Fish Pathology 15 (3/4) 257-262,1981. 3
Mechanisms of Protection in Ayu Orally Vaccinated 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 fish were challenged with organism, Vibrio anguillarum, by three ways. First, fish were exposed to organism discharged from naturally infected fish for 24 hr. Organism was isolated at high percentage from almost all part of body in the control fish, while the isolation rate was low particularly on the body surface in the immunized fish. Secondary, fish were bathed in bacterial suspension of a concentration of 107 cells per milliliter for 15 min. Twenty four hours after challenge organism was isolated from the skin of the control fish, then the number of organism increased gradually for the next 72 hr. No organism was detected in the intestine or its contents in both groups neither in the skin of the im munized fish. Finally, fish were injected intramuscularly resulting almost equal mortality in both groups. Agglutinin titer in the body surface mucus rose to 1: 64 in the immunized fish, but did not occur in the control fish. Agglutinin titer rose in neither serum nor intestinal mucus in both groups. The body surface mucus of the immunized fish prevented organism from adhering to the skin more effectively than that of the control fish did. From these results it is assumed that the defence effect by the oral immunization is attributed mainly to the agglutinin secreted in the body surface mucus.
In recent years many attempts were made to from naturally infected fish for 24 hr. Soon after control vibriosis in ayu by immunization in Japan. challenge fish were rinsed with physiological saline Three techniques, such as oral administration and sticked with filter paper on the body surface (KUSUDAet al., 1978), bath immunization (AOKI as shown in Fig. 1 and between gill lamellae. The and KITAO, 1978) and spray immunization (ITAMI filter papers were inoculated in the enrichment and KUSUDA,1978), had been used for the purpose, medium, nutrient broth with 2 % NaCl. Air blad and was found effective respectively if properly der, gullet, contents of gullet, stomach, contents applied. While immunization by above men of stomach, pyloric appendage, intestine, con tioned techniques were proved to be effective the tents of intestine, spleen and kidney were also mechanism of protecting from vibriosis has not inoculated in the same medium. Cultures after yet been explained. It was probably due to the 24 hr incubation at 25•Ž were spreaded on nutrient lack of information about the mechanism of in agar added with 2 % NaCl, 1 % sucrose and 0.004% fection and antibody taking part directly in pro bromthymol blue. After 2 day incubation yellow tection. Experiments were conducted to see the or yellow green colonies were harvested and sus entering site of fish body for pathogenic organism pended in distilled water. The suspension was and the protection reaction on the very site. used as reactive antigen for immuno-diffusion against anti V. anguillarum V-36 serum. Forma Distribution of organism after challenge tion of precipitin line indicated the existence of Experimental 20-50 g ayu were divided into two V. anguillarum within the organ or on the site of groups consisting of 10 or 20 fish. One group was body surface. Experiment was repeated three times. fed with bacterin mixed dry pellet, and another Isolatfion rate of organism in each organ of 40 was fed with pure pellet. Bacterin was admini fish in total is shown in Table 1. Between both stered at a level of 1 g wet cell/kg fish/day for ten groups of fish same isolation rate was shown in days. On the next day after vaccination, fish were the spleen and the kidney, while in other sites and challenged by exposing to organisms discharged organs high rate was recorded in the control fish 258 K. KAWAI, R. KUSUDA, and T. ITAMI especially on the body surface. Changes in number of organism in the skin, intestine and its contents after challenge Fish, 20-50 g in weight, were vaccinated for ten days as mentioned earlier. Five days after vac cination immunized fish and control fish were challenged by dipping in a bacterial suspension of a concentration of 107 cells per milliliter for 15 min. At 1, 6, 12, 24, 48, 72 and 96 hr after challenge three fish were taken respectively and employed for bacterial count in the skin, intestine and its contents. After rinsing with PBS the skin was peeled off at a width of 16 cm2.Then the intestine and its contents were separated. Each sample was homogenized and V. anguillarum cells were counted with BTB-sucrose agar plate and by slide agglutination with anti V-36 serum. Fig. 1. Sites on body surface of ayu for isolating Result is shown in Table 2. No organism was Vibrio anguillarum after challenge. Fish were detected in all samples after 96 hr in the immuni challenged by exposing to organisms discharged zed fish. While organism was detected in the skin from naturally infected fish for 24 hr. 24 hr after challenge, and the number increased
