FISH DISEASE LEAFLET 77 VIBRIOSIS IN FISH G. L. Bullock U.S. Fish and Wildlife Service, National Fisheries Center-Leetown, National Fish Health Research Laboratory, Box 700, Kearneysville, West Virginia 25430 UNITED STATES DEPARTMENT OF THE INTERIOR, Fish and Wildlife Service, Division of Fisheries and Wetlands Research, Washington, D.C. 20240. 1987 Revision of Fish Disease Leaflet 29 (1970), same title, by A. J. Ross; and Fish Disease Leaflet 50 (1977), same title, by G. L. Bullock. Introduction Vibriosis is a systemic bacterial infection of primarily marine and estuarine fishes, caused by bacteria of the genus Vibrio (Ross et al. 1968; Ghittino et al. 1972); it is a major cause of mortality in mariculture operations. It sometimes also occurs in freshwater species. Vibriosis has been known for centuries; outbreaks were recorded as early as the 1500's along the Italian coast. Terms such as "red pest," "red boil," "red plague," or "saltwater furunculosis" have been used to describe vibrio infections, but "vibriosis" is now the specific and standard name of the disease. Etiology and Diagnosis Species of the genus Vibrio occur in marine and estuarine waters and in accompanying sediments. Vibrio anguillarum was the first species to be identified as a full-fledged fish pathogen, and has now been reported in more than 42 species in widely distributed regions (Colwell and Grimes 1984). Although V. anguillarum is regarded as the dominant species causing vibriosis, several other Vibrio species are also pathogenic. Vibrio ordalii causes devastating losses among salmon propagated in cage culture in coastal waters of North America's Pacific Northwest (Crosa et al. 1977). The bacterium was first considered to be biovar II, V. anguillarum, but Schiewe et al. (1981) determined that it was a distinct species. Among other species, V. carchariae was isolated from a dead sandbar shark (Carcharhinus plumbeus), but caused mortality in spiny dogfish (Squalus acanthias) 18 h after intraperitoneal injection (Grimes et al. 1985); V. alginolyticus has been found in finfish, shellfish, and marine sediments and has been associated with acute septicemia in sea bream (Sparus aurata); V. damsela infects damselfish (Chromis punctipinnis), a tropical aquarium species, but also infects some species of sharks, and infections have been reported in man (Grimes et al. 1985; Love et al. 1981); V. vulnificus is usually encountered as a highly virulent but opportunistic human pathogen, though biogroup 2 infects eels and causes development of a red patch on the trunk or tail (Tison et al. 1982); and V. salmonicida, the most recently described species of fishpathogenic vibrios (Egidius et al. 1986), has been shown to be the cause of a septicemia in cultured salmonids in Norway-a disease characterized by a severe anemia and extensive hemorrhages. Diagnosis of fish vibriosis is based on the isolation and identification of the particular species of Vibrio involved. All of the species are gramnegative motile rods and are sensitive to the vibriostat 01129 (2,4diamino6,7diisopropylpteridine). Most vibrios ferment glucose anaerogenically; V. damsela is an exception, fermenting sugar aerogenically. Serological identification tests have been reported for V. anguillarum, V. ordalli, and V. salmonicida. Cipriano et al. (1985) described an immunoblot test for the detection of antigens of V. anguillarum in fish tissues, and Chart and Trust (1984) used an immunoblot test to distinguish between the surface antigens of V. anguillarum and V. ordalii. Egidius et al. (1986) described an enzymelinked immunosorbent assay (ELISA) plate procedure that identifies V. salmonicida but does not crossreact with V. anguillarum. Other species of fishpathogenic vibrios are now differentiated biochemically (Colwell and Grimes 1984). Clinical Signs and Pathology Vibriosis infects many marine and estuarine fishes (and sometimes freshwater species), producing either skin ulcers or a septicemia characterized by erythema, hemorrhaging, and anemia. Outbreaks caused by Vibrio anguillarum, produce red necrotic or boillike lesions in the musculature, and erythema of the fin bases and mouth of Pacific salmonids. Petechiae also occur on the body surface, and hemorrhages in the gills (Fryer et al. 1972); hemorrhages may also be present in the viscera, and the intestinal tract may be inflamed. In eels, rapidly developing septicemia characteristically occurs; victims have hemorrhages in the fins and striated muscle of the abdominal region, and skin ulcerations (Bullock et al. 1971). Dermal lesions accompanied by fin necrosis and hemorrhages are common in the winter flounder, Pseudopleuronectes americanus (Levin et al. 1972). Hacking and Budd (1971) noted that hemorrhages are the dominant finding among freshwater aquarium fishes. In Japan, external and internal pathological changes among salmonids and nonsalmonid fishes are similar (Tanaka 1975). In diseased rainbow trout (Salmo gairdneri) vibriosis results in muscle necrosis, accompanied by interfibrillar hemorrhages, congestion of interfibrillar vessels, and an absence of leucocytic response (McCarthy et al. 1974). In winter flounders, Levin et al. (1972) reported muscle necrosis and focal interstitial and tubular necrosis of the kidneys. In contrast with the generalized septicemia of Pacific salmonids caused by V. anguillarum, V. ordalii preferentially attacks skeletal and cardiac muscle, the gills, and the gastrointestinal tract. Some investigators have observed a severe leucopenia in salmonids infected with V. ordalii, thus suggesting a leucocytic factor (Schiewe 1983; Ransom et al. 1984). In sharks, V. carchariae produces vasculitis in organs of the reticuloendothelial system and in tropical aquarium species, V. damsela characteristically causes skin ulcers. In eels, V. vulnificus biogroup 2 infections are associated with red patches or swollen lesions on the trunk or tail; in the late stages of infections, histopathologic changes develop in the gills and internal organs. Salmon infected with V. salmonicida show no external pathology but severe anemia develops internally, hemorrhaging occurs in the swim bladder and rectum, and petechiae occur in the caecum and abdominal wall. Virulence Factors Different strains of vibrio have been shown to have one or more mechanisms for expressing virulence in the fish host. Crosa et al. (1977) discovered that high virulence strains of V. anguillarum contained a large plasmid that enabled the bacterium to obtain iron necessary for its metabolism, even though the host produced factors that bind iron. Trust et al. (1981) demonstrated that high virulence strains of V. anguillarum resisted the bactericidal effects of normal serum and agglutinated trout erythrocytes. Hemolysins, cytolysins, proteases, and other extracellular toxic substances have been demonstrated among some vibrios (Inamura et al. 1985; Kodama et al. 1984; Kodama et al. 1985; Kothary and Kreger 1985; Moustafa et al. 1985). Smith and Merkel (1981) postulated that the virulence of V. vulnificus and V. alginolyticus was due to the production of collagenase. Host and Geographic Range Although the seven Vibrio species reported as fish pathogens may infect many marine and estuarine fishes, cultured fishes are most susceptible because