猪丹毒丝菌-猪链球菌二联油乳剂灭活疫苗的制备及其免疫效果评价  

Preparation and immune effect of inactivated porcine Erysipelothrix rhusiopathiae and Streptococcus suis combined oil emulsion vaccine

作　　者：韩业芹 崔丽荣 邢刚 何长生[3] 孙裴[1] 刘雪兰[1] 魏建忠[1] 李郁[1] HAN Ye-qin;CUI Li-rong;XING Gang;HE Chang-sheng;SUN Pei;LIU Xue-lan;WEI Jian-zhong;LI Yu(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,Anhui Province,China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]马鞍山史记动物健康管理有限公司,安徽马鞍山238251 [3]安徽省动物疫病预防与控制中心,安徽合肥230091

出　　处：《中国生物制品学杂志》2022年第10期1160-1168,共9页Chinese Journal of Biologicals

基　　金：国家星火计划重点项目(2014GA710002);安徽省重点研究与开发计划(面上攻关)项目(201904a06020013);安徽省长三角联合科技攻关项目(1101c0603065);安徽省生猪产业体系基金项目[皖农科(2016)84号]。

摘　　要：目的制备猪丹毒丝菌(E.rhusiopathiae,ER)-猪链球菌(Streptococcus suis,SS)二联油乳剂灭活疫苗,并评价其免疫效果。方法以1株1a型ER(编号AEr31)和1株2型SS(SS2,编号HF2)为制苗菌株,甲醛为灭活剂,ISA 201VG矿物油为佐剂,通过两种方式将灭活全菌体(V-AEr31,V-HF2)与佐剂混合[(V-AEr31+V-HF2)+ISA 201VG及(V-AEr31+ISA 201VG)+(V-HF2+ISA 201VG)],制备ER-SS2二联油乳剂灭活疫苗,进行性状、无菌和安全检验。将两种灭活疫苗分别经皮下多点注射免疫小鼠(Ⅰ和Ⅱ组),同时设0.9%氯化钠对照组(Ⅲ组)。间接ELISA法检测小鼠血清中IgG抗体效价和相关细胞因子(IL-4、IL-10、IFNγ、TNF-β、MCP-1)含量,血清杀菌试验检测功能性抗体水平,MTT法测定脾淋巴细胞增殖指数(SI),流式细胞术测定CD4^(+)与CD8^(+)外周血T淋巴细胞亚群百分比;腹腔攻毒试验测定免疫保护率,组织荷菌数法测定攻毒菌株在体内的定植情况,并采集肺、肝、脾、肾脏制作病理组织切片,观察动物脏器病理变化。结果两种灭活疫苗均符合油乳剂灭活疫苗的性状、无菌、安全检测要求。Ⅰ和Ⅱ组小鼠血清IgG抗体效价、血清功能性抗体水平、相关细胞因子含量、SI及CD4^(+)与CD8^(+)T细胞亚群百分比均显著高于Ⅲ组(P均<0.05),Ⅰ和Ⅱ组间差异均无统计学意义(P均>0.05);两种灭活疫苗对小鼠的免疫保护率均为100%,Ⅰ和Ⅱ组小鼠肺、肝、脾、肾脏组织中攻毒菌株定植量显著低于Ⅲ组(P<0.05),各脏器组织病变较轻。结论采用两种不同混合方式制备的ER-SS2二联油乳剂灭活疫苗免疫小鼠后均可产生良好的体液免疫和细胞免疫,可100%抵抗ER、SS2和ER-SS2强毒株的攻击。Objective To prepare an oil emulsion-inactivated E.rhusiopathiae(ER)and Streptococcus suis(SS)combined vaccine and evaluate its immune effect.Methods Serotype 1a ER(code AEr31)and type 2 SS(SS2,code HF2)were used as vaccine strains,while formaldehyde as inactivating agent and ISA 201VG mineral oil as adjuvant.Inactivated whole bacteria(V-AEr31,V-HF2)and adjuvant were mixed by two modes,and the prepared emulsion-inactivated vaccines(V-AEr31+V-HF2)+ISA 201VG and(V-AEr31+ISA 201VG)+(V-HF2+ISA 201VG)were tested for properties,sterility and safety.Mice were divided into groupsⅠandⅡ,and immunized with inactivated vaccines(V-AEr31+V-HF2)+ISA201VG and(V-AEr31+ISA 201VG)+(V-HF2+ISA 201VG)by subcutaneous injection in several sites,using 0.9%sodium chloride solution as control(groupⅢ).The titer of IgG and contents of related cytokines(IL-4,IL-10,IFNγ,TNF-βand MCP-1)in sera of mice were determined by indirect ELISA,while the level of functional antibody by serum germicidal test,the splenic lymphocyte proliferation index(SI)by MTT assay,and the percentage of T lymphocyte subsets in CD4^(+) and CD8^(+)peripheral blood by flow cytometry.The immune protection rate was determined by intraperitoneal challenge test,while the colonization of challenge strain in vivo by the number of bacteria loaded in tissue.The lung,liver,spleen and kidney of mice were prepared into pathological sections and observed for pathological change.Results The two inactivated vaccine were qualified in tests for properties,sterility and safety.The serum IgG titer,functional antibody level,related cytokine content,SI as well as the percentage of T lymphocyte subsets in CD4^(+)and CD8^(+)peripheral blood were significantly higher in groupsⅠandⅡthan in groupⅢ(each P<0.05),while showed no significant difference in groupsⅠandⅡ(each P>0.05).Both the protective rates of the two inactivated vaccines to mice were 100%,while the bacterial colonization levels in lung,liver,spleen and kidney of mice in groupsⅠandⅡwere significantly lower than thos

关 键 词：猪丹毒丝菌 猪链球菌 油乳剂 灭活疫苗 免疫效果 免疫程序 

分 类 号：S855.3[农业科学—临床兽医学]