Virulence

infection-a possible mechanism for increased virulence of bacteria associated with rapidly destructive periodontitis. Acta Odontol Scand We have recently isolated several groups of bacteriophages infecting Actinobacillits actinomyceremcomirans from periodontal lesions in patients with rapidly destructive periodontitis. Bacteriophage infection of these bacteria in these patients was restricted to periodontal pockets showing radiographic evidence of recent bone loss and suggests an association between phage-infected A. acrinornyceremcornirans and active periodontal disease. On the basis of the biological activity of bacteriophages we propose a working hypothesis to explain the mechanism by which a phage may increase bacterial virulence in periodontal disease. 0 Hypothesis; ooeriiiew; phage infection

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef stimulates viral infectivity by facilitating an early event in the HIV-1 life cycle. Although no structural or biochemical defects in Nef-defective HIV-1 particles have been demonstrated, the Nef protein is incorporated into HIV-1 particles. To localize the function of Nef within the virus particle, we developed a novel technology involving fusion of enveloped donor HIV-1 particles bearing core defects with envelope-defective target virions bearing HIV-1 receptors.

Background Interferons (IFNs) play an important role in host antiviral responses, but viruses, including vaccinia viruses (VV), employ mechanisms to disrupt IFN activities, and these viral mechanisms are often associated with their virulence. Here, we explore an attenuation strategy with a vaccine strain of VV lacking a virus-encoded IFN-γ receptor homolog (viroceptor).

Parasites with complex life cycles are expected to manipulate the behaviour of their intermediate hosts (IHs), which increase their predation rate and facilitate the transmission to definitive hosts (DHs). This ability, however, is a doubleedged sword when the parasite can also be transmitted vertically in the IH. In this situation, as the manipulation of the IH behaviour increases the IH death rate, it conflicts with vertical transmission, which requires healthy and reproducing IHs. The protozoan Toxoplasma gondii, a widespread pathogen, combines both trophic and vertical transmission strategies. Is parasite manipulation of host behaviour still adaptive in this situation? We model the evolution of the IH manipulation by T. gondii to study the conflict between these two routes of transmission under different epidemiological situations. Model outputs show that manipulation is particularly advantageous for virulent strains and in epidemic situations, and that different levels of manipulation may evolve depending on the sex of the IH and the transmission routes considered. These results may help to understand the variability of strain characteristics encountered for T. gondii and may extend to other trophically transmitted parasites.

The Escherichia coli regulatory protein RfaH contributes to efficient colonization of the mouse gut. Extraintestinal pathogenic (ExPEC) as well as non-pathogenic probiotic E. coli strains rapidly outcompeted their isogenic rfaH mutants following oral mixed infections. LPS-core and O-...

Periodontitis is an infectious inflammatory disease that destroys the tooth-supporting tissues. It is caused by the formation of subgingival biofilms on the surface of the tooth. Characteristic bacteria associated with subgingival biofilms are the Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, collectively known as the "red complex" species. Interepithelial junctions ensure the barrier integrity of the gingival epithelium. This may however be disrupted by the biofilm challenge. The aim of this in vitro study was to investigate the effect of subgingival biofilms on the expression of inter-epithelial junctions by gingival epithelia, and evaluate the relative role of the red complex. Multi-layered human gingival epithelial cultures were challenged with a 10-species in vitro subgingival biofilm model, or its variant without the red complex, for 3 h and 24 h. A low-density array microfluidic card platform was then used for analysing the expression of 62 genes encoding for tight junctions, gap junctions, adherens junctions, and desmosomes. Although there was a limited effect of the biofilms on the expression of tight, adherens and gap junctions, the expression of a number of desmosomal components was affected. In particular, Desmoglein-1 displayed a limited and transient up-regulation in response to the biofilm. In contrast, Desmocollin-2, Desmoplakin and Plakoglobin were down-regulated equally by both biofilm variants, after 24 h. In conclusion, this subgingival biofilm model may downregulate selected desmosomal junctions in the gingival epithelium, irrespective of the presence of the "red complex". In turn, this could compromise the structural integrity of the gingival tissue, favouring bacterial invasion and chronic infection.

Marek's disease virus (MDV) isolated from poultry flocks in three states of India was monitored for the virus occurrence in the field. The MDV genome was isolated from feather follicles, spleen, and liver of the chicken (173 samples). Twenty two samples were positive for MDV genome in PCR and belonged to the serotype 1. The sequencing of MEQ gene of 11 samples revealed that nucleotide sequences of the isolate Ind-KA-01-06 was similar to the very virulent MDV, strain RB-1B. In situ hybridization studies also confirmed a presence of MDV serotype 1 in the infected liver tissues. Furthermore, the ability of the virus to induce apoptosis detected by flow cytometry showed that the virulent MDV induced apoptosis more efficiently than Turkey herpesvirus (HVT) vaccine virus. The present study showed the presence of virulent/very virulent MDV strains in the Indian poultry flocks.

