Journal of Advanced Research (2011) 2, 97–108

Cairo University

Journal of Advanced Research

REVIEW ARTICLE

Bacterial coldwater disease of fishes caused by

Flavobacterium psychrophilum
Clifford E. Starliper *

U.S. Geological Survey, Leetown Science Center, National Fish Health Research Laboratory, 11649 Leetown Road, Kearneysville,
WV 25430, USA

Received 9 January 2010; revised 26 February 2010; accepted 6 April 2010

Available online 12 October 2010

KEYWORDS Abstract Coldwater disease (CWD) is a bacterial disease that affects a broad host-species range of
Coldwater disease; fishes that inhabit cold, fresh waters. This disease occurs predominately at water temperatures of
Freshwater; 16 C and below, and is most prevalent and severe at 10 C and below. Coldwater disease occurs
Bacteria; in cultured and free-ranging populations, with hatchery-reared young trout and salmon species espe-
Flavobacterium cially vulnerable to infections. Flavobacterium psychrophilum is the etiological agent of CWD. This
psychrophilum; Gram-negative bacterium may be recovered from affected host tissues and characterized using stan-
Fish dard biochemical techniques, providing that reduced nutrient media are used. There are numerous
reports that describe sensitive and specific serologic and genomic diagnostic techniques for CWD.
The entire genome of a virulent isolate of F. psychrophilum has been sequenced and described. Rain-
bow trout (Oncorhynchus mykiss) fry syndrome is also caused by F. psychrophilum with mortalities
>50% possible among affected fish lots. Evidence suggests that pathogen transmission occurs both
horizontally and vertically. Analogous to many diseases to other animals, prevention and control are
essential to avoid losses to CWD, particularly since there is currently no commercially available vac-
cine and a limited number of antimicrobials have been approved for treating food fish worldwide.
This review provides current host and geographic ranges of the pathogen, and covers epizootiology,
transmission, pathogenicity, diagnostics, and prevention and treatment.
ª 2010 Cairo University. Production and hosting by Elsevier B.V. All rights reserved.

* Tel.: +1 304 724 4433; fax: +1 304 724 4435. Flavobacterial diseases of freshwater fishes
E-mail address: cstarliper@usgs.gov
There are three Flavobacterium spp. that are primary patho-
2090-1232 ª 2010 Cairo University. Production and hosting by gens to freshwater hatchery-reared and wild fish populations:
Elsevier B.V. All rights reserved. Flavobacterium columnare, the cause of columnaris disease,
Flavobacterium branchiophilum, the cause of bacterial gill dis-
Peer review under responsibility of Cairo University. ease, and Flavobacterium psychrophilum the cause of bacterial
doi:10.1016/j.jare.2010.04.001 coldwater disease. Combined, the diseases and mortality
caused by these pathogens constitutes one of the broadest
Production and hosting by Elsevier host- and geographic ranges of any of the bacterial pathogens
to fishes. Fish pathogenic Flavobacterium spp. are presumed
98 C.E. Starliper

