncode.com/
aligner/download.htm).
Monitoring of visible fungal growth on bats

One site situated in Northwest Germany (Latitude: 52.1; Longitude: 8.2) near
the city of Osnabrück was monitored over two consecutive winters, 2006/2007
(5th September until 19th May) and 2007/2008 (28th August until 23rd April).
The monitoring consisted of counting the total number of bats at the site as well
as the number of bats with visible white fungal growth similar to the pictures
presented in Figures A21-2 and A21-4. The counts were done by the same person
(V. Korn) every 4 days on average during the first year and every 2.5 days on
average during the second year. The procedures complied with guidelines of the
American Society of Mammalogists and were carried out under permit number
FBD7.2 60 from the Administration of the County of Osnabrück, Department of
Environment.
APPENDIX A 399
Supporting Information
FIGURE A21-S1â•‡ Monitoring of bats at an hibernaculum in Germany during (A) the win-
ter 2006/2007 (September 5th Figure
2006 untilA21-S1.eps
April 19th 2007) and (B), the winter 2007/2008
bitmap
(August 28th 2007 until April 23rd 2008). The blue line represents the total number of bats
counted whereas the green line represents the number of bats with visible white fungal
growth (Gd-suspects). Dotted vertical lines separate counts from each month. Note that
the number of counts per month was not equal between months. In (B), the black line rep-
resents the total number of bats counted whereas the blue line represents the total number
of bats bar one portion of the hibernaculum where bats grouped densely (ca. 20 individu-
als) and did not allow a reliable identification of the number of bats with white fungal
growth. The green line represents the number of bats with visible white fungal growth
(Gd-suspects) counted at the hibernaculum without considering individuals densely group-
ing at one place in the hibernaculum. The group of about 20 individuals formed while the
hibernaculum was partially flooded, likely as a result of bats changing position to avoid
drowning. Note that the right Y-axis scale is different between (A) and (B).
400 FUNGAL DISEASES
Acknowledgments
We would like to thank Dóczy Annamária, Andriy-Taras Bashta, Frédéric
Blanc, Sándor Boldogh, Gaby Bollen, Thomas Chatton, Emrah Coraman, Jére
Csaba, Simon Dutilleul, Mehmet Doker, Oliver Gebhardt, Lena Godlevska, René
Janssen, Daniel Lefèvre, Barti Levente, Vadim Martyniuk, Gerhard Mascher,
Mykola Matveev, Bernd Ohlendorf, Rian Pulles, Tony Rock, Wolfgang Rackow,
Sébastien Roué, Bücs Szilárd, Abigel Szodoray-Parádi, Farkas Szodoray-Parádi
and Julien Vittier for providing us with their field observations. The comments of
Paul Cryan, Paul Racey, Natalia Martínková and an anonymous reviewer helped
to improve a previous version of the manuscript.
Author Contributions
Conceived and designed the experiments: SJP GW VK ECT. Performed the
experiments: SJP GW HF VK KM AK. Analyzed the data: SJP GW. Contributed
reagents/materials/analysis tools: SJP GW VK HF KM AK FF WB CB TB TC
MD TG AJH FH GH MH CJ YLB LL MM BM KP MS AW UZ ECT. Wrote the
paper: SJP GW.
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Appendix B
Agenda
Fungal Diseases: An Emerging Challenge to

Human, Animal, and Plant Health
December 14–15, 2010
Keck Building, Room 100
500 Fifth Street, NW
Washington, DC
DAY 1: DECEMBER 14, 2010
8:00–8:30: Registration and Continental Breakfast
8:30–8:45: Welcoming Remarks

David A. Relman, M.D., Chair, and James M. Hughes, M.D.,
Vice Chair, Forum on Microbial Threats
8:45–10:15: Keynote Remarks

James M. Hughes, Moderator
8:45–9:30: The “Good,” the “Bad,” and the “Ugly”: Fungi Mold Your
World
Meredith Blackwell, Ph.D., Louisiana State University
403
404 FUNGAL DISEASES
9:30–10:15: Emerging Fungal Pathogens—Past, Present, and Future

Arturo Casadevall, M.D., Ph.D., Albert Einstein College of
Medicine
10:15–10:45: Discussion
10:45–11:00: Break
Session I
Environmental Factors Influencing the Emergence
and Spread of Fungal Diseases
Moderator: Steven Brickner, Ph.D.
11:00–11:30: Climate, Extreme Weather Events, and Fungal Disease

Emergence and Spread
Compton (Jim) Tucker, Ph.D., NASA Goddard Space Flight
Center
11:30–12:00: Trade-Mediated Spread of Fungal Disease and Resulting Shifts

in Amphibian Conservation Status and Practices
Ché Weldon, Ph.D., North-West University
12:00–12:30: Weather, Globalization, and Trade—Impacts on Invasion and

Dispersal of Fungal Plant Pathogens
Jim Stack, Ph.D., Kansas State University
(for Michael Jeger, Ph.D., Imperial College London)
12:30–1:00: Geography, Climate, Dust, and Disease—Epidemiology of

Valley Fever (Coccidioides Spp.) and Ways It Might Be
Controlled
John Galgiani, M.D., University of Arizona
1:20–2:00: Lunch
APPENDIX B 405
Session II
Case Studies of Emerging Fungal Diseases
of Humans, Animals, and Plants
Moderator: Erica Rosenblum, Ph.D.
2:00–2:30: Predicting and Mitigating Mycoses by Merging Deep

Sequencing with Global Mapping Projects (Amphibian
Chytridiomycosis)
Matthew Fisher, Ph.D., Imperial College London
2:30–3:00: Bat White-Nose Syndrome: An Emerging Fungal Pathogen in

New World Bats
David Blehert, Ph.D., National Wildlife Health Center, U.S.
Geological Survey
3:00–3:30: Knowing Where to Look—Environmental Sources of

Cryptococcal Disease in Human and Animal Residents in the
Pacific Northwest
Karen Bartlett, Ph.D., University of British Columbia
3:30–3:45: Break
3:45–4:15: Emergence of Phytophthora ramorum in Europe and North

America
David Rizzo, Ph.D., University of California–Davis
4:15–4:45: Rapid Global Spread of Aggressive Strains of Puccinia

striiformis on Wheat—Origins, Causes, and Consequences
Mogens Støvring Hovmøller, Ph.D., Aarhus University
5:15–6:00: Open Discussion of Day One
6:00: Meeting Adjourns

406 FUNGAL DISEASES
DAY 2: DECEMBER 15, 2010

8:30–9:00: Continental Breakfast
9:00–9:15: Summary of Day One: David A. Relman, M.D., Chair, Forum

on Microbial Threats
Session III
Host and Pathogen Factors Influencing the
Emergence and Spread of Fungal Diseases
Moderator: Jeff Duchin, M.D.
9:15–9:45: Fungal Pathogenesis in Plants and Animals: Similarities and

Differences
Barbara Howlett, Ph.D., University of Melbourne
9:45–10:15: Sexual Reproduction, Evolution, and Adaptation of C. gattii in

the Pacific Northwest
Joseph Heitman, M.D., Ph.D., Duke University
10:15–10:30: Break
10:30–11:00: Genetic Factors and Immune Responses to Fungal Infection in

Health and Disease
Steve Holland, M.D., National Institute of Allergy and
Infectious Diseases
11:00–11:30: Host–Pathogen Dynamics of Amphibian Chytridiomycosis: The

Role of the Skin Microbiome in Health and Disease
Vance Vredenburg, Ph.D., San Francisco State University
11:30–12:00: Geomyces destructans in Old World and New World Bats

Gudrun Wibbelt, D.V.M., Leibniz Institute for Zoo and Wildlife
Research
12:30–1:15: Lunch
APPENDIX B 407
Session IV
Surveillance, Detection, and Response
Moderator: Steve Morse, Ph.D.
1:15–1:45: Informal Surveillance Networks for Diseases of Humans, Plants,

and Animals
Larry Madoff, M.D., Massachusetts Department of Public
Health & University of Massachusetts Medical School
1:45–2:15: Local and National Detection Capability and Public Health

Responses for C. gattii
Julie Harris, Ph.D., Centers for Disease Control and Prevention
2:15–2:45: Global Capacity for Coordinated Surveillance, Detection, and

Response to Emerging Diseases of Wildlife
Peter Daszak, Ph.D., EcoHealth Alliance
2:45–3:15: Wildlife Conservation at the Smithsonian Conservation Biology

Institute
Luis Padilla, D.V.M., Smithsonian Conservation Biology
Institute
3:15–3:45: Break
3:45–5:15: Panel and Open Discussion

Speakers: Peter Daszak, Julie Harris, Larry Madoff, and Luis
Padilla
Discussants: David Blehert, Jacque Fletcher, and Kevin Russell
5:15–5:30: Closing Remarks

James M. Hughes and David A. Relman
5:30: Meeting Adjourns

Appendix C
Acronyms
AARK Amphibian Ark

ACAP Amphibian Conservation Action Plan
AFTOL Assembling the Fungal Tree of Life program
AM Arbuscular mycorrhizae
APHIS Animal and Plant Health Inspection Service
B.C. British Columbia

BCCDC British Columbian Centre for Disease Control
Bd Batrachochytrium dendrobatidis
BGRI Borlaug Global Rust Initiative
BWNS bat white-nose syndrome
CANV Chrysosporium anamorph of Nanniziopsis vriesii

CBD Convention on Biological Diversity
CDC Centers for Disease Control and Prevention
CFU colony-forming unit
CGB canavanine-glycine-bromothymol blue
CGD chronic granulomatous disease
CGIAR Consultative Group for International Agricultural Research
CIMMYT International Maize and Wheat Improvement Center
CITIES Convention for International Trade of Endangered Species
DNA deoxyribonucleic acid

DoD Department of Defense
409
410 FUNGAL DISEASES
DoD-GEIS Department of Defense-Global Emerging Infections

Surveillance and Response System
DOI Department of the Interior
DTRA Defense Threat Reduction Agency
EID emerging infectious disease

ELISA enzyme-linked immunosorbent assay
ENSO El Niño-Southern Oscillation
EUS epizootic ulcerative syndrome
FAO Food and Agriculture Organization of the United Nations

FDA Food and Drug Administration
FWS Fish and Wildlife Service
GAA Global Amphibian Assessment

GAO Government Accountability Office
GHI Global Health Initiative
GOARN Global Outbreak and Response Network
GPHIN Global Public Health Intelligence Network
GPM Gaussian plume model
GRACE Gravity Recovery and Climate Experiment
GRC-YR Global Reference Center for yellow rust
GRRC Global Rust Reference Center
HIV/AIDS human immunodeficiency virus/acquired immune deficiency

syndrome
ICARDA International Center for Agricultural Research in the Dry Areas

ICRISAT International Crops Research Institute for the Semi-Arid Tropics
IHR International Health Regulations
IOM Institute of Medicine
IRIS immune reconstitution inflammatory syndrome
IRRI International Rice Research Institute
ITS internal transcribed spacer
IUCN International Union for the Conservation of Nature
MIC minimum inhibitory concentration

MLST multilocus sequencing typing
NAS National Academy of Sciences

NASA National Aeronautics and Space Administration
NGO non-governmental organization
NIAID National Institute of Allergy and Infectious Diseases
APPENDIX C 411
NIH National Institutes of Health

NLM National Library of Medicine
NOAA National Oceanographic and Atmospheric Administration
NSF National Science Foundation
NWHC National Wildlife Health Center
OIE World Organisation for Animal Health

OSTP Office of Science and Technology Policy
PAMP pathogen-associated molecular pattern

PCR polymerase chain reaction
PNW Pacific Northwest
ProMED Program for Monitoring Emerging Diseases
RACE Risk Assessment of Chytridiomycosis to European Amphibian

Diversity
RVF Rift Valley fever
SCBI Smithsonian Conservation Biology Institute

SCLB Southern corn leaf blight
SOD sudden oak death
SSC Species Survival Commission
TB tuberculosis
TGen Translational Genomics Research Institute
U.K. United Kingdom

U.N. United Nations
USAID U.S. Agency for International Development
USDA U.S. Department of Agriculture
USGS U.S. Geological Survey
WHO World Health Organization

WNS white-nose syndrome
WTO World Trade Organization
Appendix D
Glossary
Abiotic: Nonliving chemical and physical factors in an environment.
Adaptive immune response: Response of the vertebrate immune system to a

specific antigen that typically generates immunological memory.
Aggressive: In plant pathology, the quality of being able to cause more disease
more quickly on susceptible host plants.
Anthropogenic: Caused or produced by humans.
Antibiotic: Class of substances that can kill or inhibit the growth of some groups
of microorganisms. Used in this report to refer to chemicals active against bacte-
ria. Originally antibiotics were derived from natural sources (e.g., penicillin from
molds), but many currently used antibiotics are semi-synthetic and modified with
additions of manmade chemical components.
Antibody therapy: Any therapeutic intervention in which a monoclonal or other

concentrated antibody is used to manage a condition, such as cancer or severe
infection.
Antifungal: Substances that can kill or inhibit the growth of fungal organisms.
Ascomycete: Any of various fungi belonging to the phylum Ascomycota, charac-

terized by the presence of sexually produced spores formed within an ascus. Like
413
414 FUNGAL DISEASES
most fungi, ascomycetes also reproduce asexually by the formation of nonsexual

spores called conidia at the ends of filaments known as hyphae.
Assembling the Tree of Life program: This National Science Foundation pro-
gram was created in 2004 with the goal of constructing the evolutionary history
for all major lineages of life. The program supports efforts to classify all major
groups of organisms and to reveal the pattern of historical relationships that
would explain similarities and differences among them.
The program has three goals: (1) Creation and support of multidisciplinary
teams of investigators to acquire and integrate molecular and morphological
evidence on both extant and extinct organisms in order to resolve phylogenetic
relationships of large, deep branches of the Tree of Life; (2) Research and de-
velopment of tools for computational phylogenetics and phyloinformatics to
improve assessment, predictive capabilities, and the visualization and navigation
of the hierarchical structure in the Tree of Life; andÂ€(3) Outreach and education
in comparative phylogenetic biology and paleontology.
Asymptomatic: Presenting no symptoms of disease.
Asymptomatic carriers: A person or animal that has contracted an infectious

disease, but displays no symptoms yet has the ability to transmit it to others.
Bacteria: Microscopic, single-celled organisms that have some biochemical and

structural features different from those of animal and plant cells.
Basidiomycetes: Any various fungi belonging to the phylum Basidomycota,

bearing sexually produced spores on a basidium. All hyphae of basidiomycetes
are divided into segments by septa and go through three stages of development.
Biofuel: Fuel produced from renewable resources, especially plant biomass,

vegetable oils, and treated municipal and industrial wastes.
Biological invasion: The process by which species (or genetically distinct popu-
lations), with no historical record in an area, breach biogeographic barriers and
extend their range.
Bioluminescence: Light produced by a chemical reaction that originates in an

organism.
Biota: The animal and plant life of a given region.
Chronic: Relating to an illness or a medical condition that is characterized by

long duration or frequent recurrence.
APPENDIX D 415
Climate: Average meteorological conditions over a specified time period, usually

at least a month, resulting from interactions among the atmosphere, oceans, and
land surface. Climate variations occur over a wide range of spatial and temporal
scales.
Climate change: A change of climate that is attributed directly or indirectly to

human activity and alters the composition of the global atmosphere; this happens
in addition to natural climate variability observed over comparable time.
Colonize: The spreading of a species into a new habitat.
Colony collapse disorder: A syndrome characterized by the disappearance of all

adult honey bees in a hive while immature bees and honey remain.
Colony-forming unit: A standard unit of measurement for environmental sam-

pling. Colonies reflect the number of “viable” organisms (i.e., organism ca-
pable of forming colonies when provided with nutritional elements necessary
for growth).
Commensals: Organisms in a mutually symbiotic relationship where both live

peacefully together while not being completely dependent on one another (e.g.,
the gut microbiome).
Communicable disease: An infectious disease transmissible (as from person

to person) by direct contact with an affected individual or the individual’s dis-
charges or by indirect means (as by a vector).
Conidia: Asexually produced fungal spore. Most conidia are dispersed by the
wind and can endure extremes of cold, heat, and dryness. When conditions are
favorable, they germinate and grow into hyphae.
Contagious: Capable of being transmitted by direct or indirect contact, as an

infectious disease.
Corn Belt: The area in the Midwestern United States—roughly covering west-
ern Indiana, Illinois, Iowa, Missouri, eastern Nebraska, and eastern Kansas—in
which corn (maize) and soybeans are the dominant crops.
Cultivar: A variety of a plant that has been created or selected intentionally and
maintained through cultivation.
Detection: The act of discovering a novel, emerging, or reemerging disease or

disease event.
416 FUNGAL DISEASES
Diagnosis: The identification of a condition, disease, or injury made by evaluat-

ing the symptoms and signs presented by an individual.
Dimorphic: The existence of two distinct types of individuals within a species,

usually differing in one or more characteristics such as coloration, size, and
shape.
Disease: A situation in which infection has elicited signs and symptoms in the
infected individual; the infection has become clinically apparent. Some exposures
to infectious disease-causing agents can also produce asymptomatic illnesses that
can be spread to others.
Disease burden: The impact of a health problem in an area measured by financial

cost, mortality, morbidity, or other indicators. It is often quantified in terms of
quality-adjusted life years (QALYs) or disability-adjusted life years (DALYs),
which combine the burden due to both death and morbidity into one index.
Ecosystem: A community of organisms together with their physical environment,

viewed as a system of interacting and interdependent relationships and including
such processes as the flow of energy through trophic levels and the cycling of
chemical elements and compounds of the system.
Ecosystem services: Benefits derived from a multitude of resources and pro-

