副猪嗜血杆菌新型微量凝集试验抗体检测方法的建立  

Establishment of the micro-agglutination test for detection of antibody against Haemophilus parasuis

作　　者：薛云[1,2] 王乐[2] 王臣[2] 赵朋超[1] 司丽芳[2] 赵战勤[1,2] XUE Yun;WANG Le;WANG Chen;ZHAO Peng-chao;SI Li-fang;ZHAO Zhan-qin(Laboratory of Veterinary biologics Engineering,College of Animal Science and Technology,Henan University of Science and Techology,Luoyang 471023,China;Lab of Medical Microbiology Engineering,College of Medical Technology and Engineering,Henan University of Science and Techology,Luoyang 471023,China)

机构地区：[1]河南科技大学医学技术与工程学院,微生物检验实验室,河南洛阳471023 [2]河南科技大学动物科技学院,兽医生物制品工程实验室,河南洛阳471023

出　　处：《中国预防兽医学报》2018年第9期806-811,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金项目(31302106;U1704117;31672530)

摘　　要：为建立国内流行血清型副猪嗜血杆菌的新型微量凝集试验抗体检测方法(MAT),本研究以副猪嗜血杆菌4型、5型、12型和13型为抗原,分别对其MAT方法进行了最佳反应条件筛选、特异性、敏感性和重复性试验,以及疫苗免疫样品和临床样本检测。MAT试验结果显示,4型、5型、12型和13型凝集抗原的最佳反应浓度介于1.5×10~9 cfu/mL～2.0×10~9 cfu/mL,最佳反应温度37℃,最佳反应时间为6h～8h。4种型的MAT方法对猪巴氏杆菌病等6种细菌性疾病和猪瘟等5种病毒性疾病阳性血清的检测结果均为阴性,4种方法之间有轻微的交叉反应,但其抗体效价均不高于1∶4 (阴性临界值)。4种型的MAT方法比相应试管凝集方法的敏感性提高了2～4倍,重复性试验结果显示该方法检测副猪嗜血杆菌4种型变异系数均小于10%,重复性良好。利用该方法检测临床样品,并与国家标准套式PCR方法对比,结果显示二者阳性符合率为82.1%。疫苗免疫抗体检测结果显示,4种型的MAT方法均能检测到商品化疫苗免疫猪14 d的血清抗体,可用于疫苗免疫的抗体评价。对临床样品的检测结果显示,断奶仔猪、保育仔猪和经产母猪的总阳性率分别为12.3%、49.3%和69.8%,提示保育期仔猪的合群可能导致了猪群的大量感染。本研究为副猪嗜血杆菌病的疫苗抗体评价和流行病学监测提供了一种简便经济可行的方法。The objective of this study was to establish micro-agglutination test （MAT） for detection of antibodies against Haemophilus parasuis. The 4 MATs were developed for detections of the antibodies against most prevalent H.parasuis serotypes of 4, 5, 12 and 13, respectively, and the reaction conditions were optimized with the standard positive and negative sera, which were determinated that antigen concentrations of inactivated whole-cell of H.parasuis serovar 4, 5, 12 and 13 were ranged from 1.5x109 to 2.0x 109cfu/mL and the inclubation temperature was 37~C for 6hrs to 8hrs. The 4 MATs were specificity for H.parasuis serovars of 4, 5, 12 and 13 detections, respectively, and the agglutination of the antibody titers were no more than 1：4 dilutions among the different H.parasuis serotypes, which was set as the cutoff value for the assays. However, the 4 assays had no cross-reactions to the positive sera of E.coli, Salmonella, P.multocida, B.bronchiscptia, A.pleuropneumoniae, S.suis2, classical swine fever virus,porcine circovirus, porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus. The sensitivities of the four MATs were 2 to 4 times higher than the tube agglutination assay. The coincidence of the nested PCR and MATs were 82.1%. The antibodies in piglets could be detected at 14 days post vaccination with five commercial H.parasuis vaccines. The positive rates were 12.3%, 49.3% and 69.8% in the samples from weaned pigs, nursery piglets and sows, respectively, indicating that piglets were infected by H.parasuis during the nursery periods. The feasibility of the MATs for detecting antibodies of H.parasuis was confirmed in this study.

关 键 词：猪 副猪嗜血杆菌 微量凝集试验 抗体 

分 类 号：S852.61[农业科学—基础兽医学]