ubiquitous in temperate freshwater aquatic environments and and Flexibacter psychrophilus [15]. This bacterial pathogen
occur in water temperatures ranging from just above freezing has been recovered from a broad geographic range and from
(F. psychrophilum) to 30 C and above (F. columnare). Most, a number of free-ranging and cultured salmonid fish species
if not all cultured freshwater fish species may be affected by and a variety of non-salmonid fish hosts (Table 1). Coldwater
at least one of these pathogens. Other members of the Family disease results in significant disease and mortality to coldwater
Flavobacteriacea have been associated with diseases of fishes. fish species, particularly to certain trout and salmon popula-
For example, Chryseobacterium piscicola is an emerging path- tions. Disease typically occurs at water temperatures below
ogen of Flavobacteriaceae having been reported from Atlantic 16 C, and is most prevalent and serious at 10 C and below
salmon (Salmo salar) and rainbow trout (Oncorhynchus my- [16]. Although all ages of fish are affected, small fish (fry and
kiss) [1,2]. fingerling size) are particularly vulnerable to infections
Columnaris disease, affects many cool- and warmwater fish [16,17]. Coldwater disease presents as different manifestations
species, typically in warm waters at 20–25 C and above; how- with the ‘classic’ or most prevalent form of disease producing
ever, it is not unusual to diagnose columnaris disease in fish, characteristic open lesions on the external body surfaces of
including trout species, in water as cool as 12–14 C. Many cul- fish. These lesions may be initially observed as areas of
tured and free-ranging fish species are considered at risk for rough-appearing skin or fin tip fraying. As the infection con-
infection and possible disease. Columnaris disease affects aqua- tinues, necrosis develops at the sites of bacterial colonization,
culture species, particularly the catfish species, as well as many often noted as dorsal and adipose fin pathology. Lesion devel-
aquarium species. F. columnare can be cultured from external opment has a predilection for the caudal peduncle and caudal
sites on fish, including lesions, skin/mucus, and gills, and inter- fin regions. Along with the external pathology, systemic bacte-
nal tissues, primarily the kidneys of fish with systemic infections. rial infections and extensive internal pathology will also be
Primary cultures can be made on Anacker and Ordal [3] Cytoph- present among many specimens. As the disease form is more
aga agar or the selective medium of Hawke and Thune [4]. The acute, the external lesions will be less prevalent and systemic
resulting colonies on primary plates are very characteristic: pale infections and internal pathology will predominate.
yellow, rhizoid and adhere tightly (i.e., sticky) to the medium F. psychrophilum was initially described and recovered in
surface. Colonies may be subcultured and confirmed using a 1948 from a die-off in coho salmon Oncorhynchus kisutch from
few relatively simple diagnostic tests [5]. the Pacific Northwest United States [18]. This disease affected
Bacterial gill disease, caused by F. branchiophilum [6–8], is the adipose-caudal fin region and in some specimens with late-
primarily a disease to young hatchery-reared salmonids; it is stage infections and prior to death, the vertebral column could
not recognized as a problem in wild fish populations [9–13]. be fully exposed. While usually fatal to fish with late-stage
In endemic areas, bacterial gill disease outbreaks in aquacul- disease signs, the prevalence and mortality in affected fish pop-
ture occur regularly and often in conjunction with increased ulations were low. Davis [19] observed slender, Gram-negative
host stressors. Although bacterial gill disease has been experi- rods 3–5 lm long and noted that overcrowding seemed to be a
mentally induced in healthy fish of various ages [14], many host predisposing factor in ‘peduncle disease’ outbreaks in
workers have noted that this disease typically occurs in associ- rainbow trout in 1941 and 1945 at a hatchery in the Eastern
ation with certain predisposing factors such as overcrowding, United States (West Virginia). To control peduncle disease,
reduced dissolved oxygen, increased ammonia, and particulate Davis [19] suggested culling out those fish with obvious clinical
matter in the water [9,10,13]. Consequently, alleviating these signs in an effort to minimize the continuous shedding of path-
host stressors has often been shown to reduce severity of active ogenic cells into the water column that served to infect other
outbreaks and prevent further outbreaks. Mortality can rise fish. It was also suggested to properly sterilize contaminated
quickly and be high if the culture conditions are not improved rearing troughs or ponds and all equipment, such as boots
or a treatment is not promptly administered. Bacterial gill and nets, which were used to handle infected fish or water.
disease is common in spring, which coincides with production The pathologies and clinical disease signs associated with
cycles at fish hatcheries when they have their greatest numbers CWD are varied and extensive [20–24]. Listlessness, loss of
of small fish after spawning and prior to stocking. A diagnosis appetite, and eroded fin tips are initial signs of CWD. Bacterial
of bacterial gill disease can often be accurately made by expe- colonization may appear as faint, white areas on the fins, with
rienced workers simply by knowing the previous bacterial gill some fish showing separation of the fin rays. Other disease
disease history of the hatchery and observing characteristic signs may include exophthalmia, abdominal distension with in-
signs displayed by affected fish. Infected fish are typically creased volumes of ascites, and pale gills. In advanced cases of
lethargic, will be high in the water column and gasping for coldwater disease, necrosis of the caudal region may be severe
air at the surface and align near and into the incoming water, and progress until caudle vertebra are exposed (Fig. 1).
all of which are obvious signs of respiration difficulty. A Lesions can also be noted on the lateral sides, snout-jaw re-
Gram-stained gill smear will show numerous Gram-negative, gion, and musculature often between the dorsal fin and back
long-thin rods. Combined, these criteria generally constitute of the head. Histological examinations show extensive pathol-
a confirmed diagnosis. Bacterial primary isolation of ogy in host tissues, including: focal necrosis in spleen, liver,
F. branchiophilum is typically not attempted because this and kidneys; increased vacuolar degeneration; increased eosin-
bacterium is particularly difficult to culture. ophilia and haemosiderin in the kidney; necrosis, pyknosis and
lymphocyte infiltration in the dermis and underlying lateral
musculature of skin lesions.
Bacterial coldwater disease Rainbow trout fry syndrome [25–30] and a relatively more
chronic form [31,32] are other disease manifestations caused by
The etiological agent of bacterial coldwater disease (CWD) is F. psychrophilum. Rainbow trout fry syndrome, as the name
F. psychrophilum, formerly known as Cytophaga psychrophila implies, affects the early life-stage fish, or the sac fry to
Bacterial coldwater disease of fishes 99

Table 1 Host and geographic records of Flavobacterium psychrophilum.

