Screening of Vibrio isolates to develop an experimental infection model in the Pacific oyster Crassostrea gigas

In an attempt to develop a reproducible experimental model of bacterial infection in Crassostrea gigas, oysters taken from very localised sub-populations suffering natural mortality outbreaks were used in cohabitation trials under laboratory conditions. From these trials, a collection of Vibrio strains was isolated from moribund and healthy oysters. In a second step, strains were experimentally tested for virulence by means of injection into healthy oysters. This screening revealed a span of virulence among isolated strains from none to medium. When pooling injected strains, results suggest increased virulence. Vibrio strains may have additive/synergistic action leading to higher C. gigas mortality rates in experimental challenges. Although the study initially aimed to develop a simple experimental model, a complex of interactions emerged between several bacterial strains during the pathogenic process in their molluscan host. Selected strains provide a suitable model of experimental disease for further studies and better understanding of bacterial interaction and pathogenesis in C. gigas.     

Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification

Vibrio vulnificus is a leading cause of seafood-related deaths in the United States. Sequence variations in the virulence-correlated gene (vcg) have been used to distinguish between clinical and environmental V. vulnificus strains, with a strong association between clinical ones and the C sequence variant (vcgC). In this study, vcgC was selected as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, specific, and quantitative detection of potentially virulent V. vulnificus strains in raw oysters. No false-positive or false-negative results were generated among the 125 bacterial strains used to evaluate assay specificity. The detection limit was 5.4 CFU per reaction for a virulent V. vulnificus strain (ATCC 33815) in pure culture, 100-fold more sensitive than that of PCR. In spiked raw oysters, the assay was capable of detecting 2.5 × 10(3) CFU/g of V. vulnificus ATCC 33815, while showing negative results for a nonvirulent V. vulnificus strain (515-4c2) spiked at 10(7) CFU/g. After 6 h of enrichment, the LAMP assay could detect 1 CFU/g of the virulent V. vulnificus strain ATCC 33815. Standard curves generated in pure culture and spiked oysters suggested a good linear relationship between cell numbers of the virulent V. vulnificus strain and turbidity signals. In conclusion, the LAMP assay developed in this study could quantitatively detect potentially virulent V. vulnificus in raw oysters with high speed, specificity, and sensitivity, which may facilitate better control of V. vulnificus risks associated with raw oyster consumption.     

Persistence,Seasonal Dynamics and Pathogenic Potential of Vibrio Communities from Pacific Oyster Hemolymph

Bacteria of the genus Vibrio occur at a continuum from free-living to symbiotic life forms, including opportunists and pathogens, that can contribute to severe diseases, for instance summer mortality events of Pacific oysters Crassostrea gigas. While most studies focused on Vibrio isolated from moribund oysters during mortality outbreaks, investigations of the Vibrio community in healthy oysters are rare. Therefore, we characterized the persistence, diversity, seasonal dynamics, and pathogenicity of the Vibrio community isolated from healthy Pacific oysters. In a reciprocal transplant experiment we repeatedly sampled hemolymph from adult Pacific oysters to differentiate population from site-specific effects during six months of in situ incubation in the field. We characterized virulence phenotypes and genomic diversity based on multilocus sequence typing in a total of 70 Vibrio strains. Based on controlled infection experiments we could show that strains with the ability to colonize healthy adult oysters can also have the potential to induce high mortality rates on larvae. Diversity and abundance of Vibrio varied significantly over time with highest values during and after spawning season. Vibrio communities from transplanted and stationary oysters converged over time, indicating that communities were not population specific, but rather assemble from the surrounding environment forming communities, some of which can persist over longer periods.     

<Emphasis Type="Italic">Rhodococcus fascians</Emphasis>: Shoot Proliferation without Elevated Cytokinins?