Table 1. Distribution of Vibrio anguillarum in ayu exposed for 24 hr to organisms discharged from naturally infected fish Mechanisms of Protection in Ayu Orally Vaccinated for Vibriosis 259
Table 2. Changes in number of Vibrio anguillarum on various sites of ayu after challenge by dipping in bacterial suspension
Table 3. Mortality and LD50 by intramuscular injection with Vibrio anguillarum
gradually for the next 72 hr in the control fish. No Mortality and LD50 by intramuscular injection organism was detected in either the intestine or its Fish, 10-20 g in weight, were vaccinated for 14 contents in the control fish for 96 hr after. challenge. days as previously mentioned. On the next day after vaccination immunized fish and control fish 260 K. KAWAI, R. KUSUDA, and T. ITAMI
were challenged by intramuscular injection with 0.24-240 cells of organism. As shown in Table 3 fish died 3-4 days after challenge in the immunized fish and 2-3 days after challenge in the control fish. Mortality in fish injected with 2.4 cells were 1/5 in the immunized fish and 2/5 in the control fish. From these re sults LD50 was calculated to 4.8 cells per fish in the immunized fish and 3.0 cells per fish in the control fish. Fig. 2. Agglutinin titers in mucus and serum in im Agglutinin titer in mucus and serum munized fish and control fish. Fish, 20-50 g in weight, were vaccinated for 10 days as mentioned earlier. Immunized fish 4 1: 2-4 and 1: 4 respectively in the control fish . days after vaccination and control fish were used. From these findings it was apparent that agglu After anesthetizing in chilled water, body surface tinin titer in body surface mucus rose by oral im mucus was rinsed away in PBS with sponge. munization. Intestinal mucus was washed out with the contents in it in PBS. Both mucus were salted out in 50% Protective effect of passive immunization by injec saturated solution with ammonium sulfate, then tion with serum of orally immunized fish dialysed and concentrated in polyethylene glycol Nine fish in one group were injected intramuscu powder. Agglutinin titer in mucus and serum larly with 0.1 ml of pooled serum of orally vacci were quantified with V-36 cell antigen. Experi nated fish used in foregoing experiment on agglu ment was repeated twice. In the first experiment, tinin titer. Another 9 fish were injected with serum 10 fish in one group were used, and only body sur of the control fish. Two days after injection fish face mucus was sampled. Samples of 5 fish were were exposed to organism discharged from natural pooled and concentrated to the ratio of 0.8 ml ly infected fish for 6 days. per fish. In the second experiment, 20 fish in one Result is shown in Table 4. Two fish died in group were used, and body surface mucus, intesti the immunized group and 9 fish died in the control mal mucus and serum were sampled . Samples of group for 6 days. 10 fish were pooled and mucus was concentrated to the ratio of 0.4 ml per fish. Adhesion-inhibition reaction of body surface mucus As shown in Fig. 2 in the first experiment, titer for organism to the skin in body surface mucus was 1 :16 in the immunized Fish, 25-40 g in weight, were vaccinated for fish, while 1: 2 in the control fish . In the second 10 days as mentioned before. On the next day of experiment, titer in body surface mucus, intestinal vaccination, body surface mucus of immunized mucus and serum were 1: 32, 1: 4 and 1: 4 respec fish and control fish were sampled and concentrated tively in the immunized fish, while results were 1: 2 , as earlier mentioned. One half ml of the bacterial
Table 4. Protective effect by passive immunization with orally vaccinated ayu serum Mechanisms of Protection in Ayu Orally Vaccinated for Vibriosis 261
Table 5. Adhesion inhibition activity of the body surface mucus for Vibrio anguillarum to the skin
Fig. 3. Schematic drawing of the method for adhe- sion-inhibition test.
suspension of a concentration of 3.5•~104 cells
per milliliter were poured into vials. Out of 20 vials, 8 were added with 0.05 ml of immunized fish mucus, another 8 were added with 0.05 ml of control fish mucus and the remaining 4 were not added any mucus. The amount of mucus added to one vial was equivalent to 1/20 of mucus re- covered from one fish. Vials were closed with screw caps which were inlaied with washed ayu skin-sheets as shown in Fig. 3. After incubation at 20 •Ž for one hour skin-sheets were washed with
PBS and homogenized. Number of adhered or- ganism to the skin was measured by counting viable cells while in the homogenates.
