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Vibriosis outbreaks due to Vibrio ordalii occur globally, but Chilean salmon aquaculture, in particular, has suffered significant monetary losses in the last 15 years. Little is known about the virulence mechanisms employed by V. ordalii. However, most Vibrio pathogens (e.g., Vibrio anguillarum, a very close taxonomic species) present outer membrane vesicles (OMVs) that are released extracellularly and implicated in the delivery of virulence factors to host cells. This study provides the first reported evidence of the fish pathogen V. ordalii producing and releasing OMVs under normal growth conditions. Analyses were conducted with the V. ordalii strain Vo-LM-18 and the type strain ATCC 33509T . For comparative purposes, the reference strain V. anguillarum ATCC 43307 was employed. The average size for the three Vibrio strains was 0.215 ± 0.6 µm (via scanning electron microscopy) or between 0.19 and 1.8 µm (via dynamic light scattering), with each bacterium presenting a wide range. SDS-PAGE revealed similarities in OMV patterns, but neither total nor external proteins were identical. Comparing V. ordalii ATCC 33509T and Vo-LM-18, bands were most evident in the total proteins, and the greatest degree of similarity in OMV profiles was between 37 and 50 kDa. The purified OMVs demonstrated haemolytic enzyme activity, which could play a role during V. ordalii infection. These data represent an initial step towards gaining new insights into this virulence factor, of which a lot is known in other pathogenic microorganisms.

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen-2 is a commercial polyvalent bath vaccine that contains formalin-inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen-2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath-immunized, bath-boost-immunized at four weeks post-primary immunization, and intraperitoneally (i.p.) boost-immunized at eight weeks post-primary immunization. The control groups were i.p. mock-immunized with PBS. Twenty-seven weeks post-primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD50 dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post-challenge. Commercial vaccine Vibrogen-2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.

The genetic heterogeneity of the close relatives Vibrio anguillarum and Vibrio ordalii , both serious pathogens of fish causing extensive losses in aquaculture, was studied. Eight housekeeping genes, i.e., atpA , ftsZ , gapA , gyrB , mreB , rpoA , topA , and pyrH , were partially sequenced in 116 isolates from diverse fish species and geographical areas. The eight genes appear to be under purifying selection, and the genetic diversity in the total data set was estimated to be 0.767 ± 0.026. Our multilocus sequence analysis (MLSA) scheme identified several widespread clonal complexes and resolved the isolates, for the most part, according to serotype. Serotype O2b isolates from diseased cod in Norway, Ireland, and Scotland were found to be extremely homogeneous. Horizontal gene transfer appears to be fairly common within and between clonal complexes. Taken together, MLSA and in silico DNA-DNA hybridization (DDH) calculations suggest that some isolates previously characterized as V . ordalii , i.e., 12B09, FF93, FS144, and FS238, are in fact V. anguillarum isolates. The precise taxonomic situation for two isolates from Atlantic cod that display several traits consistent with V. ordalii , i.e., NVI 5286 and NVI 5918, and a single environmental strain that was previously considered to represent V. ordalii , i.e., FF167, is less clear. IMPORTANCE It is still being debated whether V. anguillarum and V . ordalii represent separate bacterial species. Our study addresses this issue and elucidates the degree of genetic variability within this group of closely related bacteria, based on a substantial number of isolates. Our results clearly illustrate the existence of different populations among putative V . ordalii isolates. On the basis of additional full-length genomic analysis, we conclude that most environmental isolates previously identified as V . ordalii lie firmly within the species V. anguillarum . While bona fide fish-pathogenic V . ordalii isolates display a very close genetic relationship with V. anguillarum , they combine a clearly divergent evolutionary pattern with clear phenotypic differences. The study also highlights the need for further characterization of fish-pathogenic isolates from the northern Atlantic region that share phenotypic characteristics with V. ordalii but are genetically closer to V. anguillarum . The retention of taxonomic distinctions between the phenotypically different groups of bacteria is of practical advantage to microbial ecologists and veterinarians.

JB-1 and GC2, which were equated with Bacillus sp. and Aeromonas sobria respectively, were recovered from the digestive tract of rainbow trout, Oncorhynchus mykiss and ghost carp, Cyprinus sp. respectively, and demonstrated effectiveness as probiotics for the control of infections caused by Aeromonas salmonicida, Lactococcus garvieae, Streptococcus iniae, Vibrio anguillarum, Vibrio ordalii and Yersinia ruckeri. When administered to rainbow trout (average weight = 12 g) for 14 days in feed dosed at 2 × 108 cells g−1 of feed, JB-1 led to a reduction in mortalities to 0–13% after challenge with a range of bacterial pathogens compared to 80–100% mortalities of the controls. Similarly, use of GC2 reduced mortalities to 0–16% following the challenge compared to 80–100% mortalities of the controls. The mode of action reflected nutrition, production of inhibitory substances and stimulation of the innate immune responses. Specifically, JB-1 and especially GC2 were positive for siderophore and chitinase production, and increased lysozyme, phagocytic and respiratory burst activity.

