Dot-PPA-ELISA快速检测猪丹毒抗体方法的建立及应用研究
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摘要
本研究以猪丹毒C43004(1a型)和C43-12(2型)两株不同血清型菌株为抗原提取菌株，选用EDTA渗透法提取的抗原作为膜载抗原，首次建立了Dot-PPA-ELISA检测猪丹毒血清抗体方法，并确定了应用Dot-PPA-ELISA的最佳工作条件。通过用C43004(1a型)、C43179(1b型)和C43-12(2型)三株不同血清型的猪丹毒混合强毒同时对具有不同抗体效价的免疫猪和对照猪进行皮肤内接种攻毒，确定了猪能抵抗混合强毒攻击的抗体保护阳性标准为1：32。本试验方法用国产硝酸纤维素膜为固相载体制备的诊断膜片特异性强，不与猪瘟、猪伪狂犬病、猪肺疫、猪链球菌病、大肠杆菌、仔猪副伤寒、猪布氏杆菌病、猪衣原体病的阳性血清发生交叉反应；该方法敏感性高，能够最低检出猪血清中含2.61×10~(-9)g的猪IgG抗体；膜片保存期长，在室温可保存4个月，4℃可保存7个月其检测活性不变。通过对免疫猪免疫后6周的抗体检测，证实Dot-PPA-ELISA能用于疫苗免疫猪后的抗体效价变化监测。本试验方法操作简便、快速准确、结果客观、易于判定，结果可存档、重复性好，所用试剂材料均可做到标准化，适合基层应用和猪群的猪丹毒抗体监测以及猪丹毒的流行病学调查。
Virulent pathogenic and predominant seroepidemic E. rhusiopathiae serotypes 1a(strain C43004) and 2(strain C43-12) are used to extract diaphragm-carried antigens. An EDTA-treated antigens is selected as diaphragm-carried antigen in Dot-PPA-ELISA. This antigen has high specificy and such EDTA-treated antigenic preparations are simple. Detecting antibodies to swine erysipelas by Dot-PPA-ELISA is reported firstly. Optimum trial conditions of Dot-PPA-ELISA are picked out. The positive titre protecting pigs from swine erysipelas is establish as 1:32 by response of temperature and lesions after all immunized and unvaccinated control pigs challenge exposure with three composite virulent strains of E. rhusiopathiae(strain C43004,serotype la; strain C43179,serotype lb;strain C4312,serotype 2). The specificity is proved by the blocking and cross-reaction test. The diagnostic diaphragm does not react with antibodies to Pasteurellosis, Streptococcosis, Chlamydiosis, Salmonellosis, Colibacillosis, Brucellosis, HCV and PR
V. By Dot-PPA-ELISA detecting antibodies to swine erysipelas has good sensitivity and is sensitive more 9 times than growth agglutination test(GA), and can detect 2.61 10-9g IgG antibodies in swine serum. Stored at 4"C for 7 months or at room temperature for 4 months, the sensitivity of diagnostic diaphragms do not change. Results of this research show that Dot-PPA-ELISA is a convenient, rapid, sensitive and specific diagnostic method for detecting specific antibodies to E. rhusiopathiae in serum. Otherwise, all materials used in Dot-PPA-ELISA can be standardised. So this method can be fit for serological surveillance of swine herds vaccined and for the seroepidemiological investigation of swine erysipelas.
Virulent pathogenic and predominant seroepidemic E. rhusiopathiae serotypes 1a(strain C43004) and 2(strain C43-12) are used to extract diaphragm-carried antigens. An EDTA-treated antigens is selected as diaphragm-carried antigen in Dot-PPA-ELISA. This antigen has high specificy and such EDTA-treated antigenic preparations are simple. Detecting antibodies to swine erysipelas by Dot-PPA-ELISA is reported firstly. Optimum trial conditions of Dot-PPA-ELISA are picked out. The positive titre protecting pigs from swine erysipelas is establish as 1:32 by response of temperature and lesions after all immunized and unvaccinated control pigs challenge exposure with three composite virulent strains of E. rhusiopathiae(strain C43004,serotype la; strain C43179,serotype lb;strain C4312,serotype 2). The specificity is proved by the blocking and cross-reaction test. The diagnostic diaphragm does not react with antibodies to Pasteurellosis, Streptococcosis, Chlamydiosis, Salmonellosis, Colibacillosis, Brucellosis, HCV and PR
V. By Dot-PPA-ELISA detecting antibodies to swine erysipelas has good sensitivity and is sensitive more 9 times than growth agglutination test(GA), and can detect 2.61 10-9g IgG antibodies in swine serum. Stored at 4"C for 7 months or at room temperature for 4 months, the sensitivity of diagnostic diaphragms do not change. Results of this research show that Dot-PPA-ELISA is a convenient, rapid, sensitive and specific diagnostic method for detecting specific antibodies to E. rhusiopathiae in serum. Otherwise, all materials used in Dot-PPA-ELISA can be standardised. So this method can be fit for serological surveillance of swine herds vaccined and for the seroepidemiological investigation of swine erysipelas.
引文
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[4] Hassanein R, Sawada T, Kataoka Y, et al. Serovars of Erysipelothrix species isolated fronm the tonsils of healthy cattle in Japan[J]. Vet Microbio, 2001,82:97-100.
[5] 仇华吉，童光志．第十七届国际猪病大会述要．第十七届国际猪病会议论文选集(上海市奉贤区畜牧兽医站编译)．2002，Ⅰ-Ⅳ．
[6] 郑庆端，徐汉祥，王梦龄．猪丹毒流行病学调查与研究补报[J]．江苏农业科学，1982，12：39-41．
[7] 郑关端，徐汉祥．猪丹毒三十年来研究的成就和展望[J]．江苏农业科学，1981，5：51-55．
[8] Chin J C, Enmens G J. Immunoreactivity of fractionated antigens obtained from autoclaved extracts of an arthritogenic isolate of Erysipelothrix rhusiopathiae[J]. Aust Vet J,1986,63(11):355-359.
[9] 杨本升，刘玉斌，苟仕金等主编．动物微生物学(第二版)[M]．吉林：吉林科学技术出版社，1995，644-650．
[10] Tamura Y, Takahashi T, Zarkasie K, et al. Differentiation of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum by Sodium Dodecyl Sulfate-Poluacrylamide Gel Electrophoresis of cell proteins[J]. Int J Syst Bacterio, 1993, 43(1): 111-114.
[11] Kitajima T, Oishi E, Amimoto K, et al. Protective effect of NaOH extracted Erysipelothrix rhusiopathiae vaccine in pig[J]. J Vet Med Sci, 1998,60(4):9-14.
[12] Lachmann P G, Deicher H. Solubilization and characterization of surface antigenic components of Erysipelothrix rhusiopathiae T28[J]. Infect Immum, 1986 52:818-822.
[13] Wood R L, Haubrich D R, Harrington R J. Isolation of previously unreported serotype of Erysipelothrix rhusiopathiae from swine[J]. Am J Vet Res, 1978,39:1958-1961.
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