Study of Vibrio anguillarum strains from different sources with emphasis on ecological and pathobiological properties

A total of 317 Vibrio anguillarum strains were isolated from water, sediment, and diseased as well as healthy rainbow trout at a Danish mariculture farm and from feral fish caught close to the farm. All strains were examined serologically. Ten sera permitted determination of the O group in 66.7% of the strains from diseased rainbow trout. Furthermore, the O group could be determined in 45.1 to 65.4% of the strains from mucus, gills, and intestinal contents of healthy rainbow trout, while only 22.2 to 28.8% of the isolates from water, sediment, and gills or mucus of feral fish were groupable. Serogroup O1 and to some extent O2 appeared to be associated with trout. Strains from these serogroups were selected for analyses of hemagglutinating activity and surface hydrophobicity. Serogroup O1 comprised hemagglutinating as well as nonhemagglutinating strains; from cases of vibriosis, all O1 strains were nonhemagglutinating. The strains belonging to serogroup O2 were generally hemagglutinating. Examinations of surface hydrophobicity by salt aggregation and hydrophobic interaction chromatography suggested that the O1 strains were more hydrophobic than the O2 strains. In pathogenicity tests, O1 strains isolated from gills and mucus of healthy rainbow trout killed all trout in the test groups. A strain from the intestinal contents of healthy rainbow trout did not produce significant mortality. This strain could, however, be frequently reisolated from the pronephros of fish in the test group concerned. After challenge with strains from eel mucus and seawater, mortality was not produced, and furthermore, these strains could not be reisolated from the pronephros.     

Relationships between plasmids and phenotypes of presumptive strains of Vibrio anguillarum isolated from different fish species

On the basis of plasmid composition as well as serological and biochemical properties, 26 strains identified as Vibrio anguillarum isolated from diseased fish could be assigned to two different groups. Except for three reference strains, these++ strains were isolated from Norwegian fish. The four strains isolated from rainbow trout (Salmo gairdneri), the only strain isolated from char (Salvelinus alpinus), and three of six strains isolated from Atlantic salmon (Salmo salar) harbored a plasmid of 47 megadaltons (MDa). Restriction endonuclease analysis showed that this plasmid and the virulence plasmid pJM1, carried by V. anguillarum strain 775, were very similar but not identical. Strains harboring the 47-MDa plasmid had nearly identical biochemical properties and were serotype O1. Strains isolated from reared coastal cod (Gadus morhua), turbot (Scophthalmus maximus), halibut (Hippoglossus hippoglossus), free-living saithe (Pollachius virens), and partly from reared Atlantic salmon differed from strains harboring the 47-MDa virulence plasmid by not containing this plasmid, by having different biochemical traits, and by being serotype O2. Rainbow trout which were experimentally infected with a strain isolated from cod suffering from vibriosis developed clinical symptoms similar to those in cod but quite different from those usually seen in rainbow trout.     

Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins.

We investigated the incidence of plasmid-mediated and chromosome-mediated iron uptake systems in strains of Vibrio anguillarum that belong to serotypes O1 and O2 and were isolated from different fish species and in different geographic areas. All of the strains gave positive reactions in CAS agar medium and in the Arnow test, which indicated that catechol types of siderophores were produced. The majority of V. anguillarum serotype O1 strains harbored a 65-kb plasmid similar to plasmid pJM1 from strain 775, which encodes the siderophore anguibactin and its outer membrane receptor, protein OM2. All of the isolates harboring this plasmid promoted the growth of an anguibactin-deficient receptor-proficient mutant derived from strain 775, but none of these isolates promoted the growth of mutants lacking receptor OM2. Furthermore, under iron-limiting conditions all of these strains induced outer membrane proteins that were identical in size to protein OM2 of strain 775. In contrast, none of the serotype O2 strains contained a high-molecular-weight plasmid, but all of them induced the growth of mutants defective in the anguibactin-mediated system regardless of the presence or absence of receptor OM2. The serotype O2 strains, but not the plasmid-bearing serotype O1 strains, also induced the growth of Salmonella typhimurium enb-1 which utilizes only enterobactin as a siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival of Vibrio anguillarum and Vibrio salmonicida at different salinities

