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United States Department of Agriculture

Agricultural Research Service

Research Project: PREVENTION OF LOSSES FROM COLIBACILLOSIS AND O157:H7 AND OTHER SHIGA TOXIN-PRODUCING E. COLI (STEC) IN CATTLE AND SWINE Title: Bovine recto-anal junction squamous epithelial (RSE) cell adhesion assay for studying Escherichia coli O157 adherence

Authors
item Kudva, Indira
item Nystrom, Evelyn

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 30, 2011
Publication Date: August 29, 2011
Citation: Kudva, I.T., Nystrom, E.A. 2011. Bovine recto-anal junction squamous epithelial (RSE) cell adhesion assay for studying Escherichia coli O157 adherence. Journal of Applied Microbiology. 111(5):1283-1294.

Interpretive Summary: The bovine recto-anal junction (RAJ) is the site of Escherichia coli O157 (O157) persistence in the cattle gastrointestinal tract (GIT). Developing an O157 adherence assay using cells derived from the RAJ would facilitate host specific interaction studies and hence, we decided to standardize an adherence assay using the RAJ squamous epithelial cells or the RSEC. RSECs were selected based on our observation that O157 diffusely adhere to these cells in O157-positive cattle and that these cells can be easily collected in large numbers from cattle using recto-anal mucosal swabs (RAMS). HEp-2 cells that are routinely used to evaluate adherence capabilities of most enteric pathogens, are neoplastic human cells derived from a non-gastrointestinal site and therefore may not reflect the true bacterial-host interactions seen in the GIT of either humans or animals. In contrast, GIT-derived, freshly collected RSECs are likely to more accurately reflect interactions seen within the GIT of cattle. Moreover, the HEp-2 adherence assay works best when there is reliable recovery of stocks in culture flasks without contamination, consistent confluent growth of the cell line in chamber slides, and uniform spreading of bacteria within the wells. We found that these variables of the HEp-2 assay could be overcome using the RSEC adherence assay which worked well with both freshly recovered as well as frozen stocks of RSEC cells that could be used directly without an intermittent culture step. Additionally, being a suspension assay, growth rate of cells and distribution of bacteria did not influence the adherence observed which relied solely on strong interactions between RSEC and bacteria. The RSEC adherence patterns were also easy to interpret and quantitate compared to the HEp-2 adherence pattern that needed close study. Interestingly, the assay worked equally well in identifying other adherent E. coli and differentiating them from a poorly adherent control strain of Salmonella. Hence, RSECs have the potential to be optimal tools for assessing adherence of not only O157 but other bacteria as well. A positive result can be interpreted by taking both adherence pattern and number of bacteria/cell into account.

Technical Abstract: An adherence assay, using recto-anal junction squamous epithelial cells (RSEC), was developed for Escherichia coli O157 and related organisms. The assay was standardized in comparison with the routinely used HEp-2 cell adherence assay, in this “proof of concept” study. The novel RSEC adhesion assay provided consistent and comparable results that were easy to interpret with fewer variables. Considering the ease with which RSCE may be collected and used, these epithelial cells will provide an economical alternative to assays involving neoplastic human cell lines that have stringent culture conditions. While RSEC will provide anatomically relevant results especially for organisms associated with cattle such as E. coli O157, our results indicate potential use of these epithelial cells in routine adherence assays for other bacteria as well.

Last Modified: 4/20/2014
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