Fish Pathology 15 (3/4) 257-262,1981. 3 Mechanisms Protection Ayu Orally Vaccated for Vibriosis Kenji KAWAI*, Riichi KUSUDA* and Toshiaki ITAMI* *Fish Dis. Lab., Fac. Agr., Kochi Univ., Nankoku, Kochi, Japan Both orally immunized ayu, Plecoglossus altivelis, and control challenged, Vibrio anguillarum, by three ways. First, exposed discharged naturally fected for 24 hr. Organism was isolated at high percentage almost all part body control, while isolation rate was low particularly on body surface immunized. Secondary, bad bacterial suspension a concentration 107 per milliliter for 15 m. Twenty four hours after challenge was isolated sk control, n number creased gradually for next 72 hr. No was detected teste or its contents both groups neir sk im munized. Fally, jected tramuscularly resultg almost equal mortality both groups. Agglut titer body surface rose 1: 64 immunized, but did not occur control. Agglut titer rose neir serum nor testal both groups. The body surface immunized prevented adherg sk more effectively than that control did. From se results it is assumed that defence effect by oral immunization is attributed maly agglut secreted body surface. In recent years many attempts made control vibriosis ayu by immunization Japan. Three techniques, such as oral admistration (KUSUDA et al., 1978), bath immunization (AOKI and KITAO, 1978) and spray immunization (ITAMI and KUSUDA, 1978), had been used for purpose, and was found effective respectively if properly applied. While immunization by above men tid techniques proved be effective mechanism protectg vibriosis has not yet been explaed. It was probably due lack formation about mechanism fection and antibody takg part directly pro tection. Experiments conducted see enterg site body for pathogenic and protection reaction on very site. Distribution after challenge Experimental 20-50 g ayu divided two groups consistg 10 or 20. One group was fed bacter mixed dry pellet, and anor was fed pure pellet. Bacter was admi stered at a level 1 g wet cell/kg /day for ten days. On next day after vaccation, challenged by exposg s discharged naturally fected for 24 hr. Soon after challenge rsed physiological sale and sticked filter paper on body surface as shown Fig. 1 and between gill lamellae. The filter papers oculated enrichment medium, nutrient broth 2 % NaCl. Air blad der, gullet, contents gullet, smach, contents smach, pyloric appendage, teste, con tents teste, spleen and kidney also oculated same medium. Cultures after 24 hr cubation at 25 Ž spreaded on nutrient agar 2 % NaCl, 1 % sucrose and 0.004% bromthymol blue. After 2 day cubation yellow or yellow green colonies harvested and sus pended distilled water. The suspension was used as reactive antigen for immuno-diffusion agast anti V. anguillarum V-36 serum. Forma tion precipit le dicated existence V. anguillarum organ or on site body surface. Experiment was repeated three times. Isolatfion rate each organ 40 tal is shown Table 1. Between both groups same isolation rate was shown spleen and kidney, while or sites and organs high rate was recorded control
258 K. KAWAI, R. KUSUDA, and T. ITAMI especially on body surface. Fig. 1. Sites on body surface ayu for isolatg Vibrio anguillarum after challenge. Fish challenged by exposg s discharged naturally fected for 24 hr. Changes number sk, teste and its contents after challenge Fish, 20-50 g weight, vaccated for ten days as mentid earlier. Five days after vac cation immunized and control challenged by dippg a bacterial suspension a concentration 107 per milliliter for 15 m. At 1, 6, 12, 24, 48, 72 and 96 hr after challenge three taken respectively and employed for bacterial count sk, teste and its contents. After rsg PBS sk was peeled f at a width 16 cm<sup>2<sup>. Then teste and its contents separated. Each sample was homogenized and V. anguillarum counted BTB-sucrose agar plate and by slide agglutation anti V-36 serum. Result is shown Table 2. No was detected all samples after 96 hr immuni zed. While was detected sk 24 hr after challenge, and number creased Table 1. Distribution Vibrio anguillarum ayu exposed for 24 hr s discharged naturally fected
