11种牛分枝杆菌抗原在牛结核病诊断中的初步评价

doi:10.16431/j.cnki.1671-7236.2018.08.030

摘要/Abstract

摘要：

为筛选及评价用于牛结核病诊断的抗原，本试验将CFP-10、ESAT-6、TB10.4、TB27.4、MPT51、MPT63、MPT64、MPB70、MPB83、Rv3872和Ag85B共11种牛分枝杆菌抗原分别作为包被抗原建立间接ELISA方法，比较其对牛结核病的检出率；同时利用豚鼠和牛的皮试试验评价重组蛋白作为皮试试验刺激原的潜力。此外，将重组蛋白分别刺激结核病阳性牛和阴性牛的抗凝血24 h，检测血浆中的IFN-γ水平，评价各重组蛋白作为IFN-γ释放试验刺激原的潜力。结果显示，不同重组蛋白对结核病阳性血清的反应活性不一，MPB70总检出率最高，为59.7%；其次是Ag85B、ESAT-6和MPB83，检出率均在45%以上；MPT51的检出率最低，仅为2.2%。豚鼠和牛皮试试验均显示，单个重组蛋白作为刺激原难以产生令人满意的迟发型过敏反应（delayed type hypersensitivity，DTH），而TB10.4、TB27.4、MPT64、MPT63或Rv3872作为补充抗原，分别与CFP-10或ESAT-6混合，均可特异性地刺激结核病阳性牛产生较强的DTH反应，且与PPD-B无显著差异（P>0.05）。重组蛋白CFP-10、ESAT-6、TB10.4和MPT51均能刺激结核病牛全血释放一定的IFN-γ，其中CFP-10、CFP-10-ESAT-6串联蛋白和MPT51刺激结核病阳性牛全血释放的IFN-γ显著高于阴性牛（P<0.05）。因此，这11种牛分枝杆菌抗原并不适合单独用于牛结核病的血清学诊断、皮试试验或IFN-γ释放试验，但以CFP-10和ESAT-6为核心，TB10.4、TB27.4、MPT64、MPT63、Rv3872或MPT51作为其补充抗原，均能提高检测敏感性，有作为皮试试验和IFN-γ释放试验特异性刺激原用于牛结核病诊断的潜力。

关键词: 牛分枝杆菌; 牛结核病; 诊断; 抗体; 皮试试验; IFN-γ 释放试验

Abstract:

To screen and evaluate antigens used for diagnosis of bovine tuberculosis (bTB),eleven Mycobacterium bovis (M.bovis) antigens of CFP-10,ESAT-6,TB10.4,TB27.4,MPT51,MPT63,MPT64,MPB70,MPB83,Rv3872 and Ag85B were used as coated antigen to establish indirect ELISA,the detection rates of ELISA based on each antigen were calculated.The potential of these recombinant proteins used as stimuli in skin test were assessed by skin test in guinea pigs and cattle.Besides,to evaluate the potential of these proteins used as stimuli in IFN-γ release assay,the heparinized blood from bTB positive and negative cattle were stimulated by recombinant proteins for 24 h,and IFN-γ in plasma were detected.The results indicated recombinant proteins showed virous reactivities to bTB positive serum,MPB70-based ELISA showed highest detection rate (59.7%),and followed by Ag85B,ESAT-6 and MPB83 (>45%).MPT51-based ELISA showed the lowest detection rate,which was only 2.2%.The results of skin tests on guinea pigs and cattle showed single recombinant protein as stimulus in skin test couldn't induce satisfactory delayed type hypersensitivity (DTH),while TB10.4,TB27.4,MPT64,MPT63 or Rv3872 as supplementary antigen for CFP-10 or ESAT-6,could induce high DTH in bTB positive cattle,and showed no difference with PPD-B (P>0.05).Whole blood from bTB positive cattle could release a certain level of IFN-γ induced by the recombinant protein CFP-10,ESAT-6,TB10.4 and MPT51.The IFN-γ induced by CFP-10,CFP-10-ESAT-6 and MPT51 were significantly higher in bTB positive cattle than negative cattle (P<0.05).Therefore,these eleven M.bovis antigens were not good for singly used in serological diagnosis,skin test,or IFN-γ release assay for bTB.However,TB10.4,TB27.4,MPT64,MPT63,Rv3872 or MPT51 as supplementary antigen for CFP-10 or ESAT-6 could improve the sensitivity,and be used as specific stimuli in skin test and IFN-γ release assay for the diagnosis of bTB.

Key words: Mycobacterium bovis; bovine tuberculosis; diagnosis; antibodies; skin test; IFN-γ release assay

中图分类号:

S852.61⁺8

高新桃, 贾红, 侯绍华, 杨宏军, 郭晓宇, 袁维峰, 姜一曈, 朱鸿飞, 鑫婷, 丁家波. 11种牛分枝杆菌抗原在牛结核病诊断中的初步评价[J]. 《中国畜牧兽医》, 2018, 45(8): 2282-2292.

GAO Xintao, JIA Hong, HOU Shaohua, YANG Hongjun, GUO Xiaoyu, YUAN Weifeng, JIANG Yitong, ZHU Hongfei, XIN Ting, DING Jiabo. Preliminary Evaluation of Eleven Mycobacterium bovis Antigens in the Diagnostic Methods of Bovine Tuberculosis[J]. , 2018, 45(8): 2282-2292.

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