cesses that are supplied by natural ecosystems (e.g., decomposition of waste,
food, energy, nutrient dispersal, and cycling).
Emerging infectious disease: Infections that are rapidly increasing in incidence

or geographic range.
Emigration: To leave one’s usual country of residence to settle in another.
Endemic: Present in a community or common among a group of people; said of

a disease prevailing continually in a region.
Endophytes: Fungi that live inside the plant tissue, but without causing any
obvious negative effects.
Endosymbiont: An organism that lives inside another organism, most often

for the benefit of the two (e.g., rhizobia [nitrogen-fixing soil bacterial] that live
within root nodules—rhizobia cannot independently fix nitrogen, but need the
plant as an energy source; in turn, rhizobia supply the plant host with ammonia
and amino acids).
APPENDIX D 417
Environmental microbe: Microbe acquired from the environment (in contrast

to acquisition from other living hosts).
Enzootic: A disease of low morbidity that is constantly present in an animal

community.
Enzyme: Any of numerous proteins produced in living cells that accelerate or

catalyze the metabolic processes of an organism.
Epidemic: The condition in which a disease spreads rapidly through a com-

munity in which that disease is normally not present or is present at a low level.
Epidemiology: Study of the distribution and determinants of health-related states

or events in specified populations. Epidemiology is the basic quantitative science
of public health.
Epizootic: A disease of high morbidity that is only occasionally present in an

animal community.
Eradication: Reduction of the worldwide incidence of a disease to zero as a

result of deliberate efforts.
Etiologic agent: The organism that causes a disease.
Etiological: Of or pertaining to causes or origins.
Etiology: Science and study of the causes of diseases and their mode of operation.
Eukaryotic organism: One of the three domains of life. The two other domains,
Bacteria and Archaea, are prokaryotes and lack several features characteristic of
eukaryotes (e.g., cells containing a nucleus surrounded by a membrane and whose
DNA is bound together by proteins [histones] into chromosomes). Animals,
plants, and fungi are all eukaryotic organisms.
Expression vectors: A plasmid that is used to introduce a specific gene into a tar-
get cell. Once the expression vector is inside the cell, the protein that is encoded
by the gene is produced by the cellular-transcription and translation machinery
ribosomal complexes. The plasmid is frequently engineered to contain regula-
tory sequences that act as enhancer and promoter regions and lead to efficient
transcription of the gene carried on the expression vector.
Extreme weather: Refers to weather phenomena that are at the extremes of the
historical distribution and are rare for a particular place and/or time, especially
418 FUNGAL DISEASES
severe or unseasonal weather. Such extremes include severe thunderstorms, se-

vere snowstorms, ice storms, blizzards, flooding, hurricanes, high winds, and
heat waves.
Fermentation: The process by which complex organic compounds, such as glu-

cose, are broken down by the action of enzymes into simpler compounds without
the use of oxygen.
Food security: The availability of food and one’s access to it. A household
is considered food secure when its occupants do not live in hunger or fear of
starvation.
Fungi/fungal/fungus: For the purposes of this publication, the terms fungi, fun-
gal, and fungus are used inclusively to describe all organisms traditionally studied
by mycologists—including species that are now excluded from Kingdom Fungi
(e.g., Phytophthora ramorum and Phytophthora infestans) or whose relationship
to the fungal kingdom has yet to be determined (e.g., the microsporidia Nosemus
spp.).
Genome: The complete genetic composition of an organism (e.g., human, bac-

terium, protozoan, helminth, fungus), contained in a chromosome or set of chro-
mosomes or in a DNA or RNA molecule (e.g., a virus).
Genomics: The study of all the genes in a person, as well as interactions of those
genes with each other and with that person’s environment.
Genotype: The genetic makeup of an organism as distinguished from its physical

characteristics.
Genus: A group of species with similar characteristics that are closely related.
Germinate: The beginning of growth, as of a seed, spore, or bud.
Globalization: The increased interconnectedness and interdependence of peoples

and countries. It is generally understood to include two interrelated elements:
(1) the opening of borders to increasingly fast flows of goods, services, finance,
people, and ideas across international borders, and (2) the changes in institutional
and policy regimes at the international and national levels that facilitate or pro-
mote such flows.
Haploid: Having a single set of each chromosome in a cell or cell nucleus. In

most animals, only the gametes are haploid.
APPENDIX D 419
Hibernacula: A protective case, covering, or structure, such as a cave, in which

an organism remains dormant for the winter.
Host: Animal or plant that harbors or nourishes another organism.
Hyphae: Slender tubes that develop from germinated spores and form the struc-
tural parts of the body of a fungus. A large mass of hyphae is known as a myce-
lium, which is the growing form of most fungi.
Immunocompetence: The ability of the immune system to respond appropriately

to an antigenic stimulation.
Immunocompromised: A condition (caused, e.g., by the administration of im-

munosuppressive drugs or irradiation, malnutrition, aging, or a condition such
as cancer or HIV disease) in which an individual’s immune system is unable to
respond adequately to a foreign substance.
Incidence: As used in epidemiology, the number of new cases of a disease

that occur in a defined population within a specified time period; the rate of
occurrence.
Incubation period: The time from the moment of inoculation (exposure to the
infecting organism) to the appearance of clinical manifestations of a particular
infectious disease.
Infection: The invasion of the body or a part of the body by a pathogenic agent,
such as a microorganism or virus. Under favorable conditions the agent develops
or multiplies; the results may produce injurious effects. Infection should not be
confused with disease.
Innate immune response: Immune response (of both vertebrates and inverte-
brates) to a pathogen that involves the preexisting defenses of the body, such as
barriers formed by skin and mucosa, antimicrobial molecules, and phagocytes.
Such a response is not specific for the pathogen.
Inoculum: Collective term for microorganisms or their parts (spores, mycelial

fragments, etc.), which are capable of infection or symbiosis when transferred
to a host.
Internal transcribed spacer sequences: Internal transcribed spacer sequences

are sections of non-functional RNA that are highly variable, even between closely
related species, and are widely used for taxonomy purposes.
420 FUNGAL DISEASES
International Health Regulations (IHR): An international legal instrument that

is binding on 194 countries across the globe, including all the Member States
of the World Health Organization (WHO). Their aim is to help the international
community prevent and respond to acute public health risks that have the poten-
tial to cross borders and threaten people worldwide.
The IHR, which entered into force on June 15, 2007, requires countries to
report certain disease outbreaks and public health events to WHO. Building on
the unique experience of WHO in global disease surveillance, alert, and response,
the IHR define the rights and obligations of countries to report public health
events, and establish a number of procedures that WHO must follow in its work
to uphold global public health security.
Invasive species: Non-native plants and animals that, when introduced to new
environments, reproduce or spread so aggressively that they harm their adopted
ecosystems. Also called: exotic, alien, and non-indigenous species.
Latency: Delay between exposure to a disease-causing agent and manifestation

of the disease (onset of infectiousness).
Lichen: Symbiotic associations between fungi and photosynthetic partners

(algae).
Macrophage: Phagocytic cell derived from blood monocytes, typically resident

in most tissues. It has both scavenger and antigen-presenting functions in immune
responses.
Meiosis: The process in cell division in sexually reproducing organisms that

reduces the number of chromosomes from diploid to haploid (half the original
number).
Mendelian: A single gene disorder caused by a defect in one particular gene, and
characterized by how they are passed down in families.
Metabolites: A substance produced by the chemical processes by which cells

produce the substances and energy needed to sustain life.
Microbe: A microorganism or biologic agent that can replicate in humans (in-

cluding bacteria, viruses, protozoa, fungi, and prions).
Microbial flora: The microscopic organisms living within a particular region,

including bodily organ or body part, such as the skin.
Microbial threat: Microbes that lead to disease in humans.

APPENDIX D 421
Microbiology: A branch of biology dealing especially with microscopic forms

of life.
Microbiome: Term used to describe the collective genome of our indigenous

microbes (microflora).
Migration: The regular, usually seasonal, movement of all or part of an animal

population to and from a given area.
Mitigation: Initiatives that reduce the risk from natural and man-made hazards.
Morbidity: Diseased condition or state.
Mortality: Proportion of deaths to population or to a specific number of the

population; death rate.
Mutation: Genetic change that can occur either randomly or at an accelerated

rate through exposure to radiation or certain chemicals (mutagens) and may lead
to change in structure of the protein coded by the mutated gene.
Mycelia, mycelium: The mass of fine branching tubes (known as hyphae) that
forms the main growing structure of a fungus.
Mycorrhizal fungi: Fungi that colonize plant roots.
Natural history: The natural development of something (such as an organism or

disease) over a period of time.
Necropsy: An autopsy performed on an animal.
Neutrophil: Most common blood leukocyte; a short-lived phagocytic cell of

the myeloid series, which is responsible for the primary cellular response to an
acute inflammatory episode, and for general tissue homeostasis by removal of
damaged material.
Notifiable disease: Disease that health professionals are required to report to

state, national, or international authorities.
Obligate: Capable of existing only in a particular environment; an obligate para-

site cannot survive independently of its host.
Old World: Refers to the Western Hemisphere; in a biological context: New

World species are those from the Nearctic and Neotropic ecological zones; Old
World species are those from the Palearctic and Afrotropic ecological zones.
422 FUNGAL DISEASES
One Health: Holistic approach to preventing epizootic disease and for maintain-
ing ecosystem integrity for the benefit of humans, their domesticated animals, and
the foundation biodiversity that supports all life.
Oomycete: Not a “true fungi”; an oomycete or “water mold” that belongs to

the Kingdom Stramenopila (a major eukaryotic group that includes diatoms and
brown algae, and is distinct from plants, fungi, and animals). Like fungi, oomy-
cetes “exhibit filamentous growth, produce sexual and asexual spores, and can
feed on decaying matter or be obligate parasites of plants.”
Opportunistic: Resulting from pathogen entry via wounds or weakened state

of the host, or as a disturbance of a normally benign host–fungus relationship.
Pandemic: Disease outbreak occurring over a wide geographic area and affecting
an exceptionally high proportion of the population.
Parasite, parasitic: An organism that lives in or on and takes its nourishment

from another organism. A parasite cannot live independently. Parasitic diseases
include infections by protozoa, helminths, and arthropods.
Pathogen: Organism capable of causing disease.
Pathogenic: Capable of causing disease.
Pathogenicity: The ability of an organism, a pathogen, to produce an infectious

disease in another organism.
Pathology: The branch of medicine concerned with disease, especially its struc-
ture and its functional effects on the body.
Phagocyte/phagocytic cell: A cell that is capable of phagocytosis, or the uptake

of particulate material by a cell. The main mammalian phagocytes are neutrophils
and macrophages.
Phenology: The scientific study of cyclical biological events, such as flowering,

breeding, and migration.
Phenotype: The physical appearance of an organism as distinguished from its

genetic makeup (genotype).
Phylogeny: The connections among all groups of organisms as understood by

ancestor/descendant relationships.
APPENDIX D 423
Phylum: In taxonomy and systematics: the highest level of classification below

the kingdom.
Prevalence: Total number of cases (new as well as previous cases) of a disease

in a given population at a point in time.
Primary fungal pathogen: Pathogens able to induce symptoms of disease in

otherwise healthy individuals.
Propagules: Any of various structures that can give rise to a new individual or-
ganism. (For fungi, propagules include spores or encapsulated yeast cells.)
Psychotropic: Affecting mental activity, behavior, or perception.
Public health: The art and science of dealing with the protection and improve-
ment of community health by organized community effort and including preven-
tive medicine and sanitary and social health.
Quarantine: The enforced isolation or restriction of free movement imposed to

prevent the spread of a contagious disease.
Race (plant pathology): A subspecies group of pathogens that infect a given set
of plant varieties.
Recombination: A combining of genes or characters different from what they

were in the parents.
Recombine: The process by which the combination of genes in an organism’s

offspring becomes different from the combination of genes in that organism.
Reservoir: Any person, animal, arthropod, plant, soil, or substance (or combina-
tion of these) in which an infectious agent normally lives and multiplies, on which
it depends primarily for survival, and in which it reproduces itself in such manner
that it can be transmitted to a susceptible vector or host.
Saprophytic: Deriving nutrients from dead organic matter.
Serotype: The characterization of a microorganism based on the kinds and com-

binations of constituent antigens (a substance that stimulates the production of an
antibody when introduced into the body) present in that organism; a taxonomic
subdivision of bacteria based on the above.
424 FUNGAL DISEASES
Somatic cells: The cells of the body, with the exception of the reproductive cells
(gametes).
Species: The basic unit of taxonomy. A species is defined as a group of individu-

als that are genetically related and can interbreed to produce fertile young of the
same kind.
Species barrier: Difficulty or impossibility for an infectious agent to pass from

one species to another (due to differences between species).
Spores: Well-protected structures that can survive in adverse environmental

conditions, such as freezing or drying (better than mycelia and yeast cells), for
months and even years.
Surveillance: The continual scrutiny of all aspects of occurrence and spread of a

disease that are pertinent to effective control, involves the systematic collection,
analysis, interpretation, and dissemination of health data.
Symbiotic: The close association between two or more organisms of different

species, often but not necessarily benefiting each member. The association of
algae and fungi in lichens and of bacteria living in the intestines or on the skin
of animals are symbiotic.
Syndrome: A group or recognizable pattern of symptoms or abnormalities that

indicate a particular trait or disease.
Systematics: The classification of organisms and the evolutionary relationships

among them; taxonomy.
Thermotolerance: Garcia Solache and Casadevall (2010) define thermotolerance

as the ability to grow at mammalian (37°C) and higher temperatures. Most fungi
thrive in the range of 12°C to 30°C, but there are wide temperature tolerances
among species, with some growing at temperatures as low as –10°C or as high
as 65°C. [Garcia-Solache, M. A., and A. Casadevall. 2010. Hypothesis: global
warming will bring new fungal diseases for mammals. mBio 1(1):1–3.]
Transformation: The genetic alteration of a bacteria cell by the introduction of

genetic material from another cell or from a virus.
Transmission: Process by which a pathogen passes from a source of infection

to a new host.
APPENDIX D 425
Vaccine: A preparation of living, attenuated, or killed bacteria or viruses, frac-

tions thereof, or synthesized or recombinant antigens identical or similar to those
found in the disease-causing organism that is administered to raise immunity to
a particular microorganism.
Vector: A carrier, especially an arthropod, that transfers an infective agent from

one host (which can include itself) to another.
Vector borne: Transmitted from one host to another by a vector.
Virulence: The degree of pathogenicity of an organism as evidenced by the

severity of resulting disease and the organism’s ability to invade the host tissues.
Virulence factors: Molecules produced by a pathogen that specifically cause

disease, or that influence their host’s function to allow the pathogen to thrive.
Weather: The state of the atmosphere over a short period of time, measured
in terms of wind, temperature, humidity, atmospheric pressure, cloudiness, and
precipitation. The difference between weather and climate is a measure of time:
Climate is how the atmosphere “behaves” over relatively long periods of time.
Xylose: A white crystalline sugar extracted from wood, straw, and corn.
Yeast: Any of various one-celled fungi that reproduce by budding and can cause
the fermentation of carbohydrates, producing carbon dioxide and ethanol.
Zoonotic infection: Infection that causes disease in human populations but that
can be perpetuated solely in non-human host animals (e.g., bubonic plague); may
be enzootic or epizootic.
Appendix E
Forum Member Biographies
David A. Relman, M.D. (Chair), is the Thomas C. and Joan M. Merigan ProÂ�
fessor in the Departments of Medicine and of Microbiology and Immunology at
Stanford University, and Chief of Infectious Diseases at the VA Palo Alto Health
Care System in Palo Alto, California. He received an S.B. (biology) from MasÂ�
sachusetts Institute of Technology (1977), received his M.D. (magna cum laude)
from Harvard Medical School (1982), completed his clinical training in internal
medicine and infectious diseases at Massachusetts General Hospital, served as a
postdoctoral fellow in microbiology at Stanford University, and joined the faculty
at Stanford in 1994.
Dr. Relman’s current research focus is the human indigenous microbiota (mi-
crobiome) and, in particular, the nature and mechanisms of variation in patÂ�terns
of microbial diversity within the human body as a function of time (microÂ�bial
succession), space (biogeography within the host landscape), and in response to
perturbation, for example, antibiotics (community robustness and resilience). One
of the goals of this work is to define the role of the human microbiome in health
and disease. This research integrates theory and methods from ecology, popuÂ�
lation biology, environmental microbiology, genomics, and clinical medicine.
During the past few decades, his research directions have also included pathoÂ�
gen discovery and the development of new strategies for identifying previously
unrecognized microbial agents of disease. This work helped to spearhead the
application of molecular methods to the diagnosis of infectious diseases in the
1990s. His research has emphasized the use of genomic approaches for exploring
host–microbe relationships. Past scientific achievements include the description
of a novel approach for identifying previously unknown pathogens; the identiÂ�
fication of a number of new human microbial pathogens, including the agent of
427
428 FUNGAL DISEASES
Whipple’s disease; and some of the most extensive and revealing analyses to date
of the human indigenous microbial ecosystem.
Dr. Relman advises the U.S. government, as well as nongovernmental organiÂ�
zations, in matters pertaining to microbiology, emerging infectious diseases, and
biosecurity. He is a member of the National Science Advisory Board for BiosecuÂ�
rity, a member of the Physical and Life Sciences Directorate Review Commit-
tee for Lawrence Livermore National Laboratory, and he advises several U.S.
governÂ�ment departments and agencies on matters related to pathogen diversity,
the future life sciences landscape, and the nature of present and future biological
threats. He has served as Chair of the Board of Scientific Counselors of the Na-
tional Institute of Dental and Craniofacial Research (National Institutes of Health
[NIH]) and as a member of the Board of Directors, Infectious Diseases Society
of America (IDSA). Dr. Relman is currently vice-chair of a National Academy of
Sciences (NAS) study of the science underlying the Federal Bureau of Investiga-
tion invesÂ�tigation of the 2001 anthrax mailings, and he cochaired a 3-year NAS
study that produced a widely cited report entitled Globalization, Biosecurity, and
the Future of the Life Sciences (2006). He is a Fellow of the American Academy
of MicroÂ�biology and a member of the Association of American Physicians. Dr.
Relman received the Squibb Award from the IDSA in 2001 and was the recipient
of both the NIH Director’s Pioneer Award and the Distinguished Clinical Scientist
Award from the Doris Duke Charitable Foundation in 2006.
James M. Hughes, M.D. (Vice-Chair), is professor of medicine and public

