Comparative Serology of the Marine Fish Pathogen Vibrio anguillarum

The different serotyping systems, based on thermostable O antigens, reported for Vibrio anguillarum and V. ordalii were compared by quantitative agglutination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequent silver staining or Western blotting (immunoblotting) of purified lipopolysaccharide (LPS), using polyclonal rabbit antisera. The results demonstrate that 16 different serotypes within V. anguillarum (designated O1 to O16) can be distinguished. Each of these serotypes is characterized by a distinct polysaccharide banding pattern, as revealed by silver-stained gels of purified LPS. The comparative analysis allowed a complete alignment of the different serotypes for the first three serovars: O1, O2, and O3. Moreover, immunoblotting showed that strains belonging to each of these serotypes had the same LPS banding pattern independent of the origin of the strain. Serotype O2 contains different subtypes, O2a and O2b. While no differences were apparent between these subgroups in silver-stained gels, they could be separated by quantitative agglutination (titer determination) or immunoblotting. V. ordalii, the former biotype II of V. anguillarum, strongly reacts with anti-V. anguillarum O2a antiserum. Strains of the two species can be separated on the basis of different LPS profiles in the high-molecular-weight region of silver-stained gels of purified LPS. The silver-stained LPS profiles of the different serotypes of V. anguillarum that have been established are provided for further comparison in the future.     

Serotyping of Vibrio anguillarum

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.     

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.     

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 ).     

Characterization of ferric-anguibactin transport in Vibrio anguillarum

The fish pathogen Vibrio anguillarum is the causative agent of a fatal hemorrhagic septicemia in salmonid fish. Many serotype O1 strains harbors a 65 Kbp plasmid (pJM1 encoding an iron sequestering system essential for virulence. The genes involved in the biosynthesis of the indigenous siderophore anguibactin are encoded by both the pJM1 plasmid and the chromosome, while those involved in the transport of the ferric-siderophore complex, including the outer membrane receptor, are plasmid-encoded. This work describes the role of specific amino acid residues of the outer membrane receptor FatA in the mechanism of transport of ferric-anguibactin. FatA modeling indicated that this protein has a 22 stranded ß-barrel blocked by the plug domain, the latter being formed by residues 51–54. Deletion of the plug domain resulted in a receptor unable to act as an open channel for the transport of the ferric anguibactin complex.     

Comparative Proteomics of two Vibrio anguillarum Serotype O1 Strains with Different Virulence Phenotypes

Vibrio anguillarum is part of the natural flora in the aquatic habitat, but under certain circumstances it can cause terminal hemorrhagic septicemia in marine fish due to the action of virulence-associated proteins. In our study, V. anguillarum MN and 3010 were identified as serotype O1 by AFLP analysis, and the virulence of V. anguillarum MN was shown 50-fold higher than that of the strain 3101 by LD(50) tests with Japanese flounder Paralichthys olivaceus. Nine spots were noted as differentially expressed proteins by comparing the cellular and extracellular protein profiles of V. anguillarum MN and 3101. Mass spectrometry results showed OmpU and PrtV were highly expressed in the virulent strain MN but lowly expressed in the less virulent strain 3101. Expression level confirmed by semiquantitative RT-PCR showed that ompU and prtV were indeed highly expressed in the virulent strain MN. Together with similar amino acid sequences of both OmpU and PrtV in V. anguillarum MN and 3101, our study indicated that the expression level of OmpU and PrtV may be associated with the virulence of V. anguillarum.     

Ribotyping and plasmid profiling of Vibrio anguillarum serovar O2 and Vibrio ordalii

One hundred and twenty-nine strains of Vibrio anguillarum serovar O2 and 14 strains of Vibrio ordalii were ribotyped and examined for plasmid contents. A total of 35 different ribotypes were detected. The V. anguillarum serovar O2 strains were divided into 32 different ribotypes. The V. ordalii strains showed three different ribotypes, clearly distinct from those of the V. anguillarum strains.
Ribotypes were separated into seven clusters, of which one comprised the V. ordalii strains. Clustering of the strains indicated a genetic difference between North European and South European V. anguillarum O2 strains. Sero-subgroups O2a and O2b shared ribotypes; however, three of the clusters did not include O2a strains.
All V. ordalii strains had a plasmid of 32 kb. This plasmid was not detected in any of the V. anguillarum strains. Seventeen different plasmid profiles with 17 different sized plasmids were detected among the V. anguillarum strains. Most of the plasmids were small (< 6 kb) and found in several strains. Except for one South European strain, plasmids were detected only in the North European strains of V. anguillarum O2.     

Plasmid-mediated iron uptake and virulence in Vibrio anguillarum

The plasmid pJM1 of Vibrio anguillarum harbors genes encoding proteins that enable the bacterial cell to survive under iron limiting conditions. A subset of these proteins are involved in the biosynthesis of the siderophore anguibactin and in the internalization of the ferric-siderophore into the cell cytosol. We have identified several genes encoding non-ribosomal peptide synthetases that catalyze the synthesis of anguibactin, these genes are: angB/G, angM, angN, angR, and angT. In addition, the genes fatA, fatB, fatC, and fatD are involved in the transport of ferric-anguibactin complexes. These transport genes, together with the biosynthesis genes angR and angT, are included in the iron transport biosynthesis operon (ITBO). Both the biosynthesis and the transport genes are under tight positive as well as negative control. We have identified four regulators; two of them, a chromosomally encoded Fur and a plasmid-mediated antisense RNA, RNAbeta, act in a negative fashion, while positive regulation is facilitated by AngR and TAFr. We also have evidence that the siderophore itself plays a positive role in the regulatory mechanism of the expression of both transport and biosynthesis genes.     