Geographic origin Hosts References
Australia Rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar [59,69]
Canada Rainbow trout, brook trout Salvelinus fontinalis, Atlantic salmon, [71,84,113–115]
Arctic char Salvelinus alpinus, coho salmon O. kisutch, sea lamprey
Petromyzon marinus L.
Chile Rainbow trout, Atlantic salmon [94,106,116,117]
Denmark Rainbow trout [27,38,40]
Estonia Grayling Thymallus thymallus [118]
Finland Rainbow trout, brown trout S. trutta morpha lacustris, sea trout S. [28,38,73,86,118,119]
trutta morpha trutta, brook trout, Arctic char, whitefish Coregonus
muksun, perch Perca fluviatilis L., roach Rutilus rutilus
France Rainbow trout, common carp Cyprinus carpio, eel Anguilla anguilla [25,28,45,57]
Germany Rainbow trout, eel A. anguilla, common carp, crucian carp [120,121]
Carassius carassius, tench Tinca tinca
Japan Rainbow trout, coho salmon, chum salmon O. keta, amago salmon [48,77,85,122,123]
O. rhodurus, common carp, yamame salmon O. masou, iwana
salmon S. leucomaenis pluvius, eel A. japonica, Japanese dace (ugui)
Tribolodon hakonensis, ayu Plecoglossus altivelis, pale chub (oikawa
minnow) Zacco platypus, Japanese crucian carp (ginbuna) C.
auratus langsdorfii, and two species of goby Chaenogobius urotaenia
and Rhinogobius brunneus
Korea Ayu [124]
Northern Ireland Rainbow trout [28]
Norway Brown trout S. trutta morpha lacustris [28]
Peru Rainbow trout [71]
Scotland Rainbow trout [125]
Spain Rainbow trout, eel A. anguilla [30,126]
Sweden Rainbow trout, sea trout, Baltic (Atlantic) salmon S. salar [52,118]
Switzerland Rainbow trout [28]
Turkey Rainbow trout [127]
United Kingdom Rainbow trout, Atlantic salmon [26,29,59,116]
United States Rainbow trout, brook trout, brown trout S. trutta morpha [16,18,19,32,51,58,71,109,128,129]
lacustris, lake trout S. namaycush, steelhead trout O. mykiss
(migrating), Atlantic salmon, coho salmon, Chinook salmon O.
tshawytscha, white sturgeon Acipenser transmontanus, chum
salmon, goldfish Carassius auratus, cutthroat trout O. clarkii

early-feeding developmental stage. This disease form is acute [31]. However, with subsequent diagnostic evaluation, a whirl-
and may result in high percentages of deaths among fish lots, ing disease etiology can be eliminated and a correct diagnosis
perhaps 50% or greater total mortality. A bacteremia develops of CWD can be made based upon a case history along with
in conjunction with extensive internal pathology, including primary culture and characterization of F. psychrophilum from
anemic and pale kidneys and livers. Lethargy, exophthalmia affected tissues, including brain, spleen, kidney, liver, and le-
(often bilateral), dark skin pigmentation and pale gills are sion-skin. Kent et al. [32] showed the ataxic, spiral swimming
additional characteristic disease signs of rainbow trout fry syn- behavior was associated with F. psychrophilum infections and
drome. Lorenzen et al. [28] showed that F. psychrophilum iso- chronic inflammation of the cranium and vertebrae in coho
lates recovered from fish with rainbow trout fry syndrome salmon. Fish showing this behavior did not recover and died.
were phenotypically homogeneous with isolates recovered Based on epizootiological analyses, Kent et al. [32] concluded
from larger fish with classical CWD. Daskalov et al. [33] noted that F. psychrophilum was the cause of this disease presentation
that the effects of high oxidized lipids in fish showed similari- because it was only observed in populations that had recovered
ties in signs of rainbow trout fry syndrome. Some of the same from acute CWD. Histologic evaluations showed periostitis,
histologic characteristics of rainbow trout fry syndrome were osteitis, meningitis, and periosteal proliferation of vertebrae
also noted in nutritional diseases caused by feeding diets high at the junction of the vertebral column and cranium. This
oxidized lipids [33]. Rainbow trout fed a diet with high levels chronic CWD manifestation has occurred in fish that have
of oxidized lipids had a greater mortality, relative to controls, recovered from a previous outbreak of acute clinical CWD
by F. psychrophilum after exposure to the pathogen by scarify- [32] or it was diagnosed in fish lots with no recent history of
ing and immersion or IP challenges. CWD [31]. The bacterium may be cultured from the brain, kid-
With the chronic form of CWD, affected fish may show ney, liver, spleen and heart, but not necessarily from all tissues
spiral or erratic swimming behavior, blackened caudal (tail) re- from each specimen or from all apparently infected specimens
gions and/or spinal column deformities [31,32]. The reported [31,32].
disease signs and behavior appeared similar to those associated Concurrent infections in fish of F. psychrophilum with other
with whirling disease in fish caused by Myxobolus cerebralis fish pathogens are not uncommon. Dalsgaard and Madsen [34]
100 C.E. Starliper

