感染仔猪粪肠球菌不同分离株的鉴定及毒力基因检测

Identification and determination of virulence gene for different isolates of E. faecalis originated from infective piglet

从近年来河南省各地感染发病猪群的肠球菌分离株中,选取来源不同且具有代表性的5株分离菌进行了包括致病性和毒力基因测定在内的系统鉴定。结果表明,引起本次河南省仔猪感染发病的病原体为粪肠球菌。各地分离菌的形态、培养特性与以及对极端环境的耐受性上表现较为一致,而对各种糖的发酵上存在着的差异;对药物万古霉素、替考拉宁、利福平和氨苄西林敏感,而对临床常用药物红霉素、卡那霉素和四环素完全耐药;经16S rRNA测定,它们与粪肠球菌ATCC29212同源性在99.6%～99.8%之间,与GenBank公布的NC_004668、AJ301831的核苷酸同源性为99.7%～99.9%和99.5%～99.7%;通过对它们2种毒力表型和携带的6种主要毒力基因以及与对小鼠的LD50测定,发现5株粪肠球菌携带毒力基因不尽相同,携带全部6种毒力基因的HE1和HE5的致病力最强,而仅携带4种毒力基因的HE41致病力最小。用HE1和HE5分离菌对20日龄的断奶仔猪分别进行攻毒,2菌株均能引起仔猪的感染发病。

Among the recently isolated Enterococcal strains from some infected swine flocks in Henan province,five representatives of different origins were selected for systemic identification including pathogenicity assessment and virulent gene detection.The results indicated that the pathogen that caused swine infection was Enterococcus faecalis.The isolates showed similar morphological and cultivating characteristics and tolerance of extreme environments,whilst performed differently in carbohydrate fermentation;sensitive to vancomycin,teicoplanin,rifampicin and ampicillin,whereas totally tolerant to common clinical drugs such as erythromycin,kanamycin and tetracycline.The 16S rRNA analysis indicated that these isolates possess high homology to E.faecalis ATCC29212 （99.6%-99.8%）,and the nucleotide homology to E.faecalis NC_004668 and AJ301831 strains that are published in GenBank are 99.7%-99.9% and 99.5%-99.7% respectively.Through analysis of virulence phenotype and six main virulent genes and determination of LD50 to mice,we discovered that the five isolates carried different virulent genes,and the HE1 and HE2 strains which carried all the tested genes were the most pathogenically potential,yet the 4-gene carrying HE41 was least capable.The challenge tests were respectively performed with piglets aged 20d with isolates of HE1 and HE5 and all piglets tested were infected and showed clinical symptoms.