环丙沙星与新氟康杀灭鳗弧菌及其影响因素研究

对环丙沙星与新氟康杀灭鳗弧菌及其影响因素进行了研究,结果表明：环丙沙星（Ciproflozacin）和新氟康（Florfenicol）杀灭鳗弧菌的最高稀释度分别为10^-5和10^-4。在20℃～50℃的范围里,环丙沙星与新氟康杀灭鳗弧菌的效果随着温度升高而下降;杀菌最佳pH值分别为3和5;两种药品杀灭鳗弧菌的效果均随着可溶性淀粉浓度的增加而减弱。综合各最佳因素,环丙沙星在20℃和pH3时,杀灭鳗弧菌效果提高了19．13％;新氟康在20℃和pH5时,杀灭鳗弧菌效果提高了59．47％。     

A study of Vibrio anguillarum from farmed and wild fish using principal components analysis

Principal components analysis revealed two main groups among 163 Vibrio anguillarum cultures from diseased fish from Norwegian waters. Nearly all isolates from farmed salmonids fell in group I (arabinose positive) but those from wild fish, particularly saithe Gadus virens , more commonly appeared in group II (arabinose negative).     

Rapid identification of Vibrio anguillarum by Colony hybridization

A 562 base pair fragment of DNA from a serotype A strain of Vibrio anguillarum was cloned into pUC9 and used as a hybridization probe for the rapid identification of Vibrio anguillarum by colony hybridization. The probe was tested on nine different fish pathogens, 15 Vibrio isolates, 2 organisms closely related to Vibrio, and 9 serotypes of V. anguillarum. The probe hybridized only with the DNA of V. anguillarum serotypes A and H. The sequence of the 562 nucleotides have been determined. This probe allows rapid, reliable, and specific detection of V. anguillarum in freshwater ayu, Plecoglossus altivelis.     

Expression and processing of Vibrio anguillarum zinc-metalloprotease in Escherichia coli

The extracellular zinc-metalloprotease of Vibrio anguillarum is a secreted virulence factor. It is synthesized from the empA gene as a 611-residue preproprotease and processed to the active mature protease (EmpA) with concomitant secretion via the type II secretion pathway. Active EmpA has been found only in the V. anguillarum culture supernatant and the process of the activation seems to vary depending on strains analyzed. To better understand the mechanism of EmpA export and processing, the empA gene was cloned and expressed in Escherichia coli strains. Expression of empA did not have toxic effect on bacterial growth. Rupturing E. coli TOP10 cells by heating in gel-loading buffer resulted in activation of EmpA and severe proteolysis of the samples. In contrast, the same treatment of the E. coli MC4100A strain did not lead to the general proteolysis. In this strain, EmpA was exported into the periplasm via the Sec pathway. The periplasmic EmpA was detected in two active conformations. Therefore, in E. coli processing of EmpA precursor to an active enzyme did not require secretion to the media and the help of other V. anguillarum protein. Like in V. anguillarum, heterologous expression of empA in E. coli showed strain-specific activation process.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

A medium for presumptive identification of Vibrio anguillarum.

A medium (VAM) for differentiation of Vibrio anguillarum is described. The presence of bile salts, the high pH, and the high NaCl concentration select mainly for Vibrio species. The high salinity and the ampicillin select for a fraction of Vibrio species, and sorbitol fermentation differentiates among those vibrios still able to grow. One hundred ninety-seven of 227 strains of V. anguillarum were identified with this medium. Only 3 of 66 strains of Vibrio that were not V. anguillarum or V. anguillarum-like were recognized with this medium, and any of 7 non-Vibrio strains related to fish diseases or Escherichia coli grew on the medium. It is our contention that the medium described here constitutes an efficient instrument for presumptive detection of V. anguillarum in pathological and environmental samples.     

Mutational analysis of the zinc metalloprotease EmpA of Vibrio anguillarum

The extracellular zinc metalloprotease, EmpA, is a putative virulence factor involved in pathogenicity of the fish pathogen Vibrio anguillarum. The 611-amino acid precursor of this enzyme is encoded by the empA gene. The residues His346, His350, Glu370, Glu347, His429, Tyr361 and Asp417 are highly conserved and putatively function together at the active site of the enzyme. In this study, empA was inserted into pET24d(+) and expressed in Escherichia coli strain BL21(DE3) as a 6 x His tagged protein (r-EmpA). All the conserved residues of EmpA mentioned above were individually mutated by site-directed mutagenesis and the mutants were also expressed (m-r-EmpAs). r-EmpA and m-r-EmpAs were purified, and assayed for their proteolytic activities with azocasein as the substrate and cytotoxicities on a flounder gill cell line. m-r-EmpAs that had been mutated at His346, His350, Glu370 and Glu347 almost completely lost their proteolytic activity and cytotoxicity, pointing towards the essential roles played by these residues. In contrast, those mutated at Tyr361, His429 and Asp417 still retained a partial proteolytic activity and cytotoxicity. Our results indicate that these conserved residues play important roles in enzymatic activity and that the proteolytic activity of the enzyme is involved in the pathogenesis of V. anguillarum     

Starvation survival of the fish pathogenic bacteria Vibrio anguillarum and Vibrio salmonicida in marine environments

Abstract The possible fate in sea water of the two fish pathogenic bacteria Vibrio anguillarum and Vibrio salmonicida is discussed on the basis of microcosm experiments with these and other copiotrophic bacteria. Recent articles dealing with the survival of fish pathogens are reviewed, and the reported survival capacities are discussed in relation to ecological mechanisms, such as death, and predation, leading to the removal of bacteria from the water column.     

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.