The biological behaviour of 23 Trypanosoma cruzi isolates in Swiss mice was compared. Nineteen isolates were obtained from patients in the acute phase of Chagas disease (13), sylvatic reservoir hosts (Didelphis marsupialis) (3), and triatomine bugs (Rhodnius robustus) (3) from four regions of the State of Amazonas (AM). Four isolates were obtained from chronic chagasic patients in the State of Paraná (PR): three autochthones, and one allochthone from the State of Minas Gerais. Only one isolate was unable to infect the mice. The AM and PR isolates showed the largest number of significant differences from each other. The former had lower mean values in the pre-patent (5.4 days) and patent (4.6 days) periods (PP), with the parasitaemia (Pmax) reaching a peak of 9.9 Â 10 4 blood trypomastigotes (BT)/mL of blood by the 7th day following inoculation. The AM isolates also had higher positivity to fresh-blood examination (FBE) (84.1%) compared to haemoculture (HC) (58.7%) and polymerase chain reaction (PCR) (33.3%), in addition to higher mortality (2.9%). The PR isolates had higher values for PP (18.5 days) and Pmax (99.9 Â 10 4 BT/mL) as well as higher positivity to FBE (87.2%), HC (100%), and PCR (83.3%). The correlations between the biological behaviour of the T. cruzi isolates and the clinical and epidemiological characteristics of Chagas disease are discussed.

Sortase enzymes are found throughout Gram-positive bacteria and are responsible for the covalent attachment of specific proteins to the cell wall. Through the anchoring of these cell wall proteins, sortase enzymes are important in the ability of several Gram-positive pathogens to cause disease. Previously, deletion of srtA from Streptococcus pneumoniae (the pneumococcus) was shown to disturb the localisation of surface proteins, and decrease bacterial adherence to human pharyngeal cells in vitro. Here we present data demonstrating, for the first time, a role for srtA as a pneumococcal fitness factor in experimental models of pneumonia and bacteraemia. In addition, srtA contributed to nasopharyngeal colonisation in vivo. Furthermore, we find that the contribution of srtA varied between two pneumococcal strains. We show that the known role of srtA in adherence in vitro is dependent on capsule expression, the role of SrtA in adherence to human cells only being apparent in the absence of the pneumococcal capsule. The srtA gene was detected by PCR in all 82 clinical isolates examined and sequencing of the gene from 20 strains showed srtA to be highly conserved. The ubiquitous distribution of srtA was in contrast to the other known pneumococcal sortase genes, srtB, C and D, which were found in only 14 of the 82 tested strains (17%).

Analysis of the transcriptome of slyA mutant Salmonella enterica serovar Typhimurium revealed that many SlyA-dependent genes, including pagC, pagD, ugtL, mig-14, virK, phoN, pgtE, pipB2, sopD2, pagJ and pagK, are also controlled by the PhoP/PhoQ regulatory system. Many SlyA- and PhoP/PhoQ-co-regulated genes have functions associated with the bacterial envelope, and some have been directly implicated in virulence and resistance to antimicrobial peptides. Purified His-tagged SlyA binds to the pagC and mig-14 promoters in regions homologous to a previously proposed ‘SlyA-box’. The pagC promoter lacks a consensus PhoP binding site and does not bind PhoP in vitro, suggesting that the effect of PhoP on pagC transcription is indirect. Stimulation of pagC expression by PhoP requires SlyA. Levels of SlyA protein and mRNA are not significantly changed under low-magnesium PhoP-inducing conditions in which pagC expression is profoundly elevated, however, indicating that the PhoP/PhoQ system does not activate pagC expression by altering SlyA protein concentration. Models are proposed in which PhoP may control SlyA activity via a soluble ligand or SlyA may function as an anti-repressor to allow PhoP activation. The absence of almost all SlyA-activated genes from the Escherichia coli K12 genome suggests that the functional linkage between the SlyA and PhoP/PhoQ regulatory systems arose as Salmonella evolved its distinctive pathogenic lifestyle.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, or Johne's disease, in cattle, with potential involvement in cases of Crohn's disease in humans. Johne's disease is found worldwide and is economically important for both beef and dairy industries. In an effort to characterize this important infection in Egypt, we analysed the ecological and genomic features of recent isolates of M. paratuberculosis. In this report, we examined 26 Holstein dairy herds distributed throughout Egypt, from 2010 to 2013. Using PCR analysis of faecal samples, we estimated a mean herd-level prevalence of 65.4 %, with animallevel infection that reached a mean of 13.6 % among animals suffering from diarrhoea. Whole genome sequencing of field isolates identified numerous single nucleotide polymorphisms among field isolates relative to the standard M. paratuberculosis K10 genome. Interestingly, the virulence of M. paratuberculosis isolates from Egypt revealed diverse virulence phenotypes in the murine model of paratuberculosis, with significant differences in tissue colonization, particularly during the chronic stage of infection. Overall, our analysis confirmed that Johne's disease is a newly identified problem in Egypt and indicated that M. paratuberculosis has potentially diverse genotypes that impact its virulence. Further ecological mapping and genomic analysis of M. paratuberculosis will enhance our understanding of the transmission and evolutionary dynamics of this pathogen under natural field conditions.