they are often stressed. Serious epizootics caused by V. anguillarum, V. ordalii, or V. salmonicida occur in pink salmon (Oncorhynchus gorbuscha), chum salmon (O. keta), Atlantic salmon (Salmo salar), Japanese eel (Anguilla japonica), yellowtail (Seriola quinqueradiata), and ayu (Plecoglossus altivelis). Lewis (1985) reported a V. anguillarum septicemia in channel catfish (Ictalurus punctatus). The principal hosts of the remaining pathogenic vibrios were described in the earlier section on etiology and diagnosis. Although vibriosis is distributed worldwide, V. ordalii has thus far been limited to the Pacific Northwest and Japan, and V. salmonicida to Norway and Scotland. Control Prevention Good sanitation and management procedures should be used, and care should be taken to avoid crowding and alleviate stress when fish are handled. During the last 20 years much interest has been expressed in immunization as a means of preventing vibriosis. Intraperitoneal injection of vaccine is probably the most effective method of delivery and produces a high degree of immunity; however, it is expensive, labor-intensive, and stresses the fish. Rohovec et al. (1975) demonstrated that either wetpacked or lyophilized formalinkilled V. anguillarum cells were effective as an oral immunogen. Kusuda et al. (1978) found that formalin-killed, ultrasonicated oral bacterin protected ayu from vibriosis. The hyperosmotic infiltration technique, where fish are subjected to a concentrated sodium chloride solution immediately before exposure to antigen (Amend and Fender 1976; Lannan 1978), formed the basis of a commercial vaccine for vibriosis caused by V. anguillarum. Aoki et al. (1984a) modified the hyperosmotic technique by removing culture supernatant that contained exotoxin. Direct immersion of fish in bacterin, not preceded by hyperosmotic treatment, provided longlasting immunity in salmon (Gould et al. 1979; Johnson et al. 1982 a,b), ayu (Kusuda et al. 1980), and striped bass, Morone saxatilis (Roberson et al. 1982). Spray or shower immunization, where fish are immunized by high pressure spray, is now used to immunize Pacific salmon and ayu. Therapy A 10day treatment with either sulfamerazine at the rate of 200 mg/kg of fish, or oxytetracycline at 5075 mg/kg, has been used to control vibriosis outbreaks. Other antibacterials that have been used include trimetoprin at 30 mg/kg (Sako and Kusuda 1978), piromidic acid at 1040 mg/kg (Tashiro et al. 1979), furanace at 25 mg/L (Egidius and Anderson 1979), and the substituted quinoline halquinol at 75 mg/kg (Austin et al. 1982). Annotated Bibliography Amend, D. F., and D. C. Fender. 1976. Uptake of bovine serum albumin by rainbow trout from hyperosmotic solutions: a model for vaccinating fish. Science 192:793794. Immersion of rainbow trout in 5.32% sodium chloride for 3 min, followed by 3 min in 2% bovine serum albumin (BSA), resulted in uptake of BSA-primarily through the lateral line system and secondarily through the gills. Amend, D. F., and K. A. Johnson. 1984. Evidence for lack of antigenic competition among various combinations of Vibrio anguillarum, Yersinia ruckeri, Aeromonas salmonicida, and Renibacterium salmoninarum bacterins when administered to salmonid fishes. J. Fish Dis. 7:293300.Bacterins of the four species named were administered alone and in combination to salmonid fishes, and the level of protective immunity was compared. For each pathogen, the protection obtained with monovalent bacterins appeared comparable to that obtained with polyvalent bacterins. Vaccinations with A. salmonicida bacterin alone conferred some protection against challenges with Type II V. anguillarum and Y. ruckeri. The combination of A. salmonicida and R. salmoninarum bacterins appeared to potentiate the protection conferred against A. salmonicida. The potential of multivalent vaccines for protecting fish from several diseases seems to be real. Aoki, T., T. Kitao, M. Fukudome, S. Takahashi, and S. Egusa. 1984a. Modification of the hyperosmotic infiltration method of vaccination against vibriosis in cultured ayu Plecoglossus altivelis. J. Fish Dis. 7:149156. A vaccine solution of a formalinkilled culture of Vibrio angillarum cells was toxic to young ayu when administered by the hyperosmotic infiltration method. The toxin was present in the culture broth. After the toxin was removed from the broth by centrifugation, the fish were dipped in 5.32% NaCl solution for 2 min and then in a solution containing precipitated cells for 3 min. The immunized fish were protected against vibriosis when challenged 1 month after immersion. Aoki, T., J. Nomura, and J. H. Crosa. 1985. Virulence of Vibrio anguillarum with particular emphasis on the outer membrane components. Bull. Jpn. Soc. Sci. Fish. 51:12491254. There was a difference in lipopolysaccharide structure between the highly virulent, plasmidless strain PT479 of V. anguillarum and an isogenic strain of low virulence that had undergone 20 passages in culture medium. The highly virulent strain showed a high molecular weight band and also had a higher uptake of radioactive iron. Aoki, T., M. Sakai, and S. Takahashi. 1984b. Protective immunity in ayu, Plecoglossus altivelis, vaccinated by immersion with Vibrio anguillarum. Fish Pathol. 19:181185.Excellent protection against vibriosis was provided to ayu vaccinated by the immersion method with formalinkilled bacterin, sonicated bacterin, or lyophilized formalinkilled V. anguillarum. Lipopolysaccharides of V. anguillarum also induced protective immunity against vibriosis in fish vaccinated by the immersion method. No agglutinating antibody was detectable in the serum of fish vaccinated by immersion with vibrio bacterin; antibodies were detected in fish vaccinated by the intraperitoneal injection of formalinkilled bacterin mixed with Freund's complete adjuvant. There were no differences in serum protein pattern between fish vaccinated by immersion and unvaccinated fish, nor in the protective immunity against vibriosis between fish that received serum from immersion-vaccinated fish and those that received serum from unvaccinated fish. Austin, B. 1983. Vaccine for the control of vibriosis in eels. Vet. Rec. 113:394395.A formolized toxoid vaccine prepared from three strains of Vibrio anguillarum was given to 10,000 elvers in a fiberglass tank, in two doses 28 days apart. Administration was by bathing for 2 min. A second group of 10,000 elvers was not vaccinated. In a natural outbreak 4 months later, only 32 of the vaccinated eels died, compared with 759 (7.5%) of the controls. The bacterium was isolated from the controls that died, but not from the vaccinated eels. The vaccinated eels also resisted laboratory challenge 18 months after vaccination. Serum agglutination tests showed specific circulating antibodies within a month after the initial dose of vaccine, the titers of which were greatly increased by the booster dose. Titers remained 16 months after vaccination. It was concluded that the vaccine would protect eels for about 2 years. Austin, B., C. Johnson, and D. J. Alderman. 