In order to determine whether the disease symptoms caused by virulent strains of Rhodococcus fascians are due to increased cytokinin activity in infected tissues, germinating peas (Pisum sativum cv Novella) were inoculated with either a virulent strain or a nonvirulent strain of Rhodococcus fascians. The nonvirulent strain lacked both the ipt gene and the putative cytokinin oxidase/dehydrogenase homologue, fas5. Control peas were not inoculated. Twelve cytokinins were isolated from pea shoots 3, 6 and 9 days post-inoculation. Within 6 days of inoculation the levels of cytokinin free bases, ribosides, O-glucosides and nucleotides were decreased in shoots inoculated with the virulent strain, and were increased in shoots inoculated with the nonvirulent strain relative to the uninoculated control. The results are discussed with respect to the classic Skoog and Miller (1965) model of organogenesis and to the possible involvement of the plant cytokinin oxidase/dehydrogenase during infection by virulent strains of R. fascians.     

Changes in antigenic properties of lipopolysaccharides of Shigella sonnei phase I having lost the virulence due to transposon integration into the invasiveness plasmid PSS120

A nonvirulent strain of Shigella sonnei phase I has been obtained by integration of the transposon Tn5 into the invasiveness plasmid pSS120 in the virulent strain and designated NR18. The presence of the plasmid pSS120 in both strains results in the similar morphology and bacterial ability to agglutinate in the presence of antiserum to Shigella sonnei phase I antigen. The lipopolysaccharide preparations from the virulent and nonvirulent strains give the similar reactions with the antiserum in the reaction of hemagglutination. However, in the reaction of passive local hemolysis in the gel (Jerne reaction) the significant difference is revealed in the immunogenicity of the preparations, with the preparations from the virulent strain being 4-5 fold more immunogenic. In crossreaction, the antibodies secreted by the mouse spleen cells immunized by LPS from the virulent strain show a weak reaction with the ram erythrocytes sensitized by the LPS of the nonvirulent strain. Thus, the biological changes in the LPS of the nonvirulent strains that are, evidently, the consequence of the structural changes, are identified only by the most sensitive immunological techniques.     

Ecology and genetic structure of a northern temperate Vibrio cholerae population related to toxigenic isolates

Although Vibrio cholerae is an important human pathogen, little is known about its populations in regions where the organism is endemic but where cholera disease is rare. A total of 31 independent isolates confirmed as V. cholerae were collected from water, sediment, and oysters in 2008 and 2009 from the Great Bay Estuary (GBE) in New Hampshire, a location where the organism has never been detected. Environmental analyses suggested that abundance correlates most strongly with rainfall events, as determined from data averaged over several days prior to collection. Phenotyping, genotyping, and multilocus sequence analysis (MLSA) revealed a highly diverse endemic population, with clones recurring in both years. Certain isolates were closely related to toxigenic O1 strains, yet no virulence genes were detected. Multiple statistical tests revealed evidence of recombination among strains that contributed to allelic diversity equally as mutation. This relatively isolated population discovered on the northern limit of detection for V. cholerae can serve as a model of natural population dynamics that augments predictive models for disease emergence.     

Response of life‐history traits to artificial and natural selection for virulence and nonvirulence in a Drosophila parastitoid,Asobara tabida

Co‐evolution of host–parasitoid interactions is determined by the costs of host resistance, which received empirical evidence, and the costs of parasitoid virulence, which have been mostly hypothesized. Asobara tabida is a parasitoid, which mainly parasitizes Drosophila melanogaster and D. subobscura, the first species being able to resist to the parasitoid development while the second species is not. To parasitize resistant hosts, including D. melanogaster, A. tabida develops sticky eggs, which prevent encapsulation, but this virulence mechanism may be costly. Interindividual and interpopulation variation in the proportion of sticky eggs respectively allowed us to (i) artificially select and compare life‐history traits of a virulent and a nonvirulent laboratory strain, and (ii) compare a virulent and a nonvirulent field strain, to investigate the hypothetical costs of virulence. We observed strong differences between the 2 laboratory strains. The nonvirulent strain invested fewer resources in reproduction and walked less than the virulent one but lived longer. Concerning the field strains, we observed that the nonvirulent strain had larger wings while the virulent one walked more and faster. All together, our results suggest that virulence may not always be costly, but rather that different life histories associated with different levels of virulence may coexist at both intra‐ and interpopulation levels.     