In this work, 51 V. anguillarum isolates (42/51 of O1 serotype, 9/51 of O2) obtained from cultivated marine fish by Avsever and Un (2015) were used. These isolates were confirmed with universal primers specific to amiB genes. They were also detected successfully at a rate of 100% with a new primer couple designed according to 16S ribosomal RNA sequence. The new primers were found to have a high sensitivity and specificity but were liable to give a cross-reaction with Vibrio ordalii. The IJA appears exclusively as a peer-reviewed on-line open-access journal at http://www.siamb.org.il/. To read papers free of charge, please register online at registration form. Sale of IJA papers is strictly forbidden.

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum has been particularly devastating in the marine culture of salmonid fish. The correlation between serotype and virulence may reflect the ability of the bacterial surface antigens to interact with the host tissues. The ferric-anguibactin receptor FatA is an 86-kDa protein that is essential for anguibactin transport. The FatA amino acid sequence is similar to other receptors involved in iron transport, e.g., FhuA and FepA of Escherichia coli, and a TonB box can be identified at its amino-terminal end. V. ordalii causes vibriosis in wild and cultured marine salmonids in the Pacific Northwest of the United States and Japan. The determination and analysis of the complete nucleotide sequence could provide information about the role of pMJ101 in the pathobiology of this fish pathogen. Juvenile salmon exposed to V. ordalii by parenteral challenge developed a systemic infection, and the bacterium was recovered from liver, kidneys, spleen, and blood immediately after the infection. However, the number of bacteria in the liver declined after 1 h and then increased 22 h after infection; bacterial numbers were high in all the organs, and 100% mortality occurred 6 days after infection. The latter observations demonstrate that artificial infection of juvenile salmon is a valid experimental model to study the mechanisms and the bacterial virulence factors involved in the pathogenesis of the infections caused by V. ordalii.

Many species of pathogenic bacteria have been reported from marine fishes in Japan : Aeromonas salmonicida (atypical), clamydia like organisms (epitheliocystis organisms), Edwardsiella tarda, Flexibacter maritimus, Lactococcus garvieae (syn. Enterococcus seriolicida), Mycobacterium sp., Nocardia seriolae (syn. N. kampachi), Photobacterium damsela subsp. piscicida (syn. Pasteurella piscicida), Pseudomonas anguilliseptica, Pseudomonas putida, Renibacterium salmoninarum (in maricultured salmonids), Staphylococcus epidermidis, Streptococcus equisimilis, Streptococcus iniae, Vibrio alginolyticus, Listonella anguillarum (syn. Vibrio anguillarum), Vibrio ichthyoenteri, Vibrio ordalii, Vibrio trachuri and some other minor or unknown species. These bacteria usually give damages to some limited fish species, however, most of them have been found to be distributed in other cultured and wild fish species which will be possible hosts or carriers of pathogens. Drug resistance, deterioration of water around farms, and spread of pathogens by transporting seedling fish are the major problems for the control of disease in Japanese mariculture.

Vibrio ordalii and Vibrio anguillarum O2 express lipopolysaccharide (LPS) O antigens containing both specific and cross-reactive epitopes. The localization of these epitopes on the O antigen is not known. We have cloned and expressed the rfb gene cluster for O-antigen synthesis from V. anguillarum O2 (rfbVaO2) in Escherichia coli. E. coli DH5 alpha containing the recombinant plasmid pAM86 expressed O antigens which reacted with polyclonal antisera to V. ordalii and to V. anguillarum O2 LPS and with monoclonal antibody (MAb) 7B4, which is specific for V. anguillarum O2 O antigens. The recombinant strains were also protected from bactericidal killing by normal fish serum. Surprisingly, the LPS expressed from the cloned rfbVaO2 genes also reacted with MAb A16, which is specific for V. ordalii O antigens. Western immunoblot analysis revealed that MAb 7B4 reacted with recombinant LPS bearing shorter O-antigen repeat units, while MAb A16 reacted with the longer O antigens. Similar results were obtained when pAM86 was transformed into E. coli CLM4, which has a deletion spanning the sbcB-rfb region, indicating that the changes in antigenic profiles of O antigens from the recombinant strains were not due to genes within the E. coli rfb cluster. These data suggest that the epitope recognized by the MAb A16 is expressed by V. anguillarum O2 strains but it is apparently not accessible to the antibody in the native O polysaccharide. Cloning of the rfbVaO2 gene cluster resulted in expression of a novel O antigen. The modification(s) which leads to the alterations in antigenic profile of these recombinant LPS remains to be determined.

