猪丹毒丝菌灭活疫苗制备及其对小鼠免疫效力的评价
|
摘要
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2 mg/L甲醛灭活后,用矿物油佐剂ISA 201 VG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∶3 200、1∶6 400、1∶1 600、1∶6 400、1∶3 200(以全菌体超声裂解物为包被抗原)和1∶3 200、1∶6 400、1∶3 200、1∶25 600、1∶6 400(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ISA 201 VG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 rhusiopathiaeand 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 AEr31 and 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.
Inorder toprepare the inactivated vaccine of Erysipelothrix rhusiopathiaeand 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 AEr31 and 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.
引文
[1] 王力波,刘芳,谢江,等.猪丹毒丝菌不同年代分离菌株流行病学特征及部分生物学特性比较研究 [J].中国兽医学报,2016,36(3):423-430.WANG L B,LIU F,XIE J,et al.Comparative analysis on the epideminlogical and some biological characteristics of Erysipelothrix rhusiopathiae isolates from different years [J].Chinses Journal of Veterinary Science,2016,36(3):423-430.
[2] 万遂如.2013年规模化猪场细菌性疾病流行状况与防控对策 [J].养猪,2013(6):105-110.WAN S R.Epidemic situation and prevention and control measures of bacterial diseases in large-scale pig farms in 2013 [J].Swine Production,2013(6):105-110.
[3] 胡晓芬,王静,熊剑锋,等.一起猪丹毒杆菌病例的临床分离鉴定及诊治 [J].养猪,2014(2):107-109.HU X F,WANG J,XIONG J F,et al.Clinical isolation,diagnosis and treatment of a case of Erysipelothrix rhusiopathiae [J].Swine Production,2014(2):107-109.
[4] 罗雪刚,罗永芳,戴怡雪,等.红斑丹毒丝菌的分离鉴定及疫苗的免疫保护作用 [J].中国兽医学报,2016,36(11):1882-1886.LUO X G,LUO Y F,DAI Y X,et al.Characterization of Erysipelothrix rhusiopathiae isolates and the immunoprotection of swine erysipelas vaccine [J].Chinses Journal of Veterinary Science,2016,36(11):1882-1886.
[5] 朱良全,冯倩倩,李聪研,等.生产用培养基对猪丹毒杆菌G4T10株安全及免疫原性试验的比较研究 [J].中国兽药杂志,2013,47(12):22-24.ZHU L Q,FENG Q Q,LI C Y,et al.Comparative Study about the effect of culture media on the safety and immunogenicity of Erysipelothrix rhusiopathiaestrain G4T10 [J].Chinese Journal of Veterinary Drug,2013,47(12):22-24.
[6] DA S A,DA C I MR,LUPERNI H A C,et al.Cloning,auto-induction expression,and purification of rSpaA swine erysipelas antigen [J].Current Microbiology,2012,65(4):369-374.
[7] 吴琼娟,杨志鹏,姚焱彬,等.猪红斑丹毒丝菌制苗用菌株的筛选[J].浙江农业学报,2018,30(9):1467-1475.WU Q J,YANG ZH P,YAO Y B,et al.Screening of strains for inactivated vaccine of Erysipelothrix rhusiopathiae[J].Acta Agriculturae Zhejiangensis,2018,30(9):1467-1475.
[8] 姚焱彬,陆萍,李郁,等.检测猪丹毒杆菌抗体重组SpaA ELISA的建立[J].微生物学通报,2017,44(3):739-748.YAO Y B,LU P,LI Y,et al.Development of recombinant SpaA-based ELISA for detection of antibodies against swine erysipelas [J].Microbiology China,2017,44(3):739-748.
[9] 姚焱彬,陆萍,李郁,等.猪丹毒丝菌SpaA基因原核表达及表达蛋白质的免疫原性分析 [J].畜牧兽医学报,2017,48(3):492-500.YAO Y B,LU P,LI Y,et al.Prokaryotic expression and immunogenicity analysis of SpaA gene in Erysipelothrix rhusiopathiae [J] Acta Veterinaria et Zootechnica Sinica,2017,48(3):492-500.
[10] WANG Q,FIDALGO S,CHANG B J,et al.The deteetion and recovery of Erysipelothrix rhusiopathiae in meat and abattoir samples in Western Australia[J].Joumal of Applied Microbiology,2002,92(5):844-850.
