| Japanese flounder (Paralichthys olivaceus) is the bottom fish of the cold temperature and the important economic fish for aquaculture in China. With the development of large Japanese flounder artificial breeding, extending of breeding scale and aggravation of breeding environment, the diseases of large Japanese flounder frequently happen, which cause severe economic loss. Vibrio anguillarum can infect the majority of marine fish and is the most damaging pathogen. It is also extensively studied and reported.Vaccine is shown broad prospects of development to fight against disease, and protect the product quality and promote the development of sustainable fisheries breeding areas such as the environment. Oral immunization is safer than the traditional methods, easier to operate, but the immune effect is lower than that of the injection. The reason is that the vaccine is broken down by digestive enzymes in the stomach before absorbed by the intestinal tract, and then the antigenicity is lost. The microencapsulated oral vaccine has the merits of protecting antigen, enhancing antigen targeting, long-acting and slow-releasing antigen, and reducing the required injection amount of antigen, thus it can reduce the toxic side effects for organisms.In order to develop a specific, sensitive, rapid, and practical method for detecting effect of antigen, antibodies against V. anguillarum was detected by the improved double-antigen sandwich ELISA. V. anguillarum was labeled by HRP, and micro-agglutination was compared with double-antigen sandwich ELISA by the antibody titer. The results showed that 0.20 mg HRP was the optimal contents for labelling 2.5 mg protein antigen, and the centrifugation supernant of V. anguillarum treated with ultrasonic wave was the optimal labelling antigen, with its working concentration of 12.25mg/L. The established S-ELISA in this study might provide a novel means for detection of V. anguillarum antibody with high sensitivity and specificity.In this reaserch, the site of digestion and absorption was determined in the hintgut for Japanese flounder through the tissue slice and detecting the protease activity and chyme protein traced by GFP in the digestive tract of flounder. The results showed that the chime distributed in the whole digestive tract after 5h, the stomachic protease activity reduced to near null. The protease activity in the midgut kept at the highest level until 8h. The protease activity in the hindgue kept at the high level until 12h after ingesting. The results showed that the intestines might be more significant in protein digestion than the stomach for the fish with short digestive tract. The optimizative time of relase-time for the microencapsulated oral vaccine was 5-8h after immunized, making the lower lose for the antigen.The microencapsulated oral vaccine of V.anguillarum inactivated-whole-cell was prepared based on above conclusions and used for aquaculture of Japanese flounder immune tests. The comparison of protective effect of different vaccine groups was carried out by detection of immune serum lysozyme vitality, serum protein content, the changes in antibody titer, and challenged tests. Intraperitoneal injection group with the dose of 1×107CFU/tail and 1×106CFU/tail immunized flounder, lysozyme activity and protein content as well as the antibody levels were significantly increased, the relative protective survival (RPS) reached 89.7% and 86.2%, respectively. The microencapsulated oral vaccine with the dose of 1×108CFU/tail and 1×107CFU/tail was used for immunization, the RPS reached 41.3% and 20.7%, respectively, which was better than the treatment by the general oral vaccine with 27.6% and 13.8% of RPS, respectively. However, there were no significant differences in lysozyme activity, protein content as well as the antibody level between them. When immunization dose was 1×106CFU/tail, both the two methods of oral immunization did not have the safety, therefore, the immune-dose and the immune effect had a positive correlation. |
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