Study On The Immunogenicity Of The Inactivated Vaccine Of Duck Salmonella

There is often associated with the incidence of salmonellosis with the development of large-scale breeding industry. The sick poultry would die if there were no effective drugs, even if be cured would carry pathogen lifelong and produce contaminated eggs. This causes serious economic losses and a threat to public health. There are fewer drugs for choice because of the drug resistance. Vaccine immunity plays a decisive role in preventing diseases, but there is no standard vaccine against duck salmonellosis. According to the distribution of duck Salmonella serotypes in Sichuan and Chongqing, we used animal infection test to compare the virulence between different strains. We selected the strain as the research object, which had the strongest virulence and widely distributed. We used the traditional method to make inactivated vaccine, then inoculated experimental ducks and evaluated the vaccine immunization levels from cellular immunity and humoral immunity, with a view to provide basis for the prevention and control of ducks salmonellosis. The results were as follows:1. In this study, we chose 12 duck Salmonella strains for duck infection test in the same bacterial concentration, and filtered out the strain that has the highest mortality rate:Its serotype was identified as the Salmonella Chester (1,4, [5],12:e, h:e, n, x) by 30 kinds of Salmonella diagnostic serum, and its LD50 was 1×108.47 CFU. As the vaccine strain, DS.C2013 was made into three concentrations of propolis adjuvant inactivated vaccine (108 CFU/ml,109CFU/ml and 1010CFU/ml). There is no abnormal symptom in ducks after 5 doses of vaccine immunization, which indicated the vaccine is safe and reliable. Ducks were immunized with the three concentrations of vaccine by groups (108 CFU/ml,109 CFU/ml,1010 CFU/ml) and injected with 100LD50 of bacteria. The protection rates in turn was 50%,80%,100%, showed that the vaccine concentration of 1×1010 CFU/ml could have good effect for protection.2. We established an indirect ELISA method successfully, in which 6.64 μg/ml protein concentrations of DS.C2013 lysates was as envelope antigen. The optimum dilution of serum was 1:400, and the optimum working concentrations of HRP-IgG anti-duck is 2.50 μg/ml. The positive serums of duck plague virus, P. multocida, E. coli, R. anatipestifer, Staphylococcus aureus and DHV were negative by the method; the result of blocking test was negative; the coefficient of variation in one plate and the coefficient of variation among plates were 1.10%-2.64% and 1.53%-2.67%.3. In this study, ducks were immunized with the inactivated vaccine of DS.C2013. We analyzed the difference between the control group and the two immune groups in cellular immunity and humoral immunity to evaluate the immunogenicity of the vaccine:(1) CCK-8 test kit was used to detect the proliferation activity of ducks’lymphocyte in vitro. The proliferation of spleen lymphocytes of the immune groups was obviously higher than that of the control group stimulated with ConA in vitro (P<0.05), and the proliferation ability of the twice immune group is higher than that of the once immune group (P<0.05). The lymphocytes of peripheral blood were stimulated by ConA and vaccine antigens, both of which could enhance the activity; and the proliferation of the immune groups was significantly higher than the control group (P<0.05). The results showed that the vaccine could increase the lymphocyte proliferation ability of ducks.(2) We used Indirect ELISA kits to detect IL-4 and IFN-γ in the peripheral blood at 0 d,3 d, 7 d,10 d,15 d,20 d and 25 d after immunization. The levels of IL-4 and IFN-γ of the immune groups were shown a process of gradually increasing at 3 d after immunization; there was a significant difference between the control group and the immune groups (P <0.05). A peak was appearing at 20 d after immunization in the twice immune group, significantly higher than the once immune group (P<0.01).(3) We used staining method to detect the phagocytic activity of leukocytes at 0 d,7 d,15 d and 25 d after immunization. The phagocytic percentage and index of three groups were upward trend after immunization, the immune groups increased more obviously than the control group (P<0.05); the PP and PI of three groups were close at 15 d after immunization (P>0.05); until 25 d, the control group and the once immune group declined slightly, when the twice immune group kept on rising.(4) The antibody levels in serum of the trial ducks were detected after immunization by Indirect ELISA method. There was a certain level of antibody in serum before immunization, and all the three groups got a downward trend from 0 d to 3 d after immunization, the immune groups were declining significantly greater than the control group (P<0.01). The control group continued declining until 15 d, and then it was stabilized close to negative values. The immune groups had a upward trend within 7 d to 15 d after immunization; the once immune group began to reduce at 15 d, when the twice immune group continued to rise to reach the peak at 30 d. The twice immune group was still at a high level after 40 d (P<0.01).