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cellcycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function.

The Sabin3 mutation in the viral RNA plays an important role in directing attenuation phenotype of Sabin vaccine strain of poliovirus type 1 (PV1). We previously described that Sabin3-like mutation introduced in Coxsackievirus B3 (CVB3) genome led to a defective mutant. However, this mutation do not led to destruction of secondary structure motif C within the stem-loop V of CVB3 RNA because of the presence of one nucleotide difference (C fi U) in the region encompassing the Sabin3 mutation at nucleotides 471 of PV1 and 475 of CVB3 RNA. In order to reproduce the same sequence of PV1 sabin3 vaccine strain, we introduce in this study an additional mutation (U 475 fi C) to CVB3 Sabin3-like mutant. Our results demonstrated that Sabin3-like+C mutant displayed a decreased translation initiation defects when translated in cell-free system. This translation initiation defect was correlated with reduced yields of infectious virus particles in HeLa cells in comparison with Sabin3-like mutant and wild-type CVB3 viruses. Inoculation of Swiss mice with mutant viruses resulted in no inflammatory heart disease when compared to heart of mice infected with wild-type. Theses findings indicate that the double mutant could be exploited for the development of a live attenuated vaccine against CVB3.

Escherichia coli sequence type 131 (ST131) is a pandemic clone associated with multidrug-resistant, extraintestinal infections, attributable to the presence of the CTX-M-15 extended-spectrum ␤-lactamase gene and mutations entailing fluoroquinolone resistance. Studies on subclones within E. coli ST131 are critically required for targeting and implementation of successful control efforts. Our study comprehensively analyzed the genomic and functional attributes of the H30-Rx subclonal strains NA097 and NA114, belonging to the ST131 lineage. We carried out whole-genome sequencing, comparative analysis, phenotypic virulence assays, and profiling of the antibacterial responses of THP1 cells infected with these subclones. Phylogenomic analysis suggested that the strains were clonal in nature and confined entirely to a single clade. Comparative genomic analysis revealed that the virulence and resistance repertoires were comparable among the H30-Rx ST131 strains except for the commensal ST131 strain SE15. Similarly, seven phage-specific regions were found to be strongly associated with the H30-Rx strains but were largely absent in the genome of SE15. Phenotypic analysis confirmed the virulence and resistance similarities between the two strains. However, NA097 was found to be more robust than NA114 in terms of virulence gene carriage (dra operon), invasion ability (P < 0.05), and antimicrobial resistance (streptomycin resistance). RT 2 gene expression profiling revealed generic upregulation of key proinflammatory responses in THP1 cells, irrespective of ST131 lineage status. In conclusion, our study provides comprehensive, genome-inferred insights into the biology and immunological properties of ST131 strains and suggests clonal diversification of genomic and phenotypic features within the H30-Rx subclone of E. coli ST131.

An infectious bursal disease (IBD) outbreak occurred in the east region of Spain in the spring of 2002 and rapidly spread thorough the whole country, although proper vaccination programs were applied. In this report, 33 infectious bursal disease viruses (IBDVs) isolated from this outbreak were characterized by nucleotide sequencing of the VP2 gene hypervariable region and were compared with reference IBD strains and the 1990s Spanish IBDVs in order to determine possible emergence of IBDV isolates with modified antigenic or virulent properties. Moreover, histopathologic and immunohistochemical studies of those cases where bursal tissues were available were carried out. Of the 33 isolates, 23 were identified as very virulent IBDVs (vvIBDVs), whereas the other 10 isolates were classified as attenuated or intermediate virulence classical strains and could possibly be IBDV live vaccine strains used in the immunization of these chickens. Results of this study indicate that vvIBDV isolates from the 2002 Spanish outbreak are closely related with those from the 1990s outbreak. However, acute IBD cases have not been reported in Spain during these 10 yr. Genetic, management, and environmental factors likely related with IBD reemergence in Spain are discussed. Moreover, our results indicate that good correlation exists between the IBDV subtype present in the field and the degree of lesions in bursa tissue, as well as the immunohistochemistry staining.