health at Emory University’s School of Medicine and Rollins School of Public
Health, serving as director of the Emory Program in Global Infectious Diseases,
executive director of the Southeastern Center for Emerging Biologic Threats, and
senior advisor to the Emory Center for Global Safe Water. He is the senior sciÂ�
entific advisor for infectious diseases to the International Association of National
Public Health Institutes funded by the Bill and Melinda Gates Foundation. Prior
to joining Emory in June 2005, Dr. Hughes served as director of the National
Center for Infectious Diseases (NCID) at the Centers for Disease Control and
Prevention (CDC). Dr. Hughes received his B.A. and M.D. degrees from Stan-
ford UniverÂ�sity and completed postgraduate training in internal medicine at the
University of Washington, infectious diseases at the University of Virginia, and
preventive medicine at CDC. After joining CDC as an Epidemic Intelligence Ser-
vice officer in 1973, Dr. Hughes worked initially on foodborne and water-related
diseases and subsequently on infection control in health care settings. He served
as direcÂ�tor of CDC’s Hospital Infections Program from 1983 to 1988, as deputy
direcÂ�tor of NCID from 1988 to 1992, and as director of NCID from 1992 to 2005.
A major focus of Dr. Hughes’ career is on building partnerships among the clini-
cal, research, public health, and veterinary communities to prevent, detect, and
respond to infectious diseases at the local, national and global levels. His research
interests include emerging and reemerging infectious diseases, antimicrobial
APPENDIX E 429
resistance, foodborne diseases, health care–associated infections, vectorborne

and zoonotic diseases, rapid detection of and response to infectious diseases
and bioterrorism, strengthening public health capacity at the local, national, and
global levels, and prevention of water-related diseases in the developing world.
Dr. Hughes is a fellow and Council Delegate of the American Association for the
Advancement of Science, fellow of the American College of Physicians and the
Infectious Diseases Society of America (IDSA), President of IDSA, Councilor
of the American Society of Tropical Medicine and Hygiene, and member of the
International Board of the American Society for MicrobiolÂ�ogy. He is a member
of the Institute of Medicine.
Lonnie J. King, D.V.M. (Vice-Chair), is the 10th dean of the College of Veteri-
nary Medicine at the Ohio State University (OSU). In addition to leading this
college, Dr. King is also a professor of preventive medicine and holds the Ruth
Stanton Endowed Chair in Veterinary Medicine. Before becoming dean at OSU,
he was the direcÂ�tor of CDC’s new National Center for Zoonotic, Vector-Borne,
and Enteric DisÂ�eases (NCZVED). In this new position, Dr. King leads the Cen-
ter’s activities for surveillance, diagnostics, disease investigations, epidemiol-
ogy, research, public education, policy development, and disease prevention and
control programs. NCZVED also focuses on waterborne, foodborne, vectorborne,
and zoonotic diseases of public health concern, which also include most of CDC’s
select and bioterrorism agents, neglected tropical diseases, and emerging zoono-
ses. Before serving as director, he was the first chief of the agency’s Office of
Strategy and Innovation.
Dr. King served as dean of the College of Veterinary Medicine, Michigan
State University, from 1996 to 2006. As at OSU, he served as the CEO for acaÂ�
demic programs, research, the teaching hospital, the diagnostic center for popula-
tion and animal health, basic and clinical science departments, and the outreach
and continuing education programs. As dean and professor of large-animal clini-
cal sciences, Dr. King was instrumental in obtaining funds for the construction
of a $60 million Diagnostic Center for Population and Animal Health; he initi-
ated the Center for Emerging Infectious Diseases in the college, he served as the
campus leader in food safety, and he had oversight for the National Food Safety
and Toxicology Center.
In 1992, Dr. King was appointed administrator for the Animal and Plant
Health Inspection Service (APHIS), U.S. Department of Agriculture (USDA),
in Washington, DC. In this role, he provided executive leadership and direction
for ensuring the health and care of animals and plants, to improve agricultural
productivity and competitiveness, and to contribute to the national economy and
public health. Dr. King also served as the country’s chief veterinary officer for 5
years, worked extensively in global trade agreements within the North American
Free Trade Agreement and the World Trade Organization, and worked extensively
with the World Animal Health AssoÂ�ciation. During this time he was the Deputy
430 FUNGAL DISEASES
Administrator for Veterinary Services of APHIS, USDA, where he led national

efforts in disease eradication, imports and exports, and diagnostics in both Ames,
Iowa, and Plum Island. He spent 5 years in Hyattsville, Maryland, in staff assign-
ments in Emergency Programs, as well as Animal Health Information. While in
Hyattsville, Dr. King directed the development of the agency’s National Animal
Health Monitoring System. He left APHIS briefly to serve as the director of the
Governmental Relations Division of the American Veterinary Medical Associa-
tion (AVMA) in Washington, DC, and served as the lobbyist for the AVMA on
Capitol Hill.
Dr. King was in private veterinary practice for 7 years in Dayton, Ohio, and
Atlanta, Georgia. As a native of Wooster, Ohio, Dr. King received his bachelor of
science and doctor of veterinary medicine degrees from OSU in 1966 and 1970,
respectively. He earned his master of science degree in epidemiology from the
University of Minnesota and received his master’s degree in public administraÂ�
tion from American University in Washington, DC, in 1991. Dr. King is a board-
certified member of the American College of Veterinary Preventive Medicine and
has completed the Senior Executive Fellowship program at Harvard University.
He served as president of the Association of American Veterinary Medical ColÂ�
leges from 1999 to 2000 and was the vice-chair for the National Commission
on Veterinary Economic Issues from 2000 to 2004. He has served on four NAS
committees, including chairing the National Academies’ Committee on AssessÂ�
ing the Nation’s Framework for Addressing Animal Diseases. He is also Chair
of the IOM Committee on Lyme Disease and Other Tick-Borne Diseases and
for State of the Science, and he is also chairing the AVMA’s Commission for
AVMA Vision 2020. Dr. King is currently a member of the IOM Committee on
Microbial Threats to Health, is a past member of the Food and Drug Administra-
tion’s (FDA’s) Board of Scientific Advisors, and is past president of the Ameri-
can Veterinary Epidemiology Society. He served as the chair for the national
One Medicine Task Force for the AVMA, which helped start the country’s One
Health Initiative. Dr. King was elected as a member of the IOM of the National
Academies in 2004.
Kevin Anderson, Ph.D., serves as a Senior Program Manager in the Depart-

ment of Homeland Security’s Science and Technology Directorate, providing
oversight and requirements for science programs focused on rapid detection and
characterization of biological agents. Since joining DHS in 2003, Dr. Anderson
has provided leadership for science program development, laboratory design and
strategic planning, served as a subject matter expert and advisor to the Bioter-
rorism Risk Assessment and Biological Threat Characterization programs, and
has participated in interagency working groups and assessments which provide
guidance to medical countermeasure development, a key component of the na-
tion’s biodefense strategy. Prior to joining DHS, Dr. Anderson was a Principal
Investigator at the U.S. Army Medical Research Institute of Infectious Diseases,
APPENDIX E 431
leading research focused on understanding basic mechanisms of viral diseases

causing hemorrhagic fever and development of medical countermeasures. He
received postdoctoral training in molecular virology at the University of Alabama
at Birmingham and the University of North Carolina at Chapel Hill, performing
basic research on human respiratory syncytial viruses, and earned Ph.D. and B.S.
degrees in microbiology from Montana State University and the University of
Maryland, College Park, respectively.
Ruth L. Berkelman, M.D., is the Rollins Professor and director of the Center for
Public Health Preparedness and Research at the Rollins School of Public Health,
Emory University, in Atlanta. She received her A.B. from Princeton University
and her M.D. from Harvard Medical School. Board certified in pediatrics and
internal medicine, she began her career at CDC in 1980 and later became deputy
director of NCID. She also served as a senior advisor to the director of CDC and
as assistant surgeon general in the U.S. Public Health Service. In 2001 she came
to her current position at Emory University, directing a center focused on emergÂ�
ing infectious diseases and other urgent threats to health, including terrorism. She
has also consulted with the biologic program of the Nuclear Threat Initiative and
is most recognized for her work in infectious diseases and disease surveillance.
She was elected to the IOM in 2004. Currently a member of the Board on Life
Sciences of the National Academies, she also chairs the Board of Public and
Scientific Affairs at the ASM.
David L. Blazes, M.D., M.P.H.,1 Commander David L. Blazes is the Chief of

Global Emerging Infections Surveillance and Response System (GEIS) Division
at the Armed Forces Health Surveillance Center in Silver Spring, Maryland.
From 2004–2008, he was Director of the Emerging Infections Department at the
Naval Medical Research Center Detachment (now NAMRU-6) in Lima, Peru.
The AFHSC-GEIS network identified the first cases of the 2009 H1N1 pandemic
as well as numerous other emerging infections that threaten public health around
the world. He also serves on the faculty at the Uniformed Services University in
Bethesda, Maryland and in International Health at the Johns Hopkins Bloomberg
School of Public Health. He graduated from the U.S. Naval Academy in 1991
and the Johns Hopkins University School of Medicine in 1995 and completed his
internal medicine and infectious diseases training at the National Naval Medi-
cal Center, the President’s hospital in Bethesda. His main scientific interests are
infectious diseases surveillance strategies in developing settings, optimizing out-
break response, public health capacity building and tropical medicine training.
He has taught clinical tropical medicine at the Gorgas course within Universidad
Peruana Cayetano Heredia, at the Johns Hopkins Summer Institute of Tropical
Medicine and at the U.S. Military Tropical Medicine course in Bethesda.

432 FUNGAL DISEASES
Enriqueta C. Bond, Ph.D., is president emeritus of the Burroughs Wellcome

Fund. Dr. Bond is currently a partner in QE Philanthropic Advisors, LLC, an
organization that provides consulting services to foundations and non-profits
on matters of program, strategic planning and capacity development related to
medical sciences, international health, and science and math K–12 education. She
received her undergraduate degree from Wellesley College, her M.A. from the
University of Virginia, and her Ph.D. in molecular biology and biochemical ge-
netics from Georgetown University. She is a member of the IOM and a fellow of
the AAAS. Dr. Bond chairs the Academies’ Board on African Science Academy
Development and serves on the NRC Committee on the Future of the Research
University. She serves on the board and executive committee of the Hamner Insti-
tute, the board of the Health Effects Institute, the board of the James B. Hunt Jr.
Institute for Educational Leadership and Policy, and the NIH Council of Councils.
In addition Dr. Bond serves on a scientific advisory committee for the World
Health Organization (WHO) Tropical Disease Research Program. Prior to being
named president of the Burroughs Wellcome Fund in 1994, Dr. Bond served on
the staff of the IOM beginning in 1979, becoming its executive officer in 1989.
Roger G. Breeze, BVMS, Ph.D., MRCVS, is currently Bio-Security Deputy

Program Director, Global Security Directorate, Office of Strategic Outcomes,
Lawrence Livermore National Laboratory and serves on the senior manage-
ment team of the Defense Threat Reduction Agency’s Chemical and Biological
Defense Directorate. He received his veterinary degree in 1968 and his Ph.D. in
veterinary pathology in 1973, both from the University of Glasgow, Scotland.
He was engaged in teaching, diagnostic pathology, and research on respiratory
and cardiovascular diseases at the University of Glasgow Veterinary School from
1968 to 1977 and at Washington State University College of Veterinary MediÂ�
cine from 1977 to 1987, where he was professor and chair of the Department of
Microbiology and Pathology. From 1984 to 1987 he was deputy director of the
Washington Technology Center, the state’s high-technology sciences initiative,
based in the College of Engineering at the University of Washington. In 1987,
he was appointed director of the USDA’s Plum Island Animal Disease Center, a
Biosafety Level 3 (BSL-3) facility for research and diagnosis of the world’s most
dangerous livestock diseases. In that role he initiated research into the genomic
and functional genomic basis of disease pathoÂ�genesis, diagnosis, and control of
livestock RNA and DNA virus infections. This work became the basis of U.S.
defense against natural and deliberate infection with these agents and led to his
involvement in the early 1990s in biological weapÂ�ons defense and proliferation
prevention. From 1995 to 1998, he was South Atlantic area director for USDA’s
Agricultural Research Service before going to Washington, DC, to establish
biological weapons defense programs for USDA. He received the Distinguished
Executive Award from President Clinton in 1998 for his work at Plum Island and
in biodefense. Since 2004 he has been CEO of Centaur Science group where
APPENDIX E 433
his main commitment was to the Defense Threat Reduction Agency’s Global
Bioengagement Program.
Steven J. Brickner, Ph.D.,2 is an independent consultant based in southeastern

Connecticut. He received his Ph.D. in organic chemistry from Cornell University
and completed an NIH postdoctoral research fellowship at the University of Wis-
consin, Madison. Dr. Brickner is a synthetic organic/medicinal chemist with more
than 25 years of research experience focused entirely on the discovery of novel
antibacterial agents during his prior tenure at Upjohn, Pharmacia & Upjohn, and
Pfizer. He is co-inventor of Zyvox® (linezolid), an oxazolidinone recognized as
the first member of an entirely new class of antibiotic to reach the market in the
more than 35 years since the discovery of the first quinolone. Approved in 2000,
linezolid has annual worldwide sales of more than US$1 billion. He initiated
the oxazolidinone research program at Upjohn and led the team that discovered
linezolid and clinical candidates eperezolid and PNU-100480. While at Pfizer, he
led the early development team that placed the oxazolidinone PNU-100480 into
clinical trials, where it is being studied as a potential treatment for tuberculosis.
Dr. Brickner received an honorary doctor of science degree from the University
of Notre Dame in 2010, and he was a corecipient of the Pharmaceutical Research
and Manufacturers of America 2007 Discoverers Award and the 2007 American
Chemical Society Award for Team Innovation. He was named the 2002–2003
Outstanding Alumni Lecturer, College of Arts and Science, Miami University
(Ohio). He is an inventor or co-inventor on 21 U.S. patents, has published more
than 30 peer-reviewed scientific papers, and has given 25 invited speaker preÂ�
sentations; he has been a member of the IOM Forum on Microbial Threats since
1997. In February 2009, he established SJ Brickner Consulting, LLC, which
serves various clients in offering consulting services on all aspects of medicinal
chemistry and drug design related to the discovery and development of new
antibiotics.
Paula R. Bryant, Ph.D., is Chief of the Medical S&T Division, Chemical and
Biological Defense Program at the Defense Threat Reduction Agency (DTRA) in
Fort Belvoir, Virginia. As the Chief of the Medical S&T Division, Bryant inter-
faces with all levels of the Department of Defense and DTRA to plan, coordinate,
integrate, and execute program activities necessary to provide timely and effective
medical countermeasures against Chemical, Biological and Radiological (CBR)
threats to U.S. interests worldwide. She also served as a Senior Scientist and Se-
nior S&T Manager while at DTRA. Prior to joining DTRA, she was an Assistant
Professor in the Department of Microbiology at The Ohio State University. She
received her Ph.D. in Microbiology and Immunology from the Baylor College
of Medicine.

