江苏某地健康绵羊群产志贺毒素大肠杆菌体内分离株的分子流行病学及致病力

【目的】探讨江苏某羊场健康绵羊体内产志贺毒素大肠杆菌的带菌和流行情况,同时就分离株的致病力和对Vero细胞的毒性作用作了研究。【方法】基于本实验室已经建立的EHEC O157:H7 EDL933W株的stx1、stx2、eaeA、hlyA四个基因的多重PCR检测并配合选择性增菌、平板筛选等方法对STEC进行分离鉴定。【结果】在为期6个月的连续跟踪调查中,共分离到STEC菌株107株,分离率为19.4%(107/550)。分离株属于41种O血清型、62种O:H血清型,未定型(ONT)有22株,粗糙型(OR)1株。其中属于绵羊STEC的优势血清型有O5(2株)、O91(1株)、O103(1株)。本文检测到的优势血清型为O93,stx2阳性菌株的分离率较stx1阳性菌株的分离率高,LD50测定结果表明分离株对小鼠致病力不高,受试的3个分离株均不能致小鼠死亡。对107株stx阳性分离株噬菌斑试验表明,71株阳性菌株携带噬菌体(66.3%,71/109)。受试分离株进行Vero细胞毒性试验,其中有一个菌株stx基因阳性但不能使Vero细胞产生病变。【结论】绵羊是STEC的天然宿主,可健康带菌。虽然STEC分离株对小鼠的致病力较弱,但不能排除其对人类安全的威胁。STEC携带志贺毒素基因并不意味着一定表达志贺毒素,需对志贺毒素的表达及调控机理做进一步的研究。

Epidemiological investigation of toxin-producing Escherichia coli isolates originated from healthy sheep in one farm of Jiangsu Province and their pathogenicity

Abstract: Objective] In order to investigate the distribution of shiga toxin-producing Escherichia coli (STEC) among healthy sheep in a farm and the pathogenicity to mice and Vero cells of these STEC isolates. Methods] We used polymerase chain reaction (PCR) to detect genes of eaeA, stx1, stx2, hlyA, which had been developed in this laboratory previously, combing the selective cultivation and Chrom-Agar (CA) O157 plates to isolate STEC strains. Results] A total of 107 STEC strains were isolated in a sheep farm during six visits from August, 2008 to January, 2009. The isolation rate was 19.8% (107/550). These isolates belonged to 41 O serotypes and 60 O: H serotypes, except that 21 were O non- typable and 1 was rough. O93 was the common serotype. Some isolates such as O5, O91, O103, which are reported in other countries originated from healthy sheep were also isolated in this study. Stx2 positive rate was higher than that of stx1. 50% lethal dose assay in mice indicated the pathogenicity of isolates was low and none of the 3 tested isolates caused mice death. We selected 107 stx gene positive STEC strains to induce the lambdoid bacteriophages. The results showed that 71 out of 107 isolates formed plagues, while 28 did not after induction. Detection of Shiga toxins for three tested isolates in Vero cell assay indicated one stx gene positive strain lacked the toxigenicity to Vero cells. Conclusions] Sheep are the natural reservoirs of STEC and they are healthy to carry STEC. Although these STEC isolates experience low pathogenicity to mice, they are potential threat to human health. Shiga toxin gene positive were not equal to production of Shiga toxins, so we need to further study the Shiga toxin expression and regulation mechanism.