Resistance of Pseudomonas aeruginosa to
antibiotics is a major problem. Targeting virulence
factors is an alternative option to avoid the
emergence of resistance to antibiotics. The effect of
sub-inhibitory concentration of diclofenac sodium
on the production of virulence factors of P.
aeruginosa was investigated. The virulence factors
included protease, haemolysin, pyocyanin and
pyoverdin, in addition to pathogenic behaviors such
as swimming and twitching motilities and biofilm
formation. Diclofenac sodium showed significant
inhibition of virulence factors as compared to the
control. Diclofenac sodium decreased twitching and
swimming motilities by 29.27% and 45.36%,
respectively. The percentage of inhibition of
pyocyanin by diclofenac sodium was 42.32%. On
the other hand, pyoverdin was inhibited to a lesser
extent (36.72%). Diclofenac sodium reduced
protease by 52.58% and biofilm formation by
58.37%. Moreover, haemolytic activity in the
presence of diclofenac sodium was 15.64% as
compared to the control (100% haemolytic activity).
The inhibitory activities may be due to
inhibition of quorum sensing that regulates the
expression of virulence factors. This study suggests
the potential for the use of diclofenac sodium as an
anti-virulence agent in the treatment of
Pseudomonas aeruginosa infections.

Quantitative reverse transcription PCR (qRT-PCR) is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum) in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessed in different plant structures (leaves and inflorescences at two stages of development and fruits). These genes are the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionally used housekeeping gene GAPDH. Three software applications -geNorm, NormFinder and BestKeeper -were used to estimate expression stability and provided congruent results. The orthologs of AT4G33380 (expressed protein of unknown function, Expressed1), AT2G28390 (SAND family protein, SAND) and AT5G46630 (clathrin adapter complex subunit family protein, CACS) are revealed as the most stable. We recommend using the combination of Expressed1, SAND and CACS for the normalization of gene expression data in studies on buckwheat using qRT-PCR. These genes are listed among five the most stably expressed in Arabidopsis that emphasizes utility of the studies on model plants as a framework for other species.

The proteins belonging to the Fur family are global regulators of gene expression involved in the response to several environmental stresses and to the maintenance of divalent cation homeostasis. The Mycobacterium tuberculosis genome encodes two Fur-like proteins, FurA and a protein formerly annotated FurB. Since in this paper we show that it represents a zinc uptake regulator, we refer to it as Zur. The gene encoding Zur is found in an operon together with the gene encoding a second transcriptional regulator (Rv2358). In a previous work we demonstrated that Rv2358 is responsible for the zinc-dependent repression of the Rv2358-zur operon, favoring the hypothesis that these genes represent key regulators of zinc homeostasis. In this study we generated a zur mutant in M. tuberculosis, examined its phenotype, and characterized the Zur regulon by DNA microarray analysis. Thirty-two genes, presumably organized in 16 operons, were found to be upregulated in the zur mutant. Twenty-four of them belonged to eight putative transcriptional units preceded by a conserved 26-bp palindrome. Electrophoretic mobility shift experiments demonstrated that Zur binds to this palindrome in a zinc-dependent manner, suggesting its direct regulation of these genes. The proteins encoded by Zurregulated genes include a group of ribosomal proteins, three putative metal transporters, the proteins belonging to early secretory antigen target 6 (ESAT-6) cluster 3, and three additional proteins belonging to the ESAT-6/culture filtrate protein 10 (CFP-10) family known to contain immunodominant epitopes in the T-cell response to M. tuberculosis infection.

The genus Salmonella contains two species, S. bongori and S. enterica. Compared to the well-studied S. enterica there is a marked lack of information regarding the genetic makeup and diversity of S. bongori. S. bongori has been found predominantly associated with cold-blooded animals, but it can infect humans. To define the phylogeny of this species, and compare it to S. enterica, we have sequenced 28 isolates representing most of the known diversity of S. bongori. This cross-species analysis allowed us to confidently differentiate ancestral functions from those acquired following speciation, which include both metabolic and virulence-associated capacities. We show that, although S. bongori inherited a basic set of Salmonella common virulence functions, it has subsequently elaborated on this in a different direction to S. enterica. It is an established feature of S. enterica evolution that the acquisition of the type III secretion systems (T3SS-1 and T3SS-2) has been followed by the sequential acquisition of genes encoding secreted targets, termed effectors proteins. We show that this is also true of S. bongori, which has acquired an array of novel effector proteins (sboA-L). All but two of these effectors have no significant S. enterica homologues and instead are highly similar to those found in enteropathogenic Escherichia coli (EPEC). Remarkably, SboH is found to be a chimeric effector protein, encoded by a fusion of the T3SS-1 effector gene sopA and a gene highly similar to the EPEC effector nleH from enteropathogenic E. coli. We demonstrate that representatives of these new effectors are translocated and that SboH, similarly to NleH, blocks intrinsic apoptotic pathways while being targeted to the mitochondria by the SopA part of the fusion. This work suggests that S. bongori has inherited the ancestral Salmonella virulence gene set, but has adapted by incorporating virulence determinants that resemble those employed by EPEC.