434 FUNGAL DISEASES
John E. Burris, Ph.D., became president of the Burroughs Wellcome Fund in

July 2008. He is the former president of Beloit College. Prior to his appointÂ�
ment at Beloit in 2000, Dr. Burris served for 8 years as director and CEO of
the Marine Biological Laboratory in Woods Hole, Massachusetts. From 1984 to
1992 he was at the National Research Council/National Academies, where he
served as the executive director of the Commission on Life Sciences. A native
of Wisconsin, he received an A.B. in biology from Harvard University in 1971,
attended the University of Wisconsin, Madison, in an M.D.-Ph.D. program, and
received a Ph.D. in marine biology from the Scripps Institution of Oceanography
at the University of California, San Diego, in 1976. A professor of biology at the
Pennsylvania State University from 1976 to 1985, he held an adjunct appointment
there until going to Beloit. His research interests are in the areas of marine and
terrestrial plant physiology and ecology. He has served as president of the AmeriÂ�
can Institute of Biological Sciences and is or has been a member of a number
of distinguished scientific boards and advisory committees, including the Grass
Foundation; the Stazione Zoologica “Anton Dohrn” in Naples, Italy; the AAAS;
and the Radiation Effects Research Foundation in Hiroshima, Japan. He has also
served as a consultant to the National Conference of Catholic Bishops’ CommitÂ�
tee on Science and Human Values.
Arturo Casadevall, M.D., Ph.D.,3 is the Leo and Julia Forchheimer Professor
of Microbiology & Immunology at the Albert Einstein College of Medicine of
Yeshiva University in the Bronx, New York. He is Chairman of the Department of
Microbiology and Immunology and served as Director of the Division of Infec-
tious Diseases at the Montefiore Medical Center at the Albert Einstein College
of Medicine from 2000–2006. Dr Casadevall received both his M.D. and Ph.D.
(biochemistry) degrees from New York University in New York, New York. Sub-
sequently, he completed internship and residency in internal medicine at Bellevue
Hospital in New York, New York. Later he completed subspecialty training in
Infectious Diseases at the Montefiore Medical Center and Albert Einstein College
of Medicine. Dr. Casadevall major research interests are in fungal pathogenesis
and the mechanism of antibody action. In the area of Biodefense Dr. Casade-
vall has an active research program to understand the mechanisms of antibody-
mediated neutralization of Bacillus anthracis toxins. He has authored over 500
scientific papers. Dr. Casadevall was elected to membership in the American
Society for Clinical Investigation, the American Academy of Physicians, and the
American Academy of Microbiology. He was elected a fellow of the American
Academy for the Advancement of Science and has received numerous honors
including the Solomon A Berson Medical Alumni Achievement Award in Basic
Science from the NYU School of Medicine, the Maxwell L. Littman Award

APPENDIX E 435
(mycology award), the Rhoda Benham Award from Medical Mycology Society
of America, and the Kass Lecture of the Infectious Disease Society of America.
Dr. Casadevall is the Editor in Chief of mBio, the first open access general jour-
nal of the American Society of Microbiology. He serves in the editorial board of
the Journal of Clinical Investigation, The Journal of Experimental Medicine and
The Journal of Infectious Diseases. Previously he served as Editor of Infection
and Immunity. He has served in numerous NIH committees including those that
drafted the NIAID Strategic Plan and the Blue Ribbon Panel on Biodefense Re-
search. Dr. Casadevall served on the NAS committee that reviewed the science
behind the FBI investigation of the anthrax attacks in 2001. He is currently a
member of the National Science Advisory Board for Biosecurity and co-chaired
the NIAID Board of Scientific counselors.
Since he joined the Einstein faculty in 1992 Dr. Casadevall has mentored
dozens of graduate students, postdoctoral fellows, and junior faculty. Many of
his trainees have gone on to have highly successful careers in science and several
have currently AECOM faculty. From 2000–2006 Dr. Casadevall was director
of the Division of Infectious Diseases at AECOM-Montefiore and oversaw the
expansion of its research program. In 2001 Dr. Casadevall received the Samuel
M. Rosen outstanding teacher award and in 2008 he was recognized the American
Society of Microbiology with the William Hinton Award for mentoring scientists
from underrepresented groups.
Peter Daszak, Ph.D., is President of EcoHealth Alliance (formerly Wildlife

Trust), a U.S.-based organization which conducts research and field programs on
global health and conservation. At Wildlife Trust, Dr. Daszak manages a head-
quarters staff of 35 and a global staff of more than 700 which conducts research
and manages initiatives to prevent emerging pandemics and conserve wildlife
biodiversity. This includes research on zoonoses that spill over from wildlife in
emerging disease “hotspots,” including influenza, Nipah virus, SARS, West Nile
virus, and others. Dr. Daszak’s work includes identifying the first case of a spe-
cies extinction due to disease, the discovery of chytridiomycosis, the major cause
of global amphibian declines, publishing the first paper to highlight emerging
diseases of wildlife, coining the term “pathogen pollution,” discovery of the bat
origin of SARS-like coronaviruses, identifying the drivers of Nipah and Hendra
virus emergence, and producing the first ever emerging disease “hotspots” map.
Dr. Daszak is a member of the Council of Advisors of the One Health ComÂ�
mission, Treasurer of DIVERSITAS (ICSU), past member of the International
Standing Advisory Board of the Australian Biosecurity CRC, past member of
the IOM Committee on Global Surveillance for Emerging Zoonoses and the Na-
tional Research Council (NRC) comÂ�mittee on the future of veterinary research.
He is Editor-in-Chief of the Springer journal Ecohealth, and past treasurer and a
founding director of the International Ecohealth Association. In 2000, he won the
Commonwealth Scientific and Industrial Research Organisation medal for col-
436 FUNGAL DISEASES
laborative research in the discovery of amphibian chytridiomycosis. He has pub-

lished over 130 scientific papers and book chapters, including papers in Science,
Nature, PNAS, The Lancet, PLoS Biology, and other leading journals. His work
has been the focus of articles in the New York Times, The Wall Street Journal, The
EconoÂ�mist, The Washington Post, US News & World Report, CBS 60 Minutes,
CNN, ABC, NPR’s Talk of the Nation, and Morning Edition & Fresh Air with Terri
Gross. He is a former guest worker at the CDC, where he assisted in the pathol-
ogy activity during the 1999 Nipah virus outbreak. His work is funded by the
John E. Fogarty International Center of NIH, the National Institute of Allergy and
Infectious Diseases (NIAID), the National Science Foundation (NSF), the U.S.
Agency for International Development (USAID), Google.org, Rockefeller, and
other foundations. To date, his group is one of the few to have been awarded three
prestigious NIH/NSF Ecology of Infectious Disease awards and is one of four
partners to share a recent multi-million-dollar award from USAID (“PREDICT”)
with the goal of predicting and preventing the next emerging zoonotic disease.
Jeffrey Scott Duchin, M.D., is Chief of the Communicable Disease Epide-

miology & Immunization Section for Public Health–Seattle & King County,
Washington, and Professor of Medicine, Division of Infectious Diseases and
Adjunct Professor in the School of Public Health and Community Medicine at
the University of Washington.
Dr. Duchin trained in internal medicine at Thomas Jefferson University
Hospital. He completed a fellowship in general internal medicine and emergency
medicine at the Hospital of the University of Pennsylvania and infectious disease
subspecialty training at the University of Washington. After several years on
the faculty at the University of Pennsylvania, he joined the Centers for Disease
Control and Prevention’s (CDC’s) Epidemic Intelligence Service program where
he was assigned to the National Center for Infectious Diseases, and the CDC’s
Preventive Medicine Residency program. He worked for CDC as a medical epi-
demiologist in the Divisions of Tuberculosis Elimination and HIV/AIDS Special
Studies Branch before assuming his current position.
Dr. Duchin is a member of the Institute of Medicine’s (IOM’s) Forum on
Medical and Public Health Preparedness for Catastrophic Events and a current
member of the Centers for Disease Control and Prevention’s Advisory Commit-
tee on Immunization Practices (ACIP). He is a Fellow of the Infectious Disease
Society of America (IDSA) and of the American College of Physicians; a member
of the IDSA’s National and Global Public Health Committee and Pandemic In-
fluenza Task Force; and is past-Chair of the IDSA’s Bioemergencies Task Force.
Dr. Duchin serves on the Editorial Board and Technical Advisory Group for
Communicable Disease Alert and Response to Mass Gatherings for the World
Health Organization and previously served as a member of the Department of
Health and Human Services 2004 Tiger Team consulting with the Government of
Greece on health preparations for the 2004 Olympics, in Athens, Greece.
APPENDIX E 437
Dr. Duchin’s peer review publications and research interests focus on communi-
cable diseases of public health significance, and he has authored text book chap-
ters on the epidemiology of HIV/AIDS, bioterrorism, and outbreak investigations.
Jonathan Eisen, Ph.D., is a professor at the Genome Center at the University of

California (UC), Davis, and holds appointments in the Department of Evolution
and Ecology in the College of Biological Sciences and Medical Microbiology and
Immunology in the School of Medicine.
His research focuses on the mechanisms underlying the origin of novelty
(how new processes and functions originate). Most of his work involves the use
of high-throughput DNA sequencing methods to characterize microbes and then
the use and development of computational methods to analyze this type of data.
In particular, his computational work has focused on integrating evolutionary
analysis with genome analysis—so-called phylogenomics. Previously, he applied
this phylogenomic approach to cultured organisms, such as those from extreme
environments and those with key properties as they relate to evolution or global
cliÂ�mate cycles. Currently he is using sequencing and phylogenomic methods to
study microbes directly in their natural habitats (i.e., without culturing). In par-
ticular he focuses on how communities of microbes interact with each other or
with plant and animal hosts to create new functions. Dr. Eisen is also coordinating
one of the largest microbial genome sequencing projects to date—the “Genomic
EncyÂ�clopedia of Bacteria and Archaea” being done at the Department of Energy
(DOE) Joint Genome Institute, where he holds an Adjunct Appointment.
In addition to his research, Dr. Eisen is also a vocal advocate for “open
access” to scientific publications and is the Academic Editor-in-Chief of PLoS
Biology. He is also an active and award-winning blogger/microblogger (e.g.,
http://phylogenomics.blogspot.com, http://twitter.com/phylogenomics). Prior to
moving to UC Davis he was on the faculty of The Institute for Genomic Research
(TIGR) in Rockville, Maryland. He earned his Ph.D. in biological sciences from
Stanford University, where he worked on the evolution of DNA repair processes
in the lab of Philip C. Hanawalt and his undergraduate degree in biology from
Harvard College.
Mark B. Feinberg, M.D., Ph.D., is vice president for medical affairs and policy
in global vaccine and infectious diseases at Merck & Co., Inc., and is responsible
for global efforts to implement vaccines to achieve the greatest health benefits,
including efforts to expand access to new vaccines in the developing world. Dr.
Feinberg received a bachelor’s degree magna cum laude from the University of
Pennsylvania in 1978 and his M.D. and Ph.D. degrees from Stanford University
School of Medicine in 1987. His Ph.D. research at Stanford was supervised by
Dr. Irving Weissman and included time spent studying the molecular biology of
the human retroviruses—human T-cell lymphotrophic virus, type I (HTLV-I) and
HIV—as a visiting scientist in the laboratory of Dr. Robert Gallo at the National
438 FUNGAL DISEASES
Cancer Institute. From 1985 to 1986, Dr. Feinberg served as a project officer for
the IOM Committee on a National Strategy for AIDS. After receiving his M.D.
and Ph.D. degrees, Dr. Feinberg pursued postgraduate residency training in inter-
nal medicine at the Brigham and Women’s Hospital of Harvard MediÂ�cal School
and postdoctoral fellowship research in the laboratory of Dr. David Baltimore
at the Whitehead Institute for Biomedical Research. From 1991 to 1995, Dr.
Feinberg was an assistant professor of medicine and microbiology and immunol-
ogy at the University of California, San Francisco (UCSF), where he also served
as an attending physician in the AIDS-oncology division and as director of the
virology research laboratory at San Francisco General Hospital. From 1995 to
1997, Dr. Feinberg was a medical officer in the Office of AIDS Research in the
Office of the Director of the NIH, the chair of the NIH Coordinating Committee
on AIDS Etiology and Pathogenesis Research, and an attending physician at the
NIH Clinical Center. During this period, he also served as executive secretary of
the NIH Panel to Define Principles of Therapy of HIV Infection. Prior to joining
Merck in 2004, Dr. Feinberg served as professor of medicine and microbiology
and immunology at the Emory University School of Medicine, as an investigator
at the Emory Vaccine Center, and as an attending physician at Grady Memorial
Hospital. At UCSF and Emory, Dr. Feinberg and colleagues were engaged in
the preclinical development and evaluation of novel vaccines for HIV and other
infectious diseases and in basic research studies focused on revealing fundamenÂ�
tal aspects of the pathogenesis of AIDS. Dr. Feinberg also founded and served as
the medical director of the Hope Clinic of the Emory Vaccine Center—a cliniÂ�
cal research facility devoted to the clinical evaluation of novel vaccines and to
translational research studies of human immune system biology. In addition to
his other professional roles, Dr. Feinberg has also served as a consultant to, and
a member of, several IOM and NAS committees. Dr. Feinberg currently serves
as a member of the National Vaccine Advisory Committee and is a member of
the Board of Trustees of the National Foundation for Infectious Diseases. Dr.
Feinberg has earned board certification in internal medicine; he is a fellow of
the American College of Physicians, a member of the Association of American
Physicians, and the recipient of an Elizabeth Glaser Scientist Award from the
Pediatric AIDS Foundation and an Innovation in Clinical Research Award from
the Doris Duke Charitable Foundation.
Jacqueline Fletcher, Ph.D., Regents Professor of Plant Pathology at Oklahoma

State University, received a B.S. in biology from Emory University, Atlanta,
Georgia, an M.S. in botany from the University of Montana, and a Ph.D. in plant
pathology from Texas A&M University. She served as a postdoctoral associate at
the University of Illinois before joining OSU in 1984, where she was appointed
Sarkeys Distinguished Professor in 2001 and Regents Professor in 2008. She was
named a Fellow of the American Phytopathological Society (APS) in 2005 and
a Fellow of AAAS in 2007.
APPENDIX E 439
Dr. Fletcher is Director of the National Institute for Microbial Forensics

and Food and Agricultural Biosecurity (NIMFFAB), a multidisciplinary OSU
initiaÂ�tive that addresses high-priority national issues in research, teaching/edu-
cation, and outreach with emphases in microbial forensics applications in plant
patholÂ�ogy and produce safety. The NIMFFAB serves as a spoke laboratory for
the Department of Homeland Security (DHS)-affiliated National Bioforensic
Analysis Center, in the area of plant pathoÂ�gen forensics. Dr. Fletcher’s research
focuses on mechanisms of virulence and insect transmission of plant pathogenic
bacteria; on the relationships between human pathogens, such as Salmonella and
Escherichia coli, and plants; and on the emerging disciplines of microbial foren-
sics and agricultural biosecurity.
Dr. Fletcher served on the APS CounÂ�cil for 10 years, including the 4-year
APS presidential sequence. In the months following September 11, 2001, Dr.
Fletcher led APS responses and input to new national biosecurity initiatives. She
has served for 9 years on the APS Public Policy Board (4 years as chair) and
is currently on the APS Threatening Pathogens Advisory Committee. She also
serves on several federal biosecurity advisory panels.
S. Elizabeth George, Ph.D.,4 is director of the Biological Countermeasures

Portfolio within the Science and Technology Directorate in DHS. Until it merged
into the new department in 2003, she was Program Manager of the Chemical and
Biological National Security Program in DOE’s National Nuclear Security AdÂ�
ministration’s Office of Nonproliferation Research and Engineering. Significant
accomplishments include the design and deployment of BioWatch, the nation’s
first civilian biological threat agent monitoring system, and PROTECT, the first
civilian operational chemical detection and response capability deployed in the
Washington, DC, area subway system. Previously, she spent 16 years at the U.S.
Environmental Protection Agency (EPA), Office of Research and Development,
National Health and Ecological Effects Research Laboratory, Environmental
Carcinogenesis Division, where she was Branch Chief of the Molecular and CelÂ�
lular Toxicology Branch. She received her B.S. in biology in 1977 from Virginia
Polytechnic Institute and State University and her M.S. and Ph.D. in microbiolÂ�
ogy in 1979 and 1984, respectively, from North Carolina State University. From
1984 to 1986, she was an NRC fellow in the laboratory of Dr. Larry Claxton at
EPA. Dr. George is the 2005 chair of the Chemical and Biological Terrorism
Defense Gordon Research Conference. She has served as Councillor for the
Environmental Mutagen Society and President and Secretary of the GenotoxicÂ�
ity and Environmental Mutagen Society. She holds memberships in the ASM
and the AAAS and is an adjunct faculty member in the School of Rural Public
Health, Texas A&M University. She is a recipient of the EPA Bronze Medal and
Scientific and Technological Achievement Awards and the Department of HomeÂ�

440 FUNGAL DISEASES
land Security Under Secretary’s Award for Science and Technology. She is the
author of numerous journal articles and has presented her research at national
and international meetings.
Jesse L. Goodman, M.D., M.P.H., became chief scientist and deputy commisÂ�
sioner for science and public health of FDA in 2009. He has broad responsibility
for and engagement in leadership and coordination of FDA’s crosscutting sciÂ�
entific and public health efforts. From 2003 to 2009, Dr. Goodman was director
of FDA’s Center for Biologics Evaluation and Research, which oversees mediÂ�cal
and public health activities critical to U.S. and global preparedness and the de-
velopment, evaluation, safety, quality, and availability of biologics. A graduate
of Harvard, Dr. Goodman received his M.D. from the Albert Einstein College
of Medicine and did residency and fellowship training at the Hospital of the
UniverÂ�sity of Pennsylvania and at the University of California, Los Angeles
(UCLA), where he was also Chief Medical Resident. Prior to joining FDA, he
was professor of medicine and chief of infectious diseases at the University of
Minnesota, where he directed the multihospital infectious diseases research, train-
ing, and clinical programs, and where his NIH-funded laboratory first isolated and
characterized Anaplasma phagocytophilum, the infectious agent causing a new
tickborne disease, human granulocytic ehrlichiosis. Dr. Goodman has authored
numerous scientific papers and edited the book Tick Borne Diseases of Humans
(ASM Press, 2005). Dr. Goodman has been elected to the American Society for
Clinical Investigation and to the IOM of the NAS, where he is a longstanding
member of the Forum on Microbial Threats. He is an active clinician and teacher
who is board certified in internal medicine, oncology, and infectious diseases
and is staff physician and infectious diseases consultant at the National Naval
and Walter Reed Army Medical Centers. Dr. Goodman is adjunct professor of
medicine at the University of Minnesota.
Eduardo Gotuzzo, M.D., is principal professor of the Department of Medicine

and director of the “Alexander von Humboldt” Institute of Tropical Medicine and
InfecÂ�tious Diseases, Peruvian University Cayetano Heredia in Lima, Peru, and
head of the Department of Transmissible Diseases at the Cayetano Heredia Hos-
pital. He is also an adjunct professor of medicine at the University of Alabama,
Birmingham, School of Medicine. He is director of the International Gorgas
Course in Clinical Tropical Medicine, Universidad Peruana Cayetano Heredia,
taught jointly with the University of Alabama, Birmingham. He is an adjunct fac-
ulty member of the William J. Harrington Training Programs for Latin America,
University of Miami School of Medicine (since 1983); was associate to the Inter-
national Health DepartÂ�ment of the Johns Hopkins University (1986–1998); and
was fellow of the Center for the Americas at Vanderbilt, Vanderbilt University.
Dr. Gotuzzo is an active member in numerous international societies and has been
president of the Latin American Society of Tropical Disease (2000–2003); the
APPENDIX E 441
IDSA Scientific Program (2000–2003); the International Organizing Committee

of the International ConÂ�gress of Infectious Diseases (1994 to present); the Inter-
national Society for InfecÂ�tious Diseases (1996–1998); the PanAmerican Infec-
tious Diseases Association; the International Federation for Tropical Medicine
(2005–2008); and president of the Peruvian Society of Internal Medicine (1991–
1992). He works on several research areas and teaches on subjects including
emerging diseases, TB, HTLV-1, free-living amoebas, brucellosis, and parasites.
He has published more than 290 articles and chapters as well as six manuals and
one book. Recent honors and awards include being named an honorary member
of the American Society of Tropical Medicine and Hygiene in 2002; an honorary
member of the Society of Internal Medicine in 2000; and a distinguished visitor
at the Faculty of Medical Sciences, University of Cordoba, Argentina (1999).
In 1988, Dr. Gotuzzo received the Golden Medal for Outstanding Contribution
in the Field of Infectious Diseases awarded by Trnava University, Slovakia. In
2007, Dr. Gotuzzo received the Society Citation Award from the IDSA. He was
an honorary member of the Australian Society for Infectious Diseases (2008),
the American Society of Tropical Medicine and Hygiene (2002), Academia de
Medicina de México, Sociedad Nenzolana de Infectología, Sociedad Paraguaya
de Infectología, and the National Academy of Medicine of Mexico (2010).
Carole A. Heilman, Ph.D., serves as director of the Division of Microbiology

and Infectious Diseases (DMID) of NIAID, a component of NIH. DMID supports
research to prevent and control diseases caused by virtually all human infectious
agents (except HIV), including bacterial, viral, parasitic, and prion diseases.
DMID supports a wide variety of projects spanning the spectrum from basic
biology of human pathogens and their interaction with human hosts, through
translational and clinical research, toward the development of new and improved
diagnostics, drugs, and vaccines for infectious diseases. As director, Dr. Heilman
provides scientific direction, oversight, and management for an extramural re-
search portfolio that encompasses 300 different organisms.
DMID supports the nation’s biodefense as well as a solid research infrastrucÂ�
ture that readily responds to public health challenges, such as emerging diseases.
These resources were recently mobilized to respond to the emergence of 2009
H1N1 influenza by providing the first in-depth characterization of the H1N1 panÂ�
demic virus and conducting nine clinical trials that provided safety and efficacy
data to inform public health practice.
Dr. Heilman has a Ph.D. in microbiology from Rutgers University. She did
her postdoctoral work in molecular virology at the National Cancer Institute
(NCI) and continued at the NCI as a senior staff fellow in molecular oncol-
ogy. She later moved into health science administration, where she focused
on respiÂ�ratory pathogens, particularly vaccine development. Dr. Heilman has
received numerous awards for scientific management and leadership, including
three Department of Health and Human Services (HHS) Secretary’s Awards
442 FUNGAL DISEASES
for Distinguished Service recognizing her efforts on develÂ�opment of acellular

pertussis vaccines, AIDS vaccines, and on accelerating bioÂ�defense research and
development (R&D). Dr. Heilman serves as an infectious disease expert on the
Board of Scientific Counselors for CDC. She also serves on the scientific board
of the Fondation Mérieux’s annual Advanced Course of Vaccinology and is a
lecturer in this highly selective training program for decision makers in vac-
cinology. Throughout her career, Dr. Heilman has been a pioneer supporting the
advancement of women in biomedical careers and serves as a mentor to a number
of women within and outside of NIAID.
David L. Heymann, M.D., is currently chair of the Health Protection Agency,

United Kingdom; professor and chair, infectious disease epidemiology, at the
London School of Hygiene and Tropical Medicine; and head of the Global Health
Security Programme at Chatham House, London. Until April 2009, he was assistÂ�
ant director-general for Health Security Environment and Representative of the
director-general for Polio Eradication at WHO. Prior to that, from July 1998
until July 2003, he was executive director of the WHO Communicable Diseases
Cluster, which included WHO’s programs on infectious and tropical diseases,
and from which the public health response to severe acute respiratory syndrome
(SARS) was mounted in 2003. From October 1995 to July 1998, he was director
of the WHO Programme on Emerging and Other Communicable Diseases, and
prior to that he was the chief of research activities in the WHO Global Programme
on AIDS. Dr. Heymann has worked in the area of public health for the past 35
years, 25 of which were on various assignments from CDC, and 10 of which
have been with WHO. Before joining WHO, Dr. Heymann worked for 13 years
as a medical epidemiologist in sub-Saharan Africa (Cameroon, Côte d’Ivoire,
Malawi, and the Democratic Republic of Congo, formerly Zaire) on assignment
from CDC in CDC-supported activities. These activities aimed at strengthening
capacity in surveillance of infectious diseases and their control, with special
emphasis on the childhood immunizable diseases, including measles and polio,
African hemÂ�orrhagic fevers, poxviruses, and malaria. While based in Africa, Dr.
Heymann participated in the investigation of the first outbreak of Ebola in Yam-
buku (former Zaire) in 1976, then again investigated the second outbreak of Ebola
in 1977 in Tandala, and in 1995 directed the international response to the Ebola
outbreak in Kikwit for WHO. Prior to assignments in Africa, he was assigned for
2 years to India as a medical epidemiologist in the WHO Smallpox Eradication
Programme. Dr. Heymann’s educational qualifications include a B.A. from the
Pennsylvania State University, an M.D. from Wake Forest University, a Diploma
in Tropical Medicine and Hygiene from the London School of Hygiene and
Tropical MediÂ�cine, and practical epidemiology training in the 2-year Epidemic
Intelligence Service of CDC. He is a member of the IOM; he was awarded the
2004 Award for Excellence of the American Public Health Association, the 2005
Donald Mackay Award from the American Society for Tropical Medicine and
APPENDIX E 443
Hygiene, and the 2007 Heinz Award on the Human Condition. In 2009 he was
appointed an honorary Commander of the Most Excellent Order of the British
Empire for services to global public health, and he was recently elected a Fel-
low of the Academy of Medical Sciences in the United Kingdom. Dr. Heymann
has been visiting professor at Stanford University, the University of Southern
California, and the George Washington University School of Public Health; has
published over 145 scientific articles on infectious diseases and related issues in
peer-reviewed medical and scientific journals; and has authored several chapters
on infectious diseases in medical textbooks. He is currently the editor of the 19th
edition of the Control of Communicable Diseases Manual, a joint publication of
the American Public Health Association and WHO.
Philip Hosbach currently holds the position of vice president of immunization

policy and government relations at sanofi pasteur. The departments under his
supervision are state government affairs, federal government affairs, medical
communications, strategic advocacy, and immunization initiatives. His responsiÂ�
bilities include oversight of both public policy and immunization policy develÂ�
opment. Mr. Hosbach acts as sanofi pasteur’s principal liaison with CDC. He
is currently coordinating sanofi pasteur’s global efforts in responding to the
emerging H1N1 pandemic. He is a graduate of Lafayette College (1984); shortly
after that he began his professional career in the pharmaceutical industry with
American Home Products. That career has now spanned 25 years, including the
last 22 years focused solely on vaccines. Mr. Hosbach joined sanofi pasteur (then
Connaught Labs) in 1987 in Clinical Research and held positions of increasÂ�ing
responsibility, including Director of Clinical Operations. While in Clinical Re-
search, he also served as project manager for the development and licensure of
Tripedia, the first diphtheria, tetanus, and acellular pertussis vaccine approved by
FDA for use in U.S. infants. During his clinical research career at sanofi pasteur,
he contributed to the development and licensure of seven vaccines. Following his
work in clinical research, Mr. Hosbach took a position in the commercial operaÂ�
tions area of sanofi pasteur and quickly moved through the ranks on the busiÂ�
ness administration side of the vaccine division. During that time, Mr. Hosbach
led a number of departments within sanofi pasteur, gaining valuable business
experience within U.S. Commercial Operations. The departments he led during
that time included Public Health Sales and Marketing, Public Relations, Public
Affairs, New Product Marketing, and Business Intelligence. He has been a memÂ�
ber of the IOM Forum on Microbial Threats since 2005 and has been a Steering
Committee member of the Influenza Summit, which is jointly sponsored by the
CDC and the American Medical Association, since its inception. Since 2000
Mr. Hosbach has served on the Board of Directors for Pocono Medical Center
and Pocono Health Systems, located in East Stroudsburg, Pennsylvania. He also
serves as chairman of the Compensation Committee.
444 FUNGAL DISEASES
Stephen Albert Johnston, Ph.D., is currently director of the Center for Inno-
vations in Medicine in the Biodesign Institute at Arizona State University. His
cenÂ�ter focuses on formulating and implementing disruptive technologies for
basic problems in health care. The center has three divisions: Genomes to Vac-
cines, Cancer Eradication, and DocInBox. Genomes to Vaccines has developed
high-throughput systems to screen for vaccine candidates and is applying them to
predict and produce chemical vaccines. The Cancer Eradication group is working
on formulating a universal prophylactic vaccine for cancer. DocInBox is develÂ�
oping technologies to facilitate presymptomatic diagnosis. Dr. Johnston founded
the Center for Biomedical Inventions (also known as the Center for Translation
Research) at the University of Texas, Southwestern, the first center of its kind in
the medical arena. He and his colleagues have developed numerous inventions
and innovations, including the gene gun, genetic immunization, the tobacco
etch virus protease system, organelle transformation, digital optical chemistry
arrays, expression library immunization, linear expression elements, synbodies,
immunosignaturing diagnosis, and others. He also was involved in transcription
research for years, first cloning Gal4 and later discovering functional domains in
transcription factors and the connection of the proteasome to transcription. He has
been professor at the University of Texas Southwestern Medical Center at Dallas
and associate and assistant professor at Duke University. He has been involved
in several capacities as an adviser on biosecurity since 1996 and is a founding
member of BioChem 20/20.
Kent Kester, M.D., is currently the commander of the Walter Reed Army InstiÂ�
tute of Research (WRAIR) in Silver Spring, Maryland. Dr. Kester holds an un-
dergraduate biology degree from Bucknell University (1982) and an M.D. from
Jefferson Medical College (1986). He completed his internship and residency in
internal medicine at the University of Maryland Hospital/Baltimore VA Medical
Center (1989) and a fellowship in infectious diseases at the Walter Reed Army
Medical Center (1995). A malaria vaccine researcher with over 50 authored or
coauthored scientific manuscripts and book chapters, Dr. Kester has played a
major role in the development of the candidate falciparum malaria vaccine known
as RTS,S, having safely conducted the largest number of experimental malaria
challenge studies ever attempted to date. Dr. Kester’s previous military medical
research assignments have included director of the WRAIR Malaria Serology
Reference Laboratory; chief, Clinical Malaria Vaccine Development Program;
chief of the WRAIR Clinical Trials Center; and director of the WRAIR Division
of Regulated Activities. He currently is a member of the Steering Committee
of the NIAID/Uniformed Services University of the Health Sciences Infectious
Disease Clinical Research Program, as well as multiple NIAID Safety MonitorÂ�
ing Committees. He also serves as the consultant to the U.S. Army Surgeon
General in Medical Research and Development. Board certified in both internal
medicine and infectious diseases, Dr. Kester is also a fellow of both the AmeriÂ�
APPENDIX E 445
can College of Physicians and the IDSA. He holds faculty appointments at both
the Uniformed Services University of the Health Sciences and the University of
Maryland School of Medicine.
Gerald T. Keusch, M.D., is associate provost and associate dean for global
health at Boston University and Boston University School of Public Health. He
is a graduate of Columbia College (1958) and Harvard Medical School (1963).
After completing a residency in internal medicine, fellowship training in infecÂ�
tious diseases, and 2 years as an NIH research associate at the Southeast Asia
Treaty Organization Medical Research Laboratory in Bangkok, Thailand, Dr.
Keusch joined the faculty of the Mt. Sinai School of Medicine in 1970, where
he established a laboratory to study the pathogenesis of bacillary dysentery and
the biology and biochemistry of Shiga toxin. In 1979 he moved to Tufts MediÂ�
cal School and New England Medical Center in Boston to found the Division of
Geographic Medicine, which focused on the molecular and cellular biology of
tropical infectious diseases. In 1986 he integrated the clinical infectious diseases
program into the Division of Geographic Medicine and Infectious Diseases,
continuing as division chief until 1998. He has worked in the laboratory and
in the field in Latin America, Africa, and Asia on basic and clinical infectious
diseases and HIV/AIDS research. From 1998 to 2003, he was associate director
for international research and director of the Fogarty International Center at NIH.
Dr. Keusch is a member of the American Society for Clinical Investigation, the
Association of American Physicians, the ASM, and the IDSA. He has received
the Squibb (1981), Finland (1997), and Bristol (2002) awards of the IDSA. In
2002 he was elected to the IOM.
Rima F. Khabbaz, M.D., is deputy director for infectious diseases at CDC.

Prior to her current position, she served as director of CDC’s National Center
for Preparedness, Detection, and Control of Infectious Diseases and held other
leadership positions across the agency’s infectious disease national centers. She
is a graduate of the American University of Beirut, Lebanon, where she obtained
both her bachelor’s degree in science and her medical doctorate degree. She
trained in internal medicine and completed a fellowship in infectious diseases at
the University of Maryland, Baltimore. She joined CDC in 1980 as an epidemic
intelligence service officer, working in the Hospital Infections Program. During
her CDC career, she has made major contributions to advance infectious disÂ�
ease prevention, including leadership in defining the epidemiology of non-HIV
retroviruses (HTLV-I and II) in the United States and developing guidance for
counseling HTLV-infected persons, establishing national surveillance for hanta-
virus pulmonary syndrome following the 1993 U.S. outbreak, and developing
CDC’s blood safety and food safety programs related to viral diseases. She has
also played key roles in CDC’s responses to outbreaks of new and/or reemergÂ�ing
viral infections, including Nipah, Ebola, West Nile, SARS, and monkeypox, as
446 FUNGAL DISEASES
well as the 2001 anthrax attacks. She is a fellow of the IDSA and member of the
American Epidemiologic Society, the ASM, and the Council of State and Territo-
rial Epidemiologists. She served on IDSA’s Annual Meeting Scientific Program
Committee and currently serves on the society’s National and Global Public
Health Committee. In addition to her CDC position, she serves as clinical as-
sociate professor of medicine (infectious diseases) at Emory University. She is a
graduate of the National Preparedness Leadership Initiative at Harvard UniÂ�versity
and of the Public Health Leadership Institute at the University of North Carolina.
Stanley M. Lemon, M.D., is professor of medicine at the University of North

Carolina, School of Medicine, Chapel Hill, North Carolina. He received his
undergraduate A.B. degree in biochemical sciences from Princeton University
summa cum laude and his M.D. with honors from the University of Rochester.
He completed postgraduÂ�ate training in internal medicine and infectious diseases
at the University of North Carolina, Chapel Hill, and is board certified in both.
From 1977 to 1983 he served with the U.S. Army Medical Research and Devel-
opment Command, followed by a 14-year period on the faculty of the University
of North Carolina, School of Medicine. He moved to the University of Texas
Medical Branch in 1997, serving first as chair of the Department of Microbiology
and Immunology, then as dean of the School of Medicine from 1999 to 2004. Dr.
Lemon’s research interests relate to the molecular virology and pathogenesis of
the positive-stranded RNA viruses responsible for hepatitis. He has had a long-
standing interest in antiviral and vaccine development and has served as chair
of FDA’s Anti-Infective Drugs Advisory Committee. He is the past chair of the
Steering Committee on Hepatitis and Poliomyelitis of the WHO Programme on
Vaccine Development. He is past chair of the NCID-CDC Board of Scientific
Counselors and currently serves as a member of the U.S. Delegation to the
U.S.–Japan Cooperative Medical Sciences Program. He was co-chair of the NAS
Committee on Advances in Technology and the Prevention of Their Application
to Next Generation Biowarfare Threats, and he recently chaired an IOM study
committee related to vaccines for the proÂ�tection of the military against naturally
occurring infectious disease threats.
Edward McSweegan, Ph.D., is a program officer at NIAID. He graduated from

Boston College with a B.S. in biology in 1978. He has an M.S. in microbiology
from the University of New Hampshire and a Ph.D. in microbiology from the
University of Rhode Island. He was an NRC associate from 1984 to 1986 and
did postdoctoral research at the Naval Medical Research Institute in Bethesda,
Maryland. Dr. McSweegan served as an AAAS diplomacy fellow in the U.S.
State Department from 1986 to 1988, where he helped to negotiate science and
technology agreements with Poland, Hungary, and the former Soviet Union.
After moving to NIH, he continued to work on international health and infec-
tious disÂ�ease projects in Egypt, Israel, India, and Russia. Currently, he manages
APPENDIX E 447
NIAID’s bilateral program with India, the Indo–U.S. Vaccine Action Program,
and he represents NIAID in the HHS Biotechnology Engagement Program with
Russia and related countries. He is a member of AAAS, the ASM, and the Na-
tional AssoÂ�ciation of Science Writers. He is the author of numerous journal and
freelance articles.
Mark A. Miller, M.D., is currently the Director of the Division of International

Epidemiology and Population Studies for the Fogarty International Center at the
National Institutes of Health (NIH) in Bethesda, Maryland. He is also a Physi-
cian at the Yukon-Kuskokwim Delta Regional Hospital in Bethel, Alaska, which
primarily serves Native Americans. He previously served as a Medical Officer
on the Children’s Vaccine Initiative for WHO and the CDC, and Medical Epi-
demiologist for the CDC National Immunizations Program and Epidemiology
Program Office, Office of the Director. He also conducted research at the Armed
Forces Research Institute for Medical Studies in Bangkok, Thailand, the Yale
Arbovirus Research Unit, and Cornell University Medical College.
Dr. Miller received his B.A., magna cum laude, in neuroscience, biology, and
human ecology from Amherst College in 1983, and his M.D. from Yale Univer-
sity School of Medicine in 1990. He completed his internal medicine residency
at Yale New Haven Hospital/Hospital of St. Raphael and became board certified
in 1994. He has served as a member of many professional societies and steer-
ing committees, including the Secretary’s Advisory Council on Public Health
Preparedness Smallpox Modeling and several NSF, HHS, and NIH task forces.
He has presented and consulted nationally and internationally for organizations
includÂ�ing USAID, the Pan American Health Organization, and the World Bank.
Dr. Miller is a reviewer for nine journals, including the Journal of Infectious Dis-
eases, The Lancet, and the Journal of the American Public Health Association.
He has won many awards, including the Distinguished Service Medal, from the
U.S. Public Health Service and the CDC. He has published more than 50 scien-
tific articles in the peer-reviewed literature, nine books and/or book chapters, and
more than 50 letters and abstracts.
Paul F. Miller, Ph.D.,5 is chief scientific officer for antibacterials research. He

received his undergraduate degree in biology from LeMoyne College, and subÂ�
sequently earned a Ph.D. in microbiology and immunology from the Albany
Medical College in 1987. Following 4 years of postdoctoral studies on yeast
molecular genetics at NIH in Bethesda, Maryland, Dr. Miller joined the Parke-
Davis Pharmaceutical Research Division of Warner-Lambert Company in Ann
Arbor, Michigan, in 1990 as a senior scientist in the Infectious Diseases DepartÂ�
ment, where he developed a number of novel screens and mechanism-of-action
5 Forum member until July 31, 2011.