1982. Evaluation of substituted quinolines for the control of vibriosis in turbot (Scophthalmus maximus). Aquaculture 29:227239.Tests were conducted with 103 compounds in the treatment (by bathing or with medicated feed) of turbot experimentally infected with 13 strains of Vibrio anguillarum. The infections were controlled by four of the compounds: 5,7dichloro8-hydroxyquinoline; 5,7dichloro8quinolylNphenylcarbamate: halquinol: and oxolinic acid. Bruno, D. W., T. S. Hastings, A. E. Ellis, and R. Wootten. 1985. Outbreak of a cold water vibriosis in Atlantic salmon in Scotland. Bull. Eur. Assoc. Fish Pathol. 5:6263. An outbreak of disease caused a 3% mortality in salmon smolts in sea cages at a site in northern Scotland in January 1985. Ambient water temperature was about 7°C. There was no external sign of disease, but internally there was severe hemorrhaging in the swim bladder and rectum and petechiation of the caecum and abdominal wall; the stomach contained clear fluid. The peritoneal cavity usually contained bloodstained fluid and the spleen was often bright red. Large numbers of Vibrio sp. were isolated from internal organs of moribund fish, but their biochemical properties did not resemble those of V. anguillarum or V. ordalii. The disease was reproduced in the laboratory by injecting fish intraperitoneally with the isolate. Treatment with oxytetracycline for 14 days curbed the mortalities, but the disease recurred 2 months later. This is the first report of the disease in Scotland, although it has been described in Norway. Bullock, G. L., D. A. Conroy, and S. F. Snieszko. 1971. Bacterial diseases of fishes. Book 2A (151 pp.) in S. F. Snieszko and H. R. Axelrod, eds. Diseases of fishes. T.F.H. Publications, Inc., Neptune, N.1. A comprehensive description of vibriosis: historical aspects, pathologic changes and symptoms, etiology, diagnosis, and treatment. Chart, H. 1983. Multiflagellate variants of Vibrio anguillarum. J. Gen. Microbiol. 129:21932198. An ultrastructural examination of six strains of Vibrio anguillarum of varying virulence for eels revealed an apparent relation between pathogenicity and the possession of more than one flagellum. Chart, H., and T. J. Trust. 1984. Characterization of the surface antigens of the marine fish pathogens Vibrio anguillarum and Vibrio ordalli. Can. J. Microbiol. 30:703710. The technique of immunoblotting was used to identify the surface antigens of the two vibrios. When polyclonal antisera (prepared in rabbits) to strains representing the two most common serotypes causing vibriosis in North America were used, the lipopolysaccharides conferred antigenic specificity. Cipriano, R. C., J. B. Pyle, C. E. Starliper, and S. W. Pyle. 1985. Detection of Vibrio anguillarum antigen by the dot blot assay. J. Wildl. Dis. 21:211218. The dot blot assay, modified and adapted for detection of antigens from V. anguillarum in fish tissues, was specific for V. anguillarum and did not react with antigens of V. ordalii, Pseudomonas sp., or Yersinia ruckeri. The blot assay enabled detection of as little as 2.3 ng of a mixture of protein antigens obtained from cellfree extracts of V. anguillarum; it was about 100 times more sensitive than either the indirect fluorescent antibody technique or bacterial isolation for detecting V. anguillarum in fish tissues. Colwell, R. R., and D. J. Grimes. 1984. Vibrio diseases of marine fish populations. Pages 265287 in O. Kinne and H. P. Bulnheim, eds. Diseases of marine organisms. International Helgoland Symposium, 1983. A comprehensive review of the Vibrio species that infect fish, species of fish affected, virulence mechanisms, and control procedures for prevention and treatment of vibriosis. Crosa, J. H. 1980. A plasmid associated with virulence in the marine fish pathogen Vibrio anguillarum specifies an iron-sequestering system. Nature (Lond.) 284:566568. Many of the highvirulence strains of the marine fish pathogen Vibrio anguillarum isolated from epizootics harbor a specific plasmid class that is lacking in lowvirulence strains. Curing experiments have confirmed a link between this specific plasmic class and the ability of V. anguillarum to establish infections. In general, all bacterial virulence factors promote growth in the antagonistic environment of the host defense mechanisms. One line of defense is provided by the proteins transferrin and lactoferrin, which bind iron and render it unavailable to pathogens. A mechanism whereby invading bacteria may successfully compete for the otherwise unavailable iron could therefore become crucial in enabling them to proliferate in body fluids and tissues. The V. anguillarum virulence plasmid specifies a very efficient ironsequestering system, enabling bacteria to survive in conditions of limited iron availability. Crosa, J. H., M. H. Schiewe, and S. Falkow. 1977. Evidence for plasmid contribution to the virulence of the fish pathogen Vibrio anguillarum. Infect. Immun. 18:509513. Analysis of the plasmid deoxyribonucleic acid complement of high and low virulent strains of the fish pathogen Vibrio anguillarum showed a correlation between enhanced virulence and the presence of a 50megadalton plasmid class. All 50megadalton plasmids isolated from different high virulent V. anguillarum strains were homologous as judged by the analysis of plasmid deoxyribonucleic aciddeoxyribonucleic acid hybridization. Egidius, E., and K. Andersen. 1979. The use of Furanace against vibriosis in rainbow trout Salmo gairdneri Richardson in salt water. J. Fish Dis. 2:7980. A 10min bath treatment with 2 ppm Furanace controlled experimental vibriosis in rainbow trout. Egidius, E., O. Soleim, and K. Andersen. 1984. Further observations on coldwater vibriosis or Hitra disease (in salmon). Bull. Eur. Assoc. Fish Pathol. 4:50-51. Since its first appearance in the late 1970's, the Hitra disease haemorrhagic syndrome has persisted in salmon farms north of Stad, Norway (lat. 62°N). In 1983 the disease spread south and the Bergen region was badly affected. A slow-growing vibriolike bacterium that was isolated showed biochemical reactions similar to, but nevertheless different from, those of Vibrio anguillarum and V. ordalli. Intraperitoneal injection of the isolated bacterium in fish repeatedly induced the disease. Egidius, E., R. Wiik, K. Andersen, K. A. Hoff, and B. Hjeltnes. 1986. Vibrio salmonicida sp. nov., a new fish pathogen. Int. J. Syst. Bacteriol. 36:518520. A disease persisting in Norwegian salmonid culture since the late 1970's is caused by a bacterium that belongs to the genus Vibrio, but differs from all previously described species of the genus. The name Vibrio salmonicida sp. nov. is proposed. Evelyn, T. P. T. 1984. Immunization against pathogenic vibrios. Pages 121150 in Symposium on fish vaccination, 2022 February 1984. Paris. In preparing vaccines against Vibrio anguillarum and V. ordalii for use in salmonid fish, allowance must be made for serological diversity. The best immunogen was cellwall lipopolysaccharide. A better immune response was produced by the injection, immersion. and spraying of vaccine than by oral administration Farkas. J., and S. E. Malik. 1986. Vibrio disease of sheatfish (Silurus glanis L.) fry. Aquaculture 51:8188. In 1983, an outbreak of a fatal disease in weekold sheatfish was characterized by reddish discoloration of the upper part of the head due to infiltration of the brain tissue with erythrocytes, hemorrhages around the eyeballs, and rupture of the dorsal surface of the head. Ten isolates of a Vibrio sp. were serologically unrelated to V. anguillarum. The disease was reproduced in sheatfish fry by exposure of the eggs to the Vibrio. It could be controlled by bathing fertilized eggs in 100 ppm iodine for 10 min. Fryer. J. L., J. S. Rohovec, G. L. Tebitt, and J. S. McMichael. 