魚 病 研 究Gyobyo Kenkyu,26(1),17-20,1991.3 Injection Vaccination of White-Spotted Char, Salvelinus leucomaenis, against Furunculosis with Aeromonas salmonicida Salmolysin EijiroKawahara, Tomohiro Ueda and SetsuzoNomura LaboratoryofAquatic Microbiology,School of Fisheries Sciences, KitasatoUniversity,Sanriku, Kesen,Iwate 022-01, Japan (ReceivedAugust 22, 1990) Aeromonas salmonicida salmoysin was detoxified by heating at 37•Ž for 24 h, at 60•Ž for 30 min, at 100•Ž for 5 min, or heating at 37 or 60•Ž for 30 min with 0.4 % (v/v) formalin. These detoxified salmolysins and native salmolysin reacted with rabbit anti-native salmolysin serum. Juvenile white-spotted char (Salvelinus leucomaenis) were immunized two times at two-week intervals by intramuscular injection of salmolysin which was detoxified by heating at 60•ŽC for 30 min. Mor talities of immunized and control fishes after challenge were 28.6 and 62.5 %, respectively . An antibody to salmolysin was detected in immunized fish sera, but not detected in control fish sera. These results indicate that detoxified salmolysin is an effective immunogen for the prevention of furunculosis in white-spotted char. detoxified salmolysin for white-spotted char. Introduction Furunculosis, caused by Aeromonas salmonicida, Materials and Methods occurs particularly in farmed white-spotted char (Salvelinus leucomaenis) and coho salmon (Oncor Fish hynchus kisutch) in Japan. Damage by this Juvenile white-spotted char (Salvelinus leucoma disease in these fish is severe and economically enis) weighing about 2.8 g were used in this vaccine important. experiment. Fish erythrocytes for determination To develop an effective vaccine for the disease, of hemolytic activity were collected from yearing the protective immune response of fish against white-spotted char weighing about 100 g. These various A. salmonicida antigens have been in fish were separately maintained in flowing water vestigated by many workers. at 13-15•Ž and fed daily with commercial pellets Recently, Kawahara et al. (1990) showed that (Nihon Nosan Kogyo). A. salmonicida salmolysin had a higher lethal toxicity to rainbow trout (0. mykiss) and cytopathic Bacteria effects to coho salmon lymphocytes than extracel Aeromonas salmonicida A-1, isolated from lular protease, and that in extracellular products, diseased rainbow trout (Oncorhynchus mykiss) only salmolysin possessed immunogenicity to was used for purification of salmolysin (Nomura white-spotted char. and Saito, 1982). A. salmonicida A-5 was isolated Kawahara and Nomura (1989) reported the from diseased coho salmon (0. kisutch) obtained the humoral immune response of white-spotted from a fish farm in Tamayama, Iwate Prefecture char against salmolysin detoxified by heating or on June 23, 1987 and used for the experimental formalin treatment. After intramuscular injec challenge. tion of detoxified salmolysins, serum antibody t iter was raised and remarked 1: 16 to 1: 64 after Salmolysin Purification and Hemolytic Activity three to five weeks. Assay The present study describes on the protective Salmolysin was purified from the culture su effect of intramuscular injection vaccination using parnatant of A. salmonicida A-1. All procedures 18 E. Kawahara, T. Ueda and S. Nomura for salmolysin purification and hemolytic activity 30 min. After anaesthetization with 4-allyl-2 assay were performed as previously described by methoxyphenol (FA 100, Tanabe), fish were im Nomura et al. (1988). munized intramuscularly with 7.5 ƒÊg detoxified salmolysin in 50 ƒÊl THB. A booster immuniza Detoxification tion inoculated by the same manner two weeks Salmolysin in 20 mM Tris-hydrochloride buffer later. Control fish were intramuscularly in (THB) at pH 7.0 was incubated at 37, 60 or 100•Ž jected two times with 50 ƒÊl THB at two-week with or without 0.4 % (v/v) formalin for 5, 30, 60, intervals. 90 min, 6 or 24 h. The detoxification of the treated salmolysin was assayed by hemolytic Challenge activity. Prior to the challenge test, the median lethal dose (LD50) of A. salmonicida A-5 against the experi Antiserum mental fish was determined and calculated as Rabbit anti-salmolysin sera were prepared by 6.8 •~ 10<SUP>2<SUP>colony forming unit (CFU)/fish. Two the same method as previously reported (Nomura weeks after the second immunization, fourteen et al., 1988). vaccinated and sixteen control fish were each challenged at a dose of 9 •~ 103 CFU/fish by in Immunodiffusion Analysis tramuscular injection. Mortalities were recorded The immunodiffusion analysis was carried out for 14 d after challenge. Reisolation of the path by the method of Ouchterlony. ogen from the kidney of dead fish was performed using a trypto-soya agar medium (Nissui). From Protein determination the surviving fish, sera were obtained by the routine Protein concentration was determined by the method. method of Lowry et al. (1951) using bovine serum albumin as a standard. Results Vaccination Salmolysin was inactivated by heating at 37•Ž Salmolysin was detoxified