KATHLEEN M. HESS*, MATTHEW W. DUDLEY*, DAVID G. LYNN*t, ROLF D. JOERGERf, AND ANDREW N. BINNSt *Searle Chemistry Laboratory, 5735 Ellis Avenue, The University of Chicago, Chicago IL 60637; and tPlant Science Institute, Department of Biology, ...

Bacillus anthracis is closely related to the endospore forming bacteria Bacillus cereus and Bacillus thuringiensis. For accurate detection of the life threatening pathogen B. anthracis, it is essential to distinguish between these three species. Here we present a novel multiplex real-time PCR for simultaneous specific identification of B. anthracis and discrimination of different B. anthracis virulence types. Specific B. anthracis markers were selected by whole genome comparison and different sets of primers and probes with optimal characteristic for multiplex detection of the B. anthracis chromosome, the B. anthracis pXO1 and pXO2 plasmids and an internal control (IC) were designed. The primer sets were evaluated using a panel of B. anthracis strains and exclusivity was tested using genetically closely related B. cereus strains. The robustness of final primer design was evaluated by laboratories in three different countries using five different real-time PCR thermocyclers. Testing of a panel of more than 20 anthrax strains originating from different locations around the globe, including the recent Swedish anthrax outbreak strain, showed that all strains were detected correctly.

Navas-Cortés, J. A., Landa, B. B., Méndez-Rodríguez, M. A., and Jiménez-Díaz, R. M. 2007. Quantitative modeling of the effects of temperature and inoculum density of Fusarium oxysporum f. sp. ciceris races 0 and 5 on development of Fusarium wilt in chickpea cultivars. Phytopathology 97:564-573.

Introduction: Enterococcus faecalis is a member of the mammalian gastrointestinal microbiota but has been considered a leading cause of hospital-acquired infections. In the oral cavity, it is commonly detected from root canals of teeth with failed endodontic treatment. However, little is known about the virulence and genetic relatedness among E. faecalis isolates from different clinical sources. This study compared the presence of enterococcal virulence factors among root canal strains and clinical isolates from hospitalized patients to identify virulent clusters of E. faecalis. Methods: Multilocus sequence typing analysis was used to determine genetic lineages of 40 E. faecalis clinical isolates from different sources. Virulence clusters were determined by evaluating capsule (cps) locus polymorphisms, pathogenicity island gene content, and antibiotic resistance genes by polymerase chain reaction. Results: The clinical isolates from hospitalized patients formed a phylogenetically separate group and were mostly grouped in the clonal complex 2, which is a known virulent cluster of E. faecalis that has caused infection outbreaks globally. The clonal complex 2 group comprised capsule-producing strains harboring multiple antibiotic resistance and pathogenicity island genes. On the other hand, the endodontic isolates were more diverse and harbored few virulence and antibiotic resistance genes. In particular, although more closely related to isolates from hospitalized patients, capsuleproducing E. faecalis strains from root canals did not carry more virulence/antibiotic genes than other endodontic isolates. Conclusions: E. faecalis isolates from endodontic infections have a genetic and virulence profile different from pathogenic clusters of hospitalized patients' isolates, which is most likely due to niche specialization conferred mainly by variable regions in the genome. (J Endod 2013;39:858-864)

The Nine Mile phase II clone 4 (NMIIC4) strain of Coxiella burnetii is an attenuated phase II strain that has lost the genes for virulence determinant type 1 lipopolysaccharide. These bacteria were very virulent for severe combined immunodeficient (SCID) mice. The lethal dose 50 (LD 50 ) was~10 bacteria. Infected SCID mice died between Day 28 and Day 53 post-infection. At termination of the experiment (Day 60) only 5 of 24 mice had survived. The degree of splenomegaly was directly related to the bacterial load in the SCID mice spleens. The NMIIC4 was avirulent in immunocompetent wild mice and bacterial DNA copies in splenic tissue were extremely low. The SCID mice that were inoculated with high doses of heat inactivated NMIIC4 C. burnetii were all alive at Day 60 and without splenomegaly. It appears that the phase I lipopolysaccharide present in virulent Nine Mile phase I but not in attenuated NMIIC4 is not the only virulence factor for C. burnetii.

Moritella viscosa is considered the main aetiological agent of 'winter ulcer' disease in farmed salmonid fish. To further understand the pathogenesis of this disease, M. viscosa interaction with fish cells was studied using a Chinook salmon embryo cell line (CHSE-214). As winter ulcer appears exclusively at temperatures below 7-8 C, we attempted to identify if this connection is explained by temperature regulated bacterial virulence. Therefore, infection studies were performed at a temperature range from 4 to 15 C. At all temperatures, M. viscosa caused CHSE cells to retract and round up, lose their attachment abilities and finally disintegrate. The bacterium adhered to CHSE cells and caused changes to the cytoskeleton, however, it did not invade the cells. Increased adherence was demonstrated at 4 C compared to adherence at higher temperatures. Extracellular proteins exerted rapid pore formation and lysis of CHSE cells at a temperature range from 4 to 22 C. Furthermore, only small differences were found comparing extracellular proteomes of M. viscosa from 4 and 15 C. We propose that the pathogenic mechanisms exerted by M. viscosa on CHSE cells are disruption of the cytoskeleton which affects cell rigidity and structure, followed by pore formation and lysis caused by secreted products from the bacterium. These processes can also occur at temperatures above those experienced from winter ulcer outbreaks. However, the adhesion mechanisms appear to be temperature regulated and may contribute to temperature dependent disease outbreaks.