Mechanisms Protection Ayu Orally Vaccated for Vibriosis 259 Table 2. Changes number Vibrio anguillarum on various sites ayu after challenge by dippg bacterial suspension Table 3. Mortality and LD50 by tramuscular jection Vibrio anguillarum gradually for next 72 hr control. No was detected eir teste or its contents control for 96 hr after. challenge. Mortality and LD50 by tramuscular jection Fish, 10-20 g weight, vaccated for 14 days as previously mentid. On next day after vaccation immunized and control
260 K. KAWAI, R. KUSUDA, and T. ITAMI challenged by tramuscular jection 0.24-240. As shown Table 3 died 3-4 days after challenge immunized and 2-3 days after challenge control. Mortality jected 2.4 1/5 immunized and 2/5 control. From se re sults LD50 was calculated 4.8 per immunized and 3.0 per control. Agglut titer and serum Fish, 20-50 g weight, vaccated for 10 days as mentid earlier. Immunized 4 days after vaccation and control used. After anestizg chilled water, body surface was rsed away PBS sponge. Intestal was washed out contents it PBS. Both salted out 50% saturated solution ammonium sulfate, n dialysed and concentrated polyethylene glycol powder. Agglut titer and serum quantified V-36 cell antigen. Experi ment was repeated twice. In first experiment, 10 group used, and only body sur face was sampled. Samples 5 pooled and concentrated ratio 0.8 ml per. In second experiment, 20 group used, and body surface, testi mal and serum sampled. Samples 10 pooled and was concentrated ratio 0.4 ml per. As shown Fig. 2 first experiment, titer body surface was 1 :16 immunized, while 1: 2 control. In second experiment, titer body surface, testal and serum 1: 32, 1: 4 and 1: 4 respec tively immunized, while results 1: 2, Fig. 2. Agglut titers and serum im munized and control. 1: 2-4 and 1: 4 respectively control. From se fdgs it was apparent that agglu t titer body surface rose by oral im munization. Protective effect passive immunization by jec tion serum orally immunized Ne group jected tramuscu larly 0.1 ml pooled serum orally vacci nated used foregog experiment on agglu t titer. Anor 9 jected serum control. Two days after jection exposed discharged natural ly fected for 6 days. Result is shown Table 4. Two died immunized group and 9 died control group for 6 days. Adhesion-hibition reaction body surface for sk Fish, 25-40 g weight, vaccated for 10 days as mentid before. On next day vaccation, body surface immunized and control sampled and concentrated as earlier mentid. One half ml bacterial Table 4. Protective effect by passive immunization orally vaccated ayu serum
Mechanisms Protection Ayu Orally Table Fig. 3. Schematic drawg sion-hibition suspension milliliter vials, 8 any covered was for while Result. Vials hour ; trol 855 munized has previous immunized a function sk. or- countg viable fection work challenge exposed number as on se that is im- no serum effectively et al., suspected pro- 1978). that pro- due eir than agglut enterg or will confirm method was it found con- (KUSUDA it was results preventg which naturally fected such as occurrg cubated occurred follows: immunized cubated it was titer vibriosis This first adhered growth hibitg facr or facr preventg On washed surface agast tissue. ayu cubation Average From this phenomenon tection work orally agast washed 1130 body agglut From After sk on. crease tected re by 5. and closed cubated adherg In our Table ; that homogenates. out ml not 3. measured 0.05 Number was on ovbious Fig. 20 immunized 4 sk-sheets 304 5. Adhesion hibition activity body surface for Vibrio anguillarum sk 1/20 laied is shown adhered 261 adhe- Out remag amount sk ml homogenized. ganism 0.05 The shown and vials. equivalent which as 20 Ž PBS for 3.5 ~104 and caps sk-sheets at 8 screw. vial poured method concentration anor control a, for Vibriosis test. per Vaccated latter used discharged idea. which was designed a manner commercial ponds. At period 24 hr after challenge, isolation rate on body surface immunized was signifficantly low than that control. Difference isolation rate between two groups not so evident or organs. This dicates that enterg site body is sk. From results obtaed second challenge, it is assumed that that had entered body grows sk tissue first. This was concluded because was found grow only sk control perod 96 hr after challenge. The fact that no had been detected sk immunized dicates eir that can not penetrate sk or that y can not grow sk tissue. By third challenge method that is tramuscular jection, immunized could not be