Monoclonal antibodies (mAbs) that recognize distinct species-specific antigenic epitopes in O-antigens from Vibrio anguillarum O2, O2a and certain O2b strains (mAb 7B4) and from Vibrio ordalii strains (mAbs A16 and 7D11) were generated. Western immunoblot analysis using these mAbs revealed that vibrio strains grown in the presence of fresh rainbow trout blood expressed lipopolysaccharide (LPS) with longer (high molecular mass) O-antigens and extracellular capsular layers when compared to strains grown without rainbow trout blood. We also generated mAbs that react with O-antigens from V. anguillarum serotype O1 (mAbs 7B8, 7B5 and 1C3) and serotype O3 (mAbs 13A1 and 14C5) strains. These mAbs provide rapid and accurate diagnostic reagents for serological differentiation of V. ordalii from serotype O2 strains of V. anguillarum, and for serotyping of these pathogenic vibrios.

Thirty strains of V. anguillarum were tested for the production of inhibitory substances against closely-related bacteria using the deferred antagonism test. Only one strain, Vibrio anguillarum VL4355, inhibited strains of V. ordalii and this effect was blocked by the addition of iron salts to the culture medium. Siderophore production was investigated for this strain. Results from bioassays suggested that strain VL4355 produced a siderophore related to anguibactin, the plasmid-encoded phenolate siderophore produced by V. anguillarum strain 775. However, when plasmid DNA was compared for strains 775 and VL4355 the BamHI-generated restriction profiles were different, although hybridization experiments indicated some homology. Using the chrome-azurol sulphate assay to measure siderophore production, strain VL4355 yielded significantly higher values than other V. anguillarum strains. Amberlite XAD-2 was used to produce concentrated siderophore preparations from strains VL4355 and 775. Both preparations were inhibitory to the growth of strains of V. ordalii, but not V. anguillarum, as were solutions of the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The difference in sensitivity to iron-limiting conditions for V. ordalii and V. anguillarum, coupled with the inability of V. ordalii to utilize ferric-anguibactin, could reflect different mechanisms of iron uptake for these two organisms.

To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova.

We analysed the taxonomic position of the genus Listonella based on phylogenetic, genomic and phenotypic data. The species of the genus Listonella were nested within the genus Vibrio according to the 16S rRNA gene sequence-based phylogenetic tree. The closest neighbour of Vibrio (Listonella) anguillarum strains LMG 4437T and ATCC 68554 ( = strain 775) was Vibrio ordalii LMG 13544T, with more than 99.5 % 16S rRNA gene sequence similarity. Furthermore, Vibrio (Listonella) pelagius is highly related to Vibrio splendidus. According to average amino acid identity (AAI), multilocus sequence analysis (MLSA) and Karlin genome signature, the closest neighbour of L. anguillarum ATCC 68554 is V. ordalii LMG 13544T, with 95 % AAI, 98 % MLSA and 5 in Karlin. V. anguillarum ATCC 68554 and Vibrio cholerae N16961 had 77 % similarity in AAI, 85 % in MLSA and 14 in the Karlin signature. Phenotypic analyses of previously published data for V. (L.) anguillarum and V. (L.) pelagius revealed that the genus Listonella is extremely similar to the genus Vibrio. V. ordalii and L. anguillarum strains yielded up to 67 % DNA–DNA hybridization. There are only a few phenotypic features that might be used to discriminate these two species: L. anguillarum is positive for the Voges–Proskauer reaction, citrate utilization, starch hydrolysis, lipase activity and acid production from glycerol, sorbitol and trehalose, whereas V. ordalii is negative for these traits. We suggest that the genus Listonella is a later heterotypic synonym of the genus Vibrio and propose to use the names Vibrio anguillarum and Vibrio pelagius in place of Listonella anguillarum and Listonella pelagia, respectively.

