Differences in virulence between two serotypes of Ichthyophthirius multifiliis

Naive channel catfish Ictalurus punctatus were infected by 2 isolates of the parasitic ciliate Ichthyophthirius multifiliis that differed in virulence. The isolates, NY1 and G5, Serotypes A and D, respectively, express different surface immobilization-antigens. The virulence of the 2 isolates was compared using tail-fin infections to quantitate parasite numbers and by analysis of the survival of infected fish. Although NY1 infected fish at a lower level than G5, all NY1-infected fish died, but 51% of G5-infected fish survived. The greater virulence of NY1 is apparently a consequence of its shorter life cycle, which results in overwhelming reinfection of fish before they can develop a protective immune response. This report represents the first experimental evidence for differences in virulence between serotypes of I. multifiliis.     

Virulence and nucleotide sequence analysis of marine viral haemorrhagic septicaemia virus following in vivo passage in rainbow trout Onchorhynchus mykiss

A marine isolate of viral haemorrhagic septicaemia virus (VHSV) (860/94) was passaged in triplicate through sequential batches of rainbow trout via an intra-peritoneal infection route, without amplification in tissue culture. Following 5 passages, the VHSV glycoprotein gene was amplified directly from fish tissue homogenates and the consensus sequence compared to that of the original tissue culture isolate. Virus was also recovered directly from pools of kidney and spleen material after 5 passage events, and its virulence compared to that of unpassaged material by intra-peritoneal infection. Following passage in rainbow trout, isolate 860/94 exhibited a higher virulence for rainbow trout than unpassaged material. Sequence comparisons identified no difference in the consensus sequence of the glycoprotein gene following in vivo passage. The mechanisms responsible for the observed increase in virulence of isolate 860/94 following passage in rainbow trout thus remain unknown. The possibility that viral isolates may exhibit an increased virulence following passage in novel host species does, however, have important implications with regard to the epidemiology of this important fish pathogen.     

Iron uptake mechanisms as key virulence factors in bacterial fish pathogens

This review summarizes the current knowledge about iron uptake systems in bacterial fish pathogens and their involvement in the infective process. Like most animal pathogens, fish pathogens have evolved sophisticated iron uptake mechanisms some of which are key virulence factors for colonization of the host. Among these systems, siderophore production and heme uptake systems are the best studied in fish pathogenic bacteria. Siderophores like anguibactin or piscibactin, have been described in Vibrio and Photobacterium pathogens as key virulence factors to cause disease in fish. In many other bacterial fish pathogens production of siderophores was demonstrated but the compounds were not yet chemically characterized and their role in virulence was not determined. The role of heme uptake in virulence was not yet clearly elucidated in fish pathogens although there exist evidence that these systems are expressed in fish tissues during infection. The relationship of other systems, like Fe(II) transporters or the use of citrate as iron carrier, with virulence is also unclear. Future trends of research on all these iron uptake mechanisms in bacterial fish pathogens are also discussed.     

Mortality and kidney histopathology of chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains

An isolate of Renibacterium salmoninarum (strain MT 239) exhibiting reduced virulence in rainbow trout Oncorhynchus mykiss was tested for its ability to cause bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha, a salmonid species more susceptible to BKD. Juvenile chinook salmon were exposed to either 33209, the American Type Culture Collection type strain of R. salmoninarum, or to MT 239, by an intraperitoneal injection of 1 x 10(3) or 1 x 10(6) bacteria fish(-1), or by a 24 h immersion in 1 x 10(5) or 1 x 10(7) bacteria ml(-1). For 22 wk fish were held in 12 degrees C water and monitored for mortality. Fish were sampled periodically for histological examination of kidney tissues. In contrast to fish exposed to the high dose of strain 33209 by either injection or immersion, none of the fish exposed to strain MT 239 by either route exhibited gross clinical signs or histopathological changes indicative of BKD. However, the MT 239 strain was detected by the direct fluorescent antibody technique in 4 fish that died up to 11 wk after the injection challenge and in 5 fish that died up to 20 wk after the immersion challenge. Viable MT 239 was isolated in culture from 3 fish that died up to 13 wk after the immersion challenge. Total mortality in groups injected with the high dose of strain MT 239 (12%) was also significantly lower (p < 0.05) than mortality in groups injected with strain 33209 (73 %). These data indicate that the attenuated virulence observed with MT 239 in rainbow trout also occurs in a salmonid species highly susceptible to BKD. The reasons for the attenuated virulence of MT 239 were not determined but may be related to the reduced levels of the putative virulence protein p57 associated with this strain.     