1976. Vaccination for control of infectious diseases in Pacific salmon. Fish Pathol. 10:155164. Injectable and oral vaccines for control of furunculosis, vibriosis, and columnaris are discussed, as is live attenuated vaccine for infectious hematopoietic necrosis. Fryer. J. L., J. S. Nelson, and R. L. Garrison. 1972. Vibriosis in fish. Prog. Fish Food Sci. 5:129133. Etiology, pathologic changes, and distribution of vibriosis are reviewed and disease control in salmon by chemotherapy and peroral immunization with Vibrio anguillarum bacterin is described. Ghittino. P., S. Andruetto, and E. Vigliani. 1972. "Red mouth" enzootic in hatchery rainbow trout caused by Vibrio anguillarum. (Enzoozia di "bocca rossa" in trote iridee di allevamento sostenuata da Vibrio anguillarum). Riv. Ital. Piscic. Ittiopathol. 7:4145. Description of an outbreak of vibriosis in an Italian freshwater rainbow trout farm. Even though the condition was described as red mouth, the etiologic agent was V. anguillarum. Mortality was slight and was controlled by feeding chloramphenicol at 6 to 7 g/100 lb fish per day for 7 days. Gjedrem, T., and D. Aulstad. 1974. Selection experiments with salmon. I. Differences in resistance to vibrio disease of salmon parr (Salmo salar). Aquaculture 31:5159. Analyses of the responses of strains of salmon parr to vibriosis (Vibrio anguillarum) are reported. Significant differences in resistance among various river runs were attributed at least partly to heritage. Gould, R. W., R. Antipa, and D. F. Amend. 1979. Immersion vaccination of sockeye salmon (Oncorhynchus nerka) with two pathogenic strains of Vibrio anguillarum. J. Fish. Res. Board Can. 36:222225. Sockeye salmon were immersion-vaccinated in suspensions containing S x 107, 5 x 106, 5 x 105, or 5 x 104 bacteria/mL of bivalent or monovalent formal inkilled Vibrio anguillarum, Types I and II. Type II V. anguillarum is now recognized as V. ordalii. The fish were split two lots and held for 54 days. At that time one lot was challenged with living, virulent V. anguillarum, Type I and one with living, virulent V. anguillarum, Type II. Immunization with bivalent bacterin effectively protected the fish from vibriosis, but monovalent vaccine was effective only against the homologous challenge. Immunization with the highest concentration of Type I monovalent bacterin resulted in O% Type I and 58% Type II challenge mortality. Immunization with the highest concentration of Type II monovalent bacterin resulted in 41 % Type I and 0% Type II challenge mortality. Immunization with the highest concentration of bivalent Type I or II bacterin resulted in 2% mortality on both challenges. Protective bacterins were effective at concentrations down to 5 X 105 bacteria/mL. Grimes, D. J., S. H. Gruber, and E. B. May. 1985. Experimental infection of lemon sharks, Negaprion brevirostris (Poey), with Vibrio species. J. Fish Dis. 8:173180. Healthy lemon sharks were inoculated with Vibrio carchariae and V. damsela, two vibrios known to cause disease in other shark species. Vibrio damsela was not recovered from the inoculated sharks; V. carchariae infected the healthy lemon sharks, but did not produce clinically observable disease. However. histological evaluation revealed active splenic and hepatic disease and gramnegative bacteria associated with mononuclear cells Hacking, M. A., and J. Budd. 1971. Vibrio infections in tropical fish in a freshwater aquarium. J. Wildl. Dis. 7:273280. Vibrio anguillarum was identified as the causative agent of an epizootic in tropical freshwater fishes. It was pathogenic for selected species of other freshwater fishes, and was isolated from inoculated gravid guinea pigs. Hastein. T., and T. Refsti. 1986. Vaccination of rainbow trout against vibriosis by injection, dip, and bath. Bull. Eur. Assoc. Fish Pathol. 6:45-49. The efficacy of five commercial vibrio vaccines was assessed by single vaccination and revaccination, administered by dip, extended bathing, and injection, in rainbow trout in freshwater and seawater. Differences between vaccines were not significant; all were effective. The efficacy of vaccination methods differed significantly in the following (descending) order: injection, dip, and extended bath. Holm, K. O., E. Strom, K. Stensvag, J. Raa. and T. Jorgensen. 1985. Characteristics of a Vibrio sp. associated with the "Hitra disease" of Atlantic salmon in Norwegian fish farms. Fish Pathol. 20:125129. Vibrio sp. was isolated from the kidney tissues of moribund Atlantic salmon with "Hitra disease," a coldwater vibriosis or haemorrhagic syndrome named after the island where it was first recognized as an economic threat. Eighteen isolates from geographically distant fish farms along the Norwegian coast were similar in biochemical properties. Serotyping and DNA "fingerprinting" provided additional evidence for similarity between the isolates and showed that they differed from V. anguillarum and V. ordalii. The vibrio associated with "Hitra disease" was designated Vibrio sp. TEO. It was not found in healthy fish, even on farms with outbreaks of "Hitra disease." The Vibrio sp. TEO elicited disease with similar symptoms when injected into healthy fish, and was reisolated from them. Honda, A., H. Kodama, M. Moustafa, F. Yamada, T. Mikami, and H. Izawa. 1985. Response of rainbow trout immunized with formalinkilled Vibrio anguillarum: activity of phagocytosis of fish macrophages and opsonizing effect of antibody. Fish Pathol. 20:395402. Phagocytic activity was studied in macrophages from normal fish and fish immunized with formalinkilled Vibrio anguillarum. Phagocytosis of V. anguillarum by normal peritoneal macrophages was enhanced in the presence of antibody and complement. In rainbow trout immunized with V. anguillarum, the phagocytic activity of macrophages increased significantly 5 weeks after immunization, compared with the activity of macrophages from normal fish. Agglutinin titers increased 3 weeks after immunization, but bactericidal activity of antibody and complement was not detected. The immunized fish developed protective immunity against vibrio infection before significant levels of antibody or phagocytic activity became detectable. Horne, M. T., and A. Baxendale. 1983. The adhesion of Vibrio anguillarum to host tissues and its role in pathogenesis. J. Fish Dis. 6:461472. The adhesion of V. anguillarum to excised sections of rainbow trout gut depended on concentration and time. A standardized method for assaying adhesion was used to study the course of vibriosis in rainbow trout. The gut may be an important site of bacterial multiplication in slowly developing infections in mature fish. Adherence of bacteria to gut sections was substantially less in vaccinated than in unvaccinated fish. Horne M. T., R. J. Roberts, and M. Tatner. 