[11] 刘操,杨忠宝,刘建柱.猪肺疫菌苗种类及其使用[J].猪业科学,2009(8):35-36.LIU C,YANG ZH B,LIU J ZH.Species and use of swine plaguevaccine[J].Swine Industry Science,2009(8):35-36.
[12] 万莉,韩国全,郭万柱.猪丹毒疫苗的研究进展[J].猪业科学,2010(6):32-35.WAN L,HAN G Q,GUO W ZH.Research progress of Erysipelothrix rhusiopathiae [J].Swine Industry Science,2010(6):32-35.
[13] VAN OIRSCHOT J T.Vaccination in food animal populations[J].Vaccine,1994,12(5):415-418.
[14] 王明俊.兽医生物制品学 [M].北京:中国农业出版社,1997:205-208.WANG M J.Veterinary Biotechnology [M].Beijing:China Agricultural Press,1997:205-208.
[15] 杨汉春.动物免疫学 [M].北京:中国农业大学出版社,2003:27-29.YANG H CH.Animal Immunology [M].Beijing:China Agricultural University Press,2003:27-29.
[16] 金伯泉.细胞和分子免疫学[M].北京:科学出版社,2001:131-139.JIN B Q.Cellular and Molecular Immunology [M].Beijing:Science Press,2001:131-139.
[17] 罗满林.动物传染病学[M].北京:中国林业出版社,2013:225-227.LUO M L.Animal Epidemiology [M].Beijing:China Forestry Publishing House,2013:225-227.
[18] 赵德明.兽医病理学 [M].北京:中国农业出版社,2005:133-135.ZHAO D M.Veterinary Pathology [M].Beijing:China Agricultural Press,2005:133-135.
[19] ZUCKERMANN F A,GASKINS H R.Distribution of porcine CD4/CD8 double-positive T lymphocytes in mucosa-associated lymphoid tissues [J].Immunology,1996,87(3):493-499.
[20] TSUTSUI H,NISHIGUCHI S.Importance of Kupffer cells in the development of acute liver injuriesin mice [J].International Journal of Molecular Sciences,2014,15(5):7711-7730.
[21] 周亚飞,周梅仙.新鱼腥草素纳纳米乳佐剂对肉仔鸡免疫功能的影响[J].西北农业学报,2016,25(11):1591-1596.ZHOU Y F,ZHOU M X.Immune effect of Nano-Emulsion on SNH in broilers[J].Acta Agriculturae Boreali-occidentalis Sinica,2016,25(11):1591-1596.
[22] 邹垚.猪丹毒杆菌的分离鉴定和免疫特性分析[D].南京:南京农业大学,2015.ZOU Y.Isolation,characterization and immunological characteristic aanalysis of Erysipelothrix rhusiopathiae [D].Nanjing:Nanjing Agricultural University,2015.
[2] 万遂如.2013年规模化猪场细菌性疾病流行状况与防控对策 [J].养猪,2013(6):105-110.WAN S R.Epidemic situation and prevention and control measures of bacterial diseases in large-scale pig farms in 2013 [J].Swine Production,2013(6):105-110.
[3] 胡晓芬,王静,熊剑锋,等.一起猪丹毒杆菌病例的临床分离鉴定及诊治 [J].养猪,2014(2):107-109.HU X F,WANG J,XIONG J F,et al.Clinical isolation,diagnosis and treatment of a case of Erysipelothrix rhusiopathiae [J].Swine Production,2014(2):107-109.
[4] 罗雪刚,罗永芳,戴怡雪,等.红斑丹毒丝菌的分离鉴定及疫苗的免疫保护作用 [J].中国兽医学报,2016,36(11):1882-1886.LUO X G,LUO Y F,DAI Y X,et al.Characterization of Erysipelothrix rhusiopathiae isolates and the immunoprotection of swine erysipelas vaccine [J].Chinses Journal of Veterinary Science,2016,36(11):1882-1886.
[5] 朱良全,冯倩倩,李聪研,等.生产用培养基对猪丹毒杆菌G4T10株安全及免疫原性试验的比较研究 [J].中国兽药杂志,2013,47(12):22-24.ZHU L Q,FENG Q Q,LI C Y,et al.Comparative Study about the effect of culture media on the safety and immunogenicity of Erysipelothrix rhusiopathiaestrain G4T10 [J].Chinese Journal of Veterinary Drug,2013,47(12):22-24.