laboratory waterborne challenges, the equivalent to natural

horizontal transmission, with F. psychrophilum are equivocal
[16,40] and an abrasion artificially created on the body surface,
such as with a pre-challenge bath exposure to 0.005% forma-
lin, facilitates disease [40]. Aoki et al. [41] noted success in
F. psychrophilum laboratory challenges in 1.3 or 5.6 g rainbow
trout depended on the growth stage of the bacterial challenge
culture used to expose the fish. It was important to use
log-phase cultures for experimental bath infections to produce
typical clinical disease signs and mortality. Aoki et al. [41]
showed that 18 and 24 h F. psychrophilum cultures with chal-
lenge doses of 2.00 · 107 and 8.50 · 107 cfu/mL, respectively,
resulted in significantly greater mortalities than was obtained
with a 48 h culture, even though the 48 h culture had a greater
number of cells (3.40 · 108 cfu/mL).
Injection challenge methods are often used to expose exper-
imental groups of fish to F. psychrophilum [36,42–44]. Decoste-
re et al. [42] noted that only 10-week old rainbow trout
developed clinical signs and mortality following IP injections
with 1.00 · 106 cfu, while fish 5 or 15 months old did not. Also,
spleen phagocytes from the 10-week old fish contained viable
F. psychrophilum cells, and these cell numbers increased with
exposure time. This contrasted with the two groups of older
fish in which no F. psychrophilum cells were detected in spleen
phagocytes.
F. psychrophilum has a demonstrated ability to adapt to a
variety of environments, and not only survive, but also main-
tain pathogenicity. This bacterium has been recovered from
broad host and geographic ranges, it resists lysozyme up to
Fig. 1 Typical coldwater disease caudal lesions in rainbow trout 2 mg/mL, and a small percentage of cells survived 100 ppm
Oncorhynchus mykiss (Panel A) and coho salmon O. kisutch (Panel povidone–iodine for 30 min, a compound frequently used as
B) caused by Flavobacterium psychrophilum. Photographs courtesy an egg surface disinfectant. F. psychrophilum can survive in
of Vermont Fish and Wildlife Department, Waterbury, VT and stream water for months and adopts a different morphology
Wisconsin Department of Natural Resources, Madison, WI. apparently to withstand the conditions of starvation [45].
Madetoja et al. [46] showed that F. psychrophilum cells in
freshwater at 15 C remained culturable through 300 days.
reported a concurrent infection in rainbow trout with the Attachment to n-hexadecane and unfertilized eggs was signifi-
Gram-negative bacterium Yersinia ruckeri, the causative agent cantly greater by F. psychrophilum cells maintained in either
of enteric redmouth disease. There are other co-infections of stream water or cytophaga broth for 1 month, in contrast to
F. psychrophilum with viruses, namely, infectious pancreatic cells from 3-day-old cultures in cytophaga broth [45]. Adapt-
necrosis virus, infectious hematopoietic necrosis virus, and ability of F. psychrophilum was further demonstrated by
erythrocytic inclusion body syndrome [35–37]. F. psychrophi- Brown et al. [17] when they recovered the bacterium from
lum does not cause diseases in other animals or humans. The the brain of a newt Pleurodelinae, a non-fish host. Addition-
impact of fish losses at hatcheries reduces the numbers of fish ally, using PCR F. psychrophilum was detected from benthic
available for raising or for stocking for sport fishing purposes diatoms [47] and from algae [48]. These studies suggest that
and can impact restoration or population augmentation perhaps any number of non-fish hosts could serve as a reser-
successes of certain endangered fish species. voir for F. psychrophilum. Although the contribution of aqua-
tic non-fish hosts to the biology of CWD is not known, the
Epizootiology and transmission capability of F. psychrophilum to survive in aquatic environ-
ments is illustrated.
Since F. psychrophilum is horizontally transmitted, the water Evidence suggests that F. psychrophilum is also vertically
column is the medium in which viable cells move. The transmitted. For example, this bacterium has been recovered
reservoir(s) of F. psychrophilum include pathogen-carrier fish, from ovarian fluids, intraovum, egg surfaces, milt, mucus
bacteria-shedding diseased and dead fish, and water supplies. samples and kidneys from sexually mature chum, coho and
F. psychrophilum has a demonstrated ability to survive for long Chinook salmon, rainbow and steelhead trout, and Atlantic
periods outside fish hosts and to occur in non-fish hosts. salmon [16,17,39,49–51]. Brown et al. [17] recovered F. psy-
Madetoja et al. [38] showed that rainbow trout that died from chrophilum from the insides of fertilized and eyed eggs. Ekman
an infection with F. psychrophilum shed very high numbers of et al. [52] isolated F. psychrophilum from both male and female
bacteria. Cell shedding rates depended on water temperatures, reproductive products from Baltic salmon (S. salar) returning
and cells were shed for at least 80 days. Madsen et al. [39] from the Baltic Sea to spawn. Similar to other fish pathogens,
isolated F. psychrophilum from water samples that were F. psychrophilum can also contaminate the surface of patho-
collected near farmed rainbow trout or eggs. The results from gen-free fish eggs, which is a form of horizontal transmission
Bacterial coldwater disease of fishes 101

