Agent Of Brown Ring Disease Research Articles

Implication of the Type IV Secretion System in the Pathogenicity of Vibrio tapetis, the Etiological Agent of Brown Ring Disease Affecting the Manila Clam Ruditapes philippinarum.

Vibrio tapetis is a Gram-negative bacterium that causes infections of mollusk bivalves and fish. The Brown Ring Disease (BRD) is an infection caused by V. tapetis that primarily affects the Manila clam Ruditapes philippinarum. Recent studies have shown that a type IV secretion system (T4SS) gene cluster is exclusively found in strains of V. tapetis pathogenic to clams. However, whether the T4SS is implicated or not during the infection process remains unknown. The aim of this study was to create and characterize a V. tapetis T4SS null mutant, obtained by a near-complete deletion of the virB4 gene, in order to determine the role of T4SS in the development of BRD. This study demonstrated that the T4SS is neither responsible for the loss of hemocyte adhesion capacities, nor for the decrease of the lysosomal activity during BRD. Nevertheless, we observed a 50% decrease of the BRD prevalence and a decrease of mortality dynamics with the ΔvirB4 mutant. This work demonstrates that the T4SS of V. tapetis plays an important role in the development of BRD in the Manila clam.

MALDI-TOF MS as a promising tool to assess potential virulence of Vibrio tapetis isolates

Vibrio tapetis, the etiological agent of Brown Ring Disease, mainly affects the Manila clam Ruditapes philippinarum. Although this bacterium is mainly known as a clam pathogen, it has been isolated from several fish species. The main aim of the present study was to further explore the variability of 27 V. tapetis isolates from bivalves and fish, considering three different aspects; in vitro virulence based on the loss of clam hemocyte adhesion properties, detection of the gene virB4 encoding for an essential component of the Type IV Secretion System, and MALDI-TOF MS characterization based on whole cell extracts. Finally, these approaches were compared and evaluated for their ability to differentiate the potential pathogenicity of the 27 isolates against the Manila clams. Among the 11 V. tapetis isolates from the common dab isolated in 2018 in Belgium, only one (2BB) showed intermediate in vitro virulence against the Manila clam, and seven carried the virB4 gene while none of the V. tapetis previously isolated from fish in 2003 showed the presence of this particular gene. Finally, the peak protein profiles generated with MALDI-TOF MS analysis from all 27 V. tapetis strains showed a clear clustering of clam pathogenic and nonpathogenic isolates suggesting that a new isolate of V. tapetis that would cluster within the clam pathogenic isolates could be potentially pathogenic to the Manila clam. Thereupon, MALDI-TOF MS typing allows rapid and cost-efficient identification of V. tapetis isolates and can be defined as a complementary method of the traditional qPCR that opens new perspectives to study the virulence of V. tapetis isolates but also to perform environmental monitoring in order to prevent outbreaks.

External pH modulation during the growth of Vibrio tapetis, the aetiological agent of brown ring disease.

Brown ring disease (BRD) is an infection of the Manila clam Ruditapes philippinarum due to the pathogen Vibrio tapetis. During BRD, clams are facing immunodepression and shell biomineralization alteration. In this paper, we studied the role of pH on the growth of the pathogen and formulated hypothesis on the establishment of BRD by V. tapetis. In this study, we monitored the evolution of pH during the growth of V. tapetis in a range of pH and temperatures. We also measured the pH of Manila clam haemolymph and extrapallial fluids (EPFs) during infection by V. tapetis. We highlighted that V. tapetis modulates the external pH during its growth, to a value of 7·70. During the development of BRD, V. tapetis also influences EPFs and haemolymph pH in vitro in the first hours of exposure and in vivo after 3days of infection. Our experiments have shown a close interaction between V. tapetis CECT4600, a pathogen of Manila clam that induces BRD, and the pH of different compartments of the animals during infection. These results indicate that the bacterium, through a direct mechanism or as a consequence of physiological changes encountered in the animal during infection, is able to interfere with the pH of Manila clam fluids. This pH modification might promote the infection process or at least create an imbalance within the animal that would favour its persistence. This last hypothesis should be tested in future experiment. This study is the first observation of pH modifications in the context of BRD and might orient future research on the fine mechanisms of pH modulation associated with BRD.

Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams.

The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD) in the Manila clam Venerupis (=Ruditapes) philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600T V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 101 bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.

Vibrio tapetis, the Causative Agent of Brown Ring Disease, Forms Biofilms with Spherical Components.

Vibrio tapetis is a marine bacterium causing Brown Ring Disease (BRD) in the Manila clam Ruditapes philippinarum. V. tapetis biofilm formation remains unexplored depite the fact that it might be linked to pathogenicity. Our objectives were to characterize the in vitro biofilm formation of V. tapetis and evaluate the effects of culture conditions. Biofilm structure and its matrix composition were examined by confocal laser scanning microscopy and scanning electron microscopy. V. tapetis was able to form biofilms on a glass substratum within 24 h. Polysaccharides and extracellular DNA of the biofilm matrixes were differently distributed depending on the V. tapetis strains. Spherical components of about 1–2 μm diameter were found at the biofilm surface. They contain DNA, proteins, and seemed to be physically linked to bacteria and of cellular nature. Transmission electron microscopy showed that the spherical components were devoid of internal compartments. Temperatures >21°C inhibit BRD whereas low salinity (2%) favor it, none of the both conditions altered V. tapetis' ability to form biofilms in vitro. We suggest therefore that biofilm formation could play a role in the persistence of the pathogen in clam than in BRD symptoms.

Immune responses of phenoloxidase and superoxide dismutase in the manila clam Venerupis philippinarum challenged with Vibrio tapetis – Part II: Combined effect of temperature and two V. tapetis strains

Manila clams, Venerupis philippinarum (Adams and Reeve, 1850), were experimentally infected with two different bacterial strains and challenged with two different temperatures. Bacterial strains used in this study were Vibrio tapetis strain CECT4600T, the causative agent of Brown Ring Disease (BRD) and V. tapetis strain LP2, supposed less virulent to V. philippinarum. V. tapetis is considered to proliferate at low temperatures, i.e. under 21 °C. In a global warming context we could hypothesize a decrease of mass mortalities caused by V. tapetis but these thermal changes could also directly impact the immune system of the host V. philippinarum. Thus, the aim of this study was to investigate the effects of the extrapallial injection with V. tapetis combined with temperature challenge on two enzymes activities in V. philippinarum. More precisely, after infection, phenoloxidase (PO) and superoxide dismutase (SOD), two major enzymes involved in immune response, were studied for 30 days in two compartments: the mantle and the hemolymph. Conchyolin Deposit Stages (CDS) and Shell Repair Stages (SRS) were also determined 30 days post-injection as a proxy of the virulence of the tested strains. In this study, we highlighted that host–pathogen interaction in a varying environment affects the enzymatic response of the host. The coupled effect of V. tapetis injection and temperature challenge was detected 30 days post injection and resulted in virulence differences. These findings were supported by CDS and SRS determination in clams and lead to the conclusion that clam's immunity could be enhanced at 22 °C while V. tapetis virulence is lowered at this temperature. Another result of our study was the increase of PO and SOD basal activities as clams are exposed to warmer temperature.

Immune responses of phenoloxidase and superoxide dismutase in the manila clam Venerupis philippinarum challenged with Vibrio tapetis – Part I: Spatio-temporal evolution of enzymes' activities post-infection

Manila clams, Venerupis philippinarum (Adams and Reeve, 1850), were experimentally challenged with two Vibrio tapetis strains: CECT4600T, the causative agent of Brown Ring Disease (BRD); and LP2 supposedly non-pathogenic in V. philippinarum. Changes in phenoloxidase (PO) and superoxide dismutase (SOD), two major enzymes involved in immunity, were studied in two tissues, the mantle and hemolymph for 30 days after infection in the extrapallial cavity. Bacterial infection in V. philippinarum resulted in modulation of PO and SOD activities that was both tissue- and time-dependent. A response at early times was detected in the mantle and was associated with significant increases in PO and SOD activities in LP2- and CECT4600T-challenged clams 36 h post injection. This first response in the mantle could be explained by the proximity to the injection region (extrapallial cavity). In the hemolymph the response occurred at later times and was associated with an increase in PO activity and a decrease in SOD activity. As hemolymph is a circulating fluid, this response delay could be due to an “integration time” needed by the organism to counteract the infection. Injections also impacted PO and SOD activities in both tissues and confirmed a difference in pathogenicity between the two V. tapetis strains.