by heating at 60•Ž for for 24 h, at 60•Ž for 30 min or at 100•Ž for 5 min, Fig. 1. Detoxification of Aeromonas salmonicida salmolysin. Salmolysin was detoxified by heating at 37 (•£), 60 (•¡) or 100 (•œ) •Ž, or at 37 (•¢) or 60 (• ) •Ž with 0.4 % (v/v) formalin. Vaccination of Char against Furunculosis 19 Fig. 4. Detection of precipitating antibody against salmolysin in immunized or control white- spotted char serum by immunodiffusion. Precipitating line was stained with 1 % (w/v) Amide Black 10 B in 7 % (v/v) acetic acid. I, immunized char serum; C, control char Fig. 2. Immunodiffusion analysis of detoxified serum; S, salmolysin. salmolysin. Precipitating lines were stained with 1 % (w/v) Amide Black 10 B in 7 % (v/v) or heating at 37 or 60•Ž for 30 min with 0.4 % acetic acid. Outer well ; detoxified salmolysin by heating at 37•Ž for 24 h (A), at 60•Ž for (v/v) formalin (Fig. 1). 30 min (B) or at 100•Ž for 5 min (C), or heat- Detoxified salmolysin and native salmolysin re- ing at 37•Ž for 30 min with 0.4 % (v/v) forma- acted to rabbit anti-native salmolysin serum by lin (D), native salmolysin (E), 20 mM Tris- using the immunodiffusion test (Fig. 2). HC1 buffer at pH 7.0 (F): center well ; anti- In the experimental challenge test, mortalities native salmolysin serum. of immunized and control fish were 28.6 and 62.5 %, respectively (Fig. 3). The pathogen was reisolated from the kidney of these dead fish. Precipitating antibody against salmolysin was detected in immunized fish sera, but not detected in control fish sera (Fig. 4). Discussion The virulent factors of A. salmonicida have been investigated by many workers. At present, ex- tracellular hemolysins, termed T-lysin and salmo lysin (Titball and Munn, 1983; Nomura et al., 1788) and extracellular proteases (Shieh and MacLean, 1975; Rockey et al., 1988) are regarded as the main virulent factors of the pathogen. Fyfe et al. (1988) reported A. salmonicida ECP Fig. 3. Mortality of white-spotted char immunized with detoxified salmolysin after intramuscular and a mixture of hemolysin and protease had challenge with 9 •~ 103 CFU/fish of A. sal- similar pathologic effects in Atlantic salmon. monicida A-5. •œ; immunized, ; •›control. Lee and Ellis (1987) showed an additive relation- 20 E. Kawahara, T. Ueda and S. Nomura ship of lethality between hemolysin and protease No. 63560196. in Atlantic salmon by intraperitoneal injection. We wish to thank Mr. Teruaki Inarimori, In this study, the LD50 of hemolysin and protease staff of Iwate Prefectural Institute of Inland when injected separately were 44 and 2,400 ng/g Water Fisheries, for supplying the bacterium, and fish, respectively, and hence the lethality of the Mr. Takayuki Ishikawa in our laboratory for his. hemolysin is about fiftyfive times higher than that technical assistance. of the protease. Kawahara et al. (1990) reported the LD50 of References partial purified salmolysin or protease were each 152 and <1,514 ƒÊg/kg body weight in juvenile Fyfe, L., G. Coleman and A. L. S. Munro (1988): The combined effect of isolated Aeromonas salmonicida rainbow trout by intramuscular injection and that the lethality of the salmolysin fraction was protease and haemolysin on Atlantic salmon, Salmo salar L., compared with that of a total extracellular about ten times higher than that of the protease products preparation. J. Fish Dis., 11, 101-104. fraction. They also showed that cytopathic Hastings, T. S. and A. E. Ellis (1988): The humoral effects to coho salmon lymphocytes occurred by immune response of rainbow trout, Salmo gairdneri a combination of salmolysin and protease. Richardson, and rabbits to Aeromonas salmonicida Concerning the immunogenicity of both lethal extracellular products. ibid., 11, 147-160. factors in rainbow trout and white-spotted char, Kawahara, E. and S. Nomura (1989): Lethal toxicity immunogenicity of hemolysin was found but that and immunogenicity of salmolysin, Aeromonas of protease was very low or nonexistent (Hastings salmonicida hemolysin to white-spotted char, Sal and Ellis,1988; Kawahara and Nomura, 1989; velinus leucomaenis. Fish Pathol., 24, 149-154. Kawahara et al., 1990). Kawahara, E., S. Oshima and S. Nomura (1990): Toxicity and immunogenicity of Aeromonas sal These reports suggest salmolysin is very im monicida extracellular products to salmonids. J. portant as a vilulent factor and only possesses Fish Dis., 13, 495-503. immunogenicity to salmonids. Vaccination using Lee, K.-K. and A. E. Ellis (1989): The quantitative salmolysin toxoid may therefore be effective for relationship of lethality between extracellular pro the prevention of furunculosis. tease and extracellular haemolysin of Aeromonas Previously, after a single injection of salmolysin, salmonicida in Atlantic salmon (Salmo salar L.). detoxified by heating or formalin treatment, FEMS Microbiol. Lett., 61, 127-132. serum antibody titer of white-spotted char was Lowry, O. H., N. J. Rosenbrough, A. L. Farr and R. raised on three to five weeks later (Kawahara and J.