Screening Of Adjuvant For Inactivated Vaccine Of Rabbit Bordetella Bronchiseptica And Study Of Immune Efficacy

Rabbit Bordetella Bronchiseptica(Bb)is an important pathogen causing chronic respiratory disease in rabbits which affects development of the rabbit industry by its long pathogenicity and difficult to cure.Some research reported the bacteria could infect humans.Currently,vaccines for Bb are still at the stage of laboratory,which couldn’t offer efficient protection against Bb infections in rabbits.It is of great significance to improve the protective effect of the vaccine for rabbits and to improve the healthy development of the rabbit industry system and public health that efficient vaccine adjuvant is developed against Bb infections in rabbits.In our previous experiments,different adjuvants for inactivated vaccines were screened.ECMS800+oil adjuvant vaccine,conventional oil adjuvant vaccine and non-adjuvant vaccine were comparatively studied.And ECMS800+oil adjuvant vaccine were analyzed for its adjuvant mechanism,which could improve not only the humoral immunity but also cellular immune respose in rabbits.The research provides a technical basis for the development of rabbit Bb vaccine,and provides a new way of thinking for the development of new vaccines for veterinary.1.Optimization of direct ELISA detecting method for Bb in rabbitPolyvinyl 96-well microtiter plates were coated with the whole cell protein of Bb to establish indirect ELISA method.Optimal results as fallows:whole cell protein with 1 μg/ml incubated at 37 ℃ for 2 h;5%of skim milk blocked at 37 ℃ for 2 h;serum diluted of 1:400 incubated at 37 ℃ for 1h;(HRP)-conjugated goat anti-rabbit IgG(1:5,000)incubated at 37 ℃ for 75 min;TMB solution incubated at 37℃ for lOmin away from light.Positive and negative of the critical value was 0.19.Because of repeatability,high sensitivity and specificity,this method established can provide a new technical means for detecting antibody against Bb.2.The adjuvant screening for inactivated vaccine against Bb in rabbitsDifferent doses of ECMS,different doses of ECMS + oil,as well as conventional mineral oil and aluminum adjuvant were added to inactivated vaccine of Bb in this study.The concentration of IgG against different protein and cytokines IL-2 in serum collected at different times were detected with direct ELISA.Whole blood were collected for lymphocyte stimulation test to detect the cellular immune response of immune before challenge.Finally,all the rabbits were challenged to detect the vaccine protection.The results showed that the IgG against whole cell protein and PPP protein in ECMS800+oil group were higher than other groups inordinately.The content of cytokine IL-2 in ECMS800+oil group were higher than other groups.Stimulating index(SI)in ECMS800+oil group is higher than other groups.Protection rate in ECMS800+oil group was 100%that much higher than the other groups in challenge experiment.In summary,ECMS800+oil group screened is the best vaccine against Bb.3.Preliminary study of ECMS +oil adjuvant enhancing immune responses for Bordetella bronchiseptica vaccine on rabbitOil adjuvant vaccine and non-adjuvant vaccine as the control,ECMS800+oil vaccine screened were detected with the immunization indicators to analyze its immunogenicity to conducted a preliminary exploration of its mechanism of ECMS + oil for rabbit Bb vaccine.The concentration of IgG against whole cell protein,soluble CD4(sCD4)and CD8(sCD8)in serum and IgA in Tracheal mucosa were detected by direct ELISA.Whole blood were collected for lymphocyte stimulation test、Flow Cytometry(FCM)and the expression level of IL-2,IFN-y to detect the cellular immune response.In addition,the relative quantity of IL-2,IL-6,IFN-γ in spleen were detected with Realtime-PCR.The positive cells which secrete IgA were measured to analyze mucosal immunity.Immune organ index were detected to monitor the immune system.Finally,rabbits immuned were challenged to detect the vaccine protection.Compared with the control,ECMS800 + oil can improve the rabbit humoral and cell-mediated immunity efficiently,and achieve a better effect of mucosal immunity,in which the vaccine protection rate was significantly improved,and stimulate comprehensive and lasting immune response.The adjuvant study for the development of Bb vaccine provides a new method.