河北省部分地区犊牛腹泻大肠杆菌优势血清型筛选及兼性菌毛株的致病性

Screening of predominant serotypes of Escherichia coli from calf diarrhea and isolation and identification of facultative fimbriae strains in some areas of Hebei Province

从河北省4个地区的奶牛场采集1月龄左右腹泻犊牛的新鲜粪便,通过病原的分离培养、革兰染色、生化鉴定、16S rRNA基因PCR及测序来分离和鉴定大肠杆菌;采用11种大肠杆菌O因子定型血清对分离菌进行大肠杆菌优势血清型鉴定;利用菌毛特异性引物对优势血清型菌株进行菌毛基因PCR扩增及序列测定和分析,鉴定其所携带菌毛抗原。通过小鼠攻毒试验评价分离得到的大肠杆菌F41菌毛株、K99菌毛株及兼性F41-K99菌毛株的致病性。结果显示,采集的56份犊牛腹泻粪便样品分离鉴定出48株大肠杆菌。大肠杆菌O因子定型血清鉴定结果显示,4个地区流行的犊牛腹泻大肠杆菌优势血清型均为O101型。菌毛PCR测序结果鉴定出6株携带K99菌毛大肠杆菌,7株携带F41菌毛大肠杆菌,其中1株为兼性F41-K99菌毛大肠杆菌。小鼠致病性试验结果显示,3株分离菌对小鼠均有致病性,其中兼性F41-K99菌毛株临床症状最明显,最小致死剂量最低,为1.5×10^(10)CFU/mL,且死亡小鼠的脏器病理变化最严重,所以兼性菌毛株对小鼠的致病性最强。

In order to explore the prevalence of E.coli related calf diarrhea in dairy farms in parts of Hebei Province,fresh feces of calves about one month old with diarrhea were collected from dairy farms in 4 regions of Hebei Province.Then E.coli was isolated and identified by methods such as pathogen isolation and culture,Gram staining,PCR amplification and sequencing of 16 S rRNA gene and biochemical test,11 types of O-factor stereotype serum were used to identify the dominant serotype of E.coli,PCR amplification,sequence determination and analysis of fimbriae genes were performed on dominant serotype strains to identify the fimbriae antigens.Finally,the pathogenicity of the isolated E.coli F41 fimbriae strains,K99 fimbriae strains and facultative F41-K99 fimbriae strains was evaluated by the mouse challenge test.The results showed that 48 strains of E.coli were isolated and identified from 56 feces samples,and the dominant serotype of all E.coli isolated from feces was O101.The result of PCR sequence determination showed that 6 strains of E.coli had K99 fimbriae,7 strains of E.coli had F41 fimbriae,and one of them had K99-F41 facultative fimbriae.The mouse pathogenicity test results showed that the three isolates were pathogenic to mice,among which mice infected with the facultative F41-K99 fimbriae strain had the most obvious clinical symptoms and the lowest lethal dose was 1.5×10^(10) CFU/mL,and the organs of dead mice had the most severe pathological changes,so the facultative fimbriae strains were the most pathogenic to mice.This study revealed the prevalence of E.coli related calf diarrhea in parts of Hebei Province,and provided a theoretical basis for the prevention and control of calf diarrhea.