1984. The effects of the use of potassium alum adjuvant in vaccines against vibriosis in rainbow trout Salmo gairdneri. J. Fish Dis. 7:91100. The formolized bacterin of Vibrio anguillarum, with and without alum adjuvant, was administered intraperitoneally to rainbow trout under farm conditions. The adjuvanted preparation caused pathological changes indicative of chronic peritonitis and substantial early mortalities in fish 3 cm long (to a lesser extent in fish 20 cm long), and depressed growth rate. No additional protection against artificial challenge was obtained from its use. Inamura, H., T. Nakai, and K. Muroga. 1985. An extracellular protease produced by Vibrio anguillarum. Bull. Jpn. Soc. Sci. Fish. 51:1915-1920. The toxic extracellular product of V. anguillarum proved to be protease. The extracellular protease was purified by precipitation with ammonium sulfate and gel filtration, and showed an LD50 value of 1.7 mg protein/g in goldfish and 1.6 mg in mouse. Itami, T., and R. Kusuda. 1984. Viability and pathogenicity of Vibrio anguillarum in sodium chloride solutions of various concentrations isolated from ayu Plecoglossus altivelis cultured in freshwater. J. Shimonoseki Univ. Fish. 32:3340. The viability and pathogenicity of V. anguillarum,the etiological agent of vibriosis in cultured ayu, were investigated in diluted seawater and NaCl solutions of various concentrations prepared by using distilled water and pond water. The results suggested that V. anguillarum isolated from ayu cultured in freshwater originated in seawater, and that the high mortality of ayu was due to increases in the pathogenicity of V. anguillarum in high-concentration NaCl solutions, and to the stress caused in the fish by the transfer from freshwater to saltwater. Johnson, K. A., J. K. Flynn, and D. F. Amend. 1982a. Onset of immunity in salmonid fry vaccinated by direct immersion in Vibrio anguillarum and Yersinia ruckeri bacterins. J. Fish Dis. 5: 197205. The fry of several salmonid species were vaccinated by direct immersion in either Yersinia ruckeri or Vibrio anguillarum bacterin and the level of protective immunity was determined by the survival of fish after bath challenge with virulent organisms. The immersion time for effective vaccination was obtained within 5 s and protective immunity was demonstrated within 5 days at 18°C and within 10 days at 10°C. The minimum fish size at which protective immunity peaked was between 1.0 and 2.5 g. Immunity appeared to be a function of size and not of age, but differences in response among several species were indicated. In fish under 1.0 g, the level of protective immunity could be increased by using a more concentrated bacterin. The results were similar with both bacterins. Johnson, K. A., J. K. Flynn, and D. F. Amend. 1982b. Duration of immunity in salmonids vaccinated by direct immersion with Yersinia ruckeri and Vibrio anguillarum bacterins. J. Fish Dis. 5:207213. The level of protective immunity was determined for several salmonid species following vaccination by the direct immersion method with bacterins of commercial Vibrio anguillarum (two serotypes) and Yersinia ruckeri (Hagerman strain). The duration of protective immunity was similar for the two bacterins, but varied with the concentration of the bacterin and with the size and species of fish. In fish weighing less than 1 g, the duration of protective immunity was longer when the most concentrated bacterin was used. Immunity generally lasted about 120 days in lg fish and about 180 days in 2g fish, but lasted for a year or longer in fish weighing 4 g or more. Kawano, K., T. Aoki, and T. Kitao. 1984. Duration of protection against vibriosis in ayu Plecoglossus altivelis vaccinated by immersion and oral administration with Vibrio anguillarum. Bull. Jpn. Soc. Sci. Fish. 50:771774. Excellent protection against vibriosis was provided to ayu vaccinated by immersion with 5.32% NaCl solution containing (wet weight) either 0.94 or 9.4 g/L of lyophilized cells of V. anguillarum. Protective immunity in fish lasted at least 113 days. Strong protection against vibriosis was also demonstrated in fish immunized orally for 15 days, but this protective immunity lasted only 50 days. The serum from fish that were vaccinated by either immersion or oral administration did not show detectable levels of agglutinating antibodies against V. anguillarum. Kodama, H., M. Moustafa, S. Ishiguro, T. Mikami, and H. Izawa. 1984. Extracellular virulence factors of fish vibrio: relationships between toxic material, hemolysin, and proteolytic enzyme. Am. J. Vet. Res. 45:22032207. Biological activities of cellfree culture filtrate of three virulent Vibrio strains were examined to determine their relation to the pathogenesis of fish vibriosis. Of the three strains examined, V. anguillarum strains NCMB6 and NCMB571 produced hemolysin and protease, but V. ordalii strain N7802 did not. A culture filtrate of strain NCMB571 was lethal to rainbow trout. Extracellular products may be involved in the pathogenesis of fish vibriosis. Kodama, H., M. Moustafa, T. Mikami, and H. Izawa. 1985. Characterization of extracellular substance of Vibrio anguillarum toxic for rainbow trout and mice. Microbiol. Immunol. 29:909920. An extracellular toxic substance was separated from the cell-free culture filtrate of Vibrio anguillarum. Two fractions obtained by Sephadex G200 chromatography after DEAE-cellulose chromatography were lethal to rainbow trout and mice. Peripheral vascular disorder was observed in fish and mice that died after inoculation. The toxic substance was sensitive to potassium periodate but was resistant to trypsin and acetone. Heat inactivation of the toxic substance was almost complete at 100°C for 20 min and complete at 121°C for 20 min. The toxic activity was not associated with hemolytic or proteolytic activity. Homologous antitoxin completely neutralized the toxic activity. Kothary, M. H., and A. S. Kreger. 1985. Purification and characterization of an extracellular cytolysin produced by Vibrio damsela. Infect. Immun. 49:2531. Large amounts of an extremely potent extracellular cytolysin produced by the halophilic bacterium V. damsela were obtained free of detectable contamination with constituents of the medium or other bacterial products. The cytolysin was heat labile, protease sensitive, and active against erythrocytes from mice, rats, rabbits, and damselfish. Kusuda, R., K. Kawaii, and T. Itami. 1980. Efficacy of bath-immunization against vibriosis in cultured ayu. Bull. Jpn. Soc. Sci. Fish. 46:1053. Cultured ayu were effectively immunized by immersion for 30 s in fullstrength inactivated V. anguillarum broth culture, or for 5 h in dilutions of the culture. Kusuda, R., K. Kawai, Y. Jo, T. Akizuki, M. Fukunaga, and N. Kotake. 1978. Efficacy of oral vaccination for vibriosis in cultured ayu. Bull. Jpn. Soc. Sci. Fish. 44:2125. Efficacy of four kinds of oral vaccine for control of vibriosis in cultured ayu was investigated. The vaccines were administered daily at a level of 0.4 g wet weight of cells per kilogram of body weight. The efficacy of vaccination was tested by exposing fish to the bacteria discharged from artificially infected ayu. The formalinkilled and ultrasonicated preparations were highly effective, but no antibody was produced in the blood. The heated bacterin also was effective when administered at a level of 4.0 g. Administration of formalinkilled bacterin at a level of 4.0 g for more than 2 weeks resulted in low mortality of about 10% in contrast to about 90% for the controls. The efficacy of vaccination was maintained for 4 weeks, and was further prolonged when the booster was administered. Lannan, J. E. 1978. Vibriosis vaccination of chum salmon by hyperosmotic infiltration. Prog. FishCult. 