A Preliminary Study On Serum Immunoglobulin Of Cobia (rachycentron Canadum) And Immune Response To Vibrio Alginolyticus Vaccine

Cobia, Rachycentron canadum, has become one of the main species fish of marine aquaculture in south seaside districts of China with the characteristics of large individual size, rapid growth, adaptation to intensive culture and so on. However, the disease problems of Cobia are becoming more and more serious with the expansion of farming scale and the deterioration of culture environment. As known to us, V. alginolyticus, was the main pathogen of Vibrio diseases of marine fishes. At present, vaccination is regarded as an ideal strategy of disease resistance in aquaculture and plays an important role in fisheries increasingly. Conducted on cobia, this paper studied on the immune system of cobia, purification of serum immunoglobulin and molecular weight of its subunit, non-specific immune response and specific immune response of cobia to V. alginolyticus vaccines with using biochemistry technology and immunological technology. The results are summarized as follows:1. In order to obtain the immune serum, the adult cobia was immunized by injection with the goat IgG. And serum immunoglobulin was purified from the immune serum by ammonium sulfate precipitation and affinity chromatography. The results indicated: the serum Ig was salted out with 40～50% saturation of ammonium sulfate. Two obvious independent peaks were obtained with the affinity chromatography method and the serum Ig was in the second elution peak. SDS-PAGE showed that other proteins were not found in the purified serum antibody. The molecular weights of light and heavy chain of the antibody were 28.4 kDa and 77.2 kDa respectively; gross molecular weight would be calculated to 844.8 kDa if serum Ig of Cobia were a tetramer like IgM of other fishes. Sera anti-IgM of Cobia was prepared by immunized New Zealand rabbit repeatedly with serum Ig of cobia and the titers of anti-serum reached to 5120 at least examined by indirect ELISA.2. Juveniles cobia were immunized by injection with the V. alginolyticus Vaccine at the concentration of 1.0×106 CFU·mL-1, 1.0×108CFU·mL-1, 1.0×1010 CFU·mL-1 respectively. Non-specific immune response of the juvenile cobia were compared with examining the number of RBC, WBC and DLC in the blood, head kidney and spleen indexes, AKP activity, SOD activity, LSZ activity, antibacterial activity, the content of C3 and C- Reaction Protein in serum. The results showed that V. alginolyticus Vaccine with three kinds of different concentrations could make the juveniles cobia have nonspecific immune response. The indexes of all the immunized groups, except for RBC, were higher than the control group on day 7 after the first and enhancing immunization. This study proved that vaccination with V. alginolyticus could result in strong nonspecific immune response of the cobia and the 7th day post immunization was the most.3. Juveniles cobia were immunized by injection with the V. alginolyticus Vaccine of at the concentration of 1.0×106 CFU·mL-1, 1.0×108CFU·mL-1, 1.0×1010 CFU·mL-1 respectively. Specific immune response of the juvenile cobias were compared with examining the kinetics of antibody titers of serum tested by indirect ELISA and the Relative Percent Survivals challenged with active V. alginolyticus on the 28th day post immunization. The results showed that V. alginolyticus Vaccine could cause the juvenile cobia strong specific immune response. The antibody titers were growing with the immune time and up to the maximum 28.2, 210.8 and 29.2 one or two weeks after the vaccinations and still staying in the high level on the 28th day post the first vaccination, receptively. After 28 days of vaccination, the Relative Percent Survivals of 1.0×106 CFU·mL-1, 1.0×108 CFU·mL-1 and 1.0×1010CFU·mL-1 were 64.71%, 82.35% and 70.59% respectively. This research proved that vaccination with V. alginolyticus could result in strong specific immune response of the cobia and the concentration of 1.0×108CFU·mL-1, the 21th day post immunization was the most.