The efficacy of the disinfectant Duplalim®, a synergistic blend of superquats and glutaraldehyde, was analysedin vitroagainst 4 fish pathogens. All concentrations tested (1:200 to 1:400 dilutions) were efficacious on killingVibrio ordaliiandVibrio anguillarumin seawater after 30 s, being the level of reduction equal to 1.8 x 106CFU/ml. Higher concentration of Duplalim®(1:50 dilutions) and time of exposure (at least 5 min) is needed to kill completelyFrancisellasp, an intracellular freshwater pathogen. When Infectious Pancreatic Necrosis Virus (IPNV) was treated with 1:400 disinfectant dilution, this concentration had a significant effect after 2 minutes, regardless of the IPNV titres employed (concentration greater than 107.6TCID50/ml). Duplalim®was tested in large scale againstVibrionaceaemembers. In comparison to the controls (without the disinfectant), 1:400 dilutions of Duplalim®totally killedV. ordaliiandV. anguillarumin seawater as well as on the surface of the fishing net (used in the cages of cultured salmon) after 15 min. Cultivable bacteria remained constant in the buckets without the disinfectant (1.4 ± 0.3 × 106CFU/ml), regardless of the period sampled. In the case of the adherence on the fishing net, bacteria not exposed to the disinfectant were detected at a concentration of 7.6 ± 3.2 × 105CFU/ml. These data indicate that the use of Duplalim®againstV. ordalii,V. anguillarumand IPNV is effective in low concentration and short time of exposure (15 min at a concentration of 1:400 dilutions), while the intracellular pathogen requires higher concentration. &nbsp, En el presente trabajo se evaluó la eficaciain vitrodel desinfectante Duplalim®, una combinación sinérgica de glutaraldehído y sales de amonio cuaternario de cuarta generación, contra 4 patógenos de peces prevalentes de la salmonicultura chilena. Los resultados muestran que todas las concentraciones ensayadas (diluciones entre 1:200 a 1:400) fueron eficaces sobre los aislados deVibrio ordaliiyVibrio anguillarumpost-30 s de exposición, detectando niveles de reducción igual a 1.8 x 106UFC/ml. Concentraciones superiores de Duplalim®(dilución 1:50) y un tiempo de exposición no menor a 5 min. Fueron necesarios para eliminar completamente al patógeno intracelularFrancisellasp. Cuando el desinfectante fue ensayado contra el Virus de la necrosis Pancreática infecciosa (IPNV), se detectó que la dilución 1:400 tiene un efecto significativo después de 2 minutos sin importar los títulos de IPNV testeados (mayor concentración evaluada 107.6TCID50/ml). Duplalim®se evaluó en condiciones masivas contra los miembros de la familiaVibrionaceae. En comparación a los controles (sin adición desinfectante), la dilución 1:400 de Duplalim®eliminó completamenteV. ordaliiyV. anguillarumdespués de 15 minutos de tratamiento, tanto en el agua de cultivo como en la superficie de mallas usadas en el cultivo del salmón. Así, el análisis microbiológico del agua de los controles mostró concentraciones de 1.4 ± 0.3 × 106UFC/ml, mientras en el caso de las mallas 7.6 ± 3.2 × 105UFC/ml1. En resumen, los antecedentes obtenidos indican que el uso del desinfectante Duplalim®es efectivo contraV. ordalii,V. anguillarumy IPNV en bajas concentraciones y cortos periodos de exposición (dilución 1:400 por 15 min.), mientras que para el patógeno intracelular se requiere una concentración mayor. &nbsp

Polyclonal rabbit antisera raised against Vibrio ordalii and serotype 02 strains of Vibrio anguillarum showed extensive cross-reactivity with lipopolysaccharide from these bacterial pathogens of fish when tested in western immunoblot analysis. Results with absorbed polyclonal antisera indicated that lipopolysaccharide molecules from these strains had both common and strain-specific antigenic determinants, which allowed the antisera to be used to differentiate between V. ordalii and serotype 02 strains of V. anguillarum. Unlike rabbits, the immune response in rainbow trout to serotype 02 common antigenic epitopes was dependent on the source of the immunizing lipopolysaccharide antigens. Serum from fish immunized with V. ordalii antigens reacted more extensively with serotype 02 common antigens. In contrast, fish anti-V. anguillarum 02 serum did not interact with O antigens from the V. ordalii strains. Lipopolysaccharide from V. anguillarum serotype 02 and 02a strains showed identical antibody binding properties when interacted with rabbit or fish antiserum to either V. anguillarum 02 or V. ordalii. Lipopolysaccharide from V. anguillarum 02b strains did not interact with the tested rabbit or fish polyclonal sera. The results from this study suggest that fish and rabbits recognise different antigenic determinants in lipopolysaccharide from V. ordalii and serotpye 02 V. anguillarum strains; that V. ordalii and serotype 02 strains of V. anguillarum should be regarded as distinct serotype 02 subgroups based on the strain-specific antigenic determinants; and finally that the serological classification of V. anguillarum serotype 02b strains should be reexamined.Key words: antigenic heterogeneity, lipopolysaccharides, fish immune response.not available

