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<OL> <LI> Determine prevalence and concentrations of AHL in luminal contents collected from the rumen, small intestine, colon, and rectum of feedlot cattle at slaughter. <LI> Determine if season influences AHL concentrations by sampling feedlot cattle in the spring, summer, fall, and winter. <LI> Examine populations of E. coli O157:H7 (qualitatively and quantitatively) in above luminal content samples and determine if correlated to AHL concentrations. <LI> Utilizing growing lambs as an inexpensive cattle model, evaluate concentrations of AHL in luminal contents throughout the gastro-intestinal tract of lambs fed forage- or grain-based diets.
Approach: All samples will be collected from feedlot cattle at a single slaughterhouse in the southwestern United States. Four collections, encompassing the four seasons, will be made in July and October of 2007, and January and April of 2008. Luminal content samples (approximately 50 g) will be aseptically collected from the rumen, small intestine (jejunum), spiral colon, and rectum from 60 head at each seasonal collection (240 total samples representing each of the above GIT segments). Samples will be shipped immediately to the laboratory in Dallas (AHL analysis) or College Station, TX (E. coli O157:H7 culture), for processing as described below. Sheep study: Sixteen crossbred weaned lambs (avg. BW = 25 kg) will be purchased and transported to the livestock facilities at the USDA laboratory in College Station, TX. Equal numbers of animals will be provided Forage (90% grass hay, 10% grain/mineral supplement) or Concentrate (80% commercial sheep feed, 10% grass hay). Following a two-week adaptation period to study diets, all sheep will be individually penned and orally inoculated with a cocktail of three strains of E. coli O157:H7 made resistant to three different antibiotics. Fecal samples will be collected daily for seven days from each lamb for serial quantification of each of the inoculated strains of EHEC. All lambs will be humanely euthanized on day 8 post-inoculation and necropsied. Luminal contents and tissue will be aseptically collected from the rumen, abomasum, duodenum, jejunum, cecum, spiral colon, and rectum. Detection of AHLs in ruminal fluid and luminal contents: AHLs will be extracted from ruminal fluid and luminal contents using dichoromethanol, and subsequently tested in a reporter strain for their presence. The AHLs extracted as described above will be incubated with EHEC and used to compare expression of the LEE genes in EHEC with and without AHLs. Expression of the LEE will be assessed through real-time RT-PCR.