魚 病 研 究 Fish Pathology 20(2/3)125-129, 1985.9 Characteristics of a Vibrio sp. Associated with the "Hitra Disease" of Atlantic Salmon in Norwegian Fish Farms K. Olav HOLM*1, Ellen STROM*2, Klara STENSVAG*1, Jan RAA*2, and Trond JORGENSEN*1 *1The Research Foundation, University of Tromso, P.O. Box 3063, Guleng, N-9001 Tromso, Norway *2Institute of Fisheries ; University of Tromso A Vibrio shaped bacterium has been isolated from nephric tissues of moribund atlantic salmon suffering from the socalled "Hitra disease". Eighteen isolates of this bacterium, from geographically distant fish farms all along the Norwegian coast, were shown to be very similar in biochemical properties. Serotyping and DNA fingerprinting provided additional evidence for similarity between the isolates and that they clearly differ from Vibrio anguillarum and Vibrio ordalii. The particular Vibrio associated with the "Hitra disease" has been designated Vibrio sp. TEO, and is assumed to be involved in the disease development. The bacterium has not been found in any healthy fish, even in farms with outbreaks of "Hitra disease". The Vibrio sp. TEO elicited disease with similar symptoms when injected into healthy fish, and the same bacterium could be reisolated. Introduction of a particular Vibrio which differs from V. an guillarum and V. ordalii in moribund "Hitra dis The most serious disease of cultured salmon in eased" salmon sampled from fish farms all Norway is called "Hitra disease", a designation along the Norwegian coast. In the present paper referring to the island where it for the first time was we describe this bacterium biochemically, geneti recognized as an economic threat. A more de cally and serologically. scriptive name, however, is haemorrhagic syn drome (POPPE et al., 1984), since the affected in Materials and Methods dividuals have haemorrhages externally on the ab domen and internally on the swimming bladder, Bacterial Strains and Growth Conditions posterior gastrointestinal tract and on the abdomi The particular Vibrio bacterium described in the nal wall (external symptoms may be abscent!). present paper has been designated Vibrio sp. TEO. Microscopy has revealed damages of endothelial Strains of this bacterium have been isolated from cells in the liver, spleen and nephros (non-publish moribund salmon with typical symptoms of "Hitra ed), indicating that bacterial toxins may be involv disease" in fish farms from all along the Norwegian ed. This suggestion is supported by the fact that coast. Eighteen TEO strains from geographically moribund fish are anemic, in most cases, severely. distant fish farms have been compared with 9 strains A Vibrio bacterium has earlier been isolated from of V. anguillarum, including HI-10 (S. gairdneri), salmon with "Hitra disease" or "cold-water HI-F (S. salar) (kindly provided by Dr. E. Egidius, vibriosis" and shown to posess the ability to Institute of Marine Research, Bergen, Norway), A cause disease with similar symptoms (EGIDIUSet 3.1 (Gadus morhua), NCMB 6 (G. callarias) and al., 1981, 1984). Nevertheless, there have been sec with 2 strains of V. ordalii, DF1K and MSC2-75 ond opinions about the cause of this disease; im from Oncorhunchus kisutch. Samples from nephric balanced feed and adverse environmental condi tissue were seeded on Bacto tryptose blood agar tions have been claimed to be the major disease base (Difco) supplemented with 1.5% NaCl and 3% conditioning factors (POPPEet al., 1984). We have, human red blood cells (HRBC). The bacteria grow however, been able to demonstrate the presence ing up were maintained at 12•Ž in basal medium 126 K. Olav HOLM et al. composed of 0.27% nutrient broth (Difco), 0.5% solution (Norsk medisinaldepot, Oslo-Norway) proteose pepton (Difco), 2.0% NaCl and 1.0% (v/v) and the optical density at 540 nm recorded. The mineral solution (FORD et al., 1958). When growth haemoglobin values were estimated from a standard at various temperatures and with different carbon curve, using haemoglobin standards (DADE, W. sources were examined, the basal medium was Germany). supplemented with 5% (v/v) of the peptide fraction of a fish autolysate (CLAUSEN et al., in preparation) Endonuclease Fingerprints of Chromosomal DNA and the pH was adjusted to 7.5 using KOH. Stock The bacterial cultures were harvested in early cultures were maintained at -80•Ž on the same stationary phase. Preparation of bacterial DNA, medium supplemented with 20% sterile , glycerol. digestion with restriction endonuclease (HindIII; Bacterial cells grown for 30 hours on the blood agar New England Biolabs, Beverly, Mass.), separation were used for Gram staining. of DNA fragments by polyacrylamide gel electro phoresis and staining were done as described by Biochemical Tests BJORVATNet al. (1984). The gels were photographed The concentration of NaCl in all the growth in UV light and the negatives scanned by a LKB media was adjusted to 2.0%. For determination of (Bromma, Sweden) 2202 Ultrascan apparatus. oxidative-fermentative metabolism of glucose, 1% (final concentration) of filter sterilized glucose was Antisera added to two tubes containing the basal medium Rabbit antisera against various Vibrio bacteria and with an inverted Durham tube (gas produc were obtained by 4 to 5 subcutaneous injections of tion); one of the tubes overlayed with sterile mineral viable bacteria at 2 to 3 weeks intervals. The first oil. was given in complete Freund's adjuvant (Behring API 50 CH galleries (API SYSTEM S.A. werke AG, Marburg W. Germany), the second in France) were used for determination of oxidative incomplete Freund's adjuvant and thereafter in fermentative metabolism of different carbon sou saline. The animals were bled 10 days after the last rces. API 20 B galleries were used for the following injection. tests: oxidase (Kovacs), indole production, Voges Proskauer, nitrate reduction, catalase, growth on Radioimmunoassay citrate (Simmons), H2S production, gelatine hy Serological classification was performed using a drolysis, beta-D-galactosidase activity (ONPG) and radioimmunoassay (RIA, Fig. 1). Cells were har urease (API 20 B medium added 1.5% NaCl). vested in the midgrowth phase by centrifugation Lysine and ornithine decarboxylase, alkaline re at 4,000•~g for 10 min (4 •Ž). The pellet was resuspended action with arginine (Mollers decarboxylase me in phosphate buffered saline (PBS, pH dium) and starch hydrolysis were tested as described 7.4). All buffers used contained 0.02% Na-azide by HARRIGANand MCCANSE(1966). Growth on and were kept at 4 •Ž. citrate (Christensen) was examined using a modified Viable bacteria (OD600 = 0.20, 100ƒÊl) were mixed Christensens medium (Difco). with (100ƒÊl) inactivated (56•Ž, 30 min) rabbit anti- Lipase with 1% tributyrin as the substrate was assayed in tryptic soy agar (TSA, Difco). Salt tolerance was determined using the basal agar me dium containing various NaCl concentrations (0.0-10.0%). Haemolytic activity was tested on the blood agar (HRBC). Sensitivity to various anti biotics was examined on TSA using discs soaked with antibiotics. Haemoglobin Assay Haemoglobin values (g/100ml) were determined using a standard photometric method: 20ƒÊl un Fig. 1. The principle of the radioimmunoassay. 5ml coagulated blood was mixed with 5.0 ml Drabkin's polystyrene test tubes were used. A Vibrio sp. Associated with the "Hitra Disease" 127 vibrio antiserum (ab1) diluted (1/1,600) in PBS Table 1. The haemoglobin content (g/100ml) of blood added 5% inactivated normal sheep serum (PBSS). sampled from "Hitra diseased" and healthy The mixture was incubated for 1 hour at 12•Ž and atlantic salmon (Salmo salar) then washed by adding 4.5 ml of PBS supplemented with 0.2% bovine serum albumine (PBSA, Sigma) followed by centrifuging at 1.000•~g for 30 min (4•Ž). The supernatant was decanted and 50 ng (200ƒÊl, diluted in PBSS) of iodinated (125I), affinity purified sheep anti-rabbit immunoglobulin was added (ab2). After another one hour of incubation Table 2. Biochemical properties of representative strains of Vibrio anguillarum, Vibrio ordalii and a new washing step, the tubes were dried in an and Vibrio sp. TEO up-side-down position. Radioactivity in the tubes were recorded in a LKB 1272 Clinigamma appara tus and the radioactivity bound was calculated from the following formula: Xi and NRS represent cpm bound in triplicates using specific antiserum and normal rabbit serum respectively (ab1). Iodination of affinity purified sheep antirabbit immunoglobulin was done as described by FRAKER and SPECK(1978). Results It has been invariably demonstrated that mori bund salmon with symptoms of "Hitra disease" contained a Vibrio bacterium, here designated Vibrio sp. TEO, which differs from both V. anguil larum and V. ordalii. This bacterium could not be isolated from healthy fish, even in farms with outbreak of "Hitra disease". Diseased fish always The results were recorded as + = positive , (+) = had lower than normal levels of blood haemoglobin weakly positive and - = negative. Tests in which the (Table 1). Anemia characterized by a low haemo bacteria were identical are not shown. * From SCHIEWE globin or haematocrit value is typical for many et al (1981). bacterial fish diseases. However, whether the pres ent anemia is due to a specific haemolysin (MUNN, small (1-2 mm) circular, convex and "opaque" col 1978), non specific cytolysins (MUNROet al ., 1980) onies within 3-4 days at 12•Ž. It is a Gram or to other factors causing haemorrhages in fish, is negative, curved, nonencapsulated, oxidase po not clear. sitive, fermentative and motile rod. No spores were Injection of Vibrio sp. TEO into healthy fish kept observed. In contrast to V. anguillarum and V. in experimental tanks in the laboratory resulted in ordalii it does not grow at 25•Ž and has optimum similar symptoms as those of the "Hitra disease". growth between 12-16•Ž. Optimum NaCl con Reisolation and characterization confirmed the centration was 1.5-2.0%, whereas no growth re presence of the same bacterium in the nephros of sulted at concentrations above 5% and below 0.5%.