牛源大肠杆菌O157:H7的分离及毒力基因鉴定  

Isolation of Escherichia coli O157:H7 from cattle and detection of its virulence genes

作　　者：叶青[1,2] 张雪寒[1] 何孔旺[1] 费荣梅[2] 赵攀登[1] 栾晓婷[1] 

机构地区：[1]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《中国兽医学报》2012年第8期1148-1153,共6页Chinese Journal of Veterinary Science

基　　金：国家科技支撑计划资助项目(2012BAK08B07);江苏省农业支撑计划-社会发展资助项目(BE2011771);江苏省自然科学基金资助项目(BK2010068)

摘　　要：从2个牛场采集新鲜粪便,增菌后,免疫磁珠富集,涂布筛选性培养基,挑取可疑菌落用rfbE/fliC二重PCR和血清学方法鉴定。设计毒力基因stx1、stx2、eae、hlyA和tccp相应引物,针对O157:H7对分离株进行PCR鉴定。口服攻毒链霉素处理的BALB/c小鼠明确分离株致病性。结果显示,成功分离到7株出血性大肠杆菌O157:H7,并且有1株迟缓性发酵山梨醇麦康凯培养基。毒力基因检测显示,其中6株毒力因子表型为stx1-stx2+eae+hlyA+tccp+,另有1株表现型为stx1+stx2+eae+hlyA+tccp+,各分离株tccp基因均为阳性,但携带的重复片段数量有差异。所采集样品中肠出血性大肠杆菌O157:H7的检出率高达12%。1×1010 CFU同剂量口服接种经PBS洗涤的5株O157:H7分离株全菌,小鼠存活率有差异分别为40%,50%,60%,20%,50%,各分离株在小鼠体内排菌时间也有差异分别为攻毒后7,9,13,13,15d。To understand the epidemic status of Escherichia coli O157.H7,we collected the feces and isolated O157. H7 strains from cattle farms and detected several maior virulent genes. Fresh fecal samples were collected, cultured at 37℃ ,enriched with immunomagnetic beads and spread onto CHROM agar. After culture at 37℃ for 12 h,suspected colonies were picked and identified with rfbE/fliC PCR and serological methods. The stxl, stx2, eae, hlyA, and tccp primers were designed to amplify the fragments. Oral attack streptomycin treatment of BALB/c mice were de- termined pathogenic of strains. The results showed that seven O157..H7 isolates were successfully isolated,and No. 2 O157.-H7 gradually fermented sorbitol. After all isolates were detected major virulence genes, No. 6 phenotypic vir ulence factors were stx 1 -- stx2 ＋ eae4- hl ya 4- tcc p＋ , the remaining six isolates were stx 1 4- stx2 4- eae 4- hl yA 4- tcc p 4-. Although all the isolates have all the detected genes, they have different tccp gene that contains different numbers of repeat fragments. This study showed that the collected cattle feces carried O157 .. H7 and the detection rate is up to 12 %. In the mice inoculated with 1 X 101% CFU dose of PBS washing of five strains of O157：H7 strains isolated their survival rates were 40%,50% ,60 %,20% ,50% ,and discharge time in mice were 7,9,13,13,15 days,respectively.

关 键 词：牛 肠出血性大肠杆菌O157:H7 毒力基因 致病性 

分 类 号：S852.61[农业科学—基础兽医学] R535[农业科学—兽医学]