262 K. KAWAI, R. KUSUDA, and T. ITAMI protected effectively, and LD50 values both groups almost equivalent. It dicates that oral immunization does not produce a facr so much enough protect agst penetrated tissue. It was also observed that much time was required prior death immunized rar than control. This is probably due a facr which act prevent growg tissue a certa degree. But at least it will be said that it is adequate employ tramuscular jection estimate efficacy oral immunization agast vibriosis ayu. Agglut titer rose only body surface immunized. Titer testi nal can not be compared that body surface, for reason that surface area are not equal between two. Neverless titer testal would not be so high at any rate. From results obtaed experiments on tramuscular jection and passive immunization protectg reaction orally immunized are presumed occur on outer site body. By adhesion-hibition test function agglut detected body surface immunized was examed. The result showed lower rate number adhered cubated im munized than that control or out any. There are few studies concerng antibody immunized. Agglut detected body surface plaice by traperi neal immunization (FLETCHER and GRANT, 1969; FLETCHER and WHITE, 1973), gar by tramuscu lar immunization (BRADSHAW et al., 1971) and fected carp (HINES and SPIRA, 1974). But re is no study that had shown agglut body surface orally immunized. In this study results obtaed it is assumed that protectg mechanism orally immunized ayu can be explaed as follows. Vibrio fection begs by adhesion sk. Organism grown tissue sk or subcutaneous tissue spreads all over body (FUNAHASHI et al., 1974). Agglut secreted body surface immunized hibits move freely on surface body. Then falls f body. In this manner agglut body surface will act hibit first step fection. In this study by what mechanism does ag glut come appear body surface orally immunized is not clear. WEISZ CARRINGTON concluded that specific IgA producg cell migrates testal tissue or secre ry tissue or organ orally immunized mouse (WEISZ-CARRINGTON and LAMM, 1979). Similar mechanism may be found also orally immunized ayu. Wher agglut detected body sur face ayu is IgM or not and wher protectg facr or than agglut exists or not are problems be vestigated furr. References AOKI, T. and T. KITAO (1978) : Vibriosis ayu. Fish Pathology, 13 (1), 19-24. BRADSHAW, C. M., A. S. RICHARD and M. M. SIGEL (1971) : IgM antibodies. Proc. Soc. Exp. Biol. Med., 136, 1122-1124. FLETCHER, T. C. and P. T. GRANT (1969) : Immuno globuls serum and plaice (pleuro nectes platessa). Biochem. J., 115 (5), 65 p. FLETCHER, T. C. and A. WHITE (1973) : Antibody pro duction plaice (Pleurctes platessa) after oral and parenteral immunization Vibrio anguil larum antigens. Aquaculture, 1, 417-428. FUNAHASHI, N., T. MIYAZAKI, K. KODERA and S. KUBOTA (1974) : Hispathological study on vibrio sis ayu. Fish Pathology, 8 (2), 136-143. ITAMI, T. and R. KUSUDA (1978) : Efficacy a vac cation by spray admistration agast vibriosis cultured ayu. Bull. Jap. Soc. Sci. Fish., 44 (12), 1413. KUSUDA, R., K. KAWAI, Y. Jo, T. AKIZUKI, M. FUKU NAGA and N. KOTAKE (1978) : Efficacy oral vac cation for vibriosis cultured ayu. Bull. Jap. Soc. Sci. Fish., 44 (1), 21-25. HINES, R. S. and D. T. SPIRA (1974) : Ichthyophthiri asis mirror carp Cyprus carpio (L.). V. Aquired immunity. J. Fish Biol., 6 (4), 373-378. WEISZ-CARRINGTON, P. and M. E. LAMM (1979) : Organ and isotype distribution plasma pro ducg specific antibody after immunization: Evi dence for a generalized secrery immune system. J. Immunol., 123 (4), 1705-1708.