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The long-term goal of this proposal is to identify the genetic factors responsible for the persistence of E. coli O157:H7 in cattle. The proposed work tests our central hypothesis: Specific gene expression is required for E. coli O157:H7 to colonize the bovine gastrointestinal tract. E. coli O157:H7 clearly needs to survive and probably replicate in the rumen and upper GIT of cattle in order to establish colonization. There are conflicting opinions among various groups as to whether or not the recto-anal junction mucosa is the only site of E. coli O157:H7 colonization in cattle. However, our preliminary data, publications (34, 67), and the findings of Naylor et al. (61), strongly indicate that the recto-anal junction mucosa is at least one colonization site in cattle. Because it is the only established site, we will concentrate our investigation on the genes that affect the host-bacteria association at this location.<P>
We will test our hypothesis in three objectives: <OL> <LI> To identify E. coli O157:H7 genes required for efficient bacterial colonization of the bovine recto-anal junction mucosa. <LI> To compare the genetic repertoires of pathogenic E. coli O157:H7 with commensal E. coli that do or do not colonize the bovine recto-anal junction mucosa. <LI>To determine differential gene expression in E. coli O157:H7 and bovine intestinal cells when the bacteria colonize the bovine recto-anal junction mucosa.
NON-TECHNICAL SUMMARY: A dangerous human pathogen, E. coli O157:H7 is part of the normal bacterial flora of healthy cattle. Our inability to intervene successfully and remove slight contamination of food products with E. coli O157:H7 has put consumers at risk. Our proposed work will contribute to the basic understanding of the relationship between cattle and the human pathogen E. coli O157:H7 (O157). Dissecting the components required for these bacteria to persist in cattle will lead to successful new interventions to reduce or eliminate O157 from our food. We will identify the bovine response to colonization with O157 and the bacterial components required to persist in the bovine gastrointestinal tract. This work has implications for understanding the on-farm ecology of this pathogen and the development of effective interventions to reduce contamination of food and for interventions on the farm and for ruminant animal exhibits, particularly petting zoos and farms where children may enter animal pens.
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APPROACH: Our approach includes experimental cattle infections with E. coli O157:H7 using our novel rectal swab application procedure and our highly sensitive RAMS (recto-anal mucosa swab) culture technique to determine animals carrying O157. RAMS samples will be cultured by direct plating that yields quantitative culture data (cfu/g) or will be selectively-enriched by incubation overnight prior to plating that yields qualitative (positive or negative) culture data. To identify E. coli O157:H7 genes required for efficient bacterial colonization of the bovine recto-anal junction mucosa we will focus on well known and extensively studied virulence factors including the Shiga toxins, enterohemolysin, intimin, the translocated intimin receptor, and the pO157. We will test a set of isogenic derivatives that lack specific virulence genes or have been disabled in specific gene clusters to investigate the contribution of these factors to the colonization ability at the bovine recto-anal junction mucosa. Bacterial colonization and persistence will be compared to wild-type. To identify genetic repertoires of pathogenic E. coli O157:H7 that differ from commensal E. coli that do or do not colonize the bovine recto-anal junction mucosa, we will compare a very interesting E. coli O not typeable:H25 (designated E. coli SMN13) that behaves like E. coli O157:H7 in the bovine recto-anal junction mucosa niche. Comparing these strains will be straightforward. The E. coli SMN13 genome sequence will be compared to the known genomes of E. coli O157:H7 EDL 933 (ATCC 43895) and E. coli K12. To determine differential gene expression of E. coli O157:H7 and bovine intestinal cells when the bacteria colonize the bovine recto-anal mucosa, we will use standard DNA microarray expression analysis. To minimize variation, all analyses will use E. coli O157:H7 ATCC 43895 (the sequenced EDL 933 strain) and 4 to 6 month-old Holstein steers. Both E. coli O157:H7 cells and bovine intestinal mucosa cells will be harvested from the rectums of live animals.
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PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: The results from this basic research was disseminated to the scientific community through peer-reviewed publications in journals and through presentations at scientific meetings. <BR> PARTICIPANTS: Haiqing Sheng, Linda Liou, Ji Youn Lim, Jie Li, Lonie Austin, Greg Bohach, Roland Cobbald, Witold Ferens, Kurt Gustin <BR> TARGET AUDIENCES: The target audiences are other scientists and our efforts to exchange knowledge with them involves publishing results in refereed scientific journals and giving oral and poster presentations at meetings. <BR> <BR>
IMPACT: 2007/01 TO 2007/12<BR>
The findings from this work contributes to our understanding of the relationship between healthy cattle and the human pathogen E. coli O157:H7. This science-based information will lead to interventions to eliminate or reduce the food-borne illness caused by this bacteria.