[17,53–55]. Kumagai et al. [54] exposed F. psychrophilum to lesions, mucus, kidney and spleen and reproductive products
groups of eggs before and after water hardening, as well as of spawning adults. However, not all apparently affected fish
to eyed eggs. All of the groups were then disinfected with could have sufficient number of viable cells in internal tissues
50 mg/L povidone-iodine for 15 min. F. psychrophilum was for successful primary culture. Recovery of the pathogen from
subsequently recovered from only those eggs that were exposed lesions is often more challenging than from internal sample
to the pathogen prior to water hardening. Cipriano [49] recov- sites due to the presence of environmental bacteria or oomyce-
ered between 5.00 · 102 and 2.50 · 108 cfu F. psychrophilum tes that will readily grow on primary isolation bacteriological
per gram from Atlantic salmon eggs that were treated with media. Taking cultures from a greater number of fish or sam-
50–100 mg/L povidone–iodine at fertilization, post-water ples will enhance the chance to recover the bacterium. With
hardened and eyed egg stages. Further evidence that F. psy- some diagnostic cases, it may be possible to observe character-
chrophilum is internalized within eggs was reported by Kuma- istic F. psychrophilum cells from infected tissues on histologic
gai et al. [53] who demonstrated that disinfection with 50 mg/L slides, yet be unsuccessful in culturing the bacterium from
povidone-iodine for 15 min was not effective in eliminating the those same tissues, or vice versa, particularly from asymptom-
bacterium from either eyed- or fertilized eggs that had been atic fish having reduced infection levels. The pathology to fish
pathogen-exposed prior to the water hardening process. caused by F. psychrophilum can be extensive, for example,
Kumagai et al. [54] showed the importance of water hardening focal necrosis in various organs, and periostitis, osteitis, men-
the eggs in pathogen-free water to prevent (egg) surface ingitis, ganglioneuritis and pyknotic nuclei are possible [26,32].
contamination. Particularly with chronic coldwater disease, masses of
F. psychrophilum may be seen in the cranial area and anterior
Diagnosis and isolate characterization vertebra as well as inflammation and cartilage necrosis along
the vertebral column.
A successful diagnosis of CWD considers all relevant informa- Homogenization of sample tissues prior to the inoculations
tion. Important factors include facility disease history, the may enhance recovery, especially from fish with low-level
rearing conditions for the fish, water temperature, host(s) in- infections. Primary culture plates can be inoculated using
volved and their ages, presence of characteristic clinical disease one of several techniques, such as direct streak-plating or
signs, the observation of characteristic bacterial cells in Gram- preparing a dilution series and drop-inoculating specific
stained tissue preparations, and confirmation of F. psychrophi- volumes on the medium surface to yield viable cell numbers
lum as the causative agent from moribund or freshly dead spec- (i.e., cfu/g). Several bacteriological media may be used for
imens through primary culture and biochemical identifications, primary culture of F. psychrophilum. Cytophaga medium [3]
serological, or genotypic assays. is frequently employed in diagnostic laboratories; the recipe
Microscopic examination of F. psychrophilum cells in in- consists of 0.05% tryptone, 0.05% yeast extract, 0.02%
fected tissues reveals long, thin, rod-shaped cells typically in sodium acetate, 0.02% beef extract, and pH 7.0–7.2. Agar
a size range of 0.75–1.0 lm wide by 3–5 lm long (Fig. 2). Some may be added if desired. Cytophaga medium was developed
cells may be attached end-to-end and consequently will appear to support the growth of bacteria that require a reduced nutri-
longer. ent load requirement. Holt et al. [21] described tryptone yeast
F. psychrophilum can be recovered from a number of exter- extract salts (TYES) consisting of 0.4% tryptone, 0.04% yeast
nal and internal sites including skin/mucus, gills, brain, ascites, extract, 0.05% magnesium sulfate, 0.05% calcium chloride,
and pH 7.2 as an excellent liquid medium, that diagnosticians
routinely supplement with agar for use as a primary isolation
medium for F. psychrophilum. Other reduced nutrient concen-
tration media have also been used [16,56–59]. Some authors re-
port improved growth of F. psychrophilum after supplementing
the medium with serum, a component typically used for slow
growing or fastidious bacteria that will grow on rich nutrient
media. Lorenzen [60] and Brown et al. [17], for example,
incorporated 5.0% and 0.5%, respectively, of new born calf
serum. Obach and Baudin Laurencin [61] supplemented
Cytophaga medium with 10% fetal calf serum for recovery
of F. psychrophilum from rainbow trout. Daskalov et al. [62]
utilized Cytophaga medium as a basal medium to which they
added galactose, glucose, rhamnose and skimmed milk.
Rangdale et al. [59] modified cytophaga medium by increasing
the tryptone concentration ten-fold (to 0.5%) and the beef
extract from 0.02% to 0.05%. Increased tryptone (to 0.5%)
in Cytophaga medium has since been used by various research-
ers who reported excellent growth of laboratory cultures.
Lorenzen [60] showed the importance of the brand of beef
Fig. 2 Simple stain (crystal violet; 1000·) of Flavobacterium extract to culture F. psychrophilum, with optimal results using
psychrophilum cells. External lesion material smear from a the semi-solid form. Kumagai et al. [63] suggested the incorpo-
rainbow trout Oncorhynchus mykiss affected with coldwater ration of 5 lg/mL tobramycin to primary culture media to aid
disease. Photomicrograph courtesy of Vermont Fish and Wildlife recovery of F. psychrophilum by retarding the growth of
Department, Waterbury, VT. environmental bacterial contaminants.
102 C.E. Starliper

