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

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

O157 and non-O157 VTEC are important zoonotic pathogens. Cattle contaminated with VTEC are the major reservoir for human infection. However, little is currently known about the mechanisms involved colonisation of the bovine gut. Progress has been slow because adherent VTEC bacteria are seen only infrequently following experimental challenges. The ability of VTEC to cause disease is associated with the capacity to cause attaching and effacing (A/E) lesions and to elaborate Shiga toxin. Little is currently known about A/E lesion formation during colonisation of the bovine gut. The genes required for A/E lesion formation by VTEC O157:H7 in vitro are carried on the LEE, which encodes the effector Tir and the adhesin intimin, and prophage CP-933U, which encodes TccP. Binding of intimin to the extracellular domain of Tir and of TccP to the C-terminus of Tir activates N-WASP and trigger A/E lesion. In vivo evidence show that intimin and Tir are essential for colonisation of the bovine gut while TccP have an accessory function. In VTEC O26, phosphorylation of Tir leads to recruitment of Nck and activation of N-WASP. However, as VTEC O26 express TccP2 it can induce A/E lesions by alternative pathways. Intestinal in vitro organ cultures (IVOC) provide a powerful model to study bacterial interaction with mucosal surfaces. We recently developed a robust bovine IVOC (bIVOC) of terminal ileum, terminal colon and terminal rectum, infection of which resulted in colonisation and A/E lesion formation. In this project we aim to exploit the bIVOC to study the bovine phase of VTEC infection cycle. We will investigate: the association of prototype VTEC O157 and O26 strains; the affects of animal age; colonisation characteristics of field O157 and O26 VTEC isolates; how passage through the GI affects colonisation dynamics; the composition of the actin-rich pedestal in bIVOC infected with VTEC O26 and O157 and the role Tir and TccP/TccP2 in colonisation and A/E lesion formation.