[6] DA S A,DA C I MR,LUPERNI H A C,et al.Cloning,auto-induction expression,and purification of rSpaA swine erysipelas antigen [J].Current Microbiology,2012,65(4):369-374.
[7] 吴琼娟,杨志鹏,姚焱彬,等.猪红斑丹毒丝菌制苗用菌株的筛选[J].浙江农业学报,2018,30(9):1467-1475.WU Q J,YANG ZH P,YAO Y B,et al.Screening of strains for inactivated vaccine of Erysipelothrix rhusiopathiae[J].Acta Agriculturae Zhejiangensis,2018,30(9):1467-1475.
[8] 姚焱彬,陆萍,李郁,等.检测猪丹毒杆菌抗体重组SpaA ELISA的建立[J].微生物学通报,2017,44(3):739-748.YAO Y B,LU P,LI Y,et al.Development of recombinant SpaA-based ELISA for detection of antibodies against swine erysipelas [J].Microbiology China,2017,44(3):739-748.
[9] 姚焱彬,陆萍,李郁,等.猪丹毒丝菌SpaA基因原核表达及表达蛋白质的免疫原性分析 [J].畜牧兽医学报,2017,48(3):492-500.YAO Y B,LU P,LI Y,et al.Prokaryotic expression and immunogenicity analysis of SpaA gene in Erysipelothrix rhusiopathiae [J] Acta Veterinaria et Zootechnica Sinica,2017,48(3):492-500.
[10] WANG Q,FIDALGO S,CHANG B J,et al.The deteetion and recovery of Erysipelothrix rhusiopathiae in meat and abattoir samples in Western Australia[J].Joumal of Applied Microbiology,2002,92(5):844-850.
[11] 刘操,杨忠宝,刘建柱.猪肺疫菌苗种类及其使用[J].猪业科学,2009(8):35-36.LIU C,YANG ZH B,LIU J ZH.Species and use of swine plaguevaccine[J].Swine Industry Science,2009(8):35-36.
[12] 万莉,韩国全,郭万柱.猪丹毒疫苗的研究进展[J].猪业科学,2010(6):32-35.WAN L,HAN G Q,GUO W ZH.Research progress of Erysipelothrix rhusiopathiae [J].Swine Industry Science,2010(6):32-35.
[13] VAN OIRSCHOT J T.Vaccination in food animal populations[J].Vaccine,1994,12(5):415-418.
[14] 王明俊.兽医生物制品学 [M].北京:中国农业出版社,1997:205-208.WANG M J.Veterinary Biotechnology [M].Beijing:China Agricultural Press,1997:205-208.
[15] 杨汉春.动物免疫学 [M].北京:中国农业大学出版社,2003:27-29.YANG H CH.Animal Immunology [M].Beijing:China Agricultural University Press,2003:27-29.
[16] 金伯泉.细胞和分子免疫学[M].北京:科学出版社,2001:131-139.JIN B Q.Cellular and Molecular Immunology [M].Beijing:Science Press,2001:131-139.
[17] 罗满林.动物传染病学[M].北京:中国林业出版社,2013:225-227.LUO M L.Animal Epidemiology [M].Beijing:China Forestry Publishing House,2013:225-227.
[18] 赵德明.兽医病理学 [M].北京:中国农业出版社,2005:133-135.ZHAO D M.Veterinary Pathology [M].Beijing:China Agricultural Press,2005:133-135.
[19] ZUCKERMANN F A,GASKINS H R.Distribution of porcine CD4/CD8 double-positive T lymphocytes in mucosa-associated lymphoid tissues [J].Immunology,1996,87(3):493-499.
[20] TSUTSUI H,NISHIGUCHI S.Importance of Kupffer cells in the development of acute liver injuriesin mice [J].International Journal of Molecular Sciences,2014,15(5):7711-7730.
[21] 周亚飞,周梅仙.新鱼腥草素纳纳米乳佐剂对肉仔鸡免疫功能的影响[J].西北农业学报,2016,25(11):1591-1596.ZHOU Y F,ZHOU M X.Immune effect of Nano-Emulsion on SNH in broilers[J].Acta Agriculturae Boreali-occidentalis Sinica,2016,25(11):1591-1596.
[22] 邹垚.猪丹毒杆菌的分离鉴定和免疫特性分析[D].南京:南京农业大学,2015.ZOU Y.Isolation,characterization and immunological characteristic aanalysis of Erysipelothrix rhusiopathiae [D].Nanjing:Nanjing Agricultural University,2015.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |