牛源A型多杀性巴氏杆菌(Pm12)灭活疫苗的制备及小鼠攻毒保护试验  

Preparation of Bovine type A Pm12 inactivated vaccine and attack test in mice

作　　者：陶乔孝慈 邬琴[1] 顾晓晓 马雪 李劼[1] 周霞 张星星[2] 黄新[2] 吴桐忠[2] 韩猛立[2] 钟发刚[2] TAO Qiaoxiaoci;WU Qin;GU Xiaoxiao;MA Xue;LI Jie;ZHOU Xia;ZHANG Xingxing;HUANG Xin;WU Tongzhong;HAN Mengli;ZHONG Fagang(Animal Science and Technology College,Shihezi University,Shihezi 832003,China;State Key Laboratory for Sheep Genetic Improvement and Healthy Production/Institute of Animal Husbabdry and Veterinary,Xinjiang Academy of Agricultural Reclamation,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]新疆农垦科学院畜牧兽医研究所/省部共建绵羊遗传改良与健康养殖国家重点实验室,新疆石河子832000

出　　处：《黑龙江畜牧兽医》2021年第5期127-130,135,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：省部共建绵羊遗传改良与健康养殖国家重点实验室优秀中青年人才培养引导计划专项(SKLSGIHP2017A03);兵团区域创新引导计划项目(2017BA038;2018BB036);兵团国际科技合作计划项目(2019BC004);兵团重点领域科技攻关项目(2019AB029)。

摘　　要：为了防控新疆北疆A型多杀性巴氏杆菌(Pm)引起的牛纤维素性化脓性肺炎,分析制备的A型Pm12灭活疫苗在试验小鼠体内抗体产生情况和保护作用,试验按细菌灭活疫苗制备程序将分离筛选自新疆北疆部分地区发病牛场的1株毒性强、免疫原性好的牛源A型Pm12制备成灭活疫苗,并进行安全检验。根据棋盘法对全菌抗原包被浓度、封闭液、血清稀释度、酶标二抗工作浓度、封闭时间、一抗孵育时间、二抗孵育时间及底物反应时间等检测条件进行筛选,建立检测小鼠血清抗体的间接ELISA方法,用建立的间接ELISA方法测定免疫小鼠血清抗体产生规律,对免疫小鼠以不同半数致死量(LD50)的Pm12进行攻毒保护试验,同时用A型和B型Pm对免疫小鼠进行交叉保护试验。结果表明:制备的牛源A型Pm12灭活疫苗含菌量为8×109cfu/mL,安全检测合格。根据棋盘法确定全菌抗原包被浓度为1∶25,最佳血清稀释度为1∶320~1∶640,最适封闭液为5%BSA,最适封闭时间为1 h,血清最适作用时间为1 h,酶标抗体最适稀释度为1∶3 000,底物最适作用时间为15 min;用制备的疫苗免疫小鼠后,60天的血清抗体效价均高于28天水平;Pm12灭活苗对不同LD50攻毒小鼠的保护率为80%、80%、50%,对A型Pm保护率为80%,对B型Pm的交叉保护率为60%。说明可用试验建立的方法检测小鼠血清抗体水平,制备的牛源A型Pm12灭活疫苗能够诱导小鼠产生较高水平且持续较长时间的血清抗体,能够有效保护小鼠对抗A型Pm感染,且对B型Pm呈现部分交叉保护作用。In order to prevent and control bovine cellulosic pyogenic pneumonia caused by Pasteurella multocida type A(Pm) in northern Xinjiang, the antibody production and protective effect of the prepared inactivated Pm12 vaccine in the experimental mice were analyzed. In the experiment, a highly toxic and immunogenic bovine type A Pm12 isolated and screened from the diseased cattle farms in parts of northern Xinjiang was prepared into an inactivated vaccine according to the bacterial inactivated vaccine preparation procedure and safety test was carried out. The chessboard method was used to screen the detection conditions such as antigen coating concentration, blocking solution, serum dilution, enzyme-labeled secondary antibody working concentration, blocking time, primary antibody incubation time, secondary antibody incubation time and substrate reaction time. Indirect ELISA method was established to detect serum antibody in mice. The production rule of serum antibody in immunized mice was determined by the established indirect ELISA method. The attack protection test was carried out on immunized mice with Pm12 with different lethal dose(LD50), and cross protection test was carried out on immunized mice with type A and type B Pm. The results showed that the bacterial content of the prepared inactivated bovine type A Pm12 vaccine was 8×109 cfu/mL, and the safety test was qualified. According to the chessboard method, the concentration of whole bacterial coating antigen was 1∶25, the optimal serum dilution was 1∶320-1∶640, the optimal blocking solution was 5% BSA, the optimal blocking time was 1 h, the optimal serum action time was 1 h, the optimal enzyme antibody dilution was 1∶3 000, and the optimal time of substrate was 15 min. After the mice were immunized with the prepared vaccine, serum antibody levels were monitored by ELISA, and serum antibody titers at 60 days were all higher than 28 days. The protective rates of Pm12 inactivated vaccine against different LD50 attacked mice were 80%, 80%, 50%, 80% for ty

关 键 词：牛A型多杀性巴氏杆菌 灭活疫苗 小鼠 抗体水平 免疫保护 交叉保护 

分 类 号：S852.6112[农业科学—基础兽医学] S823[农业科学—兽医学]