40:4345. Fry of chum salmon starting to feed were vaccinated with Vibrio anguillarum bacterin by hyperosmotic infiltration, and mortalities of vaccinated and unvaccinated fish were compared in ambient temperature, seawater flow, and controlled challenge experiments. In the controlled challenge experiment, cumulative mortalities after 10 days were 6% in the vaccinated group and 63% in the unvaccinated group; in the ambient challenge experiment the cumulative mortalities after 70 days in the respective groups were 6% and 96%. The differences were statistically significant (P < 0.005), indicating that survival was highly contingent on vaccination under the conditions observed. Levin, M. A., R. E. Wolke, and V. J. Cabelli. 1972. Vibrio anguillarum as a cause of disease in winter flounder (Pseudopleuronectes americanus). Can. J. Microbiol. 18:15851592. Vibrio anguillarum was repeatedly isolated from skin and muscle lesions of winter flounder. In the acute phase, dermal lesions were usually accompanied by fin necrosis, petechia, and ecchymoses; however, frank ulceration was usual in more chronic cases. Anemia was evidenced by lowered hematocrit values and increased renal hematopoiesis. The disease was reproduced experimentally by intradermal injection of as few as 640 cells of V. anguillarum. Differential identification of the V. anguillarum from aeromonads, plesiomonads, and other marine vibrios is described and discussed. Lewis, D. H. 1985. Vibriosis in channel catfish Ictalurus punctatus. J. Fish Dis. 8:539-546. In a Vibrio anguillarum epizootic in channel catfish, lesions in infected fish included ulceration and petechiae on the body surfaces, vent, and caudal peduncle. Internally, haemorrhages were present in liver and kidney and the intestinal tract was filled with a clear viscous fluid. Under conditions of the study, vibrios were apparently sequestered in kidney and liver during initial stages of infection (8 12 h after exposure). Later, bacterial numbers in blood were comparable to those in kidney and liver. Clinical signs in infected channel catfish were suggestive of cellular and tissue destruction and renal dysfunction. On the basis of data from this study and others, vibriosis appears to be a disease in which the agent is localized in certain tissues. Secondary septicemia may be incidental to factors that compromise host defenses. Love, M., D. TeebkenFisher, J. E. Hose, J. J. Farmer III, F. W. Hickman, and G. R. Fanning. 1981. Vibrio damsela, a marine bacterium causes skin ulcers on the damselfish (Chromis punctipinnis). Science 214:11391140. The previously undescribed marine bacterium Vibrio damsela was isolated from naturally occurring skin ulcers on the blacksmith, a species of temperatewater damselfish. Laboratory infection of the blacksmith with Vibrio damsela produced similar ulcers; V. damsela was pathogenic for four other species of damselfish but not for fish of other families. McCarthy, D. H., J. P. Stevenson, and M. S. Roberts. 1974. Vibriosis in rainbow trout. J. Wildl. Dis. 10:27. Clinical signs and histopathological changes related to epizootic vibriosis in rainbow trout during their acclimation to seawater are described, as are the biochemical characteristics of the Vibrio anguillarum isolate. Source of infection was believed to be the seawater used in acclimation. Moustafa, M., H. Kodama, T. Mikami, and H. Izawa. 1985. Toxic substance in culture filtrate of Vibrio sp. strain N7802, a poor producer of hemolysin and protease. Fish Pathol. 20:181-186. The culture filtrate of pathogenic Vibrio sp. (strain N7802) was fractionated by DEAEcellulose ion exchange chromatography. The fraction showing lethal activity in rainbow trout and mice was eluted from the column with a linear gradient of NaCl (DII fraction). The first peak (Gl fraction) obtained by purification of the DII fraction by Sephadex G200 gel chromatography was lethal for rainbow trout and mice. Neither protease nor hemolytic activity was observed in the GI fraction. In sodium dodecylsulfatepolyacrylamide disc gel electrophoresis, the GI fraction revealed one protein band with a molecular weight of 35 K and one periodic acidSchiff positive band in the same position as the protein band. These results indicate that Vibrio sp. strain N7802 produced an extracellular toxic substance that was distinct from hemolysin or protease, and that may play a role in the pathogenesis of vibriosis in rainbow trout. Muroga, K., M. Iida, H. Matsumoto, and T. Nakai. 1986. Detection of Vibrio anguillarum from water. Bull. Jpn. Soc. Sci. Fish. 52:641648. Vibrio anguillarum from freshwater ayu culture ponds and seawater of the Inland Sea of Japan was detected by a two-step culture method consisting of alkaline peptone water and BTB teepole agar. The bacterium was often cultured from seawater, especially at temperatures below 20°C, but not from freshwater, in spite of the occurrence of vibriosis in some ponds. It was demonstrated by in vitro experiments that the organism persisted in seawater but died within 3 to 5 h in freshwater. Muroga, K., H. Yamanoi, Y. Hironaka, S. Yamamoto, M. Tatani, Y. Jo, S. Takahashi, and H. Hanada. 1984. Detection of Vibrio anguillarum from wild fingerlings of ayu Plecoglossus altivelis. Bull. Jpn. Soc. Sci. Fish. 50:591596. From 1979 to 1983, V. anguillarum was isolated from wild fingerlings of ayu by selective enrichment in alkaline peptone water and subsequent plating on BTB teepole agar. The detection rate from fingerlings was 0.08, 2.5, 6, and 17% in Lake Biwa (freshwater lake), Lake Hamana (seawater lake), Hiwasa coastal sea, and the estuary of the Hiwasa River (Japan), respectively. The fingerlings caught in seawater or brackish water harbored V. anguillarum at higher rates than those caught in the freshwater lake, and the pathogen was detected exclusively in the later period of the season for fingerling collection in both freshwater and marine environments. Of 54 isolates of V. anguillarum, 32 were confirmed to be virulent for eels. Nelson, J. S., J. S. Rohovec, and J. L. Fryer. 1985. Location of Vibrio anguillarum in tissues of infected rainbow trout (Salmo gairdneri) using the fluorescent antibody technique. Fish Pathol. 