Vaccination of fish for the prevention of specific bacterial diseases afffecting commercially reared fish species has had a significant impact on this industry. Almost all of the vaccines available at this time are bacterins or formalin-inactivated whole cell suspensions, some with adjuvants. The first vaccines to be successfully commercialized were those against Vibrio anguillarum, Vibrio ordalii , and Yersinia ruckeri in the late 1970s. Developed initially for the salmonid industry, these bacterins are now routinely used worldwide on many species of fish. Though in some areas salmon farming has flourished without the use of these vaccines, in most areas they have been essential to the economic viability of aquaculture operations. Vaccines against Vibrio salmonicida , a pathogen of salmonids, Aeromonas salmonicida , a pathogen of salmonids and carp, and Edwardsiella ictaluri , a pathogen of channel catfish have also been commercialized and are in widespread use. A number of other bacterins have been the subject of research and some of them may eventually be available. Though a bacterin against Vibrio parahaemolyticus , a pathogen affecting species of fish reared in warmwater has been successfully tested, as have bacterins against Aeromonas hydrophila and Edwardsiella tarda , the serologic heterogeneity of these groups of organisms make it unlikely that widely utilizable vaccines will be available in the near future. Those pathogens that appear to be serologically more homogeneous, including Flexibacter columnaris, Pasteurella piscicida and Streptococcus species affecting fish, will likely end up in commercially available bacterins in the not too distant future. The use of a new generation of adjuvants in conjunction with automated injection methods could result in vaccines that will protect against diseases that conventional methods may not be successful against, such as bacterial kidney disease (BKD) caused by Renibacterium salmoninarum .

We examined genetic variation in mortality and mean times to death for three British Columbia populations of chinook salmon Oncorhynchus tshawytscha, subgroups of which were challenged with one of three pathogens: Vibrio anguillarum, which causes vibriosis; Aeromonas salmonicida, which causes furunculosis; or Renibacterium salmoninarum, which causes bacterial kidney disease. In each population, 10 males were mated to 20 females in a nested breeding design, the progeny were divided into groups, and each group was challenged with a pathogen. Heritability estimates (sire component, binary character) of mortality rate were 0.00 for vibriosis, 0.00–0.34 for furunculosis, and 0.00–0.38 for bacterial kidney disease. Estimates of heritabilty for time to death were 0.00 for vibriosis, 0.00–0.02 for furunculosis, and 0.06–0.50 for bacterial kidney disease. There was little correlation among mortality rates for the different pathogens for the families examined in each population. Kitimat River chinook salmo...

We examined variation in mortality and mean time to death for four British Columbia populations of pink salmon Oncorhynchus gorbuscha, subgroups of which were challenged with Vibrio anguillarum, V. ordalii, or Aeromonas salmonicida. Two hundred fish in each population were challenged with one of the three pathogens. Mortality in the four populations ranged from 84 to 91% after challenge with V. anguillarum, from 13 to 26% after challenge with V. ordalii, and from 51 to 63% after challenge with A. salmonicida. A significant difference in mortality among populations was observed only in the V. ordalii challenge. No significant differences in time to death were observed.

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 108 cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 106 ml−1 from 3·7 ± 0·8 × 106 ml−1), erythrocytes (1·2 ± 0·1 × 108 ml−1 from 0·8 ± 0·1 × 108 ml−1), protein (23 ± 4·4 mg ml−1 from 16 ± 1·3 mg ml−1), globulin (15·7 ± 0·2 mg ml−1 from 9·9 ± 0·1 mg ml−1) and albumin (7·3 ± 0·2 mg ml−1 from 6·1 ± 0·1 mg ml−1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml−1 from 40 ± 8·0 units ml−1), lysozyme (920 ± 128·8 units ml−1 from 760 ± 115·3 units ml−1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.

Isolates of Vibrio ordalii were recovered from salmon (Oncorhynchus tshawytscha and Oncorhynchus nerka) cultured in New Zealand. The fish suffered from clinical vibriosis, from seven outbreaks in five geographically distinct areas. Affected fish showed gross signs and histopathology similar to those reported from North America. The presence of a plasmid, designated pVO1, approximately 30 kilobases (kb) in size, was seen in all V. ordalii strains examined This plasmid was identical in size and restriction endonuclease cleavage analysis to a plasmid (pMJ101) previously found in V. ordalii strains elsewhere. After togging pVO1 with the transposon Tn1 from the R plasmid RP4, attempts to isolate plasmid‐less derivatives using a variety of curing agents were unsuccessful. All of the New Zealand isolates possessed identical chromosomal DNA cleavage patterns. These patterns were only slightly different from that of the type strain but substantially different from that of the Vibrio anguillarum type strain.

