规模化奶牛场犊牛腹泻产气荚膜梭菌的分离与鉴定

Isolation and Identification of Clostridium perfringens from Diarrhea of Calves in Large-scale Dairy Farms

为了确定规模化奶牛场犊牛腹泻的发病原因,采集腹泻犊牛和同群犊牛血清及肛门拭子样品各15份,并采集同舍泌乳母牛所产大罐奶样品100 mL。血清采用牛病毒性腹泻病毒(BVDV)抗原检测试剂盒检测,肛门拭子和大罐奶样品用牛腹泻五联病原体快速诊断试剂盒检测,对肛门拭子和大罐奶样品进行增菌培养、克隆纯化,并对克隆用毒力基因多重PCR鉴定其毒力型,并用多位点序列分型(MLST)分析其菌群结构。结果显示:血清中未检测到BVDV,而仅腹泻犊牛肛门拭子为产气荚膜梭菌弱阳性,腹泻犊牛及同群犊牛共3份肛门拭子分离到产气荚膜梭菌(阳性率20.0%),大罐奶中未分离到活菌。经毒力基因多重PCR鉴定全部为A型。说明A型产气荚膜梭菌感染是引起犊牛腹泻的主要原因,但经MLST分群,3个克隆不在同一分支,说明产气荚膜梭菌在牛群中存在基因多态性。本研究为规模化奶牛场预防产气荚膜梭菌引起的犊牛腹泻病提供一定科学依据。

In order to determine the pathogenesis of calf diarrhea in large-scale dairy farms,15 serum and 15 anal swab samples were collected from diarrhea calves and co-herd calves,and 100 mL samples from milk cans of cows in the same house were collected.Serum samples were detected by Bovine viral diarrhea virus(BVDV)antigen detection kit,anal swabs and milk samples from large cans were detected by the rapid diagnostic kit targeting on five pathogens of bovine diarrhea.Bacteria from anal swabs and large cans of milk samples were cultured,cloned,and then virulence types were identified by multiplex PCR for the typical clones.Multilocus sequence typing(MLST)was used to cluster these strains.The results showed that no BVDV was detected in serum samples,and only Clostridium perfringens(C.perfringens)was weak positive for anal swabs of diarrhea calves.C.perfringens was isolated from three anal swabs of diarrhea calves and their cohorts(positive rate was 20.0%).No live bacteria was isolated from large cans of milk.All three clones were identified as type A by analyzing virulence genes with multiplex PCR.The results indicated that C.perfringens type A infection was the main cause of diarrhea in calves,but the three clones were not in the same branch in MLST clustering,indicating that C.perfringens gene polymorphism exists in the farm.This study will provide some reference for the prevention of the disease in large-scale dairy farms.