20:229235. Vibriosis was induced in rainbow trout by intraperitoneal injection or waterborne infection with strain LS174. Every 3 h for 48 h, three fish were sampled and fixed for histological examination. The progression of the infection and the fate of the invading pathogen were determined by staining sections with antiV. anguillarum fluoresceinlabeled rabbit immunoglobulin G (IgG). The tissue location of the pathogen in both groups of infected fish was similar; the bacterium was initially sequestered in the spleen, where the numbers increased, followed by proliferation into the kidney. Death resulted from bacteremia, after most tissues of the fish were infected. Necrosis was extensive in kidneys, spleen, posterior intestine, and liver. The gills were congested with microorganisms and the epithelial cells destroyed. Extracellular bacterial antigen was observed in the musculature, kidneys, liver, intestine, and spleen. No phagocytosis by macrophages was observed. Park, S. W., and S. K. Chun. 1986. Characteristics of pathogenic Vibrio sp. isolated from cultured yellowtail Seriola quinqueradiata. Bull. Korean Fish. Soc. 19:147154. Vibriosis has caused severe losses among cultured yellowtail at some cage farms in Korea in recent years. Among the isolated bacteria from the diseased yellowtail, Vibrio sp. isolated from the kidney was considered to be the causative organism. Tetracycline, chloramphenicol, and gentamycin were bacteriostatic for the pathogenic strain, but sulfisomezole and sulfisoxazole were not. When the isolated strain was injected intramuscularly into yellowtail, red seabream, rock-bream, and common carp, it was virulent to all fish examined at 25°C but there was no virulence at 15°C, except in yellowtail. Ranson, D. P., C. N. Lannan, J. S. Rohovec, and J. L. Fryer. 1984. Comparison of histopathology caused by Vibrio anguillarum and Vibrio ordalli in three species of Pacific salmon. J. Fish Dis. 7:107116. The histopathology associated with naturally acquired vibriosis in chum salmon fingerlings caused by V. anguillarum was compared with that caused by infection with V. ordalii. Pathogenesis of the two forms differed. Bacteremia caused by V. anguillarum in the early stages involved 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. ordalli, bacteremia developed only in the late stages of the disease; the concentration of bacterial cells per milliliter of blood was less than in the V. anguillarum infection, by a factor of 102103. Tissues with the most pronounced changes were skeletal and cardiac muscle, anterior and posterior gastrointestinal tract, and the gills. V. ordalii observed in the tissues was not evenly dispersed but was present in 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, and chinook salmon. Severe decreases in circulating leukocytes accompanied bacteremia caused by either bacterial species. Roberson, B. S., S. Wolski, and F. M. Hetrick. 1982. Anti Vibrio anguillarum antibody among striped bass Morone saxatilis following injection with viable bacteria or ambient exposure to isolated Oantigen or environmental vibriosis. Abstr. Annu. Meet. Am. Soc. Microbiol. 82:B91. Striped bass held in filtered and unfiltered water drawn from the Chesapeake Bay were bled at intervals after they were injected with viable Vibrio anguillarum or were immersed for 2 min in Oantigen (50 mg/L) extracted from this bacterium. Serum antibody levels were estimated by thinlayer immunoassay and ELISA procedures by using the Oantigen as the test antigen. Enhanced antibody levels were detected in survivors of virulence titrations as well as in fish exposed to the Oantigen. Antibody in control fish (untreated or treated with phosphate buffered saline) was found after minor, latespring outbreaks attributable to vibrios entering the holding tanks by way of the water supply, and was accompanied by increased resistance to experimental challenge with viable V. anguillarum. In contrast, fish in freshwater failed to develop antibody unless intentionally exposed to vibrio antigens. Rohovec, J., R. L. Garrison, and J. L. Fryer. 1975. Immunization of fish for the control of vibriosis. Pages 105112 in Proceedings of the third U.S.Japan meeting on aquaculture, 1516 October 1974, Tokyo. Orally and parenterally administered vaccines were described. Fish injected with 2 x 108 formalinkilled cells mixed with Freund's complete adjuvant or fed a ration containing 0.5 mg formalinkilled whole cell bacterin for 15 days were protected against a natural challenge of Vibrio anguillarum. Oral immunization was effective at 4 to 21°C. Increasing the amount of vaccine fed or the period of feeding did not increase protection. Ross, A. J., J. E. Martin, and V. Bressler. 1968. Vibrio anguillarum from an epizootic in rainbow trout (Salmo gairdneri) in the U.S.A. Bull. Off. Int. Epizoot. 69:11391148. Isolants of Vibrio anguillarum from a freshwater hatchery were characterized and compared with a known isolate of V. anguillarum. Rucker, R. R. 1959. Vibrio infection among marine and freshwater fish. Prog. FishCult. 21:2225. A review of the literature and a report of the first documented cases of vibriosis in North American hatcheries. Sakai, M., T. Aoki, T. Kitao, J. S. Rohovec, and J. L. Fryer. 1984. Comparisons of the cellular immune response of fish vaccinated by immersion and injection of Vibrio anguillarum. Bull. Jpn. Soc. Sci. Fish. 50:11871192. Immune responses, measured by plaqueforming cell assay and rosetteforming cell assay in anterior kidney and spleen, and serum hemagglutinating antibody titers, were stronger in rainbow trout given formolized V. anguillarum vaccine by intraperitoneal injection along with Freund's complete adjuvant than in those given the vaccine by immersion, without adjuvant. Sakai, M., T. Aoki, T. Kitao, J. S. Rohovec, and J. L. Fryer. 1986. Fluctuation in the number of bacterial cells in organs of vaccinated fish after artificial challenge. Bull. Jpn. Soc. Sci. Fish. 52:249255. After coho salmon were challenged with Vibrio anguillarum, the organism appeared in the organs of both vaccinated and unvaccinated fish within 30 min. In vaccinated fish, the numbers of the organism did not change for 10 h, then gradually decreased, and were completely eliminated at 72 h. In unvaccinated fish, the numbers steadily increased and all fish had died by 72 h. The organism was eliminated more rapidly by fish vaccinated by injection than by those vaccinated by immersion. Sako, H., and R. Kusuda. 1978. Chemotherapeutical studies on trimethoprim against vibriosis of pondcultured ayu-I. Microbiological evaluation of trimethoprim and sulfonamides on the causative agent Vibrio anguillarum. Fish Pathol. 13:9196. A 3day feeding of trimethoprim at 30 mg/kg controlled both experimental and natural vibriosis in ayu. Schiewe, M. H. 1983. Vibrio ordalii as a cause of vibriosis in salmonid fish. Pages 3140 in J. H. Crosa, ed. Bacterial and viral diseases of fish. Washington Sea Grant Program, Seattle. A review of the current knowledge of V. ordalii as a cause of vibriosis; the cultural, biochemical. and serological properties that separate V. ordalii from V. anguillarum; and the pathogenesis of V. ordalli. Schiewe, M. H., T. J. Trust, and J. H. Crosa. 1981. Vibrio ordalii sp. nov.: a causative agent of vibriosis in fish. Curr. Microbiol. 6:343348. Vibrio ordalli sp. nov. is the name proposed for the bacterium previously designated as V. anguillarum biotype 2. The change in classification is based on differences between the classical V. anguillarum and V. ordalii in cultural and biochemical characteristics, and in similarity of the deoxyribonucleic acid sequence. Schreck, C. B., R. Patino, C. K. Pring, J. R. Winton, and J. E. Holway. 1985. Effects of rearing density on indices of smoltification and performance of coho salmon, Oncorhynchus kisutch. Aquaculture 45:345358. Effects of raceway rearing density on coho salmon were evaluated at Eagle Creek (Washington) National Fish Hatchery at the time production fish were released from the facility. The production rearing density, compared with lower densities (onethird and twothirds), appeared to delay or impair smoltification as judged by lower plasma thyroxine levels, gill (Na+K)-ATPase activity, and blood sodium regulatory ability in fish at the higher density. High rearing density also lowered the capacity of the fish to resist Vibrio anguillarum. Smith, G. C., and J. R. Merkel. 