448 FUNGAL DISEASES
tools. He then moved to Pfizer in 1997 as manager of the Antibacterials Biology

Research group within the Antibacterials, Immunology, and Cancer Zone at the
Groton, Connecticut, research labs, and has taken on increasing responsibility
since that time. In his current role, he is responsible for all antibacterial research
activities through early clinical development, as well as collaboratively estabÂ�
lishing R&D strategies in this disease area. His specific research interests and
expertise include genetic mechanisms of intrinsic antibiotic resistance in bacteria
as well as the use of novel genetic technologies for the elucidation of antibiotic
mechanisms of action.
Stephen S. Morse, Ph.D.,6 is Professor of Epidemiology at the Mailman School

of Public Health of Columbia University, and Director of the PREDICT project of
the USAID Emerging Pandemic Threats program. He was also founding Director
of the Columbia University Center for Public Health Preparedness. He returned
to Columbia in 2000 after four years in government service as Program Manager
at the Defense Advanced Research Projects Agency, where he codirected the
Pathogen Countermeasures Program and subsequently directed the Advanced
Diagnostics Program. Before going to Columbia, he was Assistant Professor of
Virology at the Rockefeller University in New York, where he remains an adjunct
faculty member. He is the editor of two books, Emerging Viruses (Oxford UniÂ�
versity Press, 1993; paperback, 1996), which was selected by American Scientist
for its list of 100 Top Science Books of the 20th Century, and The Evolutionary
Biology of Viruses (Raven Press, 1994). He was a founding Section Editor of the
CDC journal Emerging Infectious Diseases and was formerly an Editor-in-Chief
of the Pasteur Institute’s journal Research in Virology. Dr. Morse was Chair and
principal organizer of the 1989 NIAID-NIH Conference on Emerging Viruses, for
which he originated the term and concept of emerging viruses/infections. He has
served as a member of the IOMâ•‚NAS Committee on Emerging Microbial Threats
to Health, chaired its Task Force on Viruses, and was a contributor to the resultÂ�
ing report Emerging Infections (1992). He has served on a number of NAS and
IOM committees, including the IOM Committee on Xenograft Transplantation.
Dr. Morse also served as an adviser to WHO and several government agencies.
He is a fellow of the AAAS, the New York Academy of Sciences (and a past
Chair of its microbiology section), the American Academy of Microbiology, the
American College of Epidemiology, and an elected life member of the Council
on Foreign Relations. He was the founding Chair of ProMED, the nonprofit interÂ�
national Program to Monitor Emerging Diseases, and was one of the originators
of ProMED-mail, an international network inaugurated by ProMED in 1994 for
outbreak reporting and disease monitoring using the Internet. Dr. Morse received
his Ph.D. from the University of Wisconsin, Madison.

APPENDIX E 449
George Poste, Ph.D., D.V.M., is chief scientist, Complex Adaptive Systems

Initiative, and Del E. Webb Professor of Health Innovation at Arizona State UniÂ�
versity (ASU). He assumed this post in 2009. From 2003 to 2009 he directed and
built the Biodesign Institute at ASU. In addition to his academic post, he serves
on the Board of Directors of Monsanto, Exelixis, Caris Life Sciences, LGC, and
the Scientific Advisory Board of Synthetic Genomics. From 1992 to 1999 he
was Chief Science and Technology Officer and President, R&D, of SmithKline
Beecham (SB). During his tenure at SB he was associated with the successful
registration of 31 drug, vaccine, and diagnostic products. In 2004 he was named
“R&D Scientist of the Year” by R&D Magazine, in 2006 he received the Einstein
award from the Global Business Leadership Council, and in 2009 he received the
Scrip Lifetime Achievement award voted by the leadership of the global pharÂ�
maceutical industry.
He has published over 350 research papers and edited 14 books on pharÂ�
maceutical technologies and oncology. He has received honorary degrees in sciÂ�
ence, law, and medicine for his research contributions and was honored in 1999
by Her Majesty Queen Elizabeth II as a Commander of the British Empire for
his contributions to international security. He is a Fellow of the Royal Society,
the Royal College of Pathologists, and the U.K. Academy of Medicine; a DistinÂ�
guished Fellow at the Hoover Institution, Stanford University; and a member of
the Council on Foreign Relations. He has served on numerous government panels
related to biosecurity and national competitiveness.
John C. Pottage, Jr., M.D., has been vice president for Global Clinical DevelopÂ�
ment in the Infectious Disease Medicine Development Center at GlaxoSmith-
Kline since 2007. Previously he was senior vice president and chief medical
officer at Achillion Pharmaceuticals in New Haven, Connecticut. Achillion is
a small biotechnology company devoted to the discovery and development of
medicines for HIV, hepatitis C virus, and resistant antibiotics. Dr. Pottage ini-
tially joined Achillion in May 2002. Prior to Achillion, Dr. Pottage was medical
director of Antivirals at Vertex Pharmaceuticals. During this time he also served
as an assoÂ�ciate attending physician at the Tufts New England Medical Center in
Boston. From 1984 to 1998, Dr. Pottage was a faculty member at Rush Medical
College in Chicago, where he held the position of associate professor, and also
served as the medical director of the Outpatient HIV Clinic at Rush-Presbyterian-
St. Luke’s Medical Center. While at Rush, Dr. Pottage was the recipient of several
teaching awards and is a member of the Mark Lepper Society. Dr. Pottage is a
graduate of St. Louis University School of Medicine and Colgate University.
David Rizzo7 received his Ph.D. in plant pathology from the University of Min-
nesota and joined the faculty of the University of California-Davis, Department

450 FUNGAL DISEASES
of Plant Pathology and the Graduate Group in Ecology in 1995. Research in his
lab focuses on the ecology and management of forestÂ€tree diseases, including dis-
eases caused by both native and introduced pathogens. Research in the lab takes
a multiscale approach ranging from experimental studies on the basic biology of
organisms to field studies across forest landscapes. Active collaborations include
projects with landscape ecologists, epidemiologists, molecular biologists, ento-
mologists, and forest managers. The primary research effort in the lab is currently
PhytophthoraÂ€species in California coastal forests, with an emphasis onÂ€Sudden
Oak Death. As part of his research on Sudden oak Death, Dr. Rizzo also serves
as the scientific advisor for the California Oak Mortality Task Force.In conifer
forests of the Sierra Nevada Mountains, the lab studies a variety of diseases and
their relationship to past and present forest management and conservation issues.
In addition to research, Dr. Rizzo teaches undergraduate and graduate courses in
mycology as well as introductory biology. Since 2004, he has been director of the
Science and Society program in the College of Agricultural and Environmental
Sciences. Science and Society is an academic program designed to offer students
the opportunity to discover the interdisciplinary connections that link the biologi-
cal, physical and social sciences with societal issues and cultural discourses.
Gary A. Roselle, M.D., is program director for infectious diseases for the VA
Central Office in Washington, DC, as well as the chief of the medical service at
the Cincinnati VA Medical Center. He is a professor of medicine in the DepartÂ�
ment of Internal Medicine, Division of Infectious Diseases, at the University of
Cincinnati College Of Medicine. Dr. Roselle serves on several national adviÂ�
sory committees. In addition, he is currently heading the Emerging Pathogens
Initiative for the VA. He has received commendations from the undersecretary
for health for the VA and the secretary of VA for his work in the Infectious DisÂ�
eases Program for the VA. He has been an invited speaker at several national
and international meetings and has published more than 90 papers and several
book chapters. Dr. Roselle received his medical degree from the OSU School of
Medicine in 1973. He served his residency at the Northwestern University School
of Medicine and his infectious diseases fellowship at the University of Cincinnati
School of Medicine.
Alan S. Rudolph, Ph.D., M.B.A., has led an active career in translating inter-
disciplinary life sciences into useful applications for biotechnology develÂ�opment.
His experience spans basic research to advanced development in academia, gov-
ernment laboratories, and most recently in the nonprofit and private sectors. He
has published more than 100 technical publications in areas including molecular
biophysics, lipid self-assembly, drug delivery, blood subÂ�stitutes, medical imag-
ing, tissue engineering, neuroscience, and diagnostics. As a National Research
Council Post-Doctoral Fellow, his earliest work at the U.S. Naval Research
Laboratory (NRL) demonstrated the translational value of strategies used by or-
APPENDIX E 451
ganisms that survive environmental extremes to preserve Defense products such

as biosensors and blood products for field deployment. After a decade at NRL he
was recruited to join the Defense Advanced Research Projects Agency, to lead
new strategic efforts to extract and exploit useful prinÂ�ciples and practices in life
sciences and technology and establish an agency-wide strategy for investments in
biosciences and biotechnology. As Chief of Biological Sciences and Technology,
Dr. Rudolph established a framework for investments that continue today. These
include new programs in broad areas of bioscience and technology such as sen-
sors, diagnostics, materials, robotics, biomolecular, cell and tissue engineering,
medical devices, and neuroscience and technology, including the current efforts
in revolutionizing prosthetics. He received a meritorious civil service citation
from the Office of the Secretary of Defense for his contributions to defining and
implementing a new generation of life sciences and national security investments.
In 2003, he left civil service for the private sector and starting new corporate
biotechnology efforts. As Chief Executive Officer of Adlyfe Inc., a diagnostic
platform company, and Board Chairman of Cellphire Inc., focused on developÂ�
ment of novel hemostatic biologics for bleeding injury, he took nascent technolÂ�
ogy demonstrations and secured venture capital funding and pharmaceutical
partnerships while managing all aspects of development toward first human use.
These efforts included managing early manufacturing and regulatory strategies
required for FDA approval of diagnostics and therapeutics. Most recently, he
started a new international nonprofit foundation and, as Director of the InternaÂ�
tional Neuroscience Network Foundation, he has secured corporate and private
philanthropic donors to fulfill the mission of the organization focused on brain
STEM efforts and clinical trial management in underserved populations. He has
a doctorate degree in zoology from the University of California at Davis and an
M.B.A. from the George Washington University.
Kevin Russell, M.D., M.T.M.&H., F.I.D.S.A. CAPT MC USN, is the Director,

Department of Defense Global Emerging Infections Surveillance and Response
System, and Deputy Director, Armed Forces Health Surveillance Center, in the
U.S. Department of Defense. In this position, his priorities have been standardÂ�
ization, greater affiliations with world militaries, continuing to introduce sciÂ�entific
rigor into the network, and synchronization with other U.S. government global
surveillance programs. He graduated from the University of Texas Health Science
Center San Antonio Medical School in 1990; after a family practice internship,
he was accepted into the Navy Undersea Medicine program. He was stationed in
Panama City, Florida, at the Experimental Diving Unit where he worked in div-
ing medicine research from 1991 to 1995. After a preventive medicine residency
with a masters in tropical medicine and hygiene, he was transferred to Lima, Peru,
where he became head of the Virology Laboratory. His portfolio included febrile
illness (largely arboviral in origin) and HIV surveillance studies in eight different
countries of South America, as well as prospective dengue transmission studies.
452 FUNGAL DISEASES
In 2001, he moved back to the United States and became the director of the Re-
spiratory Disease Laboratory at the Naval Health Research Center in San Diego,
California. Febrile respiratory illness surveillance in recruits of all services was
expanded into shipboard populations, Mexican border populations, support for
outbreaks, and deployed settings. Validation and integration of new and emerging
advanced diagnostic capabilities, utilizing the archives of specimens maintained
at the laboratory, became a priority. A BSL-3-Enhanced was constructed. Projects
expanded in 2006 to clinical trials support as Dr. Russell became the Principal
InvestigaÂ�tor for the Navy site in the FDA Phase III adenovirus vaccines trial, and
more recently to support the Phase IV post-marketing trial of the recently FDA-
approved ACAM2000 smallpox vaccine.
Janet Shoemaker is director of the ASM’s Public Affairs Office, a position she
has held since 1989. She is responsible for managing the legislative and regulaÂ�
tory affairs of this 42,000-member organization, the largest single biological sciÂ�
ence society in the world. Previously, she held positions as assistant director of
public affairs for the ASM; as ASM coordinator of the U.S.–U.S.S.R. Exchange
Program in Microbiology, a program sponsored and coordinated by the NSF and
the U.S. Department of State; and as a freelance editor and writer. She received
her baccalaureate, cum laude, from the University of Massachusetts and is a
graduate of the George Washington University programs in public policy and in
editing and publications. She is a member of Women in Government Relations,
the American Society of Association Executives, and AAAS. She has coauthored
articles on research funding, biotechnology, biodefense, and public policy issues
related to microbiology.
P. Frederick Sparling, M.D., is professor of medicine, microbiology, and im-

munology at the University of North Carolina (UNC), Chapel Hill. He is director
of the SouthEast Sexually Transmitted Infections Cooperative Research Center
and also the Southeast Regional Centers of Excellence in Biodefense and Emerg-
ing Infections. Previously he served as chair of the Department of MediÂ�cine and
chair of the Department of Microbiology and Immunology at UNC. He was
president of the IDSA from 1996 to 1997. He was also a member of the IOM
Committee on Microbial Threats to Health (1990–1992) and the IOM Committee
on Emerging Microbial Threats to Health in the 21st Century (2001–2003). Dr.
Sparling’s laboratory research has been on the genetics and molecular biology
of bacterial outer membrane proteins, with a major emphasis on gonococci and
meningococci. His work helped to define the genetics of antibiotic resistance in
gonococci and the role of iron-scavenging systems in the pathogenesis of human
gonorrhea. Current interests include pathogenesis of gonococcal infections and
development of a vaccine for gonorrhea and managing a large multi-institution
interactive research group focused on emerging infections and biodefense.
APPENDIX E 453
Terence Taylor is the founding president of the International Council for the
Life Sciences (ICLS). The ICLS is an independent nonprofit organization regÂ�
istered in the United States and in the European Union. The ICLS is designed
to promote best practices and codes of conduct for safety and security in relaÂ�
tion to biological risks. Terence Taylor also served as the vice president, Global
Health and Security, at the Nuclear Threat Initiative. Prior to these appointments
Terence Taylor was assistant director at the International Institute for Strategic
Studies (IISS) in London and was president and executive director of IISS-US in
Washington, DC. At IISS, in addition to his overall program responsibilities, he
led the Institute’s work on life sciences and security. He has substantial experiÂ�
ence in international security policy matters as a U.K. government official (both
military and diplomatic) and for the United Nations (UN) both in the field and
at UN Headquarters. He was a commissioner and one of the Chief Inspectors
with the UN Special Commission on Iraq, with particular responsibilities for
biologiÂ�cal issues. His government experience is related to both military field
operations and to the development and implementation of policies in relation to
arms control and nonproliferation treaties and agreements for both conventional
weapons and weapons of mass destruction and the law of armed conflict aspects
of International HumanÂ�itarian Law. He has also conducted consulting work on
political risk assessment and studies of the private biotechnology industry. He
was a Science Fellow at Stanford University’s Center for International Security
and Cooperation. He was an officer in the British Army with experience in many
parts of the world includÂ�ing UN peacekeeping, counterinsurgency, and counter-
terrorism operations.
Murray Trostle, Dr.P.H., is a foreign service officer with USAID, presently