Expression of common fluorescent reporters may modulate virulence for Mycobacterium marinum: dramatic attenuation results from Gfp over-expression

Mycobacterium marinum is an established surrogate pathogen for Mycobacterium tuberculosis because of its strong conservation of thousands of orthologous genes, lower risk to researchers and similar pathology in fish. This pathogen causes TB-like chronic disease in a wide variety of fish species. As in human TB, the microbe grows within the host macrophages, can mount life-long chronic infections and produces granulomatous lesions in target organs. One of the fish species known to manifest chronic "fish TB" is the small laboratory fish, Japanese ricefish (medaka; Oryzias latipes). Our laboratory is currently characterizing the disease progression in medaka using fluorescent reporter systems that are introduced into engineered strains of M. marinum. While conducting these studies we observed differences in growth, plasmid stability, and virulence depending on which fluorescent reporter construct was present. Here, we describe large negative effects on virulence and organ colonization that occurred with a commonly used plasmid pG13, that expresses green fluorescent protein (Gfp). The studies presented here, indicate that Gfp over-expression was the basis for the reduced virulence in this reporter construct. We also show that these negative effects could be reversed by significantly reducing Gfp expression levels or by using low-expression constructs of Rfp.     

Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh

Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans. In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates. The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of A. veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.     

The Influence of Infective Dose on the Virulence of a Generalist Pathogen in Rainbow Trout (Oncorhynchus mykiss) and Zebra Fish (Danio rerio)

Pathogen density and genetic diversity fluctuate in the outside-host environment during and between epidemics, affecting disease emergence and the severity and probability of infections. Although the importance of these factors for pathogen virulence and infection probability has been acknowledged, their interactive effects are not well understood. We studied how an infective dose in an environmentally transmitted opportunistic fish pathogen, Flavobacterium columnare, affects its virulence both in rainbow trout, which are frequently infected at fish farms, and in zebra fish, a host that is not naturally infected by F. columnare. We used previously isolated strains of confirmed high and low virulence in a single infection and in a co-infection. Infection success (measured as host morbidity) correlated positively with dose when the hosts were exposed to the high-virulence strain, but no response for the dose increase was found when the hosts were exposed to the low-virulence strain. Interestingly, the co-infection resulted in poorer infection success than the single infection with the high-virulence strain. The rainbow trout were more susceptible to the infection than the zebra fish but, in both species, the effects of the doses and the strains were qualitatively similar. We suggest that as an increase in dose can lead to increased host morbidity, both the interstrain interactions and differences in infectivity in different hosts may influence the severity and consequently the evolution of disease. Our results also confirm that the zebra fish is a good laboratory model to study F. columnare infection.     

Identification and Characterization of Pathogenic Aeromonas veronii Biovar Sobria Associated with Epizootic Ulcerative Syndrome in Fish in Bangladesh

Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans. In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates. The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of A. veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.     

Functional Characterization of <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> Twin-Arginine Translocation System: Its Roles in Biofilm Formation,Extracellular Protease Activity,and Virulence Towards Fish

The bacterial twin-arginine translocation (Tat) system contributes to translocate folded proteins and plays pleiotropic roles in growth, motility, and the secretion of some virulent factors. In this study, the authors identified the Tat gene cluster in fish pathogen Vibrio alginolyticus and explored its roles in pathogenesis toward fish. Vibrio alginolyticus Tat mutants showed growth deficiency in TMAO medium, while the complement strain restored the ability to grow in the medium, demonstrating the conservative function of the Tat system in translocation of redox enzymes or cofactors in this bacterium. In V. alginolyticus, deletion of the tatABC genes led to a drastic decrease in biofilm biogenesis. Interestingly, the secretion of extracellular protease Asp, an established exotoxin of the bacterium, was significantly decreased in the TatC mutant, suggesting that TatC might play a part in the production of virulence factors in the bacterium. Furthermore, the Tat mutants displayed attenuated virulence toward the fish model and EPC cells. These findings suggest that the Tat secretion related to the extracellular protease activity as well as virulence in V. alginolyticus provided new insights into the pathogenesis of vibriosis in fish.     

Streptococcus iniae capsule impairs phagocytic clearance and contributes to virulence in fish

Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient DeltacpsD strains. Compared to wild-type S. iniae, the DeltacpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The DeltacpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae DeltacpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.     