History and epizootiology of Haplosporidium nelsoni (MSX), an oyster pathogen in Delaware Bay, 1957–1980

Between 1957 and 1959, a previously unknown sporozoan parasite, now designated as Haplosporidium nelsoni (formerly Minchinia nelsoni), or MSX, killed 90–95% of the oysters in lower Delaware Bay. Native oysters have been studied for more than 20 years since then to determine long-term disease and mortality patterns resulting from this host-parasite association. Development of resistance to MSX-kill in native oysters has reduced disease mortality to about half the original level, even though the pathogen continues to be very active in the bay. Since the initial epizootic, MSX levels have fluctuated in a cyclic pattern with peaks every 6 to 8 years. Periods of low disease pressure follow very cold winters, while average or above average winter temperatures correlate with high MSX activity. During peak years, every oyster in the lower bay may become infected. Although the parasite is salinity limited, salinities in the lower bay, the area from which oysters are marketed, are nearly always favorable for MSX, and fluctuations in river flow have almost no effect on MSX in this region. Infection periods recur each summer. Some oysters die soon after becoming infected; others survive through winter, but die in spring as the pathogen compounds normal overwinter stresses. Many survivors are able to suppress or rid themselves of infections when temperatures approach 20°C in late spring. Resistance to MSX-kill in native oysters is not correlated with an ability to prevent infection, but with restriction of parasites to localized, nonlethal lesions. The persistence of “hot spots” for infection in areas where oysters are sparse, the lack of spores in infected oysters, and failure to transmit the disease experimentally lead to the hypothesis that an alternate or reservoir host produces infective stages of MSX.     

Hemolymph microbiome of Pacific oysters in response to temperature,temperature stress and infection

Microbiota provide their hosts with a range of beneficial services, including defense from external pathogens. However, host-associated microbial communities themselves can act as a source of opportunistic pathogens depending on the environment. Marine poikilotherms and their microbiota are strongly influenced by temperature, but experimental studies exploring how temperature affects the interactions between both parties are rare. To assess the effects of temperature, temperature stress and infection on diversity, composition and dynamics of the hemolymph microbiota of Pacific oysters (Crassostrea gigas), we conducted an experiment in a fully-crossed, three-factorial design, in which the temperature acclimated oysters (8 or 22 °C) were exposed to temperature stress and to experimental challenge with a virulent Vibrio sp. strain. We monitored oyster survival and repeatedly collected hemolymph of dead and alive animals to determine the microbiome composition by 16s rRNA gene amplicon pyrosequencing. We found that the microbial dynamics and composition of communities in healthy animals (including infection survivors) were significantly affected by temperature and temperature stress, but not by infection. The response was mediated by changes in the incidence and abundance of operational taxonomic units (OTUs) and accompanied by little change at higher taxonomic levels, indicating dynamic stability of the hemolymph microbiome. Dead and moribund oysters, on the contrary, displayed signs of community structure disruption, characterized by very low diversity and proliferation of few OTUs. We can therefore link short-term responses of host-associated microbial communities to abiotic and biotic factors and assess the potential feedback between microbiota dynamics and host survival during disease.     

Identification of genes involved in the host response to neurovirulent alphavirus infection

Single-amino-acid mutations in Sindbis virus proteins can convert clinically silent encephalitis into uniformly lethal disease. However, little is known about the host gene response during avirulent and virulent central nervous system (CNS) infections. To identify candidate host genes that modulate alphavirus neurovirulence, we utilized GeneChip Expression analysis to compare CNS gene expression in mice infected with two strains of Sindbis virus that differ by one amino acid in the E2 envelope glycoprotein. Infection with Sindbis virus, dsTE12H (E2-55 HIS), resulted in 100% mortality in 10-day-old mice, whereas no disease was observed in mice infected with dsTE12Q (E2-55 GLN). dsTE12H, compared with dsTE12Q, replicated to higher titers in mouse brain and induced more CNS apoptosis. Infection with the neurovirulent dsTE12H strain was associated with both a greater number of host genes with increased expression and greater changes in levels of host gene expression than was infection with the nonvirulent dsTE12Q strain. In particular, dsTE12H infection resulted in greater increases in the levels of mRNAs encoding chemokines, proteins involved in antigen presentation and protein degradation, complement proteins, interferon-regulated proteins, and mitochondrial proteins. At least some of these increases may be beneficial for the host, as evidenced by the demonstration that enforced expression of the antiapoptotic mitochondrial protein peripheral benzodiazepine receptor (PBR) protects neonatal mice against lethal Sindbis virus infection. Thus, our findings identify specific host genes that may play a role in the host protective or pathologic response to neurovirulent Sindbis virus infection.     

Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli

A multidimensional chromatographic method has been applied for the differential analysis of proteins from different strains of Escherichia coli bacteria. Proteins are separated in the first dimension using chromatofocusing (CF) and further separated by nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second dimension. A 2-dimensional (2-D) expression map of bacterial protein content is created for virulent O157:H7 and nonvirulent E. coli strains depicting protein isoelectric point (pI) versus protein hydrophobicity. Differentially expressed proteins are further characterized using electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) determination and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. Using this method, no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 strains could be distinguished from nonvirulent E. coli. Several proteins differentially expressed between O157:H7 and nonvirulent E. coli are identified as potential markers for detection and treatment of O157:H7 infection.     

Mortalities of Eastern and Pacific Oyster Larvae Caused by the Pathogens Vibrio coralliilyticus and Vibrio tubiashii

Vibrio tubiashii is reported to be a bacterial pathogen of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) and has been associated with major hatchery crashes, causing shortages in seed oysters for commercial shellfish producers. Another bacterium, Vibrio coralliilyticus, a well-known coral pathogen, has recently been shown to elicit mortality in fish and shellfish. Several strains of V. coralliilyticus, such as ATCC 19105 and Pacific isolates RE22 and RE98, were misidentified as V. tubiashii until recently. We compared the mortalities caused by two V. tubiashii and four V. coralliilyticus strains in Eastern and Pacific oyster larvae. The 50% lethal dose (LD₅₀) of V. coralliilyticus in Eastern oysters (defined here as the dose required to kill 50% of the population in 6 days) ranged from 1.1 × 10⁴ to 3.0 × 10⁴ CFU/ml seawater; strains RE98 and RE22 were the most virulent. This study shows that V. coralliilyticus causes mortality in Eastern oyster larvae. Results for Pacific oysters were similar, with LD₅₀s between 1.2 × 10⁴ and 4.0 × 10⁴ CFU/ml. Vibrio tubiashii ATCC 19106 and ATCC 19109 were highly infectious toward Eastern oyster larvae but were essentially nonpathogenic toward healthy Pacific oyster larvae at dosages of ≥1.1 × 10⁴ CFU/ml. These data, coupled with the fact that several isolates originally thought to be V. tubiashii are actually V. coralliilyticus, suggest that V. coralliilyticus has been a more significant pathogen for larval bivalve shellfish than V. tubiashii, particularly on the U.S. West Coast, contributing to substantial hatchery-associated morbidity and mortality in recent years.     

Pesticides and Ostreid Herpesvirus 1 Infection in the Pacific Oyster,Crassostrea gigas

Since 2008, mass mortality outbreaks have been reported in all French regions producing Pacific oysters, and in several Member States of the European Union. These mass mortality events of Pacific oysters are related to OsHV-1 infection. They occur during spring and summer periods leaving suspect the quality of the marine environment and the role of seasonal use of pesticides associated with the arrival of freshwater in oyster rearing areas. Pesticides have been also detected in French coastal waters, especially in areas of oyster production. Using PMA real-time PCR we showed that a mixture of 14 pesticides has no effect on the integrity of virus capsids from viral suspension in the conditions tested. A contact of oysters with this pesticide mixture was related to higher mortality rates among experimentally infected animals in comparison with control ones (no previous pesticide exposure before experimental infection). We therefore suggest that pesticides at realistic concentration can exert adverse effects on Pacific oysters and causes an increased susceptibility to the viral infection in experimental conditions.     