Sixty-eight strains of Vibrio anguillarum, five of V. ordalii and the type strains of V. alginolyticus, V. carchariae, V. damsela and V. parahaemolyticus were compared using the API 20E gallery. Within the V. anguillarum strains, distinct groups could be separated mainly on the basis of their reaction on indole production and the fermentation of amygdalin and arabinose. Vibrio ordalii, the former V. anguillarum biotype 2, could easily be separated from V. anguillarum and from the other fish pathogenic Vibrio spp.

A heterogeneous sialic acid-binding lectin with affinity for bacterial LPS was isolated and partially characterized from hemolymph of the horse mussel Modiolus modiolus.(1) Using two-dimensional electrophoresis with immobilized pH gradients, the lectin revealed three subunits with different molecular weight and isoelectric points (pI); Mr14 (pI approximately 5.1 and approximately 5.5), 17.5 (pI approximately 5.5) and 20 (pI approximately 4.9) kDa. The affinity purified lectin existed in its native state as aggregates, and by stepwise centrifugation it could be fractionated into molecular entities with distinct specificities towards human and/or horse erythrocytes (modiolin H and/or E activity, respectively). While the medium size entities (rangeor = 30 and100 kDa) exhibited only modiolin E activity and the lowest size entities (rangeor = 5 and10 kDa) demonstrated only modiolin H activity, the largest aggregates (or = 100 kDa-)expressed both activities. Antibacterial activity of the lectin has been observed against various marine bacteria, whereas the whole hemolymph was less effective. The lectin exhibited strong antibacterial effect against all tested strains of Vibrio anguillarum, Vibrio salmonicida, Vibrio viscosus, Vibrio wodanis, and Vibrio ordalii, slight effect on Aeromonas salmonicida salmonicida and Shewanella putrefaciens, and no inhibitory effect with Alteromonas sp. Hemolymph of the horse mussel demonstrated no antibacterial effect against A. salmonicida salmonicida, Alteromonas sp., Sh. putrefaciens and some strains of V. anguillarum, but slight effects against some strains of V. anguillarum and both strains of V. ordalii, and more predominantly against V. wodanis, V. salmonicida and V. viscosus. These results indicate that the lectin plays a role in elimination of bacteria. Circulating hemocytes were demonstrated to be the source of the lectins since granules of the hemocytes were immunoreactive to anti-hemolymph lectin antibody and protein A/gold labelling.

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.

No systematic classification of fish-pathogenic vibrios has been accomplished previously despite the use of serological, physiological, and genetical classification systems. In this study, a comparative 16S rRNA analysis of 34 strains (representing seven species) of fish-pathogenic vibrios was performed. The 16S rRNA sequences were obtained by using reverse transcriptase. Nearly complete sequences were obtained for nine strains. On the basis of the results of this analysis, the remaining strains were investigated by analyzing selected stretches containing a total of 560 nucleotides. With the exception of a few strains, including ATCC 43313 (serovar O9), our comparative 16S rRNA analysis confirmed that strains preliminarily identified as Vibrio anguillarum were phylogenetically closely related. Strains of V. anguillarum could be divided into groups, with the main group containing serotype O1 and O2 strains isolated from Atlantic salmon, rainbow trout, turbot, cod, and saithe. The other distinctive group was represented by type strain NCMB 6. This strain was nearly indistinguishable from the type strains of Vibrio ordalii and Vibrio damsela on the basis of the 16S rRNA stretches compared. The results of a comparative 16S rRNA analysis justified the status of Vibrio salmonicida as a distinct species. Originally, this species was characterized biochemically as a very homogeneous species. However, two strains, which were isolated from diseased halibut and from the intestines of healthy cod, could not be distinguished from V. salmonicida strains phylogenetically, although they differed from the original species description in several phenotypic traits. Our results indicate that V. salmonicida and Vibrio fischeri form a cluster that is clearly separated from the cluster that includes V. anguillarum.