荧光假单胞菌TonB依赖型外膜受体P698的研究

目的:鱼类病原菌荧光假单胞菌是一种革兰氏阴性菌,在水产上可以引起多种经济鱼类的疾病,作为一种鱼类病原菌目前对其致病机理还知之甚少。本研究从鱼类病原菌荧光假单胞菌TSS中克隆得到了一个Ton B依赖型的外膜受体(命名为P698),分析了其与细菌致病性的关系,研究了P698作为亚单位疫苗的免疫保护效应。方法:通过序列分析、荧光定量PCR、基因敲除、体外蛋白重组等方法研究P698的蛋白结构、表达情况以及其与细菌毒力的联系和免疫原性。结果:研究发现P698具有Ton B依赖型外膜受体家族的典型结构。与正常培养条件相比,在缺铁条件下培养的TSS中p698基因的表达没有明显变化。通过插入失活获得p698缺失的突变株TSSP,生长分析发现跟野生株相比,突变株TSSP的生长能力有明显降低。在以大菱鲆为模型的活体侵染实验中发现,与野生株相比,突变株的体内侵染复制能力都有明显降低。为检测P698的免疫保护性,我们对P698进行了体外重组表达,获得了重组蛋白。用重组P698蛋白作亚单位疫苗免疫大菱鲆,在免疫后一个月的鱼体检测到了特异抗体的存在,并且受免疫鱼对于致死剂量的TSS攻毒表现出了显著的保护效应。结论:我们的研究结果表明P698与细菌的侵染有一定相关性,并且作为亚单位疫具有一定的免疫保护效应。     

Intensive aquaculture selects for increased virulence and interference competition in bacteria

Although increased disease severity driven by intensive farming practices is problematic in food production, the role of evolutionary change in disease is not well understood in these environments. Experiments on parasite evolution are traditionally conducted using laboratory models, often unrelated to economically important systems. We compared how the virulence, growth and competitive ability of a globally important fish pathogen, Flavobacterium columnare, change under intensive aquaculture. We characterized bacterial isolates from disease outbreaks at fish farms during 2003–2010, and compared F. columnare populations in inlet water and outlet water of a fish farm during the 2010 outbreak. Our data suggest that the farming environment may select for bacterial strains that have high virulence at both long and short time scales, and it seems that these strains have also evolved increased ability for interference competition. Our results are consistent with the suggestion that selection pressures at fish farms can cause rapid changes in pathogen populations, which are likely to have long-lasting evolutionary effects on pathogen virulence. A better understanding of these evolutionary effects will be vital in prevention and control of disease outbreaks to secure food production.     

Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum

The role of the flagellum and motility in the virulence of the marine fish pathogen Vibrio anguillarum was examined. Non-motile mutants were generated by transposon mutagenesis. Infectivity studies revealed that disruption of the flagellum and subsequent loss of motility correlated with an approximate 500-fold decrease in virulence when fish were inoculated by immersion in bacteria-containing water. However, the flagellar filament and motility were not required for pathogenicity following intraperitoneal injection of fish. The transposon-insertion site for six mutants was determined by cloning and sequencing of the Vibrio DNA flanking the transposon. V. anguillarum genes whose products showed strong homology to proteins with an established role in flagellum biosynthesis were identified. One of the aflagellate mutants had a transposon insertion in the rpoN gene of V. anguillarum . This rpoN mutant failed to grow at low concentrations of available iron and was avirulent by both the immersion and intraperitoneal modes of inoculation. A chemotaxis gene, cheR , was located upstream of one transposon insertion and an in-frame deletion was constructed in the coding region of this gene. The resulting non-chemotactic mutant exhibited wild-type pathogenicity when injected intraperitoneally into fish but showed a decrease in virulence similar to that seen for the non-motile aflagellate mutants following immersion infection. Hence, chemotactic motility is a required function of the flagellum for the virulence of V. anguillarum     

Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids,outer membrane protein profiles and siderophore production

The virulence of 18 strains of Vibrio anguillarum serogroup O1 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only strains that carried the 67 kbp virulence plasmid or derivatives of it produced the outer membrane protein, OM2. All virulent strains harboured the 67 kbp plasmid or derivatives of it, indicating its importance for virulence. However, some strains carrying the virulence plasmid or a derivative of it, produced siderophores as well as OM2 but were non-pathogenic to fish. Likewise, among the virulent strains, considerable variation in LD50 values was recorded. Plasmid profiling and restriction analysis showed that the virulence plasmid existed in various molecular weights from 26 to 80 kbp, with 65-67 kbp being the most common, and that this plasmid displayed various restriction profiles. The presence of other plasmids did not seem to affect the pathogenic properties.     

Development of a gene transfer system for curing of plasmids in the marine fish pathogen Vibrio salmonicida.

