Association of VTEC O157 and O26 strains with the bovine intestinal mucosa ex vivo

在英国，产毒性大肠杆菌（VTEC）与严重的人类疾病有关。这些致病菌中最著名的成员是大肠杆菌O157。最致命的疫情发生在1996年的苏格兰，但此后几乎每年都有O157大肠杆菌出现在新闻中，英格兰和威尔士也报道了疫情。最近，可以引起与大肠杆菌O157类似疾病的大肠杆菌O26已成为对人类健康的重大威胁。感染VTEC的牛是人类感染的主要宿主。人们可因食用受污染的食品、与动物直接接触或从环境中感染。虽然牛是感染的主要来源，但目前对VTEC与牛肠道的关系知之甚少。为了制定控制措施以尽量减少传播的风险，更好地了解是必不可少的。进展受到阻碍，不仅因为大型动物实验繁琐和昂贵，而且还因为在对牛进行实验性口服攻毒后，很少看到附着的VTEC细菌。肠道外植体的体外器官培养（IVOC）为研究病原菌与宿主粘膜表面的相互作用提供了一个强有力的系统。几年来，人体IVOC系统被认为是评估VTEC与人体肠道黏膜相互作用的金标准。技术上的困难阻碍了bIVOC系统的开发，该系统将允许常规、可重复和大规模地调查VTEC与牛肠道粘膜的关系。在过去的12个月里，Francis Girard博士在猪IVOC模型方面拥有丰富的经验，他优化了培养和感染条件，从而在我们的实验室建立了常规使用的bIVOC系统。牛肠道不同区域的双挥发性有机化合物在离体培养8小时后具有良好的超微结构保存效果。bIVOC与VTEC O157和O26的孵育导致了有效的定植和典型的“附着和消退”病变的形成，特别是在末端直肠的外植体上，这表明它复制了完整牛定植过程中的关键事件。我们实验室的这一进展为有效研究牛VTEC感染阶段开辟了一条实用的途径。由于一种动物可以产生数百种双挥发性有机化合物，我们的模型大大减少了动物的使用。该项目的目的是：1。评价和比较VTEC O157和O26菌株与bIVOC的相关性。2. 确定VTEC是否与不同年龄的动物有不同的相互作用（因为流行病学研究表明年轻的小牛更容易受到影响）。3. 为了确定从故意感染的小牛身上恢复的细菌是否表现出与实验室培养的同类细菌不同的定植动态、模式和特异性。这可能有助于解释为什么VTEC在农场的动物之间有效地传播。4.

Verotoxigenic Escherichia coli (VTEC) are associated with severe human disease in the UK. The best known member of these pathogenic bacteria is E. coli O157. The most deadly outbreak occurred in Scotland in 1996 but almost every year since E. coli O157 has appeared in the news with outbreaks also reported in England and Wales. Recently, E. coli O26, which can cause similar disease to E. coli O157, has emerged as a significant risk to human health. Cattle infected with VTEC are the major reservoir for human infection. People can be infected from consumption of contaminated food products, by direct contact with animals or from the environment. Although the fact that cattle are the main source of infection, little is currently known about the association of VTEC with the bovine gut. Better understanding is essential in order to develop control measures to minimise the risk of spread. Progress has been hampered not only because large animal experimentation is cumbersome and expensive, but also due to the fact that adherent VTEC bacteria are seen only infrequently following experimental oral challenge of cattle. In vitro organ cultures (IVOC) of gut explants provide a powerful system to study the interaction of pathogenic bacteria with mucosal surfaces of their respective hosts. For several years now the human IVOC system is recognised as the gold standard for assessment of VTEC interactions with human gut mucosa. Technical difficulties stood in the way of developing a bIVOC system that would allow routine, reproducible and large scale investigation of VTEC association with the bovine gut mucosa. Over the past 12 months Dr. Francis Girard, who has vast experience in the porcine IVOC model, optimised culture and infection conditions which led to establishment of a bIVOC system for routine use in our laboratories. bIVOC of different regions of the bovine gut resulted in excellent ultra-structural preservation over 8 h ex vivo incubation. Incubation of bIVOC with VTEC O157 and O26 resulted in efficient colonisation and formation of typical 'attaching & effacing' lesions, particularly on explants from the terminal rectum, indicating that it replicates crucial events during colonisation of intact cattle. This development in our laboratories opens a practical way to efficiently study the bovine phase of VTEC infection. As hundreds of bIVOC can be made from a single animal, our model significantly reduces animal usage. The aims of this project are: 1. To evaluate and compare association of VTEC O157 and O26 strains with bIVOC. 2. To determine if VTEC interact differently with animals of different ages (as epidemiological studies show that young calves are more susceptible). 3. To determine if bacteria recovered from deliberately infected calves show altered colonisation dynamics, patterns and specificities to lab-grown counterparts. This may help to explain why VTEC is efficiently spread between animals on farms. 4. To investigate the molecular and cellular mechanisms involved in binding of VTEC O157 and O26 to bIVOC and the contribution of two critical factors, Tir and TccP, in this process