猪胸膜肺炎放线杆菌ApxⅣ-ELISA鉴别诊断试剂盒的研制与应用  

Development and Application of ApxⅣ-ELISA Differentiating Diagnostic Kit for Actinobacillus pleuropneumoniae

作　　者：贾凡[1] 胡军勇[1] 王建银[1] 石碧[1] 李树清[2] 陈焕春[1] 周锐[1] 

机构地区：[1]华中农业大学动物医学院农业微生物学国家重点实验室,武汉430070 [2]上海出入境检验检疫局,上海200135

出　　处：《农业生物技术学报》2007年第6期911-917,共7页Journal of Agricultural Biotechnology

基　　金：国家科技支撑计划(No.2006BAD06A01);湖北省科技攻关计划(No.2006AA205A02);教育部新世纪优秀人才支持计划(No.NCET-04-0740)资助

摘　　要：胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)是一种严重危害世界养猪业的重要病原菌,至少有15种血清型,根据所有血清型菌株仅在感染猪体内产生外毒素ApxⅣ的特点,建立了一种能够区分感染猪与免疫猪的鉴别诊断方法ApxⅣ-ELISA。研究在反应条件方面加以优化,将该诊断方法进一步研制成试剂盒,对其特异性、敏感性、重复性和稳定性进行了测试,与间接血凝试验及IDEXX公司的CHEKIT-APP-APXⅣ试剂盒进行了对比研究,并用该试剂盒检测了来自中国、英国、丹麦、美国、加拿大、澳大利亚等国的临床猪血清1453份。结果表明,ApxⅣ-ELISA试剂盒具有很高的特异性和敏感性,与CHEKIT-APP-APXⅣ试剂盒的检测符合率为91.37%;3批试剂盒的批内和批间重复性良好(变异系数均<15%);试剂盒在2～8℃保存9个月内稳定性良好。所检种猪血样中,来自中国猪场1、中国猪场2、美国、加拿大、丹麦、澳大利亚和英国的ApxⅣ抗体阳性率分别为30.05%、70.69%、39.72%、0.64%、42.22%、1.85%和93.94%;所检我国2个规模化猪场的仔猪ApxIV抗体阳性率分别为21.77%和27.15%,育肥猪的阳性率分别为5.51%和11.07%。研究表明,ApxⅣ-ELISA试剂盒能够有效区分APP感染猪与三价灭活疫苗或亚单位疫苗免疫猪。Actinobacillus pleuropneumoniae （APP） is the causative agent of porcine contagious pleuropneumonia which causes great economic losses in the pig industry worldwide. There are at least 15 serotypes of APP so that it is very difficult to diagnose and control this disease. An ApxⅣ-ELISA which could distinguish the vaccinated pigs from APP-infected ones was established based on the fact that the toxin Apx Ⅳ expresses only in vivo in APP-infected pigs. A differentiating diagnostic kit was developed by the optimization of conditions, whose specificity, sensitivity, repeatability and stability were evaluated and compared with an analogous ELISA kit （CHEKIT-APP-APX Ⅳ ） from the company IDEXX and the indirect haemagglutination assay （IHA）. A total of 1 453 clinical sera from China, UK, Denmark, USA, Canada and Australia were screened using the Apx Ⅳ-ELISA kit. The results showed that the ApxⅣ-ELISA kit had very high specificity, sensitivity, repeatability [the inter- and intmbatch imprecision （CV%） 〈 15%] and stability （very stable at 4～8 ℃ for 9 months）. Compared with CHEKIT-APP-APXⅣ, the overall agreement rate was up to 91.37%. The ApxIV antibody positive ratios of sows from China Farm 1 （CN1）, China Farm 2 （CN2）, USA, Canada, Denmark, Australia and UK were 30.05%, 70.69%, 39.72%, 0.64%, 42.22%, 1.85% and 93.94%, respectively; The positive ratios of piglets were 21.77% （CN1） and 27.15% （CN2）, and those of finishing pigs were 5.51% （CN1） and 11.07% （CN2）. The data confirm that the ApxⅣ-ELISA kit can be used to distinguish the vaccinated pigs from APP-infected ones.

关 键 词：胸膜肺炎放线杆菌 ApxⅣ-ELISA 鉴别诊断试剂盒 

分 类 号：S188[农业科学—农业基础科学]