牛副结核分枝杆菌的分离及鉴定

Isolation and identification of Mycobacterium avium subsp.paratuberculosis from cattle

为进一步确定新疆某奶牛场疑似感染副结核病的病原菌,本研究采集了37头疑似患病奶牛的直肠刮取物,应用卵黄琼脂培养基对其进行分枝杆菌的分离培养,经菌落形态观察、抗酸染色以及多重PCR等方法对分离菌进行病原学鉴定,并利用副结核分枝杆菌亚型鉴定引物进行PCR扩增及测序分析。结果显示,分离培养的7株分离菌均为抗酸染色阳性的分枝杆菌;经多重PCR鉴定,其中3株谱型与副结核分枝杆菌k10参考株一致;进一步通过基因分型的PCR扩增及测序分析,确定这3株分离菌均为牛副结核分枝杆菌。本研究为进一步开展该菌病原学和流行病学研究提供了必要的研究资料。

To determine the pathogens of suspected bovine paratuberculosis from a dairy farm in Xinjiang, the rectal scrapes of 37 dairy cows were collected for Mycobacteria isolation using yolk agar medium. All the isolates were identified by morphological observation, microscopicall examination after acid-fast staining, multiplex PCR assays for M.avium subsp. paratuberculosis. Then, we identified subtypes of the isolates by PCR amplification and sequence analysis of typing gene. The results showed that all 7 isolates were positive in acid-fast staining, and the multiplex PCR results from 3 isolates of them were consistent with the control strain M.avium subsp. paratuberculosis k10. Therefore, the 3 isolates were identified as M.avium subsp. paratuberculosis. Furthermore, the PCR amplification and sequencing analysis of the typing genes indicated that the 3 isolates belonged to cattle type of M.avium subsp. paratuberculosis. This study provides necessary materials for etiological and epidemiological research of M.avium subsp. paratuberculosis.