Evaluation of different culture media for the isolation and growth of the fastidious Vibrio tapetis, the causative agent of brown ring disease

Thirteen culture media were evaluated at two temperatures for the growth and isolation of Vibrio tapetis. The bacterium showed similar growth dynamics at 15°C or 25°C, being faster at 15°C regardless the general media employed. Best growth of V. tapetis was obtained on Agar Seawater (ASWT) (1.7×106cfu/ml), Mannitol Marine Agar (MMA) (2.6×106cfu/ml), and Mannitol Trypticase Soy Agar (MTSA-1) (1.9×106cfu/ml), being slightly lower on Marine Agar (MA) (5.0×105cfu/ml). Growth was poor on TCBS and nule in the other media containing bile salts, indicating their inhibitory effect on the V. tapetis growth. Recovery of V. tapetis from mixed Vibrio populations, differing in acid production from sucrose and mannitol, was only possible using the selective medium MMA at both temperatures. The use of ASWT or MA at 15°C for the routinary growth of V. tapetis, and MMA for isolation of V. tapetis from bivalve samples is recommended.

Transcriptomic analysis of Ruditapes philippinarum hemocytes reveals cytoskeleton disruption after in vitro Vibrio tapetis challenge

The Manila clam, Ruditapes philippinarum, is an economically-important, commercial shellfish; harvests are diminished in some European waters by a pathogenic bacterium, Vibrio tapetis, that causes Brown Ring disease. To identify molecular characteristics associated with susceptibility or resistance to Brown Ring disease, Suppression Subtractive Hybridization (SSH) analyzes were performed to construct cDNA libraries enriched in up- or down-regulated transcripts from clam immune cells, hemocytes, after a 3-h in vitro challenge with cultured V. tapetis. Nine hundred and ninety eight sequences from the two libraries were sequenced, and an in silico analysis identified 235 unique genes. BLAST and “Gene ontology” classification analyzes revealed that 60.4% of the Expressed Sequence Tags (ESTs) have high similarities with genes involved in various physiological functions, such as immunity, apoptosis and cytoskeleton organization; whereas, 39.6% remain unidentified. From the 235 unique genes, we selected 22 candidates based upon physiological function and redundancy in the libraries. Then, Real-Time PCR analysis identified 3 genes related to cytoskeleton organization showing significant variation in expression attributable to V. tapetis exposure. Disruption in regulation of these genes is consistent with the etiologic agent of Brown Ring disease in Manila clams.

Two-dimensional proteome reference map of Vibrio tapetis, the aetiological agent of brown ring disease in clams

Vibrio tapetis is the etiological agent of brown ring disease (BRD) in clams, one of the most threatening diseases affecting this commercially important bivalve. In this study we have constructed a proteome reference map of the V. tapetis type strain CECT 4600(T). Eighty-two proteins, consistently present in all 2D-gels, were identified by mass spectrometry or by de novo sequencing. The majority of the proteins identified (66%) belonged to four COG categories: 'Carbohydrate transport and metabolism', 'Post-translational modification, protein turnover and chaperones', 'Energy production', and 'Amino acid transport and metabolism'. Glyceraldehyde-3-phosphate dehydrogenase, enolase, fructose-bisphosphate aldolase, phosphoglycerate kinase. molecular chaperones Dnak and GroEL, alkyl hydroperoxide reductase, peptidyl-prolyl cis-trans isomerase B and factor Tu, were identified among the 20 most abundant proteins. A comparison of this reference map with that obtained for the V. tapetis strain GR0202RD, with different origin and pathophysiological characteristics, was performed. Under the culture conditions employed in this study, glucose degradation is one of the major pathways for energy production in Vibrio tapetis. In addition, the two strains studied, although with remarkable differences at genetic and pathophysiological levels, showed a high similarity under laboratory conditions. The results obtained here can be considered as a first step to gather valuable information on protein expression, related not only to diverse cellular functions and regulation but also to pathogenesis and bacterium-host interactions in the disease process.