1981. Collagenolytic activity of Vibrio vulnificus: potential contribution to its invasiveness. Infect. Immun. 35:11551156. Vibrio vulnificus (a lactosepositive vibrio) produced collagenase when grown in 2% synthetic sea salts supplemented with hydrolyzed casein. The addition of collagen or peptone to the medium increased the level of collagenase production. Collagenase activity was inhibited by EDTA but not by fetal calf serum Sorensen, U. B. S., and J. L. Larsen. 1986. Serotyping of Vibrio anguillarum. Appl. Environ. Microbiol. 51:593597. A serotyping scheme based on the detection of Oantigens by slide agglutination in fishpathogenic 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 and divided into 10 distinct serotypes (01 through 010). More than 90% of the fishpathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype 01, whereas 78% of the strains isolated from feral fish were serotype 02. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. Tanaka, J. 1975. Vibrio infection of marine fishes. Pages 113114 in Proceedings of the third U.S.Japan meeting on aquaculture, 1516 October 1974, Tokyo. Vibriosis in marine fishes cultured in Japan is described, and the characteristics of vibrio anguillarum and control of vibriosis are discussed. Tashiro, F., S. Moridawa, A. Montonishi, I. Sanjo, N. Kimura, K. Inque, T. Nomura, M. Ushiyama, Y. Jo, F. Hayashi, and I. Kunimine. 1979. Studies on chemotherapy of fish diseases with piromidic acid-II. Its clinical studies against bacterial infections in cultured salmonids and eels. Fish Pathol. 14:93101. Piromidic acid controlled vibriosis, furunculosis, and edwardsiellosis when administered for 5 to 11 days at 10, 20, or 40 mg/kg. Tatner, M. F., and M. T. Horne. 1986. Correlation of immune assays with protection in rainbow trout Salmo gairdneri immersed in vibrio bacterins. J. Appl. Ichthyol. 2:130139. Rainbow trout weighing 80100 g each and totaling 100 kg were immersed in a formolized, bivalent vaccine of Vibrio anguillarum and Vibrio ordalli, at a 1/10 dilution for 30 s at 10°C. The protection levels developed at 8 and 10 weeks post vaccination were assessed by intraperitoneal challenge with 105 cells of live V. anguillarum or V. ordalli. No decline in protection due to antigen depletion was found between batches throughout the procedure. The most likely immune mechanism responsible for the observed protection was suggested to be cellular and to involve the action of serum antibody as an opsonin to increase phagocytosis. Tison, D. L., M. Nishibuchi, J. D. Greenwood, and R. J. Seidler. 1982. Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels. Appl. Environ. Microbiol. 44:640646. Clinical and nonclinical isolates of the lactosepositive Vibrio vulnificus were compared with Vibrio strains isolated from lesions on eels cultured commercially in Japan. Strains were compared phenotypically and antigenically, for pathogenicity to mice and eels and for genetic relatedness. The strains isolated from diseased eels differed phenotypically from the original species description of V. vulnificus in that they were negative for indole production, ornithine decarboxylase activity, growth at 42°C, and acid production from mannitol and sorbitol. No relation between the surface antigens of V. vulnificus strains from environmental and clinical sources and the strains from diseased eels was observed. Typical V. vulnificus strains and the eel isolates were pathogenic to mice; however, only the strains originally isolated from diseased eels were pathogenic to eels. It was proposed that strains phenotypically similar to the type strain of the species be classified as V. vulnificus biogroup 1, and the strains phenotypically similar to those isolated from diseased eels be classified as V. vulnificus biogroup 2. Tolmasky, M. E., L. A. Actis, A. E. Toranzo, J. L. Barja, and J. H. Crosa. 1985. Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain. J. Gen. Microbiol. 131:19891998. Vibrio strains isolated from diseased turbot at an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates harbored a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were closely similar but not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitations. Tolmasky, M. E., and J. H. Crosa. 1984. Molecular cloning and expression of genetic determinants for the iron uptake system mediated by the Vibrio anguillarum plasmid pJM1. J. Bacteriol. 160:860866. The high pathogenicity of this vibrio for fish is conferred by plasmid pJM1, which mediates an efficient iron transport system. Experimental infection showed that the pathogenicity increased when the ability of the vibrio to grow under conditions of iron limitation was restored by introducing a recombinant clone into a strain of low virulence. Toranzo, A. E., J. L. Barja, and S. Devesa. 1985. First isolation of Vibrio anguillarum biotype I causing an epizootic in reared turbot Scophthalmus maximus in Galicia northwest Spain. Invest. Pesq. 49:6166. An epizootic of vibriosis in juvenile turbot reared in an experimental system in Galicia, northwest Spain, is described. Vibrio anguillarum biotype I was isolated as pure culture from the internal organs of diseased fish. Pathogenicity assays confirmed that this vibrio strain was the causative organism of the epizootic. Trust, T. J., I. D. Courtice, A. G. Khouri, J. H. Crosa, and M. H. Schiewe. 1981. Serum resistance and hemagglutination ability of marine vibrios pathogenic for fish. Infect. Immun. 34:702707. Representative strains of marine vibrios pathogenic for fish were shown to be resistant to the bactericidal activity of normal (nonimmune) rainbow trout serum, and loss of this resistance coincided with a marked reduction in virulence. Thermal lability and a requirement for Mg, but not for Ca, suggested that a mechanism for bactericidal serum activity was the alternative complement pathway. In Vibrio anguillarum, serum resistance was not coded for by the virulence plasmid pJM1. Umbreit, T. H., and M. R. Tripp. 1975. Characterization of the factors responsible for the death of fish infected with Vibrio anguillarum. Can. J. Microbiol. 21:12721274. An extracellular toxin produced by Vibrio anguillarum affected goldfish; it was toxic after being heated to 100°C. Wertheimer, A. C., and R. M. Martin. 1986. Growth and survival to adulthood of chum salmon Oncorhynchus keta vaccinated as juveniles against vibriosis. Trans. Am. Fish. Soc. 115:343346. Three consecutive year classes of juvenile chum salmon were vaccinated with Vibrio sp. bacterin, marked, and released into the marine environment. The return rates of adults from these releases were similar to those of unvaccinated control groups for two of the three year classes and were significantly lower for one year class. The vaccinated and control groups showed no difference in size or age distribution at return. Thus vaccination procedures did not increase survival or growth of these freely migrating chum salmon.

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