Stomoxys calcitrans control is primarily based on the use of chemical products; however, new methods for control have been investigated. Biological control has been proposed as a promising alternative to traditional chemicals. The current study evaluated three methods in vitro to treat S. calcitrans eggs with the fungus Metarhizium anisopliae and assessed the virulence of this fungus to immature stages of this fly. In the first method of egg exposure, eggs were immersed in conidial suspensions and transferred to Petri plates with high humidity. A high mortality was observed in both treated and control groups. In the second method, the eggs were transferred to rearing medium right after immersion in conidial suspensions; the suspensions with highest conidial concentrations, 10 7 and 10 8 conidia mL −1 , caused 96.25% and 100% mortality, respectively. In the third method, the eggs were sprayed with conidial suspensions on the rearing medium. A high mortality was observed at 10 7 and 10 8 conidia mL −1 , 78.3% and 100%, respectively. The second method had the lowest mortality in the control group and was considered the most useful method for evaluating the effect of entomopathogenic fungi on S. calcitrans eggs. While eggs were very susceptible to M. anisopliae infection, larvae and pupae were not.

The HN878 strain of Mycobacterium tuberculosis is regarded as "hypervirulent" due to its rapid growth and reduced survival of infected mice when compared with other clinical isolates. This property has been ascribed due to an early increase in type I IFNs and a failure to generate TH1-mediated immunity, induced by a response to an unusual cell wall phenolic glycolipid expressed by the HN878 isolate. We show, however, that although type I IFN does play an inhibitory role, this response was most apparent during the chronic disease stage and was common to all M. tuberculosis strains tested. In addition, we further demonstrate that the HN878 infection was associated with a potent TH1 response, characterized by the emergence of both CD4 and CD8 T cell subsets secreting IFN-␥. However, where HN878 differed to the other strains tested was a subsequent reduction in TH1 immunity, which was temporally associated with the rapid emergence of a CD4 ؉ CD25 ؉ FoxP3 ؉ CD223 ؉ IL-10 ؉ regulatory T cell population. This association may explain the paradoxical initial emergence of a TH1 response in these mice but their relatively short time of survival.

Purpose: Acanthamoeba keratitis is a severe, often sight threatening, corneal infection which in Western countries is predominantly seen in daily wear of contact lenses. This review aims to summarise the pathobiology and epidemiology of contact lens-related Acanthamoeba keratitis, and to present strategies for prevention, particularly with respect to modifiable risk factors in contact lens wear. Recent Findings: The virulence of Acanthamoeba and resistance to treatment in keratitis appears to be linked with the production of a low molecular weight protease MIP133 by the organism, in response to binding to corneal epithelial cells through a mannose binding protein, and to the ability of the organism to convert from the trophozoite to the resistant cyst form. Recent epidemiological studies in contact lens relate disease have confirmed the link between solution topping up and Acanthamoeba keratitis and have reinforced the importance of avoidance of tap water, either as part of the care for the contact lens or storage case, handling lenses with wet hands or showering while wearing lenses. In the most recent analysis from the USA, there were no strong effects for solution type, water source or water disinfection process. Wearer age, lens wear time and history to appear to be linked with Acanthamoeba keratitis. Daily disposable contact lens use would be expected to reduce the prevalence of Acanthamoeba disease although this is unproven. Summary: While Acanthamoeba keratitis remains challenging to diagnose and manage, strategies to limit the disease severity in contact lens wearers should include attention to recently identified risk factors, particularly those related to water contact. Public health awareness measures, the use of daily disposable contact lenses, a better understanding of the contribution of the host immunity and the development of standardised methods for culture of amoeba and testing of contact lens care systems against Acanthamoeba in the licensing process may be of value. Alternative treatments for the future may include those which target the mannose binding protein or the genes which control conversion to the cyst form.