Identification of an Entamoeba histolytica serine-, threonine-, and isoleucine-rich protein with roles in adhesion and cytotoxicity

Entamoeba histolytica is a leading cause of parasitic death globally. However, the molecular framework regulating pathogenesis is poorly understood. We have previously used expression profiling to identify Entamoeba genes whose expressions were strictly associated with virulent strains (R. C. MacFarlane and U. Singh, Infect. Immun. 74:340-351, 2006). One gene, which we have named EhSTIRP (Entamoeba histolytica serine-, threonine-, and isoleucine-rich protein), was exclusively expressed in virulent but not in nonvirulent Entamoeba strains. EhSTIRP is predicted to be a transmembrane protein and is encoded by a multigene family. In order to characterize its function in amebic biology, we used a double-stranded RNA-based approach and were able to selectively down-regulate expression of this gene family. Upon EhSTIRP down-regulation, we were able to ascribe cytotoxic and adhesive properties to the protein family using lactate dehydrogenase release and Chinese hamster ovary cell adhesion assays. EhSTIRP thus likely represents a novel determinant of virulence in Entamoeba histolytica. This work validates the fact that genes expressed exclusively in virulent strains may represent virulence determinants and highlights the need for further functional analyses of other genes with similar expression profiles.     

Prior Inoculation with Type B Strains of Francisella tularensis Provides Partial Protection against Virulent Type A Strains in Cottontail Rabbits

Francisella tularensis is a highly virulent bacterium that is capable of causing severe disease (tularemia) in a wide range of species. This organism is characterized into two distinct subspecies: tularensis (type A) and holarctica (type B) which vary in several crucial ways, with some type A strains having been found to be considerably more virulent in humans and laboratory animals. Cottontail rabbits have been widely implicated as a reservoir species for this subspecies; however, experimental inoculation in our laboratory revealed type A organisms to be highly virulent, resulting in 100% mortality following challenge with 50–100 organisms. Inoculation of cottontail rabbits with the same number of organisms from type B strains of bacteria was found to be rarely lethal and to result in a robust humoral immune response. The objective of this study was to characterize the protection afforded by a prior challenge with type B strains against a later inoculation with a type A strain in North American cottontail rabbits (Sylvilagus spp). Previous infection with a type B strain of organism was found to lengthen survival time and in some cases prevent death following inoculation with a type A2 strain of F. tularensis. In contrast, inoculation of a type A1b strain was uniformly lethal in cottontail rabbits irrespective of a prior type B inoculation. These findings provide important insight about the role cottontail rabbits may play in environmental maintenance and transmission of this organism.     

Production of IL-12 by macrophages infected with Toxoplasma gondii depends on the parasite genotype

Three clonal strain types (I, II, and III) of Toxoplasma gondii predominate worldwide. The outcome of infection in mice is highly dependent on the parasite genotype with type I strains being uniformly virulent, while types II and III are nonvirulent. Interactions with the innate immune response play a major role in determining the outcome of infection in the murine model. To identify key early differences in the innate immune response that contribute to pathogenesis, we examined the cytokine production of macrophages after in vitro infection with parasites of virulent type I and nonvirulent type II genotypes. Infection with type II strain parasites stimulated the production of proinflammatory cytokines, and particularly high levels of the Th1-polarizing cytokine, IL-12. Infection with type II strain parasites stimulated NF-kappaB nuclear translocation at early time points and led to the up-regulation of mRNA levels of IL-12 and other proinflammatory cytokines that was dependent on the myeloid differentiation factor 88 signaling pathway. Induction of IL-12 required active invasion by live parasites and was not blocked by infection with virulent type I strain parasites, arguing against an active inhibition of signaling. Our findings suggest that early induction of high levels of IL-12 by macrophages infected with type II strain parasites may contribute to more effective control.     

Analysis of Vibrio vulnificus from Market Oysters and Septicemia Cases for Virulence Markers

Representative encapsulated strains of Vibrio vulnificus from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all V. vulnificus strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical V. vulnificus isolates demonstrates the need for alternative subtyping approaches and virulence assays.     

Cryptococcus Strains with Different Pathogenic Potentials Have Diverse Protein Secretomes

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.