serving as the deputy director of the Avian and Pandemic Influenza PreparedÂ�
ness and Response Unit. Dr. Trostle attended Yale University, where he received
a master’s in public health in 1978, focusing on health services administration.
In 1990, he received his doctorate in public health from UCLA. His research
involved household survival strategies during famine in Kenya. Dr. Trostle has
worked in international health and development for approximately 38 years. He
first worked overseas in the Malaysian national malaria eradication program in
1968 and has since focused on health development efforts in the former Soviet
Union, Africa, and Southeast Asia. He began his career with USAID in 1992 as a
postdoctoral fellow with AAAS. During his career he has worked with a number
of development organizations, such as the American Red Cross, Project Concern
International, and the Center for Development and Population Activities. With
USAID, Dr. Trostle has served as director of the child immunization cluster,
where he was chairman of the European Immunization Interagency CoordinatÂ�
ing Committee and USAID representative to the Global Alliance on Vaccines
and Immunization. Currently, Dr. Trostle leads the USAID Infectious Disease
Surveillance Initiative as well as the Avian Influenza Unit.
454 FUNGAL DISEASES
Mary E. Wilson, M.D., is Associate Professor of Global Health and PopulaÂ�tion

at the Harvard School of Public Health. Her academic interests include the ecol-
ogy of infections and emergence of microbial threats, travel medicine, tuberculo-
sis, and vaccines. Her undergraduate degree in French, English, and philosophy
was awarded by Indiana University; she received her M.D. from the University of
Wisconsin and completed an internal medicine residency and infectious disease
fellowship at the Beth Israel Hospital in Boston (now Beth Israel-Deaconess
Medical Center). She was Chief of Infectious Diseases at Mount Auburn Hos-
pital, a Harvard-affiliated community teaching hospital in CamÂ�bridge, Massa-
chusetts, for more than 20 years. She is a Fellow in the IDSA and the American
College of Physicians. She has served on ACIP of the CDC, the Academic Ad-
visory Committee for the National Institute of Public Health in Mexico, and on
four committees for the IOM of the National Academies, including the Commit-
tee on Emerging Microbial Threats to Health in the 21st Century, whose report
(Microbial Threats to Health: Emergence, Detection, and Response) was released
in March 2003. She has worked in Haiti at the Albert Schweitzer HosÂ�pital and
leads the Harvard-Brazil Collaborative Course on Infectious Diseases, which is
taught in Brazil. In 1996 she was a resident scholar at the Bellagio Study Center,
Italy, and in 2002 she was a Fellow at the Center for Advanced Study in the Be-
havioral Sciences in Stanford, California. She was a member of the Pew National
Commission on Industrial Farm Animal Production, whose report Putting Meat
on the Table: Industrial Farm Animal Production in America was released in the
spring of 2008. A former GeoSentinel Site Director (Cambridge), she now serves
as a Special Advisor to the GeoSentinel Surveillance Network, a global network.
She has lectured and published widely, serves on several editoÂ�rial boards, and is
an associate editor for Journal Watch Infectious Diseases. She is the author of A
World Guide to Infections: Diseases, Distribution, Diagnosis (Oxford University
Press, New York, 1991); senior editor, with Richard Levins and Andrew Spiel-
man, of Disease in Evolution: Global Changes and Emergence of Infectious
Diseases (New York Academy of Sciences, 1994); and editor of the volume New
and Emerging Infectious Diseases (Medical Clinics of North America) published
in 2008. She joined the Board of Trustees for ICDDR, B (International Centre for
Diarrheal Disease Research, Bangladesh) in 2009 and is a member of the Board
of Scientific Counselors for the CDC, the FXB-USA Board, and the APUA Board
of Directors.
Appendix F
Speaker Biographies
Karen Bartlett, Ph.D., M.Sc., received her B.A. from the University of Victo-
ria, her M.Sc. in Occupational Hygiene from the University of British Columbia
(UBC), and her Ph.D. in Interdisciplinary Studies (Environmental Health) also
from UBC. She completed postdoctoral training in Inhalation Toxicology at the
University of Iowa. She is an associate professor in the School of Environmental
Health, in the College of Interdisciplinary Studies at UBC. Dr. Bartlett’s current
research interests are in four thematic areas: environmental sources of infectious
disease; mold and building material interactions (the built environment); animal
models of lung infections for therapeutic protocols; and occupational and envi-
ronmental exposure to airborne biologic particles. Examples of recent research
are bioaerosol exposures in the built environment (including First Nations hous-
ing); exposures to compost workers; and environmental sources of Cryptococcus
gattii. Dr. Bartlett is an adjunct faculty in the Department of Pathology, UBC,
and an associate faculty member in the Institute of Resource and Environmental
Sustainability and the School of Populations and Public Health.
Meredith Blackwell, Ph.D., is interested in phylogeny, evolution, and life history

studies of fungi associated with arthropods. Her current research focuses on the
interactions between gut yeasts of fungus- and wood-feeding beetles. She and
her colleagues have sampled yeasts from a largely unexplored habitat—the gut
of beetles in the United States, Latin America, and Thailand—and discovered
more than 200 undescribed species, about 20 percent of all known yeasts. Fur-
thermore, gene cloning and imaging studies have led to the discovery of a larger
community of gut organisms in wood-feeding beetles, including parabasalids and
bacteria as well as the yeasts that produce enzymes that degrade plant cell walls.
455
456 FUNGAL DISEASES
Dr. Blackwell, who is Boyd Professor at Louisiana State University, has been
involved in several projects involving the fungal systematics community. The
recent Deep Hypha and Assembling the Fungal Tree of Life projects involved
more than 100 mycologists from 25 countries in phylogenetic studies of major
groups of fungi and a phylogenetic classification used in many major publica-
tions. Dr. Blackwell is a Distinguished Mycologist of the Mycological Society of
America and Fellow of the British Mycological Society. She served as president
of the International Mycological Association and the Mycological Society of
America and is coauthor of Introductory Mycology, a widely used textbook cur-
rently under revision.
David Blehert, Ph.D., earned his doctorate degree in bacteriology in 1999 from
the University of Wisconsin–Madison where he studied the biotransformation
of munitions manufacturing wastes as mediated by soil bacteria. He then com-
pleted a postdoctoral fellowship at the National Institutes of Health in Bethesda,
Maryland where he investigated bacterial communication mechanisms among
constituents of the human dental plaque community. His research emphasis was
on the role of the signaling molecule autoinducer-2 in the formation of bacterial
biofilms. Dr. Blehert joined the U.S. Geological Survey–National Wildlife Health
Center in Madison, Wisconsin, in 2003 as the head of the diagnostic microbiology
laboratory. Current research projects under way in his laboratory include charac-
terization of microbial aspects of the pathogenesis and epidemiology of bat white-
nose syndrome; the use of molecular markers to understand the epidemiology of
avian cholera in wild waterfowl; and development of rapid in vitro techniques
for the detection of botulinum neurotoxins. His laboratory’s collaborative efforts
to identify the fungus that causes the skin infection hallmark of bat white-nose
syndrome were published in the January 9, 2009, issue of Science.
Arturo Casadevall, M.D., Ph.D., is the Leo and Julia Forchheimer Profes-
sor and Chair of Microbiology & Immunology at the Albert Einstein College
of Medicine. Dr. Casadevall received both his M.D. and Ph.D. (biochemistry)
degrees from New York University in New York. Subsequently, he completed
internship and residency in internal medicine at Bellevue Hospital in New York.
Later he completed subspecialty training in infectious diseases. Dr. Casadevall’s
major research interests are in fungal pathogenesis and the mechanism of anti-
body action. Dr. Casadevall has authored more than 470 scientific papers. He has
been elected to membership in the American Society for Clinical Investigation,
American Academy of Physicians, and American Academy of Microbiology. He
was elected a Fellow of the American Academy for the Advancement of Science
(AAAS) and has received numerous honors. In 2005, he received the Alumni
Award in basic science from his alma mater, New York University. Dr. Casadevall
is editor in chief of mBio and serves on the editorial board of the Journal of Clini-
APPENDIX F 457
cal Investigation and the Journal of Experimental Medicine. In 2008, he received

the Hinton Award from the American Society for Medicine for his efforts in train-
ing scientists from underrepresented minority groups.
Peter Daszak, Ph.D., is president of EcoHealth Alliance (formerly Wildlife

Trust), a U.S.-based organization that conducts research and field programs on
global health and conservation. At Wildlife Trust, Dr. Daszak manages a head-
quarters staff of 35 and a global staff of more than 700. The staff conduct research
and manage initiatives to prevent emerging pandemics and to conserve wildlife
biodiversity. This includes research on zoonoses that spill over from wildlife in
emerging disease “hot spots,” including influenza, Nipah virus, Severe Acute
Respiratory Syndrome (SARS), West Nile virus, and others. Dr. Daszak’s work
includes identifying the first case of a species extinction due to disease; the dis-
covery of chytridiomycosis, the major cause of global amphibian declines; pub-
lishing the first paper to highlight emerging diseases of wildlife; coining the term
“pathogen pollution”; discovery of the bat origin of SARS-like coronaviruses;
identifying the drivers of Nipah and Hendra virus emergence; and producing the
first emerging disease “hot spots” map.
Dr. Daszak is a member of the Council of Advisors of the One Health ComÂ�
mission, Treasurer of DIVERSITAS, past member of the International Standing
Advisory Board of the Australian Biosecurity CRC, and past member of the
Institute of Medicine (IOM) Committee on Global Surveillance for Emerging
Zoonoses and the National Research Council (NRC) comÂ�mittee on the future
of veterinary research. He is editor in chief of the Springer journal Ecohealth
and past treasurer and a founding director of the International Ecohealth Asso-
ciation. In 2000, he won the Commonwealth Scientific and Industrial Research
Organisation medal for collaborative research in the discovery of amphibian
chytridiomycosis. He has published more than 130 scientific papers and book
chapters, including papers in Science, Nature, PNAS, The Lancet, PLoS Biology,
and other leading journals. His work has been the focus of articles in the New
York Times, The Wall Street Journal, The EconoÂ�mist, The Washington Post, US
News & World Report, CBS, 60 Minutes, CNN, ABC, NPR’s Talk of the Nation,
and Morning Edition & Fresh Air with Terri Gross. He is a former guest worker
at the Centers for Disease Control and Prevention (CDC), where he assisted in
the pathology activity during the 1999 Nipah virus outbreak. His work is funded
by the John E. Fogarty International Center of NIH, the National Institute of Al-
lergy and Infectious Diseases, the National Science Foundation (NSF), the U.S.
Agency for International Development (USAID), Google.org, Rockefeller, and
other foundations. To date, his group is one of the few to have been awarded three
prestigious NIH/NSF Ecology of Infectious Disease awards and is one of four
partners to share a recent multimillion-dollar award from USAID (“PREDICT”)
with the goal of predicting and preventing the next emerging zoonotic disease.
458 FUNGAL DISEASES
Matthew C. Fisher, Ph.D., currently holds the appointment of reader in the

Department of Infectious Disease Epidemiology at the Imperial College London.
He received his B.S. and Ph.D. in biology from Edinburgh University and was a
Postdoctoral Fellow at the University of California, Berkeley.
Global health necessitates the adoption of a broad perspective: Anthro-
pogenic activity is accelerating global changes, with inevitable decreases in
human welfare as ecosystems deteriorate. His research program focuses on the
kingdom Fungi, whose impact on global health is increasing as a consequence of
global changes. Broadly Dr. Fisher’s research investigates the changing impact
of fungal disease by focusing on two themes: The first, fungal disease ecology,
ascertains the environmental envelopes that are associated with mycoses, and
the occurrence of fungal pandemics and panzootics. This theme has, at its core,
the idea that anthropogenic activity is widely mixing fungal pathogens across
global scales. However, the manifestation of disease only occurs if a match oc-
curs between the invader-fungus and the recipient-host/biome: He investigates
this within current and future-climate scenarios for several important human,
animal, and plant pathogens. The second theme is fungal evolution, and here he
investigates the adaptive stored potential that exists within different fungal patho-
gens. Dr. Fisher’s combined use of population genetics, comparative genomics,
and molecular epidemiology are used to decipher the evolutionary histories of
mycoses, to investigate their origins, and to predict their future trajectories as
pathogens.
John N. Galgiani, M.D., was born in San Francisco, received his B.A. from
Stanford University, his M.D. from Northwestern University, and a fellowship in
Infectious Diseases from Stanford. In 1978, Dr. Galgiani joined the faculty of the
University of Arizona currently he is Professor of Medicine. Dr. Galgiani has fo-
cused his career primarily on the special problems of coccidioidomycosis (Valley
Fever) and its impact on the general population and special groups such as organ
transplant recipients and patients with AIDS. For 19 years, he was project direc-
tor of an NIH-sponsored coccidioidomycosis clinical trials group. Dr. Galgiani’s
laboratory has collaborated in efforts to develop vaccines to prevent Valley Fever.
For the past 5 years, Dr. Galgiani has led a development program for nikkomycin
Z, a possible cure for Valley Fever, now in clinical trials. In 1996, Dr. Galgiani
founded the Valley Fever Center for Excellence to disseminate information about
Valley Fever, help patients with the severest complications of this disease, and
encourage research into the biology and diseases of its etiologic agent.
Julie Harris, Ph.D., M.P.H., received her bachelors’ degree from Rensselaer
Polytechnic Institute, her Ph.D. in microbiology from Columbia University and
her M.P.H. from Johns Hopkins in epidemiology before joining the CDC’s Epi-
demic Intelligence Service in 2007, where she worked on the prevention and
control of enteric infections in the United States and in several countries in Africa.
APPENDIX F 459
She joined the Mycotic Diseases Branch at the CDC in 2009, where her first task
was to create a surveillance system for Cryptococcus gattii, an emerging fungal
infection in the Pacific Northwest. She also works on mycotoxins in Bangladesh
and Guatemala, cryptococcal infections in Thailand, and coccidioidomycosis in
the United States.
Joseph Heitman, M.D., Ph.D., is chair and James B. Duke Professor, Depart-
ment of Molecular Genetics and Microbiology, Duke University. He received his
B.S. and M.S. in chemistry and biochemistry at the University of Chicago, and
M.D. and Ph.D. from Cornell and Rockefeller Universities. He was an EMBO
fellow at the Biocenter in Switzerland where he pioneered yeast as a model to
study immunosuppressive drugs. He elucidated the role of FKBP12 in forming
complexes with FK506 and rapamycin that inhibit cell signaling and growth
and discovered the targets of rapamycin TOR1/TOR2, pathways conserved from
yeasts to humans. Dr. Heitman has been at Duke since 1992, and focuses on
model and pathogenic fungi, studying the evolution of sex and roles of sexual
reproduction in microbial pathogens; how cells sense and respond to nutrients
and the environment; the targets and mechanisms of action of immunosuppres-
sive and antimicrobial drugs; and the genetic and molecular basis of microbial
pathogenesis and development. Their discovery of fungal unisexual mating has
implications for how sex might create diversity de novo with implications for
pathogen evolution and emergence.
Dr. Heitman received the Burroughs Wellcome Scholar Award in Molecular
Pathogenic Mycology, the ASBMB AMGEN award, and the Infectious Diseases
Society of America (IDSA) Squibb Award. He has taught the Molecular Mycol-
ogy Course at the MBL since 1998. He is an editor for Eukaryotic Cell, Fungal
Genetics and Biology, and PLoS Pathogens; a board member for PLoS Biology,
Current Biology, and Cell Host & Microbe; an advisory board member for the
Broad Institute Fungal Genome Initiative and the Department of Energy/JGI Fun-
gal Kingdom project; and cochair/chair for the FASEB Microbial Pathogenesis
conference (2011, 2013). He is a Fellow of the American Society for Clinical
Investigation, the IDSA, the American Academy of Microbiology, AAAS, and
the Association of American Physicians.
Steven Holland, M.D., received his M.D. and Medicine and Infectious Disease
training from Johns Hopkins. He came to NIH in 1989 as a National Research
Council fellow in the Laboratory of Molecular Microbiology, working on tran-
scriptional regulation of HIV. In 1991, Dr. Holland joined the Laboratory of
Host Defenses, shifting his research to the host side, with a focus on phagocyte
defects and their associated infections. His work centered on the pathogenesis
and management of chronic granulomatous disease, as well as other congenital
immune defects affecting phagocytes, including those predisposing to mycobac-
terial and fungal diseases. In 2004, he became chief of the Laboratory of Clinical
460 FUNGAL DISEASES
Infectious Diseases. The laboratory takes a fully integrated approach to infectious

disease, incorporating the molecular genetics of the host and the pathogen as well
as mechanisms of pathogenesis that allow the development and study of novel
therapeutics. The integrated bench-to-bedside model adds clinical insight into
mechanisms of action and therapy. The laboratory has been engaged in the human
genetics of fungal susceptibility for several years, identifying specific Mendelian
associations with genes in the NADPH, STAT3, DOCK8, and interferon gamma/
IL-12 pathways.
Mogens Støvring Hovmøller, Ph.D., is senior plant pathologist at Aarhus Uni-

versity, Faculty of Agricultural Sciences, Department of Integrated Pest Manage-
ment in Denmark. He completed his Ph.D. in 1991 at the Royal and Veterinary
and Agricultural University (now Copenhagen University) in population genetics
of fungal crop pathogens. His research interests expand from host–pathogen and
pathogen–environment interactions to population genetics, evolutionary biol-
ogy, and epidemiology of crop pathogens. He has been involved in multiple
international research projects focusing on dispersal and evolution of Puccinia
striiformis, a basidiomycete fungus causing yellow rust on wheat. He is a board
member of the European and Mediterranean Cereal Rust Foundation and member
of the Technical Advisory Committee of the Borlaug Global Rust Initiative. He is
leading a Global Rust Reference Center (GRRC) located in Denmark, which was
launched in 2009 by Aarhus University, the International Center for Agricultural
Research in the Dry Areas (ICARDA), and the International Maize and Wheat
Improvement Center (CIMMYT). GRRC is complementing existing wheat rust
surveillance efforts by the Food and Agricultural Organisation of the United Na-
tions, CIMMYT, ICARDA, and national rust diagnostic laboratories in Europe,
Australia, North America, and elsewhere, and extends and maintain a wheat rust
gene bank to support international resistance breeding and research.
Barbara Howlett, Ph.D., has a B.Sc. (Honors) in biochemistry and a Ph.D. in

botany from University of Melbourne, Australia. She has also spent time at the
Australian National University, Canberra, University of California, Berkeley, and
Stanford University. She has worked in a diverse range of research areas, includ-
ing immunology, memory in bacteria chemotaxis, nitrogen fixation, and plant
disease. Her current research is on blackleg, the major disease of the oilseed crop,
canola. Her approach to this topic is multidisciplinary and holistic, ranging from
developing disease management strategies for farmers, to coleading an initiative
to sequence and annotate the genome of the blackleg fungus. She is also inter-
ested in parallels and differences between fungal pathogenesis of plants and ani-
mals. These experiences and her membership on a panel of the Australian Grains
Research and Development Corporation, which invests $AUD120 million pa into
grains research, have familiarized her with disease threats to agriculture and food
production. Dr. Howlett has published more than 110 refereed scientific articles.
APPENDIX F 461
She is an associate editor of PLoS Pathogens, a senior editor of Molecular Plant