The fish pathogenic bacteria Vibrio anguillarum and V. salmonicida showed the capacity to survive for more than 50 and 14 months, respectively, in seawater microcosms. A salinity of 5% proved lethal to V. anguillarum harvested in the late-exponential growth phase, whereas a salinity of 9% was lethal to the bacterium after it had been starved at a salinity of 30% for 67 days. The lethal salinity for V. salmonicida harvested in the late-exponential growth phase was probably in the vicinity of 10%. V. anguillarum and V. salmonicida were very sensitive to nalidixic acid. Direct determination of viable cells after incubation with nalidixic acid was not possible, since the cells did not elongate. Samples of V. salmonicida were double stained with fluorescein isothiocyanate-labeled antibodies and 4',6-diamidino-2-phenylindole. After 3 or 4 days of starvation, there was a discrepancy between the total numbers of cells as determined by immunofluorescence versus by staining with 4',6-diamidino-2-phenylindole. The immunofluorescence counts remained high, which indicated the presence of intact cell envelopes but leakage of DNA and other cytoplasm components. After 2 weeks of starvation, for some of the cells, the region stained with 4',6-diamidino-2-phenylindole (i.e., DNA) was markedly smaller than the cell envelope. I attributed this to a shrinkage of the cytoplasm or a confined nucleoid or both. V. anguillarum lost its exoproteolytic activity before 11 days of starvation.     

Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum.

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguillarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguillarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasmid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.     

Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain

Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross-reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components.     

Differences in virulence genes in Vibrio cholerae eltor strains isolated from different sources in Turkmenistan territory

Polymerase chain reaction (PCR) detected the presence of various genes associated with virulence in genome of strains V. cholerae eltor isolated in Turkmenistan territory during epidemic and epidemic-free perios. It was found that a complete set of virulence genes (ctxA+, tcpA+ and toxR+) contained strains isolated from patients, carriers and environment only in cholera epidemics. Strains isolated from the environment in the period free of epidemics did not contain ctxA and tcpA in 78.2% of cases, but 5.2% of the strains carried a complete set of virulence genes. There were also nontoxigenic strains containing genes tcpA and toxR. Such strains were isolated from the environment (16.6%) and vibrion carriers (42.9%). Isolated were also strains V.cholerae eltor carrying bacteriophage CTX phi with incomplete set of virulence genes and having genotype ctxA-, ace+ and zot+. Almost all the strains ctxA-, tcpA+ carry attRS1-site in genome. This shows that such strains may transform into toxigenic as a result of infection with bacteriophage CTX phi.     

Purification and some properties of a chloramphenicol acetyltransferase mediated by plasmids from Vibrio anguillarum

Vibrio anguillarum strains were isolated from chloramphenicol-resistant bacteria in diseased fish. Plasmid Rms418, which confers chloramphenicol resistance, was transferred from V. anguillarum GN11379 to Escherichia coli K12 by conjugation. The Rms418-encoded chloramphenicol acetyltransferase (CAT) [EC 2.3.1.99] was isolated and purified to homogeneity using affinity chromatography on immobilized p-amino-chloramphenicol or ATP. The general CAT could be adsorbed by a matrix with a chloramphenicol base ligand (Zaidenzaig, Y. & Shaw, W.V. (1976) FEBS Lett. 62,266-271), but the Rms418-encoded CAT was not bound under these conditions. The specific activity of the enzyme, when measured by the spectrophotometric assay, was 71.4 units/mg protein at 37 degrees C. The molecular weight of the enzyme treated with SDS and 2-mercaptoethanol was shown to be approximately 22,000. The molecular weight of the native enzyme, as determined by gel filtration, was approximately 69,000, and the optimal pH was 7.8. The Km values for chloramphenicol and CoASAc were 34.5 and 150 microM, respectively. Enzyme activity was inhibited by HgCl2, p-chloromercuribenzoate (p-CMB), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and ethylendiaminotetraacetic acid (EDTA). The half life at 53 degrees C was approximately 100 min.     

Monoclonal antibodies for a comparative serological study of strains of Vibrio anguillarum

Murine monoclonal antibodies against characteristic determinants of heat stable somatic antigens of typical serovar I (820/15/8 Kop) and serovar II (134/82/1 Kiel) of European Vibrio anguillarum strains were produced. Three stable hybridoma cell lines, called aVaI/1F4, aVaI/6F4 and aVaI/2H5, produced monoclonal antibodies each against a typical serovar I determinant of strain 820/15/8 Kop. One hybridoma cell line (aVaII/5A4) producing monoclonal antibodies against Vibrio anguillarum strain 134/82/1 Kiel (serovar. II) was established. Fourteen Vibrio strains and Aeromonas salmoniáda strains were serologically compared by Enzyme-Linked Immunosorbent Assay (Elisa ).     