Proteomics and multilocus sequence analysis confirm intraspecific variability of Vibrio tapetis

Vibrio tapetis is the etiological agent of brown ring disease (BRD) in clams. Phenotypic, antigenic and genetic variability have been demonstrated, with three groups being established associated with host origin. In this work we analyze the variability of representative strains of these three groups, CECT 4600(T) and GR0202RD, isolated from Manila clam and carpet-shell clam, respectively, and HH6087, isolated from halibut, on the basis of the whole proteome analysis by 2D-PAGE and multilocus sequence analysis (MLSA). A quantitative analysis of the proteome match coefficient showed a similarity of 79% between the clam isolates, whereas fish isolate showed similarities lower than 70%. A preliminary mass spectrometry (MS) assay allowed the identification of 27 proteins including 50S ribosomal protein L9, riboflavin synthase β subunit, ribose-phosphate pyrophosphokinase and succinyl-CoA synthase α subunit. The MLSA approach gave similar results, showing a 99.4% similarity of the clam isolates, which was higher than that observed between the fish isolate and either clam strain (98.2%). The topology of the maximum parsimony tree, obtained from 2D-PAGE analysis, and the phylogenetic tree, constructed with the maximum likelihood algorithm from concatenated sequences of 16S rRNA gene and five housekeeping genes (atpA, pyrH, recA, rpoA and rpoD), was very similar, confirming the closer relationship between the two clam isolates.

First Isolation of Vibrio tapetis, the Etiological Agent of Brown Ring Disease (BRD), in Manila Clam Ruditapes philippinarum in Japan

Mass mortality occurred in Manila clam Ruditapes philippinarum under a hanging cultivation condition from April to July 2008 in Japan. A bacterium was isolated from the affected clams showing brown conchiolin deposits on the inner shell surface and identified as Vibrio tapetis, the causal agent of brown ring disease (BRD) in clams, by bacteriological characterization and genetic analysis. Experimental infection of clams confirmed pathogenicity of the isolated bacterium. These results indicate that the present mortality of cultured clams was caused by BRD. This is the first report on isolation of V. tapetis from diseased R. philippinarum in Japan.

DjlA, a Membrane-Anchored DnaJ-Like Protein, Is Required for Cytotoxicity of Clam Pathogen Vibrio tapetis to Hemocytes

DjlA is an inner membrane cochaperone belonging to the DnaJ family, which has been shown to be involved in Legionella sp. pathogenesis. In this study, we explored the role of this protein in the physiology and virulence of Vibrio tapetis, the etiological agent of brown ring disease (BRD) in Manila clam (Ruditapes philippinarum). Analysis of the djlA locus in V. tapetis revealed a putative organization in an operon with a downstream gene that we designated duf924(Vt), which encodes a conserved protein with an unknown function and has homologues in bacteria and eukaryotes. djlA mutants displayed a reduced growth rate and showed an important loss of cytotoxic activity against R. philippinarum hemocytes in vitro, which could be restored by extrachromosomal expression of wild-type djlA(Vt) but not duf924(Vt). These results are in keeping with the potential importance of DjlA for bacterial pathogenicity and open new perspectives for understanding the mechanism of action of this protein in the novel V. tapetis-R. philippinarum interaction model.