The optimum incubation temperature for primary isolation employed to determine the results. An example of this is the
and culture growth of F. psychrophilum is 15–16 C. Colonies unique phenotype of some F. psychrophilum isolates from
on Cytophaga agar are pale-yellow and about 2–3 mm in Australia, which produce brown pigment when grown on a
diameter after 2–3 days of incubation. Colonies form a charac- medium containing tyrosine [69]. Lorenzen et al. [28] showed
teristic fried egg appearance with a slightly raised center and that the concentration of certain medium supplements, or
mild spreading, irregular margin (Fig. 3). Colonies do not biochemical test substrates, may affect the test results. If the
adhere to the medium surface in the similar manner that concentration of a substrate in a medium is too low, this could
F. columnare colonies do. Suspect F. psychrophilum colonies result in a false-negative interpretation. Furthermore, they
can readily be subcultured onto fresh media, e.g., Cytophaga emphasized the need to use fresh growth cultures as the
agar, for characterization and identification using standard inoculum for biochemical characterization tests, and the use
biochemical and physiological methods [9,15,28,57–58,64– of sensitive test procedures for certain characters, such as the
69]. Unless growth/no growth on select media is to be evalu- use of lead acetate to detect weak production of hydrogen
ated, the basal medium for biochemical testing must be sulfide.
reduced nutrient to support bacterial growth, even for negative Other sensitive diagnostic techniques in addition to bacte-
test reactions. For example, the basal medium of Pacha [70], rial culture have been employed to detect F. psychrophilum in
which consists of 0.2% peptone, 0.2% sodium chloride, water, in fish, and fish sex products, or to diagnose or confirm
0.03% potassium phosphate, 0.00015% bromothymol blue, standard culture diagnostics for coldwater disease. A number
and 0.3% agar, pH 7.0–7.2, is an excellent choice as a basal of clinicians have used antisera raised against F. psychrophilum
medium to evaluate acid production from assimilation of in the immunofluorescence antibody technique [41,48,71–74]
sugars. and for immunohistochemistry [35,38,75]. Enzyme-linked
Isolates typically do not grow, or grow poorly on high- immunosorbent assays have been developed using antibodies
nutrient concentration media routinely used in fish disease F. psychrophilum cell surface components for detection of the
diagnostic laboratories, including brain heart infusion agar, pathogen in fish [71,76]. Misaka et al. [77] used nitrocellulose
tryptic soy agar, triple sugar iron agar and blood agar. Most bacterial colony blotting off culture media plates and immuno-
F. psychrophilum isolates are reported to produce oxidase staining to quantify viable F. psychrophilum from kidneys and
and catalase, hydrolyze gelatin and casein, produce flexiru- ovarian fluids of chum salmon Oncorhynchus keta.
bin-like pigments (chromogenic shift from yellow to orange Fish disease diagnosticians are increasingly employing and
in 10% KOH), degrade tyrosine, and lyse killed Escherichia relying on nucleic acid genotype based assays to detect fish
coli cells. Most isolates are negative for assimilation of a suite pathogens, including F. psychrophilum, or to confirm the
of sugars (production of acid indicated by a pH drop in a basal identifications made using other methods, such as standard
medium with a pH indicator), indole production, starch hydro- phenotypic characterizations. A number of procedures using
lysis, and degradation of tributyrin and xanthine. Variable polymerase chain reaction assays (PCR), and particularly the
results are reported for elastin hydrolysis, nitrate reduction, more specific nested PCR assays, have been described
and chondroitin sulfate AC lyase. Some of the variability [47,51,72–74,78–89]. Amita et al. [48] detected F. psychrophi-
reported in line-data for certain biochemical tests might be lum in a water sample and in algae using PCR. Izumi et al.
attributed to differences in isolate origins or the methods [47] used a nested PCR to detect F. psychrophilum from benthic
diatoms samples from surfaces of stones. Suzuki et al. [90]
compared the sensitivities of various PCR primers for
F. psychrophilum and found that the primer targeting the 16S
rDNA was the more sensitive; however, this primer resulted
in a level of false-positive reactions. Because of this, they
concluded that PCR primers targeting the DNA gyrase
subunit gene gyrB and the peptidyl-prolyl cis–trans isomerase
C gene ppiC were the preferred primers for F. psychrophilum.
A multiplex PCR was developed by del Cerro et al. [82] to
detect three fish pathogens simultaneously, which included
F. psychrophilum.