Chemical properties of lipopolysaccharide (LPS) isolated from Vibrio anguillarum PT514

Vibrio anguillarum, one of the causative agents of fish vibriosis, is serologically and biochemically divided into three groups (A, B and C). The chemical composition and molecular architecture of lipopolysaccharide (LPS) isolated from V. anguillarum PT 514, which belongs to serogroup B, were investigated. The LPS contained glucose (Glc), fructose (Fru), L-glycero-D-mannoheptose (L-D Hep), glucosamine (GlcN) and 4-amino-4,6-dideoxyglucose as sugar constituents in molar ratios of 8.9:0.7:3.0:1.1:1.6. Sephadex G-50 gel-chromatography of a degraded polysaccharide fraction separated from the LPS by 5% acetic acid hydrolysis suggested that the O-specific polysaccharide region consists of, in average, as much as 29 moles of Glc per 3 moles of L-D Hep, while the core polysaccharide contains at least Glc, L-D Hep and GlcN in molar ratios of 3.2 : 3.0 : 0.2. Fru and 4-amino-4,6-dideoxyglucose components were released from LPS on weak-acid hydrolysis, indicating that PT 514 LPS is distinguishable from those of Vibrio anguillarum belonging to the other serogroups. 2-Keto-3-deoxyoctonate (KDO), a common sugar constituent of gram-negative bacterial LPS, was not detected by Weissbach's color reaction under the conventional hydrolysis condition, but O-phosphoryl KDO was found in the strong-acid hydrolysate (4 M HCl, 100 C, 45 min). This substance was identical, at least in high-voltage paper electrophoresis, to 5-O-phosphoryl KDO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clonality of Vibrio anguillarum strains isolated from fish from the Scandinavian countries, Sweden, Finland and Denmark

In order to investigate whether outbreaks of vibriosis in the Baltic region were caused by the spread of certain pathogenic clones, 291 Vibrio anguillarum isolates from Finland (n = 156), Sweden (n = 88) and Denmark (n = 47) were studied with respect to serogroup, ribotype, plasmid content, and biochemical phenotypes as expressed with the PhenePlate (PhP) typing system. For comparison, 54 V. anguillarum serogroup O1 from other countries worldwide were included. Most isolates from Finland, Sweden and Denmark belonged to serogroup O1 (255), followed by O2 (30). Four Finnish isolates cross-reacted strongly with antisera against two new serogroups VaNT2 and VaNT4, whereas two strains were non-typeable. The serogroup O1 isolates displayed ten different ribotype patterns, whereas the other strains were considerably more diverse with respect to ribotypes. Most of the O1 isolates carried the 67 kb virulence plasmid and a group of Finnish isolates, in addition, carried an 86 kb plasmid. Additional plasmids with molecular weights of 63, 76, 135 or 260-290 kb were found in single O1 isolates. With few exceptions, strains of serogroup O2 either had no plasmids or carried one or two small plasmids. PhenePlate typing revealed considerable diversity within the species, serogroup O1 being the most homogeneous. A few PhP types were dominant, whereas other types were observed only in one to four isolates. The prevalence of the different types changed significantly from one year to another but in Finland, one clonal lineage became increasingly important from 1992 (20% of isolates) to 1996 (80%). Remaining clones were mostly restricted to specific geographic areas. By cluster analysis, it was demonstrated that most of the isolates from Finland, Sweden and Denmark belonged to two clusters, and most of the strains from Southern Europe fell into two other, distinct clusters. Most isolates from the UK, North America, Chile and Tasmania grouped together in a distinct cluster. For the typing of V. anguillarum, O-serotyping should be the primary method. For isolates belonging to serogroups other than O1, plasmid profiling in combination with ribotyping gives a very good discrimination between strains, whereas for serogroup O1, another method is required. It is concluded that PhP typing is a tool that provides a good discrimination between O1 isolates.     