COMPARISON OF TECHNIQUES FOR DIAGNOSIS OF BROWN RING DISEASE AND DETECTION OF PHILIPPINARUM

Abstract The bacterium, Vibrio tapetis, is the aetiological agent of Brown Ring Disease (BRD), which affects the Manila clam, Venerupis (Ruditapes) philippinarum. Two PCR assays for detection of V. tapetis were applied to a sample of 52 Manila clams, and evaluated in comparison with the more traditional V. tapetis detection method of microbiological isolation and characterization, as well as the traditional BRD diagnostic technique of shell valve analysis. The pathogen was detected in 15.4% of the sample using the PCR assay of Rodriguez et al. (2003, 2006) in 50% of the sample using the PCR assay of Paillard et al. (2006) and in 36.5% of the sample by microbiological methods. Whereas shell valve analysis was the least sensitive technique, detecting BRD in 7.7% of the sample, it was an essential diagnostic tool because it was the only technique that identified the disease, rather than the aetiological agent. None of the four techniques was sufficient on its own for effective BRD diagnosis; rather various c...

Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species: II. Cellular and biochemical changes following in vivo challenge

This work compared the effect of challenge with Vibrio tapetis, the etiologic agent of brown ring disease (BRD) in clams, and other bacterial strains on defence-related factors in four bivalve species: Ruditapes philippinarum (highly susceptible to BRD), R. decussatus (slightly susceptible to BRD), Mercenaria mercenaria and Crassostrea virginica (both non-susceptible to BRD). Results show that bacterial challenge modulated defence-related factors, namely total and differential haemocyte counts, percentage of viable haemocytes, and lysozyme activity, both in haemolymph and extrapallial fluid. Injection with bacteria induced a response that was dependent upon the bacterial and bivalve species investigated, and upon the site of inoculation: external (pallial cavity), pseudo-internal (extrapallial space), or internal compartment (adductor muscle). The most conspicuous changes were systematically measured in R. philippinarum injected with V. tapetis, indicating a bacterial pathogenicity particular to the host in which it causes a specific disease syndrome. Alterations of defence-related factors were maximal in haemolymph of clams injected with V. tapetis in the muscle, and in the extrapallial fluid when the bacteria were injected into the pallial or the extrapallial cavity. Resistance to the development of the BRD symptom was not related to the extent of the haemocyte reaction measured following in vivo challenge.

Pathogenicity of Vibrio tapetis, the etiological agent of brown ring disease in clams.

Brown ring disease (BRD) causes high mortalities in the introduced Manila clam Ruditapes philippinarum cultured in western Europe. The etiological agent of BRD, Vibrio tapetis, adheres to and disrupts the production of the periostracal lamina, causing the anomalous deposition of periostracum around the inner shell. Because the primary sign of BRD is found outside the soft tissues, the processes leading to death are not as obvious as those for internal pathogens. This study was designed to evaluate the pathogenicity of V. tapetis, in an attempt to help explain the mechanisms of mortality. We found high mortalities (up to 100%) for clams following the inoculation of V. tapetis into the extrapallial space (between mantle and inner shell) or the posterior adductor muscle of healthy R. philippinarum. Microscopy and immunological detection methods showed that the pathogen was rapidly eliminated from tissues and hemolymph of animals that survived the inoculation. In clams that died, the bacteria were found to have proliferated, resulting in severe tissue disruption. Bacteria were able to penetrate into tissues from the extrapallial space through the external epithelium of the mantle. In contrast, no mortalities were observed following injection of V. tapetis in the native European clam Ruditapes decussatus, which is resistant to BRD. This clam rapidly eliminated the bacterium from hemolymph and soft tissues. Clam mortality associated with BRD in the field is likely to result from the penetration of V. tapetis into the clam's extrapallial space through the disrupted periostracal lamina and from there into the soft tissues through the irritated mantle epithelium. Some bacteria also penetrate through the digestive epithelia. In either case, bacteria proliferate rapidly in the soft tissues, causing severe damage and subsequent death.