Pathogenicity and immunity

The genome of a virulent F. psychrophilum isolate has been

delineated [91]. The circular chromosome consists of
2,861,988 base pairs, which is relatively small compared to
other environmental bacteria within the family; the average
genome size for the genus Flavobacterium, estimated by
DNA reassociation assays, is 4.1 ± 1 Mb [92]. The G + C
content of F. psychrophilum is 32.54% [64].
Fig. 3 Flavobacterium psychrophilum colonies on Cytophaga Potential gene products related to virulence for F. psychro-
agar [3] supplemented with 0.2% gelatin. The bacterial colonies philum were described [91]. Proteases are considered to be
were gelatinase positive, as indicated by clear zones adjacent to essential virulence components, and potential secreted
and surrounding the colonies. proteases were identified in the genome [93]. Genes coding
Bacterial coldwater disease of fishes 103

for cytolysins and haemolysin-like proteins are considered F. psychrophilum may be an antigen for the development
important virulence determinants, while fibronectin-type adhe- of a vaccine for protection against CWD and rainbow trout
sins may have an essential role in the bacterium’s attachment fry syndrome. Johnson et al. [44] showed that the major his-
capability. Other enzymes act to negate host defense mecha- tocompatibility gene region MH-IB was linked to survivabil-
nisms. Avendaño-Herrera et al. [94] employed pulsed-field ity to CWD in rainbow trout that were IP injection
gel electrophoresis of Sac I restriction patterns of Chilean F. challenged to F. psychrophilum.
psychrophilum field isolates and demonstrated two distinct ge-
netic groups that correlated with host of origin, rainbow trout Prevention, control, and treatment
and Atlantic salmon.
Innate immunity to F. psychrophilum in rainbow trout has As with all fish diseases, including CWD, management strate-
been correlated with spleen size [95]. Hadidi et al. [95] screened gies that minimize the risks of pathogen introductions or trans-
71 full-sibling crosses and found that the resistant or suscepti- mission, and reduce the severity of overt disease outbreaks are
ble phenotypes were stable. The spleen-somatic indices of 103 desired alternatives to chemical or antimicrobial treatment
fish created high, medium, and low spleen-index groups. Spec- therapies. Prevention of diseases is the most prudent form of
imens having the larger spleen indices were significantly more disease control and treatment; this especially pertains to cul-
resistant to F. psychrophilum. Acute serum amyloid A (A- tured fish populations, and ultimately to wild fish populations
SAA) is normally thought to be a major acute-phase reactant restored or augmented with fishes reared at hatcheries. Proper
and effector of innate immunity in vertebrates. When chal- fish husbandry will alleviate host stressors that are often in-
lenged with whole cell F. psychrophilum, lipopolysaccharides volved or suspected in the disease processes, such as factors
(LPS), or CpG oligonucleotides, A-SAA was strongly induced that compromise the integrity of the mucus covering the fin
in many immune-relevant rainbow trout tissues [96]. Unlike tips [106,107]. Disease preventative techniques include rearing
mammalian A-SAA, trout A-SAA does not increase in the small (i.e., most susceptible) fish in pathogen-free water, main-
plasma of diseased fish. Therefore, the role of this molecule taining safe carrying capacities for the water supply and flow,
in protection against F. psychrophilum is perhaps more impor- the use and proper storage of quality fish food, cleanliness of
tant in localized defense mechanisms. the fish holding tanks, minimizing organic material and nitrite
Numerous studies have been done that demonstrate pro- [108], and effective sanitization of equipment used in fish pro-
tective immune responses in an effort to develop a vaccine duction [109]. High numbers of F. psychrophilum cells are shed
for CWD. Passive immune protection to F. psychrophilum into the water column by fish that died from CWD. It was
with serum from convalescent, and previously immunized shown to be very important to quickly remove dead fish from
rainbow trout was demonstrated (in rainbow trout) by LaF- the population thereby reducing re-infection [38]. Periodic
rentz et al. [97]. Protection to specific molecular mass F. psy- health and pathogen inspections on statistically significant
chrophilum cell fractions was shown by LaFrentz et al. [36], numbers of specimens from each fish lot to detect a pathogen
also to the P18 surface antigen [98], and to formalin- and prior to the expression of clinical disease are an essential part
heat-inactivated F. psychrophilum cells [99]. Additionally, of a disease prevention strategy. If a pathogen is detected early,
protection against F. psychrophilum was shown by vaccina- the affected fish and therefore, the pathogen can be confined
tion with an outer membrane fraction [100] and a 70– (i.e., quarantined) within a designated area of a facility and a
100 kD cell fraction [36] composed of O-polysaccharide com- containment and treatment strategy begun. Caution should al-
ponents of LPS. Aoki et al. [101] showed that membrane ways be exercised when moving fish between culture facilities,
vesicles were released in F. psychrophilum stationary phase especially if fish are suspected to be diseased or if the source
growth cultures. Stationary phase F. psychrophilum cells or facility has a disease history.
membrane vesicles alone provided no protection to rainbow Povidone–iodine is commonly used as a fish egg surface dis-
trout; however, host survival to challenge was 94–100% infectant to fertilized and eyed eggs [107]. Although this treat-
when these two components were combined in experimental ment is not 100% effective to inactivate F. psychrophilum in all
vaccines. Analysis of virulent and avirulent strains of F. psy- situations, it reduces egg-associated pathogen transmission.
chrophilum by comparative immunoproteomic methods dem- Brown et al. [17] showed that 2% of F. psychrophilum cells sur-
onstrated eight proteins that were unique to the virulent vived an exposure to 100 ppm povidone–iodine for 30 min.
strain [102]. Two highly immunogenic heat shock proteins Kumagai et al. [53] treated fertilized rainbow trout, coho
(HSP 60, HSP 70) shared extensive homology with the heat and masu salmon eggs with 50 ppm povidone–iodine for
shock proteins of other, related bacteria. LaFrentz et al. 15 min and subsequently recovered F. psychrophilum from
[103] developed an attenuated strain of F. psychrophilum 60% to 80% of the treated eggs; additionally, they treated eyed
through repeated passage on increasing concentrations of rif- coho salmon eggs with up to 1000 ppm povidone-iodine for
ampicin. Intraperitoneal injection with the attenuated strain 15 min or 200 ppm for up to 120 min and both resulting data
conferred significant protection in rainbow trout to challenge sets for treated eggs were comparable to infected, but un-
with the virulent parent strain. The protected fish showed treated controls. At the 1000 ppm concentration, for example,
elevated specific antibody titers. More importantly, LaFrentz 8.0 · 104 cfu/g egg were recovered. Results clearly show that
et al. [103] showed that immersion exposure to the attenu- standard egg treatment protocols may not be relied upon to
ated strain also elicited a protective immune response in fish. effectively disinfect salmonid eggs and control the spread of
Álvarez et al. [104] also demonstrated protection in rainbow F. psychrophilum [17,53,110].
trout fry using an attenuated strain of F. psychrophilum; this In the United States, antimicrobial agents or other drugs to
strain was attenuated using transposon insertion mutagene- be used in fish destined for human consumption must be ap-
sis. LaFrentz et al. [105] suggested that the glycocalyx of proved by the U.S. Food and Drug Administration and used
104 C.E. Starliper