In this study, we evaluated the potential use of entomopathogenic nematodes as a control for the beetle Aethina tumida Murray (Coleoptera: Nitidulidae). In particular, we conducted 1) four screening bioassays to determine nematode (seven species, 10 total strains tested) and application level effects on A. tumida larvae and pupae, 2) a generational persistence bioassay to determine whether single inoculations with nematodes would control multiple generations of A. tumida larvae in treated soil, and 3) a Þeld bioassay to determine whether the nematodes would remain efÞcacious in the Þeld. In the screening bioassays, nematode efÞcacy varied signiÞcantly by tested nematode and the infective juvenile (IJ) level at which they were applied. Although nematode virulence was moderate in screening bioassays 1Ð3 (0 Ð 68% A. tumida mortality), A. tumida mortality approached higher levels in screening bioassay 4 (nearly 100% after 39 d) that suggest suitable applicability of some of the test nematodes as Þeld controls for A. tumida. In the generational persistence bioassay, Steinernema riobrave Cabanillas, Poinar & Raulston 7-12 strain and Heterorhabditis indica Poinar, Karunaka & David provided adequate A. tumida control for 19 wk after a single soil inoculation (76 Ð94% mortality in A. tumida pupae). In the Þeld bioassay, the same two nematode species also showed high virulence toward pupating A. tumida (88 Ð100%) mortality. Our data suggest that nematode use may be an integral component of an integrated pest management scheme aimed at reducing A. tumida populations in bee colonies to tolerable levels.

SUMMARY Over the last few years, dramatic increases in our knowledge about diffusely adhering Escherichia coli (DAEC) pathogenesis have taken place. The typical class of DAEC includes E. coli strains harboring AfaE-I, AfaE-II, AfaE-III, AfaE-V, Dr, Dr-II, F1845, and NFA-I adhesins (Afa/Dr DAEC); these strains (i) have an identical genetic organization and (ii) allow binding to human decay-accelerating factor (DAF) (Afa/Dr DAF subclass) or carcinoembryonic antigen (CEA) (Afa/Dr CEA subclass). The atypical class of DAEC includes two subclasses of strains; the atypical subclass 1 includes E. coli strains that express AfaE-VII, AfaE-VIII, AAF-I, AAF-II, and AAF-III adhesins, which (i) have an identical genetic organization and (ii) do not bind to human DAF, and the atypical subclass 2 includes E. coli strains that harbor Afa/Dr adhesins or others adhesins promoting diffuse adhesion, together with pathogenicity islands such as the LEE pathogenicity island (DA-EPEC). In this review, the f...

In this study, we evaluated the prevalence of primary resistance of Brazilian H. pylori isolates to metronidazole, clarithromycin, amoxicillin, tetracycline, and furazolidone. In addition, the vacA, iceA, cagA and cagE genotypes of strains isolated from Brazilian patients were determined and associated with clinical data in an effort to correlate these four virulence markers and antibiotic resistance.

Pseudomonas fuscovaginae was first reported as a pathogen of rice causing sheath rot in plants grown at high altitudes. P. fuscovaginae is now considered a broad-host-range plant pathogen causing disease in several economically important plants. We report what is, to our knowledge, the first draft genome sequence of a P. fuscovaginae strain.

Multihost parasites can infect different types of hosts or even different host species. Epidemiological models have shown the importance of the diversity of potential hosts for understanding the dynamics of infectious disease (e.g., the importance of reservoirs), but the consequences of this diversity for virulence and transmission evolution remain largely overlooked. Here, I present a general theoretical framework for the study of life-history evolution of multihost parasites. This analysis highlights the importance of epidemiology (the relative quality and quantity of different types of infected hosts) and between-trait constraints (both within and between different hosts) to parasite evolution. I illustrate these effects in different transmission scenarios under the simplifying assumption that parasites can infect only two types of hosts. These simple but contrasted evolutionary scenarios yield new insights into virulence evolution and the evolution of transmission routes among different hosts. Because many of the pathogens that have large public-health and agricultural impacts have complex life cycles, an understanding of their evolutionary dynamics could hold substantial benefits for management.

Aims: To determine the presence of Vibrio cholerae in different areas of Argentina in three sample types, to determine the composition of planktonic communities in areas at which this pathogen was detected and to characterize the virulence properties and antimicrobial resistance of the recovered environmental isolates. Methods and Results: Water and plankton samples were collected in marine, brackish and freshwater environments. Vibrio cholerae non-O1, non-O139 was isolated in 36AE1% of the samples analysed. The microorganism was detected in freshwater but not in marine or brackish samples. No relationship was found between isolation of V. cholerae and presence of any species of plankton. All the isolates presented very similar virulence profiles by PCR, lacking ctxA and tcpA El Tor and containing hlyA (98AE7%), rtxA (99AE0%), toxR (98AE7%) and stnsto (1AE9%). Resistance to ampicillin was found in both Tucumán (21%) and Buenos Aires isolates (45%). Conclusions: We identified two geographic areas in Argentina where V. cholerae was present: freshwaters of the rivers from Tucumán and the Río de la Plata. Significance and Impact of the Study: The identification of V. cholerae strains in the environment, carrying both virulence factors and resistance to antimicrobial agents, highlight the need for a continuous and active surveillance of this pathogen.