Phagocytic and chemotactic responses of manila and carpet shell clam haemocytes against Vibrio tapetis, the causative agent of brown ring disease

Phagocytic and chemotactic activities of haemocytes from two clam species, Tapes decussatus andT. philippinarum have been studied using bacterial challenge. Three strains of Vibrio tapetis, the causative agent of the brown ring disease affecting these clam species, and one strain of Escherichia coli were used in the study. All extracellular products of the bacterial strains were demonstrated to be chemoattractants to clam haemocytes, however, significant differences (P<0·025) in migration indices of haemocytes were obtained between the E. coli and V. tapetis strains. Phagocytosis by T. decussatus and T. philippinarum haemocytes is apparently a non-specific process, although affected by the viability of the microorganisms. Soluble haemolymph proteins are not required for identification of non-self or in the internalisation process.

Intraspecific characterization of Vibrio tapetis strains by use of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling.

A total of twenty-two strains of Vibrio tapetis, the causative agent of brown ring disease affecting cultured clams, were compared and evaluated in an investigation of strain heterogeneity using pulsed-field gel electrophoresis (PFGE), ribotyping, and plasmid profile analysis. A total of 90.9% of V. tapetis strains tested by using NotI showed the same PFGE pattern, consisting of 15 bands. In contrast, the V. tapetis strains showed a low degree of similarity with six reference Vibrio species tested. All V. tapetis strains harbored a large plasmid of 74.5 kb. This plasmid was not detected in any of the other Vibrio species. In addition, endonuclease restriction analysis of the plasmid content of the strains using EcoRI and HindIII clearly showed that all the strains of V. tapetis possessed the same cleavage pattern. The three enzymes used for ribotyping, PvuII, SmaI, and SalI, yielded patterns with 8 to 12 bands ranging in size from 2 to 23 kb. The application of the SalI and SmaI endonuclease rendered the separation of the strains tested in two ribotypes, while all the V. tapetis strains belonged to the same ribotype when the enzyme PvuII was used.

Determination of the etiological agent of brown ring disease in southwestern Spain

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 29:181-188 (1997) - doi:10.3354/dao029181 Determination of the etiological agent of brown ring disease in southwestern Spain Castro D, Santamaría JA, Luque A, Martínez-Manzanares E, Borrego JJ Since 1989 the presence of organic brown deposits on the inner surface of the shells of cultured manila clams Tapes philippinarum associated with episodes of high mortality has been recorded on the southern Atlantic coast of Spain. The gross signs observed in diseased clams were identical to those previously described in France as brown ring disease (BRD), caused by strains of Vibrio tapetis sp. nov. This bacterial pathogen has been isolated in several French clam production areas, and recently in BRD-affected clams cultured in Galicia (NW Spain) and Portugal, using bacteriological procedures. However, this methodology has failed to detect the pathogen in microbiological studies carried out in southwestern Spain. In the present study, an immunodetection test, based on the indirect immunofluorescence (IIF) technique, was developed, using polyclonal antisera to V. tapetis whole cells and against outer membrane (OM). This method provided evidence for the presence of the bacterium in all the BRD-affected clams analysed. V. tapetis was also detected by the same method in healthy (i.e. asymptomatic) clams, which indicates its opportunistic character. Brown ring disease · Vibrio tapetis · Immunodetection · Microbiological analyses Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 29, No. 3. Publication date: June 19, 1997 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1997 Inter-Research.

Antigenic characterization of the etiological agent of the brown ring disease affecting manila clams

Summary Vibrio P1 is considered as the causative agent of Brown Ring Disease, an epizootic disease of cultured clams in Europe. Although its pathogenic implication has been clearly established, its taxonomical inclusion and virulence mechanisms are not still completely elucidated. In the present work, we have studied the outer membrane protein (OMP) and lipopolysaccharide (LPS) patterns and other antigenic characteristics of Vibrio P1 strains, and we have compared to those both reference strains of Vibrio and Vibrio strains isolated from diseased clams, by means of SDS-PAGE, immunoblotting, Western blot, slide agglutination and dot-blot techniques. The present results strongly suggest that Vibrio P1 constitutes a homogeneous group on the basis of its antigenic characteristics. Although this microorganism shares common epitopes with several Vibrio species, antigenic heterogeneity with regard to both LPS-structure and OMP-pattern is presented.