Analysis of 16S–23S rRNA gene internal transcribed spacer of Vibrio anguillarum and Vibrio ordalii strains isolated from fish

The basis of the bactericidal action of antibiotics and the mechanisms of antibiotic tolerance are largely unknown. To elucidate one of the mechanisms of antibiotic tolerance, the present study investigated the role of Pseudomonas aeruginosa quorum sensing (QS) and the rpoS gene in antibiotic tolerance. The survival rates of the lasR and lasI mutants were observed to be lower than that of the parental strain in time-dependent killing studies with 8 μg mL⁻¹ ofloxacin, but the survival rates of the rhlR and rhlI mutants were not different from that of the parental strain. Moreover, a lasR -overexpressing strain was more tolerant to ofloxacin than the parental strain, but this was not the case for an rhlR -overexpressing strain. The mRNA expression levels of lasR , lasI , and rpoS in the wild-type strain in the presence of bactericidal concentration of ofloxacin were lower than that in the absence of ofloxacin. In addition, the significant loss of antibiotic tolerance in the lasR mutant was recovered by the overexpression of rpoS . These results suggest that the Las QS system in P. aeruginosa is involved in the development of ofloxacin tolerance, and the tolerance induced by the Las-system is regulated by rpoS gene.     

The epidemiological importance of Vibrio cholerae isolated from different ecological systems

The analysis of the data on the isolation of V. cholerae from different ecological systems indicates that V. eltor do not constantly inhibit the rivers and sea at the territory under control. Hemolytically active V. cholerae without the vct gene, found to be faintly virulent and avirulent when studied on suckling rabbits used as a model and when evaluated by the complex method, show no tendency towards epidemic spread in the presence of conditions for the realization of the transmission of vibrios by the water route.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum.

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Note: Molecular cloning of chitinase genes from Vibrio anguillarum and V. parahaemolyticus

Chitinase genes from Vibrio anguillarum KV9001 and V. parahaemolyticus ATCC17802 were cloned into Escherichia coli. Open reading frames of chitinase genes from V. anguillarum (vac) and V. parahaemolyticus (vpc) are 1755 bp and 1890 bp, respectively. The deduced amino acid sequences of these genes have 71·6% identity. There are two consensus sequence regions in the VAC and VPC proteins. The vac gene was highly prevalent in V. anguillarum , and the DNA probe of the vac gene hybridized to V. alginolyticus and Beneckea proteolytica DNA. The DNA probe of the vpc gene hybridized to V. alginolyticus, V. harveyi and V. ordalii DNA.     

Cloning and nucleotide sequence analysis of a chloramphenicol acetyltransferase gene from Vibrio anguillarum.

The chloramphenicol resistant gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a transferable R plasmid (pJA7324) isolated from the fish pathogen Vibrio anguillarum strain PT24 was cloned into the plasmid vector pUC19. The nucleotide sequence analysis of 1,348 base pair DNA identified an open reading frame encoding a protein of 216 amino acid residues with a calculated molecular mass of 25,471 daltons. The predicted amino acid sequences for this cat gene are 37-69% homologous with other CAT proteins of both Gram-negative and -positive bacteria. Colony hybridization performed with a PvuII-BamHI fragment including this cat gene as a probe, revealed that the same or similar chloramphenicol resistance genes existed among V. anguillarum isolates.     

Vibrio anguillarum: Prevalence of typical and atypical strains in marine recipients with special reference to carbohydrate pollution

Primary isolates of Vibrio anguillarum-like organisms could be separated into typical V. anguillarum (VA) and atypical V. anguillarum (AVA) by biochemical tests. The prevalence of the fish pathogenic V. anguillarum was highly influenced by carbohydrate pollution as compared to the AVA. Water an sediment counts of VA generally increased at the polluted sites during April-May and persisted at a level of approx. 100/ml water and 1,000/g sediment until October-November. A further increase in VA counts could be registered locally at the time when the sugar beet processing season started (September-October). At the control site VA counts increased during June-July to a level of 10/ml persisting until August, while the only increase in sediment counts occurred in September (100/g). The maximum counts in water and sediment were at the control site 10/ml and 100/g and the polluted sites 100,000/ml and 50,000/g, respectively.

     

鳗弧菌毒力质粒DNA序列的测定

采用亚克隆法与引物步移法相结合的测序战略 ,对海洋鱼类重要病原菌鳗弧菌毒力质粒pEIB1进行序列测定 ,测得整个质粒序列长度为 6 6 16 4bp。序列的初步分析结果表明 ,G C含量为 4 2 .7% ,共有 4 4个可读框 (ORF) ,其中包括与铁载体合成、调节、运输以及质粒复制相关的基因。