猪丹毒丝菌灭活疫苗制备及其对小鼠免疫效力的评价

Preparation of Inactivated Vaccine against Erysipelothrix rhusiopathiae and Evaluation of Immune Efficacy in Mice

为制备猪丹毒丝菌灭活疫苗并评价其对小鼠的免疫效力,将3株受试菌株(AEr21、AEr31和AEr32)培养至稳定期,经终质量浓度为2mg/L甲醛灭活后,用矿物油佐剂ISA201VG制备成猪丹毒丝菌灭活疫苗,并与商品化疫苗分别免疫小鼠。应用ELISA方法检测小鼠血清中IgG抗体水平和细胞因子含量(IL-4、IL-10、MCP-1、TNF-β、IFN-γ),流式细胞术测定CD4^+/CD3^+、CD8+/CD3^+T细胞亚群百分比,采用灌胃和腹腔注射方式测定免疫保护率,并采集小鼠脏器(肺脏、肝脏、脾脏、肾脏)制备病理组织切片,观察病理变化。结果显示,AEr21、AEr31、AEr32全菌体灭活疫苗组和商品化弱毒疫苗组、灭活疫苗组二次免疫小鼠后的血清IgG抗体效价分别为1∶3200、1∶6400、1∶1600、1∶6400、1∶3200(以全菌体超声裂解物为包被抗原)和1∶3200、1∶6400、1∶3200、1∶25600、1∶6400(以重组SpaA为包被抗原);商品化弱毒疫苗组诱导小鼠产生的细胞因子水平和CD4^+/CD3^+、CD8+/CD3^+T细胞比例最高,与灭活疫苗组差异显著,各灭活疫苗组间差异均不显著;AEr21、AEr31、AEr32全菌体灭活疫苗组和商品化弱毒疫苗组、灭活疫苗组对灌胃和腹腔注射攻毒小鼠的免疫保护率分别为80%、100%、100%、100%、100%和80%、100%、60%、100%、100%;AEr21和AEr32全菌体灭活疫苗组的病理组织变化较AEr31全菌体灭活疫苗组和商品化弱毒疫苗组、灭活疫苗组更显著。表明受试菌株(AEr31)与矿物油佐剂ISA201VG制备成的猪丹毒丝菌灭活疫苗免疫小鼠后不仅可产生较高水平的细胞免疫和体液免疫,还可完全抵抗强毒株的攻击。

Inorder toprepare the inactivated vaccine of Erysipelothrix rhusiopathiae and evaluate its immune efficacy in mice,the three testedstrains(AEr21,AEr31,AEr32)were cultured in to stable phase,and it was inactivated at a final concentration of 2 mg/L formaldehyde and then it was prepared into inactivated vaccine against Erysipelothrix rhusiopathiae with mineral oil adjuvant ISA 201 VG and compared with commercial vaccines.Serum antibody(IgG)and cytokines(IL-4,IL-10,MCP-1,TNF-β,IFN-γ)levels were measured by ELISA and the subpopulation of T cells of CD4^+/CD3^+,CD8+/CD3^+were determined by flow cytometry.Calculating the protective rate of each immunization group after challenge by gavage and intraperitoneal injection,and collecting mouse organs(lung,liver,spleen,kidneys)to perform pathological examination.The results showed that,the titers of serum IgG antibodies in the AEr21,AEr31,AEr32 inactivated whole cell vaccines and commercial attenuated vaccines and inactivated vaccines were 1∶3 200,1∶6 400,1∶1 600,1∶6 400,1∶3 200(using whole cell sonolysate as coating antigen)and 1∶3 200,1∶6 400,1∶3 200,1∶25 600,1∶6 400(using recombinant SpaA as coating antigen)respectively.The level of cytokines and the proportion of CD4^+/CD3^+,CD8+/CD3^+T cells induced by commerical attenuated vaccine were the highest,which were significantly different from the inactivated vaccine(P>0.05),but therewas no significant difference between each inactivated vaccine(P>0.05).The immune protection rates of the AEr21,AEr31,AEr32 inactivated vaccine group and commercial attenuated,inactivated vaccine group were 80%,100%,100%,100%,100%and 80%,100%,60%,100%,100%for gavage and intraperitoneal injection respectively.The histopathological changes of AEr21 and AEr32 inactivated vaccine groups were more significant thanAEr31 inactivated vaccine group and the commercial attenuated and inactivated vaccine group.The results showed that the inactivated vaccine prepared by AEr31and the mineral oil adjuvant ISA 201 VG could not only produce higher level of cellular and humoral immunity,but also completely resist the attack of virulent strains.