The function of the rorf2 gene located on the locus of enterocyte effacement (LEE) pathogenicity island of enteropathogenicEscherichia coli (EPEC) has not been described. We report that rorf2 encodes a novel protein, named EspG, which is secreted by the type III secretory system and which is translocated into host epithelial cells. EspG is homologous withShigella flexneri protein VirA, and the clonedespG (rorf2) gene can rescue invasion in a Shigella virA mutant, indicating that these proteins are functionally equivalent in Shigella. An EPECespG mutant had no apparent defects in in vitro assays of virulence phenotypes, but a rabbit diarrheagenic E. coli strain carrying a mutant espG showed diminished intestinal colonization and yet diarrheal attack rates similar to those of the wild type. A second EspG homolog, Orf3, is encoded on the EspC pathogenicity islet. The clonedorf3 gene could also rescue invasion in aShigella virA mutant, but an EPEC espG orf3 double mutant was not diminis...

Several Gram negative bacteria use a complex system called``type III secretion system'' (TTSS) to engage their host. The archetype of TTSS is the plasmid-encoded``Yop virulon'' shared by the three species of pathogenic Yersinia (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica). A second TTSS, called Ysa (for Yersinia secretion apparatus) was recently described in Y. enterocolitica 8081, a strain from serotype O:8. In this study, we describe the ysa locus from A127/90, another strain of serotype O:8, and we extend the sequence to several new genes encoding Ysp proteins which are the substrates of this secretion system, and a putative chaperone SycB. According to the deduced protein sequences, the ysa system from A127/90 is identical to that of 8081. It is dierent from the chromosome-encoded TTSS of Y. pestis but is instead closely related to the Mxi-Spa TTSS of Shigella and to the SPI-1 encoded TTSS of Salmonella enterica. We further demonstrated that the ysa locus is only present in biotype IB strains of Y. enterocolitica. Including this new Ysa system, a phylogenetic analysis of the 26 known TTSSs was carried out, based on the sequence analysis of three conserved proteins. All the TTSSs fall into ®ve dierent clusters. The phylogenetic tree of these TTSSs is completely dierent from the evolu-tionary tree based on 16S RNA, indicating that TTSSs have been distributed by horizontal transfer.

. White spot syndrome virus WSSV is one of the most important pathogens of penaeid shrimp. It is widely distributed in most Asian countries where penaeid shrimp are cultured, as well as in the Gulf of Mexico and SE USA. The virulence of six geographic isolates of WSSV was compared using Litopenaeus Õannamei postlarvae and Farfantepenaeus duorarum juveniles. The six geographic isolates of WSSV originated from China, India, Thailand, Texas, South Carolina, as well as from crayfish maintained at the USA National Zoo. For challenge studies, virus infected tissues were given per os to L. Õannamei postlarvae and Fa. duorarum juveniles. Resultant WSSV infections were confirmed by histological examination. The cumulative mortality of L. Õannamei postlarvae reached 100% after challenge with each of the six geographic isolates of WSSV. However, the Texas isolate caused mortalities more rapidly than did the other shrimp isolates; the crayfish WSSV isolate was the slowest. In marked contrast, cumulative mortalities of juvenile Fa. duorarum reached only 35-60%, and varied among the geographic isolates of WSSV. Interestingly, in Fa. duorarum, the Texas WSSV isolate was also the most virulent, while the crayfish WSSV was the least virulent. The findings suggest that slight differences in virulence exist among geographic isolates of WSSV, and that susceptibility may vary with species and lifestages of the host. q 0044-8486r99r$ -see front matter q 1999 Elsevier Science B.V. All rights reserved.

Staphylococcus haemolyticus is an opportunistic bacterial pathogen that colonizes human skin and is remarkable for its highly antibiotic-resistant phenotype. We determined the complete genome sequence of S. haemolyticus to better understand its pathogenicity and evolutionary relatedness to the other staphylococcal species. A large proportion of the open reading frames in the genomes of S. haemolyticus, Staphylococcus aureus, and Staphylococcus epidermidis were conserved in their sequence and order on the chromosome. We identified a region of the bacterial chromosome just downstream of the origin of replication that showed little homology among the species but was conserved among strains within a species. This novel region, designated the "oriC environ," likely contributes to the evolution and differentiation of the staphylococcal species, since it was enriched for species-specific nonessential genes that contribute to the biological features of each staphylococcal species. A comparative analysis of the genomes of S. haemolyticus, S. aureus, and S. epidermidis elucidated differences in their biological and genetic characteristics and pathogenic potentials. We identified as many as 82 insertion sequences in the S. haemolyticus chromosome that probably mediated frequent genomic rearrangements, resulting in phenotypic diversification of the strain. Such rearrangements could have brought genomic plasticity to this species and contributed to its acquisition of antibiotic resistance.