Development And Application On Polyvalent Vaccine Of Bacterial Pathogens Of Marine Fish

Culture of marine fish progresses to the level of high density and intensity, accompanied by a prominence of disease problem. Bacterial diseases are the main pathogens, which can cause large-scale mortalities of marine fishs and mass economical loss in the world. Control of fish diseases using therapeutics such as antibiotics have met problems including potential development of resistant bacteria, concern over drug residues and environmental impacts. In the present study, the optimization of cultural condition of Vibrio anguillarum, V. harveyi, V. parahaemolyticus and Edwardsiella tarda was studied. A polyvalent vaccine was prepared and its immune efficiency was estimated. An expressed outer membrane protein ompU from the pathogenic V.anguillarum W-1 was purified and its immune efficiency on Flounder was also studied.The influence of temperature, salinity, initial pH, and media on the growth of V. anguillarum, V. harveyi, V. parahaemolyticus and Edwardsiella tarda were studied. The optimum culture condition to V. anguillarum were temperature 28℃, salinity 20, pH6.0～8.0. The optimum culture condition to V. harveyi were temperature 28℃, salinity10～30, pH5.0～9.0. The optimal growing condition of V. parahaemolyticus was concluded: temperature 28℃, salinity 20～40, pH7.0～9.0. The optimal growing condition of E. tarda was as follows: temperature30℃, salinity 10～20, pH6.0～7.0 on the basis of LB medium.A polyvalent vaccine was prepared by mixing equal mount of formalin killed cells of V.anguillarum, V.harveyi,V.parahaemolyticus and E. tarda. Turbot juveniles were immunized with the polyvalent vaccine by intraperitoneal injection. A booster injection was conducted on day 14 after the prime vaccination. Serum was collected from 5 turbot per week and the specific antibody was determined by use of a microtitration agglutination test. The vaccinated fish were intraperitoneally injected with 106-108 CFU/mL of V. anguillarum, V. harveyi, V. parahaemolyticus and E. tarda respectively on day 70 post the prime vaccination. The results showed that specific immune reaction could be stimulated with the vaccine. Specific antibodies of the four bacteria could be detected in 7 days after the prime vaccination. The agglutinating antibody titers reached peaks of 28.17, 27.07, 26.85 and 28.81 respectively within 21-57days. The serum antibody titers maintained high level in 70 days. The vaccine induced efficient protection against V.anguillarum and V. harveyi, with relative percentage survival(RPS)of 66.67% and 44.44%. The protection efficiency against V. parahaemolyticus and E. tarda was much lower, and the RPS was 20% and 8.33% respectively.An expressed outer membrane protein ompU from the pathogenic V.anguillarum W-1 was purified . The molecular weight of the purified protein was 38kDa when detected by SDS-PAGE and Western blot analysis. Flounder were vaccinated with the purified outer membrane protein via intraperitoneally injection.A booster injection was conducted on day 14 after the prime vaccination, A dosage of 0.2 ml of W-1 (3.29×108 CFU/ml) was intraperitoneally injected to the vaccinated fish and control groups 4 weeks after immunization to evaluate the protective efficiency of the vaccine. The results showed the relative percentage survival(RPS)was 20.00% and 33.33% respectively.