in accordance with product label information. Certain factors the fish. Plant et al. [43] concluded that these proteins did
should be considered when using a therapeutic agent, such as not seem to be useful for further vaccine development. LaF-
tissue clearance time, toxicity to fishes in different water chem- rentz et al. [130] identified and analyzed specific proteins of
istries, and the organic load in the water. If it is unclear F. psychrophilum cultures grown in vivo and in vitro in an
whether a drug will result in adverse effects to fish in a certain iron-limited medium. Through evaluations using 2-D poly-
water chemistry profile, it may be advisable to initially try the acrylamide gel electrophoresis, numerous proteins from the
treatment in a pilot study on a small number of individuals to cultures showed increased intensities, while others showed les-
identify a potential problem, rather than simply treating large ser intensities. The expressed (upregulated) proteins may be
numbers of fish and discovering toxicity with no means to important in the course of CWD in fish (LaFrentz et al.
quickly stop the treatment. [130] and perhaps warrant utilization in the development of
For fish bacterial diseases treated with oral delivery of med- a fish vaccine.
icated food, early intervention is paramount to achieve a suc-
cessful treatment for CWD. This is especially true since one of Disclaimer
the earliest disease signs is the fish’s loss of appetite, which will
directly affect the efficacy of treatment. A successful antimicro- Any use of trade, product, or firm names is for descriptive pur-
bial treatment is dependant on an early and accurate diagnosis poses only and does not imply endorsement by the U.S.
of F. psychrophilum as the causal agent of disease. However, Government.
prophylactic or indiscriminate antimicrobial therapy should
be avoided because of the risk to develop antimicrobial-resis-
tant bacterial strains [59,111,112]. Prior to the use of an anti- References
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