Pathology, and on the editorial board of Eukaryotic Cell.
Mike Jeger, Ph.D., is a professor in the Division of Biology, Imperial College

London, based at the Silwood Park campus, near Ascot, U.K. His research in-
terests are in quantitative plant disease epidemiology, mathematical modeling,
and disease management. He has worked on a wide range of plant–pathogen
systems, including fungal (and other) pathogens of agricultural and horticultural
crops, forest trees, and plants in natural grassland communities and has published
extensively in the related scientific literature. He currently works on theoretical
models concerning the spread of exotic plant pathogens in networks, such as in
the horticultural nursery trade, where invasion criteria and the potential size of
disease outbreaks depend critically on network structure. He is involved profes-
sionally and internationally in issues relating to plant health and is currently
chair of the Plant Health Panel of the European Food Standards Agency, which
provides independent advice to the European Commission. He will soon take up
an emeritus professorship and become a senior research investigator in the Centre
for Environmental Policy at Imperial College, where he will work on policy and
technical issues relating to biosecurity.
Lawrence C. Madoff, M.D., serves as director of epidemiology and immuni-

zation for the Massachusetts Department of Public Health, where he oversees
programs related to infectious disease threats in the commonwealth. He is an
academic infectious disease physician specializing in the epidemiology of emerg-
ing pathogens, bacterial vaccine development, and international health. He is a
professor of medicine at the University of Massachusetts Medical School and is
on the attending staff at University of Massachusetts Memorial Medical Center.
In addition, Dr. Madoff has been the editor of ProMED-mail, the Program for
Monitoring Emerging Diseases, since 2002. In this capacity, he has expanded
the program to more than 50,000 participants and extended its reach through the
development of regional projects in the Mekong Basin, Africa, and the newly
independent states of the former Soviet Union. Dr. Madoff chaired the organiz-
ing committees for the International Meetings on Emerging Diseases and Sur-
veillance in 2007, 2009, and 2011. He is a member of the American Society for
Microbiology, the International Society for Infectious Diseases, and the Council
of State and Territorial Epidemiologists; past president of the U.S. Lancefield
Streptococcal Research Society; and a Fellow of the IDSA and the American
College of Physicians.
Luis Padilla, D.V.M., has served as the staff clinical veterinarian for the Smith-
sonian Conservation Biology Institute of the National Zoological Park in Front
Royal, Virginia, since 2007. He received his Doctorate of Veterinary Medicine
from Cornell University, where he also obtained a B.S. degree in biology. Dr.
462 FUNGAL DISEASES
Padilla completed postdoctoral clinical training at the Oradell Animal Hospital

in New Jersey and at the Saint Louis Zoo, in Missouri. After serving as associate
veterinarian at the Oklahoma City Zoo and as adjunct professor of zoological
medicine at Oklahoma State University, Dr. Padilla joined the Smithsonian’s
National Zoo as a supervisory veterinarian in 2006. His academic interests are
in the anesthesia of wildlife and non-domestic species, ungulate medicine and
advanced disease diagnostics, and the use of captive animals as models to under-
stand disease dynamics in free-ranging populations. He serves as the veterinary
advisor for the clouded leopard species survival plan and is a Diplomate of the
American College of Zoological Medicine.
David Rizzo, Ph.D., received his Ph.D. in plant pathology from the University of
Minnesota and joined the faculty of the University of California-Davis, Depart-
ment of Plant Pathology and the Graduate Group in Ecology in 1995. Research
in his lab focuses on the ecology and management of forest tree diseases, includ-
ing diseases caused by both native and introduced pathogens. Research in the
lab takes a multiscale approach ranging from experimental studies on the basic
biology of organisms to field studies across forest landscapes. Active collabo-
rations include projects with landscape ecologists, epidemiologists, molecular
biologists, entomologists, and forest managers. The primary research effort in
the lab is currently Phytophthora species in California coastal forests, with an
emphasis on sudden oak death. In conifer forests of the Sierra Nevada Mountains,
the lab studies a variety of diseases and their relationship to past and present
forest management and conservation issues. In addition to research, Dr. Rizzo
teaches undergraduate and graduate courses in mycology. Since 2004, he has
been director of the Science and Society program in the College of Agricultural
and Environmental Sciences. Dr. Rizzo also serves as the scientific advisor for
the California Oak Mortality Task Force.
Erica Bree Rosenblum, Ph.D., is an evolutionary biologist focused on under-

standing the molecular mechanisms of evolution. She is currently an assistant
professor in the Department of Biological Sciences at the University of Idaho.
Dr. Rosenblum’s research emphasizes understanding the processes that generate
and impact biological diversity (i.e., speciation and extinction). She uses genetic
and genomics tools in the lab, but also works with endangered species in their
natural habitats. Much of her current work focuses on understanding mechanisms
of host–pathogen interaction between frogs and the chytrid fungus responsible
for amphibian declines. She collaborates with an international, multidisciplinary
working group to understand the catastrophic impacts of this emerging fungal
pathogen. In addition to her research, Dr. Rosenblum contributes to a variety of
educational initiatives, and her work has been featured in a number of forums,
including the New York Times, the Discovery Channel, Science, Ranger Rick, and
Natural History Magazine. Dr. Rosenblum received her B.A. from the Ecology
APPENDIX F 463
and Evolutionary Biology Department at Brown University and completed her

Ph.D. in the Integrative Biology Department at the University of California–
Berkeley. She conducted her postdoctoral research as an NSF Bioinformatics Fel-
low in the Department of Genome Sciences at the Lawrence Berkeley National
Laboratory. Her work is funded by the NSF and NIH.
Jim Stack, Ph.D., is director of the Great Plains Diagnostic Network (GPDN)
and a professor of plant pathology at Kansas State University. As director of
GPDN, Dr. Stack coordinates a nine-state project for the rapid detection and ac-
curate diagnosis of high-consequence pathogens and pests. He is the Principal
Investigator of a plant biosecurity project at the National Agriculture Biosecurity
Center and has collaborated on several international projects regarding plant
biosecurity. Prior to joining Kansas State, Dr. Stack was on the faculty at the
University of Nebraska and at Texas A&M University. He formerly worked for
EcoScience Corporation as the director of applied research, leading the discov-
ery, development, and commercialization of microbe-based products to protect
fruit from storage decay pathogens. His research interests include pathogen detec-
tion and diagnostics, pathogen ecology, and epidemiology.
Compton Tucker, Ph.D., is a senior earth scientist in the Biospheric and

Hydrospheric Sciences Laboratory at the National Aeronautics and Space Ad-
ministration’s (NASA’s) Goddard Space Flight Center in Maryland. A native of
Carlsbad, New Mexico, he holds a B.S. degree in biology and M.S. and Ph.D.
degrees in forestry, all from Colorado State University. Upon completing his
B.S. in biology, he worked at Colorado National Bank in Denver and the First
National Bank of Albuquerque, New Mexico. Realizing that banking was not
his calling, he entered graduate school at Colorado State University and was as-
sociated with the Natural Resource Ecology Laboratory for his graduate work.
After completing his Ph.D. degree in 1975, he was a National Academy of Sci-
ences Postdoctoral Fellow at the Goddard Space Flight Center before joining
NASA as a physical scientist. He is the author of approximately 165 journal
articles on the use of remote sensing to study vegetation that have been cited
more than 14,000 times. In collaboration with coworkers, he is presently study-
ing tropical deforestation and fragmentation, global variations in photosynthetic
capacity, climatically coupled diseases, tropical glacier variation from Bolivia
to Mexico, and climate using satellite and ground data. He was on NASA detail
to the Climate Change Science Program from 2006 to 2009. He is the recipient
of NASA’s Exceptional Scientific Achievement Medal, the Henry Shaw Medal
from the Missouri Botanical Garden, National Air and Space Museum Trophy,
the William Nordberg Memorial Award for Earth Sciences, the Mongolian
Friendship Medal, the William T. Pecora Award from the U.S. Geological Sur-
vey, and the Galathea Medal from the Royal Danish Geographical Society. He
is a fellow of the American Geophysical Society.
464 FUNGAL DISEASES
Vance Vredenburg, Ph.D., is an assistant professor of biology at San Fran-

cisco State University. His research includes studies on the ecology, evolution,
and conservation of amphibians. His current research investigates the impacts
of emerging infectious disease on amphibian hosts. With a collaborative team,
he studies chytridiomycosis, the lethal amphibian disease caused by the fungal
pathogen Batrachochytrium dendrobatidis (Bd), which is implicated in mass die-
offs of amphibians globally. Dr. Vredenburg and colleagues recently documented
the spread of Bd through susceptible frog populations in the protected parks of
the Sierra Nevada, California. While most populations are driven completely
to extinction after pathogen arrival, a few populations survive. His most recent
work investigates the role that symbiotic skin microbes (e.g., Janthinobacterium
lividim, a bacterium) may play in frog host immunity.
Dr. Vredenburg received his Ph.D. from the University of California, Berke-
ley, in 2002. He is a cofounder of AmphibiaWeb (www.AmphibiaWeb.org), an
online conservation resource for the world’s amphibians that receives an average
of more than 20,000 successful searches per day from students, research biolo-
gists, and conservationists worldwide. Dr. Vredenburg is a research associate at
the California Academy of Sciences and the Museum of Vertebrate Zoology. His
research is funded by the NSF.
Ché Weldon, Ph.D., M.Sc., holds research interests that have always been cen-
tered on amphibians, which started in 1997 with host–parasite interactions (B.Sc.
Honors project) and was followed by an in-depth assessment of the sustainable
global use of African clawed frogs (M.Sc. project, 1998–1999). The following
year, as a field biologist with the Southern African Frog Atlas and Red Data Proj-
ect, Dr. Weldon developed a more focused interest in amphibian conservation. It
was during this time that the amphibian chytrid fungus was first detected and a
global surge started to investigate its role in amphibian declines. For his studies
on amphibian chytrid in South Africa, Dr. Weldon received the W.O. Neitz medal
for best Ph.D. thesis in parasitology. With then-supervisor L. H. du Preez, Dr.
Weldon established the African Amphibian Conservation Research Group. Dr.
Weldon specialized in amphibian diseases in Africa as a Postdoctoral Fellowship
at North-West University, and later became a zoology lecturer at the university
where he has continued his research on the amphibian chytrid and amphibian
conservation. From the 12 peer-reviewed articles that Dr. Weldon has authored
in the past 6 years, 6 have been cited a total of 130 times.
Gudrun Wibbelt, D.V.M., M.R.C.V.S., graduated from University of Veterinary

Medicine Hannover, Germany, followed by a residency for veterinary pathol-
ogy at University of Liverpool, United Kingdom. Since 2005 she is a certified
veterinary pathologist with a special focus on wildlife and zoo animal diseases.
She heads the wildlife pathology and electron microscopy unit of the Leibniz
Institute for Zoo and Wildlife Research, Berlin, which has a unique standing in
APPENDIX F 465
wildlife and nature conservation research in Germany/Europe. For more than 5

years, one of her main research interests has been the diseases of European bats,
with special emphasis on the correlation of histopathology and bacteriology/
virology, an aspect largely neglected by investigations on bats and emerging dis-
eases. With the emergence of white-nose syndrome of bats in the United States,
she leads collaborative European research efforts on fungal infections in native
bats from Europe.

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An Emerging Threat to Human,

LeighAnne Olsen, Eileen R. Choffnes, David A. Relman,

Forum on Microbial Threats

Advising the Nation. Improving Health.

4 Forum member until July 31, 2011.

Beth Bell, Centers for Disease Control and Prevention

The Forum on Emerging Infections was created by the Institute of Medicine

planning committee1: Gerald Keusch (Boston University), Rima Khabbaz (Cen-

A Contributed Manuscripts 101

A1 The Emergence of Cryptococcus gattii in British Columbia and the

A7 Surveillance for Emerging Diseases in Wildlife, 188

A21 Pan-European Distribution of White-Nose Syndrome Fungus

A2-1 Examples of Fungal Associations with Plants, 127

A9-1 Characteristics of C. gattii Patients in the United States,

A13-1 General Similarities and Differences Between Fungal Pathogens of

A20-1 Bats Tested for Geomyces destructans by Using Microscopy, Fungal

A21-1 Confirmed Records of Geomyces destructans on Hibernating Bats in

WO-22 Yellow “stripe” rust on wheat, 61

A1-1 Map of the forecasted ecologic niche and region of emergence of C.

A2-1 Diagrammatic representation of relationships of fungal taxa, examples

A3-7  Peniphorella baculorubrensis, a bark-decaying basidiomycete common

A4-1 Occurrence of white-nose syndrome and/or Geomyces destructans in

A5-1 Organism fitness as a function of body temperature, 180

A6-1 Frequency histogram of thermal growth tolerance for 4802 fungal

A7-1 Proportion of emerging infectious diseases caused by different

A8-1 Annual cases of coccidioidomycosis, 199

A9-1 Human infections with C. gattii, United States, December 2004–

A11-1 Mechanisms of fungal sensing and control, 250

A12-1 Typical macroscopic symptoms of rust infections on adult wheat

A16-1 Current distribution of Phytophthora ramorum in California and

A18-1 Decline of (A) Sierra Nevada mountain yellow-legged frog, Rana

A19-1 Maps indicating (A) the global prevalence of Batrachochytrium

A21-1 Distribution of confirmed and suspected records of G. destructans on

FUNGAL DISEASES: AN EMERGING THREAT TO

Fungi are the only group of organisms that have been

At the beginning of the 20th century, the American chestnut population

Organization of the Workshop Summary

THE HIDDEN KINGDOM

see contributed manuscripts by Blackwell in Appendix A (pages 116–167).

(98 000 species)

Ecosystem Services8 and Interactions

• Half of all ascomycetes (Phylum Ascomycota) are lichens [symbiotic

13â•›â•›Commonly referred to as Panic Grass.

FIGURE WO-1-1â•‡ Leafcutter ants tending their fungal garden.

For more information on leafcutterbitmap

• Recognize and attach to the host;

pendently of its host (Science dictionary).

Fungal Pathogens of Plants

• Irish Potato Famine: The mid-19th-century epidemic of potato late

Fungal Pathogens of Humans and Animals

Fungal Pathogens as “Invasive Species”

a normally benign host–fungus relationship.

within a specified time period; the rate of occurrence (IOM, 1992).

the organism’s ability to invade the host tissues (IOM, 1992).

The range of factors identified as influencing the interactions between these

Thirteen factors of emergence of infectious diseases were elucidated in a 2003

SOURCE: IOM (2003).

Human Activity and Behavior

A1 The Emergence of Cryptococcus gattii in British Columbia and the

A21 Pan-European Distribution of White-Nose Syndrome Fungus

A9-1 Characteristics of C. gattii Patients in the United States,

A13-1 General Similarities and Differences Between Fungal Pathogens of

A20-1 Bats Tested for Geomyces destructans by Using Microscopy, Fungal

A21-1 Confirmed Records of Geomyces destructans on Hibernating Bats in

WO-22 Yellow “stripe” rust on wheat, 61

A1-1 Map of the forecasted ecologic niche and region of emergence of C.

A2-1 Diagrammatic representation of relationships of fungal taxa, examples

A3-7 Peniphorella baculorubrensis, a bark-decaying basidiomycete common

A4-1 Occurrence of white-nose syndrome and/or Geomyces destructans in

A5-1 Organism fitness as a function of body temperature, 180

A6-1 Frequency histogram of thermal growth tolerance for 4802 fungal

A7-1 Proportion of emerging infectious diseases caused by different

A8-1 Annual cases of coccidioidomycosis, 199

A9-1 Human infections with C. gattii, United States, December 2004–

A11-1 Mechanisms of fungal sensing and control, 250

A12-1 Typical macroscopic symptoms of rust infections on adult wheat

A16-1 Current distribution of Phytophthora ramorum in California and

A18-1 Decline of (A) Sierra Nevada mountain yellow-legged frog, Rana

A19-1 Maps indicating (A) the global prevalence of Batrachochytrium

A21-1 Distribution of confirmed and suspected records of G. destructans on