All reported natural isolates of the marine fish pathogen Vibrio salmonicida contain plasmids, and in another marine fish pathogen, Vibrio anguillarum, it has been shown that a plasmid is important for expression of virulence by the organism. To study the function of the plasmids in V. salmonicida, we developed a gene transfer system based on the plasmid RSF1010 replicon. The gene transfer system was used to construct a plasmid-free strain, and this strain was found to behave similarly to the wild type in a fish pathogenicity test based on intraperitoneal injection of the bacteria. We were unable to detect any other phenotypic differences between the two strains. It could therefore be concluded that at least in the V. salmonicida strain tested, extrachromosomal DNA is not required for expression of virulence.     

Association of Type III secretion genes with virulence of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida possesses a number of potential virulence factors, including a recently identified plasmid-encoded Type III secretion system. A number of field isolates of A. salmonicida subsp. salmonicida were examined for the presence of Type III secretion genes. Using in vitro experiments, it was found that field isolates containing such genes are cytotoxic to fish cell lines, whereas those that lack these genes are not. Using a rainbow trout in vivo model, the virulence of a wild type A. salmonicida subsp. salmonicida strain (Strain JF2267), which possesses Type III secretion genes, was compared to that of a laboratory derivative of the same strain that has lost these genes. While Strain JF2267 was virulent towards rainbow trout, its derivative was not. The A. salmonicida subsp. salmonicida Type Strain ATCC 33658T, which also lacks Type III secretion genes, was also found to be avirulent by this challenge model. The findings from both the in vitro and in vivo experiments suggest that the presence of Type III secretion genes is associated with the virulence of this important fish pathogen.     

Development of a gene transfer system for curing of plasmids in the marine fish pathogen Vibrio salmonicida.

All reported natural isolates of the marine fish pathogen Vibrio salmonicida contain plasmids, and in another marine fish pathogen, Vibrio anguillarum, it has been shown that a plasmid is important for expression of virulence by the organism. To study the function of the plasmids in V. salmonicida, we developed a gene transfer system based on the plasmid RSF1010 replicon. The gene transfer system was used to construct a plasmid-free strain, and this strain was found to behave similarly to the wild type in a fish pathogenicity test based on intraperitoneal injection of the bacteria. We were unable to detect any other phenotypic differences between the two strains. It could therefore be concluded that at least in the V. salmonicida strain tested, extrachromosomal DNA is not required for expression of virulence.     

Iron uptake by Pasteurella piscicida and its role in pathogenicity for fish.

We evaluated the iron uptake mechanisms in Pasteurella piscicida strains as well as the effect of iron overload on the virulence of these strains for fish. With this aim, the capacity of the strains to obtain iron from transferrin and heme compounds as well as their ability to overcome the inhibitory activity of fish serum was analyzed. All the P. piscicida strains grew in the presence of the iron chelator ethylene-diamine-di (O-hydroxyphenyl acetic acid) or of human transferrin, which was used by a siderophore-mediated mechanism. The chemical tests and cross-feeding assays showed that P. piscicida produced a siderophore which was neither a phenolate nor a hydroxamate. Cross-feeding assays as well as preliminary chromatographic analysis suggest that this siderophore may be chemically related to multocidin. All the P. piscicida isolates utilized hemin and hemoglobin as an iron source, since the virulence of the strains increased when the fish were preinoculated with these compounds. This effect was stronger in the avirulent strains (50% lethal dose was reduced by 4 logs when fish were pretreated with hemin or hemoglobin). Only the pathogenic P. piscicida isolates were resistant to the bactericidal action of the fresh fish serum. The nonpathogenic strains grew in fish serum only when it was heat-inactivated or when it was supplemented with ferric ammonium citrate, hemin, or hemoglobin. In all the strains, at least three iron-regulated outer membrane proteins (IROMPs) (105, 118, and 145 kDa) were increased when the strains were cultured in iron-restricted medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence, characterisation and detection of potential virulence determinants of emerging aquatic bacterial pathogens from the Philippines and Thailand

Strains of Aeromonas spp., 'non-cholera vibrios' (NCVs) and Plesiomonas shigelloides isolated from aquatic environments, fish and human diarrhoeal cases in the Philippines and Thailand were characterised for potential virulence markers. Thus, the production of cytotoxin, cell-associated and cell-free haemolysin and their capacity to adhere to human intestinal (Henle 407) cells in vitro was investigated. In addition, the occurrence of tlh and tdh haemolysin genes and urease activity among V. parahaemolyticus strains was investigated. The results showed that strains recovered from clinical sources (human and fish) produced these virulence factors, whereas these are absent in environmental strains.