The plasmid profiles of oxytetracycline- and streptomycin-resistant isolates of Aeromonas salmonicida, Vibrio anguillarum, and Vibrio ordalii were examined by agarose gel electrophoresis. Bacterial isolates were from disease outbreaks in fish on the Atlantic and Pacific coasts. Resistant isolates were examined when grown in the presence and absence of antibiotic. Alkaline lysis methods were used for plasmid isolation. Vibrio spp. were predominantly plasmidless, except for a 47-kilobase (kb) plasmid. Atlantic coast isolates of A. salmonicida possessed four or six plasmids, with four smaller plasmids ranging in size from 4.3 to 8.1 kb being consistently observed. The plasmid profiles of antibiotic-sensitive ATCC strains were identical. The plasmid profiles of the Pacific coast isolates of A. salmonicida varied slightly from those of the Atlantic coast isolates with six plasmids observed, ranging in size from 4.2 to 8.9 kb. Resistance to the antibiotics was not altered following plasmid curing experiments and resistance was not transferable to Escherichia coli. Thus, resistance to oxytetracycline and streptomycin did not appear to be plasmid mediated.Key words: plasmids, antibiotics, Aeromonas, Vibrio, fish.

An oligonucleotide DNA probe based on 5S rRNA sequence data was constructed for the identification of the fish pathogens, Vibrio anguillarum and Vibrio ordalii . Specificity of the probe was tested in a colony blot hybridization assay. The respective probe was found to be specific for both V. anguillarum and V. ordalii . No cross hybridization was observed against other fish pathogens and the closely related Vibrionaceae genera. This specific probe may be useful rapid identification of V. anguillarum and V. ordalii .

Sixty-eight strains of Vibrio anguillarum, five of V. ordalii and the type strains of V. alginolyticus, V. carchariae, V. damsela and V. parahaemolyticus were compared using the API 20E gallery. Within the V. anguillarum strains, distinct groups could be separated mainly on the basis of their reaction on indole production and the fermentation of amygdalin and arabinose. Vibrio ordalii, the former V. anguillarum biotope 2, could easily be separated from V. anguillarum and from the other fish pathogenic Vibrio spp.

The histopathology associated with naturally acquired vibriosis in chum salmon, Oncorhynchus keta (Walbaum), fingerlings caused by Vibrio anguillarum was compared with that caused by infection with Vibrio ordalii. Pathogenesis of the two forms was found to be different. Bacteraemia caused by V. anguillarum occurred in the early stages with pronounced histopathological changes in blood, loose connective tissue, kidney, spleen, gills and posterior gastrointestinal tract. Bacterial cells appeared uniformly dispersed throughout the affected tissues but were most abundant in the blood. With V. ordalii, bacteraemia developed only in late stages of the disease and the concentration of bacterial cells per ml of blood was less than in the V. anguillarum infection by a factor of 102–103. Tissues with most pronounced changes were skeletal and cardiac muscle, anterior and posterior gastrointestinal tract and the gills. Vibrio ordalii observed in the tissues was not evenly dispersed but was present within tissue as colonies or aggregates of cells. The differences in pathology observed in naturally infected chum salmon were produced experimentally with each pathogen by waterborne exposure of chum; coho, Oncorhynchus kisutch (Walbaum); and chinook salmon, Oncorhynchus tshawytscha (Walbaum). Severe decreases in circulating leucocytes accompanied bacteraemia caused by either bacterial species.

An examination of 41 fish pathogenic and 51 environmental strains of Vibrio anguillarum from Danish coastal areas, and of 8 reference strains of Vibrio anguillarum, biotype 1, showed that these strains shared rather broad-spectered activities towards many carbohydrates, amino acids, proteins and lipids. They also exhibited rather uniform growth characteristics, including parameters as salinity (NaCl), temperature, antibiotic resistance, and growth on specific media. A computer analysis of these strains and 3 strains of Vibrio anguillarum, biotype 2 (Vibrio ordalii), showed that the Danish strains all belonged to Vibrio anguillarum, biotype 1, and that no basis existed for setting up new biotypes. Mean values of the G+C content in DNA of strains of the various categories of Vibrio anguillarum, biotype 1, ranged from 45.5 to 46, while the mean value for the Vibrio ordalii strains was 48.9. The biochemically active Vibrio anguillarum seems more suitable for environmental studies than the more fastidious Vibrio ordalii.

Examination of polynucleotide sequence relationships among 11 strains of Vibrio anguillarum biotype 2 isolated from moribund fish in North America and Japan demonstrated the highly conserved character of this group of fish pathogens and, moreover, confirmed its discrete nature from V. anguillarum biotype 1. Additional molecular analyses of the V. anguillarum biotype 2 strains revealed the universal presence of a multicopy plasmid with a molecular mass of approximately 20 × 106 daltons and a mole percent guanine plus cytosine of 44. The plasmid of strain DF3K was representative of this molecular species and was designated pMJ101. Subsequent DNA–DNA hybridizations using nick translation-labeled pMJ101 as a probe indicated all the 20 × 106 dalton plasmids were either identical or highly conserved, and, furthermore, that pMJ101 was apparently unrelated to either the virulence plasmid, pJM1, of V. anguillarum biotype 1 or to representatives of common plasmid incompatibility groups. The lack of relatedness between pMJ101 and pJM1 was further supported by differences in their restriction endonuclease cleavage patterns.

