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

在英国，产毒大肠杆菌(VTEC)与严重的人类疾病有关。这些致病菌中最著名的成员是O157大肠杆菌。最致命的一次疫情发生在1996年的苏格兰，但自从O157大肠杆菌出现在新闻中以来，几乎每年都会出现疫情，英格兰和威尔士也报告了疫情。最近，可引起与O157大肠杆菌类似的疾病的O26大肠杆菌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。人们可以通过食用受污染的食品、通过直接接触动物或从环境中感染。虽然牛是主要的感染源，但目前对VTEC与牛肠道的联系知之甚少。为了制定控制措施，将传播风险降至最低，更好的了解是至关重要的。进展受到阻碍，不仅是因为大型动物实验繁琐和昂贵，还因为粘附性VTEC细菌在牛的实验性口腔挑战后才罕见地出现。肠道外植体的体外器官培养(IVOC)为研究病原菌与其各自宿主的粘膜表面的相互作用提供了一个强大的系统。多年来，人类IVOC系统一直被认为是评估VTEC与人类肠道粘膜相互作用的金标准。技术上的困难阻碍了BIVOC系统的开发，该系统将允许对VTEC与牛肠道粘膜的关联进行常规、可重复性和大规模的研究。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，他对培养和感染条件进行了优化，从而建立了我们实验室常规使用的BIVOC系统。牛肠道不同部位的BIVOC在体外孵育8h后具有良好的超微结构保存效果。BIVOC与VTEC O157和O26孵育后，可以有效地定植并形成典型的“附着和消失”病变，特别是在直肠末端的外植体上，这表明它复制了完整牛的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VTEC感染的牛阶段开辟了一条实用的途径。由于一只动物可以制造数百个BIVOC，我们的模型极大地减少了动物的使用。本项目的目的是：1.评估和比较VTEC O157和O26菌株与bIVOC的相关性。2.确定VTEC与不同年龄的动物是否有不同的相互作用(因为流行病学研究表明，小牛犊更容易受到影响)。3.为了确定从故意感染的牛犊中提取的细菌是否显示出与实验室培养的细菌相比，细菌的定植动态、模式和特异性发生了变化。这可能有助于解释为什么VTEC在农场的动物之间有效传播。4.探讨VTEC O157和O26与bIVOC结合的分子和细胞机制以及TIR和TccP两个关键因素在此过程中的作用

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