西藏牦牛源EHEC的MLST聚类和致病性分析  

MLST clustering and pathogenicity analysis of EHEC from Tibetan yaks

作　　者：娄永志 卢姊豪 陈姝宇 常攀 辛娇娇 索南顿珠 索朗斯珠 贡嘎 LOU Yong-zhi;LU Zi-hao;CHEN Shu-yu;CHANG Pan;XIN Jiao-jiao;SUONAN Dun-zhu;SUOLANG Si-zhu;GONG Ga(College of Animal Science,Tibet Agriculture&Animal Husbandry University,Linzhi 860000,China)

机构地区：[1]西藏农牧学院动物科学学院,林芝860000

出　　处：《中国人兽共患病学报》2023年第6期563-569,共7页Chinese Journal of Zoonoses

基　　金：财政部和农业农村部-国家肉牛/牦牛产业体系项目(No.CARS-37)。

摘　　要：目的掌握西藏牦牛源肠出血性大肠杆菌核酸多位点序列分型、聚类情况和致病性。方法对实验室已分离鉴定的14株肠出血性大肠杆菌采用分子生物学和动物实验的方法,进行多位点序列分型鉴定及聚类分析,动物实验后挑选致病性最强菌株进行生长曲线测定和半数致死量(LD 50)计算,HE染色制作组织病理切片。结果14株牦牛源肠出血性大肠杆菌中共出现了3种ST型,分别为ST 33、ST 226、ST 446,各ST型中西藏阿里菌株占比最多为57%(8/14);各ST型中ST 33占比最多为57%(8/14),其次是ST 226占比14%(2/14)该型菌株的致病性最强,ST 446占比最少为7%(1/14)。此外,还有3株占比22%(3/14)未定型肠出血性大肠杆菌。MLST聚类分析表明管家基因在不同ST型中携带等位基因的数量不同,ST 33型中Mdh 22个最多,ST 446型中icd 26个最多,ST 226型中Fumc 27个最多。14株菌株攻毒80只小鼠表现出不同程度的致病性,其中ST 226型致病性最强。将致死小鼠解剖后发现肠上皮出血水肿,脾脏、肝脏有淤血等病理变化。14株菌株中的阿里株(TBY 7)是ST 226型其致病性最强,其生长曲线显示14 h达最大生长数,浓度为1×10^(11)cfu/mL;60只小鼠差浓度梯度攻毒试验计算出该菌的LD_(50)为2.4×10^(7)cfu/mL。阿里株(TBY 7)ST 226型感染小鼠后经HE染色制作组织病理切片后,结果显示小鼠的十二指肠、盲肠、结肠和直肠黏膜上皮坏死脱落,盲肠肠壁内可见寄生虫,空肠、回肠绒毛结构坏死萎缩、固有层充血;肝细胞轻微空泡变性,脾脏红髓轻微淤血。结论西藏牦牛源肠出血性大肠杆菌存在不同ST型,且各地区均有分布;不同ST型的致病性与携带等位基因数量相关,且主要引起肠组织病变,提示相关部门应加强对该致病菌的宣传。To grasp the multilocus sequence typing(MLST),clustering,and pathogenicity of enterohemorrhagic Escherichia coli of Tibetan yak origin,molecular biology and animal experiments were performed on 14 strains of enterohemorrhagic E.coli.The results showed the following:14 strains of enterohemorrhagic E.coli included 3 ST types(ST 33,ST 226,and ST 446),with ST 33,ST 226,and ST 446 accounting for 57%(8/14),14%(2/14),and 7%(1/14),respectively,and 3 untyped enterohemorrhagic E.coli;MLST clustering results showed that the maximum number of Mdh alleles was 22 in ST 33,26 in ST 446,and 27 in ST 226;among the 14 strains,ST 226(TBY 7)was the most pathogenic,and pathologic changes,such as hemorrhagic edema in the intestinal epithelium and bruising in the spleen and liver,were noted during necropsy of the mice;for ST 226(TBY 7),the growth curve showed that the maximum growth of 1×10^(11)cfu/mL was reached at 14 h,and the LD_(50)of the bacterium was calculated to be 2.4×10^(7)cfu/mL by the differential concentration gradient test in 60 attacked mice;and histopathologic sections of mice killed by TBY 7 showed that the mucosal epithelium of the duodenum,cecum,colon,and rectum had necrotic parasites in the intestinal wall of the cecum,and the jejunum and ileum had necrotic atrophy and congestion of the lamina propria.The hepatocytes had slight vacuolar degeneration,and the spleen had slight red marrow bruising.The experimental results showed that there were different ST types of enterohemorrhagic E.coli of Tibetan yak origin that were distributed in various regions.The pathogenicity of different ST types was related to the amount of carrying alleles,which mainly caused intestinal tissue lesions.

关 键 词：牦牛源肠出血性大肠杆菌(EHEC) ST分型 MLST聚类分析 致病性 

分 类 号：R378.2[医药卫生—病原生物学] S852.61[医药卫生—基础医学]