Lipopolysaccharides(LPS)were prepared from different serotypes of Vibrio anguillarum, J-O-1, J-O-2 and J-O-3, and Vibrio ordalii. The LPS fractions were obtained by phenol-water extraction followed by Cetavlon treatment and high speed centrifugation.Chemical analysis of V. anguillarum J-O-1, J-O-2 and J-O-3 showed 2.5%, 4.8%and 1.3% protein;36.3%, 30.0% and 20.0% sugar and 7.1%, 11.3% and 6.6%fatty acids. Chemical analysis of V. ordalii showed 17.5%protein, 13.8% sugar and 1.4% fatty acids. The monosaccharide composition of V. anguillarum J-O-1, J-O-2 and J-O-3 was respectively:0.1%, 0.1% and0.1% 2-keto-3-deoxyhexose(KDO);3.0%, 1.9% and 0.6% pentose;32.6%, 21.6% and 12.6% hexose;10.8%, 7.6% and 2.8% 6-deoxyhexose;4.6%, 7.7% and 7.7% heptose;5.1%, 6.0% and10.2% amino sugar. The monosaccharide composition of V. ordalii LPS was:0.0% KDO, 1.5%pentose, 6.6%hexose, 4.0%6-deoxyhexose, 0.2%heptose and 3.1%amino sugar. The principal fatty acids of the V. anguillarum lipid A were:myristic acid, palmitic acid and stearic acid. The principal fatty acids constituents V. ordalii lipid A were: lauric acid, palmitic acid, stearic acidand arachidic acid.

Agglutinating activity was widely distributed among 101 Vibrio anguillarum strains of different origin and three Vibrio ordalii strains from salmonids. The spectrum of cells which were agglutinated comprised yeast cells and human (type O), poultry, guinea pig, and trout erythrocytes, whereas ovine, bovine, and tanned bovine erythrocytes were not affected. Mannose-sensitive hemagglutination, mannose-resistant hemagglutination, and non-agglutinating strains were recognized. The three V. ordalii strains showed mannose-resistant hemagglutination, whereas V. anguillarum exhibited either mannose-sensitive hemagglutination or was non-agglutinating. Among V. anguillarum , sensitivity to d -galactose and l -fucose occurred sporadically. An agglutination typing scheme was developed for strains of V. anguillarum based on the agglutination pattern of human, poultry, guinea pig, and trout erythrocytes and yeast cells. Eight different agglutination types (A through H) were defined. The distribution of these types among fish pathogenic and environmental V. anguillarum strains were studied. The application of the typing scheme in ecological and epidemiological studies and for preventive medical purposes is discussed.

Strains of Vibrio salmonicida that were isolated from different localities on the Norwegian west coast and strains of Vibrio anguillarum, Vibrio ordalii, and Vibrio parahemolyticus were compared genetically. Our results showed that strains of V. salmonicida constitute a homogeneous group (82 to 100% deoxyribonucleic acid homology), which is distinct from the other species, and that V. anguillarum is a rather diverse species. This diversity seems to be related to the host specificity of the bacterial strains.

The technique of immunoblotting was used to identify the surface antigens of the marine vibrios pathogenic for fish, Vibrio anguillarum and Vibrio ordalii. Polyclonal antisera raised in rabbits to strains representing the two most common serotypes causing Vibriosis in fish in North America were used. The results demonstrated that antigenic specificity was conferred by the lipopolysaccharides, with three serotypes being displayed among the strains examined. The lipopolysaccharides of strains chosen as type species for V. anguillarum and V. ordalii displayed antigenic cross-reactivity. The morphological heterogeneity of the Vibrio lipopolysaccharides was also analyzed in silver-stained polyacrylamide gels and by intrinsic 32P-radiolabelling. Two distinct lipopolysaccharide morphologies were exhibited, one with 0 polysaccharide chains of heterogeneous chain length, the other having 0 polysaccharide chains of more uniform chain length but displaying microheterogeneity. These lipopolysaccharide morphologies corresponded to different serogroups. Two minor proteins of apparent molecular weights 49 000 – 51 000 present in outer membrane preparations isolated by the sarcosinate extraction procedure were also strong antigens, and common to all strains of V. anguillarum tested and to several strains of V. ordalii. The major outer membrane protein was a weak antigen common to both species.

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.