和牛源致病性大肠杆菌的分离鉴定

Isolation and Identification of Wagyu-Originated pathogenicity Escherichia coli

为查明和牛犊牛腹泻死亡原因,该试验采集齐齐哈尔市龙江县和牛腹泻犊牛的新鲜粪便10份进行细菌的分离培养、生化鉴定、16S rRNA基因测序。选择1株腹泻样品的纯化菌为供试菌株进行致病性试验、药敏试验、毒力基因检测和同源性分析。结果显示,从10份样品中分离的10株病原菌与大肠埃希氏菌生长形态及生化特征一致,分离率为100%;供试菌株命名为Wagyu.1;毒力基因检测结果显示,K88、CS31A、BfpA、EAST1和hlyA基因为阳性;16S rRNA序列与GenBank中大肠埃希氏菌同源性为93.6%~99.9%,系统进化树显示,Wagyu.1与人源大肠埃希氏菌62-b pink菌株(Genbank登录号:MN208129)在同一个分支,亲缘关系最近;体外药敏试验结果显示,仅对头孢他啶和头孢吡肟最敏感。结果表明,分离株Wagyu.1为一种毒力较强的致病性大肠埃希氏菌,其与人源大肠埃希氏菌的亲缘关系最近,建议加强人畜分流管理,减少疫病的发生。

To investigate the cause of Wagyu calf diarrhea mortality,this study collected ten fresh fecal samples from diarrheic Wagyu calves in Longjiang County Qiqihar City,for bacterial isolation and culture,biochemical identification,and 16S rRNA gene sequencing.A single purified bacterial strain isolated from a diarrhea sample,was selected for pathogenicity tests,antimicrobial susceptibility testing,virulence gene detection,phylogenetic tree construction,and homology analysis.The results showed that all 10 pathogenic strains isolated from the samples were consistent with the growth morphology and biochemical characteristics of Escherichia coli,with an isolation rate of 100%.The test strain is named Wagyu.1,virulence gene detection revealed that K88,CS 31A,BfpA,EAST 1,and hlyA were positive.The 16S rRNA sequence showed 93.6%~99.9%homology with E.coli in GenBank.The phylogenetic tree indicated that Wagyu.1 and the human-derived E.coli strain 62-b pink(GenBank accession number:MN208129)in the same branch,exhibiting the closest phylogenetic relationship.The in vitro antimicrobial susceptibility test showed that the strain was most sensitive to cefotaxime and ceftazidime,The findings suggest that the isolate Wagyu.1 is a highly virulent pathogenic E.coli strain.The close phylogenetic relationship and homology with human-derived E.coli for Human-animal separation management and reduce disease incidence.