多重PCR和间隔区寡核苷酸分型技术应用于不同流行地区的牛结核病研究

目的 在牛结核病监测中联合应用菌株的多重PCR鉴定和间隔区寡核苷酸分型(spoligotyping),快速鉴定分离菌株并分析不同地区流行菌株的特点。方法 分别在牛结核病清净地区和流行地区进行牛型PPD变态反应试验,扑杀阳性牛后进行病理检查,并采集病料分离细菌。获取分离菌株,进行多重PCR鉴定和spoligotyping分型,分析不同地区的菌株特征。结果 结核病清净地区监测到16头牛型PPD阳性牛,扑杀后未见有病变,采集病料分离到4株分枝杆菌,经多重PCR鉴定均为非典型分枝杆菌。流行地区监测牛型PPD阳性23头,扑杀后发现14头有病变,采集病料后共有11头分离到疑似菌,经多重PCR鉴定均为牛型分枝杆菌。用spoligotyping分析共分为4个型,其中2个为未见报道的新型,报英国AHVLA牛结核spoligotyping数据库后获取通用编号SB1903和SB1904。结论 分离菌株中的优势菌株为首次报道的SB1903,但也检出有国际流行菌株SB0140,证实该地区牛结核病的流行是以“独特菌型地域内相互传播为主、输入性感染为辅”为特征。本次监测发现国内有SB0140菌株分布,提示应调查该型菌是否已经传染至我国人间。

Application of multiple-PCR and spoligotyping for investigation of bovine tuberculosis from different epidemic areas

To characterize the Mycobacterium bovis in the different epidemic areas, we combined the multiple-PCR and spoligotyping to investigate the bacteria isolated in the bovine tuberculosis surveillance. The bovine PPD skin tests were performed in lower-prevalence and higher-prevalence areas in China. The positives were slaughtered and their carcasses were examined for the tubercles. Isolated pathogens were identified by using multiple-PCR and then genotyped by using spoligotyping. No lesion was found from the carcasses of 16 positives in the lower-prevalence area. The 4 strains Mycobacteria were isolated from the lymph nodes collected from the positives and then identified as nontuberculosis Mycobacteria by using multiple-PCR. The 23 positives were slaughtered and examined in the higher-prevalence area. The lesions were found from 14 cattle and then the pathogens were isolated from the 11 of them. The bacteria were identified as Mycobacterium bovis by using multiple-PCR. Totally, 4 spoligotypes were classified and 2 of them were new types. Number SB1903 and SB1904 were given after the patterns were submitted to the bovine tuberculosis spoligotyping database. It was revealed that the character of "domestic typing SB1903" was predominant but the world-spread typing SB0140 still be insistent in the area. It suggests that the scientists should pay attention on the transmission of the typing SB0140 to human beings as the typing has been found from cattle in China.