牦牛源志贺菌分离鉴定、毒力基因检测与分型

doi:10.11843/j.issn.0366-6964.2020.05.020

摘要/Abstract

摘要： 本试验旨在阐明牦牛源志贺菌致病性及分子流行特性，为探索志贺菌流行途径，制定合理的防控策略提供新思路。2017年在甘肃、青海、西藏三省（区）共采集牦牛肛门棉拭子样品1 396份，通过选择培养基筛选、生化鉴定、血清凝集试验对分离菌株进行系统鉴定，应用PCR方法检测分离株中ipaH、ipaBCD、ial、sen、set1A、set1B和stx七种毒力基因流行情况。参照McMLST网站数据库提供的15对管家基因序列进行MLST分型；并参考美国CDC的PulseNet实验方法，用限制性内切酶NotⅠ和XbaⅠ分别对福氏志贺菌和宋内志贺菌染色体进行酶切，对这些分离菌株进行PFGE分析。结果显示，41株分离株符合志贺菌生化特征，分为4个生化表型，B3（36.59%）和B4（32.35%）为主要生化表型。血清凝集试验鉴定23株为福氏志贺菌，包括四个血清型1a（n=2）、2a（n=16）、2b（n=3）、Xv（n=2）；18株为宋内志贺菌，分为Ⅰ相（n=12）和Ⅱ相（n=6）。共检测到6种毒力基因ipaH、ipaBCD、ial、sen、set1A、set1B，携带率分别为100%、92.68%、73.17%、70.73%、26.83%、26.83%。具有7种毒力基因型，其中VT5和VT7型为主要流行型，分别占43.9%和24.39%，同时携带两种及以上毒力基因的志贺菌占92.68%。41株志贺菌共分为10个ST型，其中ST100、ST116、ST155型为主要流行型。Not Ⅰ酶切的福氏志贺菌分为13个PT型，而Xba Ⅰ酶切的宋内志贺菌分为14个PT型。综上所述，牦牛源志贺菌生化表型、血清型、ST型和PT既存在多态性，又有优势流行型。本试验分离的志贺菌与人源志贺菌携带相同的毒力基因，其中ipaH、ipaBCD、ial、sen基因携带率较高，对公共安全具有潜在的威胁。

关键词: 志贺菌, 牦牛, 毒力基因, 多位点序列分型, 脉冲场凝胶电泳

Abstract: The purpose of this experiment is to clarify the pathogenicity and molecular epidemic characteristics of Shigella from yak, and to provide new ideas for exploring the epidemic ways of Shigella and formulating reasonable control strategies. In 2017, 1 396 samples of yak anal cotton swabs were collected in Gansu, Qinghai and Tibet, and isolates were systematically identified by selective medium screening, biochemical identification and serum agglutination test. Seven virulence genes (ipaH, ipaBCD, ial, sen, set1A, set1B and stx) were detected by PCR. MLST typing of 15 pairs of housekeeping gene sequences provided by Mc MLST website database was carried out. Referring to the PulseNet experimental method of CDC in the United States, The chromosome of S. flexneri and S. sonnei were digested by restriction endonuclease NotⅠ and XbaⅠ, and PFGE analysis was performed on these isolates. The results showed that 41 strains were consistent with the biochemical characteristics of Shigella. They were divided into 4 biochemical phenotypes, B3 (36.59%) and B4 (32.35%) were the main biochemical phenotypes. Serum agglutination test identified 23 strains of S. flexneri, including four serotypes 1a (n=2), 2a (n=16), 2b (n=3), Xv (n=2); 18 strains were S. sonnei bacteria are divided into phase Ⅰ (n=12) and phase Ⅱ (n=6). A total of six virulence genes ipaH, ipaBCD, ial, sen, set1A, and set1B were detected, and the carrying rates were 100%, 92.68%, 73.17%, 70.73%, 26.83%, and 26.83%, respectively. There are 7 virulence genotypes, of which VT5 and VT7 are the main epidemic types, accounting for 43.9% and 24.39%, respectively, while Shigella carrying two or more virulence genes account for 92.68%. Fourty-one strains of Shigella were divided into 10 types of ST, of which ST100, ST116 and ST155 were the main epidemic types. S. flexneri of NotⅠ enzyme digestion was divided into 13 PTs, while S. sonnei was divided into 14 PTs by XbaⅠ enzyme digestion. The biochemical phenotype, serotype, ST, and PT type of Shigella isolated from yaks are both polymorphic and prevalent. The Shigella isolates carried the same virulence genes as human, and carrying rates of ipaH, ipaBCD, ial, and sen genes were higher, which posing a potential threat to public safety.

Key words: Shigella, yak, virulence genes, multilocus sequence typing, pulsed field gel elect-rophoresis

中图分类号:

张立伟, 张逸博, 郝贺, 汪文雅, 时欣欣, 韩旺, 仝星, 张永英, 石玉祥, 朱阵. 牦牛源志贺菌分离鉴定、毒力基因检测与分型[J]. 畜牧兽医学报, 2020, 51(5): 1091-1100.

ZHANG Liwei, ZHANG Yibo, HAO He, WANG Wenya, SHI Xinxin, HAN Wang, TONG Xing, ZHANG Yongying, SHI Yuxiang, ZHU Zhen. Isolation, Identification, Virulence Gene Detection and Typing of Shigella from Yak[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 1091-1100.

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