Result is shown in Table 5. Average number of adhered organism to the skin were as follows: naturally infected fish was designed in a manner 304 cells on the incubated in the immunized such as occurring in commercial ponds. At the fish mucus; 855 cells on the incubated in the con- period of 24 hr after challenge, isolation rate on the trol fish mucus; and 1130 cells on the incubated body surface of the immunized fish was signi- one without mucus. From these results it is fficantly low than that of the control fish. Dif- ovbious that the body surface mucus of the im- ference of isolation rate between two groups were munized fish has a function preventing organism not so evident in other organs. This indicates from adhering to the skin. that the entering site of organism into the body is the skin. From the results obtained in the second challenge, In our previous work it was found that no in- it is assumed that organism that had entered into crease in agglutinin titer occurred in the serum of the body grows in the skin tissue first. This was orally immunized fish which were effectively pro- concluded because organism was found to grow tected against vibriosis (KUSUDA et al., 1978). only in the skin of the control fish within the perod From this phenomenon it was suspected that pro- of 96 hr after challenge. The fact that no or- tection against infection was due to either the ganism had been detected in the skin of the im- growth inhibiting factor other than agglutinin or munized fish indicates either that organism can the factor preventing organism from entering into not penetrate the skin or that they can not grow in the tissue. This work will confirm the latter idea. the skin tissue. On the first challenge method used in which By the third challenge method that is intramu- fish were exposed to organism discharged from scular injection, the immunized fish could not be 262 K. KAWAI, R. KUSUDA, and T. ITAMI protected effectively, and LD50 values of both from the body. In this manner the agglutinin in groups were almost equivalent. It indicates that the body surface mucus will act to inhibit the first oral immunization does not produce a factor so step of infection. much enough to protect aginst organism penetrated In this study by what mechanism does the ag into the tissue. It was also observed that much time glutinin come to appear in the body surface mucus was required prior to death in the immunized fish in orally immunized fish is not clear. WEISZ rather than in the control fish. This is probably CARRINGTONconcluded that specific IgA producing due to a factor which act to prevent organism from cell migrates from intestinal tissue to other secre growing in the tissue to a certain degree. But at tory tissue or organ in orally immunized mouse least it will be said that it is inadequate to employ (WEISZ-CARRINGTONand LAMM, 1979). Similar intramuscular injection to estimate efficacy of oral mechanism may be found also in orally immunized immunization against vibriosis in ayu. ayu. Agglutinin titer rose only in the body surface Whether the agglutinin detected in the body sur mucus of the immunized fish. Titer in the intesti face mucus of ayu is IgM or not and whether the nal mucus can not be compared to that in the body protecting factor other than agglutinin exists or surface mucus, for the reason that surface area are not in the mucus are problems to be investigated not equal between the two. Nevertheless titer in further. the intestinal mucus would not be so high at any rate. References From the results obtained in experiments on intramuscular injection and passive immunization AOKI, T. and T. KITAO (1978) : Vibriosis of ayu. protecting reaction in orally immunized fish are Fish Pathology, 13 (1), 19-24. BRADSHAW,C. M., A. S. RICHARD and M. M. SIGEL presumed to occur on the outer site of the body. By adhesion-inhibition test the function of the (1971) : IgM antibodies in fish mucus. Proc. Soc. Exp. Biol. Med., 136, 1122-1124. agglutinin detected in the body surface mucus of FLETCHER,T. C. and P. T. GRANT (1969) : Immuno the immunized fish was examined. The result globulins in the serum and mucus of plaice (pleuro showed lower rate in the number of adhered nectes platessa). Biochem. J., 115 (5), 65 p. organism incubated with the mucus of the im FLETCHER,T. C. and A. WHITE (1973) : Antibody pro munized fish than with that of the control fish or duction in plaice (Pleuronectes platessa) after oral without any mucus. and parenteral immunization with Vibrio anguil There are few studies concerning antibody in larum antigens. Aquaculture, 1, 417-428. the mucus of immunized fish. Agglutinin detected FUNAHASHI,N., T. MIYAZAKI, K. KODERA and S. in the body surface mucus in plaice by intraperi KUBOTA(1974) : Histopathological study on vibrio toneal immunization (FLETCHERand GRANT,1969; sis in ayu. Fish Pathology, 8 (2), 136-143. ITAMI, T. and R. KUSUDA (1978) : Efficacy of a vac FLETCHERand WHITE,1973), in gar by intramuscu cination by spray administration against vibriosis in lar immunization (BRADSHAWet al., 1971) and in cultured ayu. Bull. Jap. Soc. Sci. Fish., 44 (12), infected carp (HINES and SPIRA, 1974). But 1413. there is no study that had shown agglutinin in the KUSUDA,R., K. KAWAI, Y. Jo, T. AKIZUKI, M. FUKU body surface mucus of orally immunized fish. In NAGAand N. KOTAKE(1978) : Efficacy of oral vac this study from the results obtained it is assumed cination for vibriosis in cultured ayu. Bull. Jap. that the protecting mechanism in orally immunized Soc. Sci. Fish., 44 (1), 21-25. ayu can be explained as follows. Vibrio infection HINES, R. S. and D. T. SPIRA (1974) : Ichthyophthiri begins by adhesion of organism to the skin. asis in the mirror carp Cyprinus carpio (L.). V. Organism grown in the tissue of skin or in the Aquired immunity. J. Fish Biol., 6 (4), 373-378. subcutaneous tissue spreads all over the body WEISZ-CARRINGTON,P. and M. E. LAMM (1979) : Organ and isotype distribution of plasma cells pro (FUNAHASHIet al., 1974). Agglutinin secreted ducing specific antibody after immunization: Evi in the body surface mucus of the immunized fish dence for a generalized secretory immune system. J inhibits organism to move freely on the surface of . Immunol., 123 (4), 1705-1708. the body. Then the mucus falls off with organism