The goal of this research is to increase our understanding of the relationship between cattle and the human food-borne pathogen, E. coli O157:H7 (O157). Healthy cattle are transiently colonized with O157 and there is a strong seasonal variation with the highest prevalence in the warm months. Although O157 must survive passage through the bovine gastrointestinal site, colonization occurs at a unique site, the recto-anal junction mucosa. The factors that lead to efficient colonization, persistence, and clearance of O157 carriage in cattle are not known. Also, the mechanism(s) that contribute to seasonal variation in O157 prevalence are unknown. <P>The proposed work has three objectives that build on the previous findings by and expertise of our program. <br/>First, ongoing work to identify the O157 bacterial genes required for efficient colonization will be continued; second, E. coli isolates (ONT:H25) that are similar to the O157 pathogen and efficiently colonize the bovine recto-anal junction mucosa will be compared to O157 using full genome sequencing and bioinformatics; and third, O157 challenge of cattle under controlled conditions will be used to assess factors that contribute to seasonal variation in O157 carriage. To accomplish these objectives we will use established bacterial culture, molecular biology, and bioinformatics techniques. <P>The outputs from our work include conducting and analyzing experiments and teaching undergraduate and graduate students. We will generate data and conclusions that will be vetted through scientific review. We will disseminate our findings to advance knowledge through publication, presentations at scientific and community meetings, and through the training and mentoring of students in our research program and in the classroom.
E. coli O157:H7 (O157) is an important zoonotic pathogen estimated to cause more than 70,000 cases of human infection annually in the United States. The disease is characterized by bloody diarrhea and in 5 to 10% of the patients, a severe life-threatening sequel, the hemolytic uremic syndrome, results. The most important reservoir for O157 is healthy cattle, which carry O157 transiently and sporadically in their gastrointestinal tract. We do not understand how cattle become colonized with O157 or how they spontaneously clear the bacteria. It is well established however, that the prevalence of O157 carriage among cattle is greater in the summer than in the winter. O157 is transmitted to humans by ingestion of contaminated foodstuffs or water or by direct contact with cattle. Because healthy cattle are the source of human infection, practices that reduce or eliminate O157 from cattle will significantly reduce illness in people. Such practices have not been established. In fact, just reducing the level of O157 carriage to the winter levels would have a significant positive effect. This proposal outlines experiments that will add to our basic understanding of the relationship between cattle and O157 and includes three objectives. First we will continue our ongoing work to analyze specific bacterial genes that contribute to the microbes' ability to efficiently colonize cattle by analyzing the effects of removing specific traits of O157. Second, we will use the power of DNA sequencing and genetic analysis to compare other, non-O157 E. coli (previously isolated and characterized in our laboratory) that also efficiently colonize the same unique bovine GI tract niche as O157. These comparisons may identify previously overlooked bacterial traits that allow O157 to persist in cattle. Third, we will determine if seasonal variation in O157 carriage is due to intrinsic or extrinsic factors. This information will begin to solve the puzzle of seasonal variation in O157 carriage. The ultimate goal of this basic research is to contribute to our knowledge and to the development of science-based effective interventions to reduce or eliminate O157 from cattle. In turn, this will positively impact food safety and human health.
This project will use established methods. For the first objective, we will continue to characterize O157 engineered deletion mutations for their ability to efficiently pass through the bovine gastrointestinal tract and/or colonize the recto-anal junction mucosa. We will test a set of isogenic derivatives that lack specific genes of interest or have been disabled in specific gene clusters to investigate the contribution of these factors to the bacteria's ability to pass through the rumen and colonization the bovine recto-anal junction mucosa. The derivatives will be engineered using the PCR-based one step gene inactivation system described by Datsenko and Wanner and antibiotic resistance genes will be removed before strains are used in animals. Cattle will be challenge by oral or rectal application of bacteria and bacterial carriage will be monitored by culture. All animals will be challenged with a single quantified dose of O157 and assessed for the bacteria by recto-anal swab samples. Briefly, swabs will be obtained and placed into trypticase soy broth for transport to the laboratory. The broth will be directly plated to give quantitative culture data (cfu/swab) or will be enriched by incubation overnight prior to plating to give qualitative (positive or negative) culture data. Sorbitol MacConkey agar containing cefixime, potassium tellurite, vancomycin, and MUG will be used. The numbers of sorbitol- and ?-glucuronidase-negative colonies will be counted and confirmed to be O157 by a latex agglutination test. Resulting bacterial counts will be compared to wild-type and among mutant O157 for statistical differences in concentrations and duration of O157 carriage. For the second objective, E. coli ONT:H25, a previously identified prevalent non-O157 that transiently colonizes the same gastrointestinal niche as O157 will be characterized. Whole genome sequencing and standard in silico bioinformatic comparisons will be used to analyze at least three E. coli ONT:H25 isolates with the three fully sequenced O157 strains (EDL933, Sakai, and the spinach out-break strain) and other relevant E. coli. For the third objective, the same group of 20 cattle will be challenged with O157 over a two year period, twice in the summer and twice in the winter. The carriage of O157 will be monitored by the bacterial culture technique and data analyses similar to those described for objective 1. Our efforts to cause a change in knowledge will include one-on-one training and mentoring of technical staff and students in our program; presentations at scientific meetings, and publication in reviewed scientific journals. In addition, the PI will give presentations to the public, industry, and government entities as requested. The success of the project will be measured by these activities to increase our basic understanding of the relationship between healthy cattle and the O157 human pathogen. Although our objectives do not include specific testing of interventions to reduce O157 in the food supply, an important outcome and impact of this work may guide such efforts.
2012/01 TO 2012/12<br/>
OUTPUTS: The activities on this project included conducting experiments to address the objectives in the project and analyzing the results. Both graduate and undergraduate students were trained and had a hands-on laboratory research experience through this process. Also, technical staff expanded their experience. The target audiences for this work 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 scientific meetings. We accomplished this, as our results are or are in the process of publication and were disseminated in the scientific community through peer-reviewed publications in journals and through invited seminars, and presentations at scientific meetings.
<br/>PARTICIPANTS: Individuals who worked on this project included faculty, staff, and graduate students. The work provided training and professional development for all involved. Partner organizations included Washington State University and USDA ARS.
<br/>TARGET AUDIENCES: Target audiences for this work include other scientists and professionals in industry that are affected by the human pathogen, E. coli O157:H7.
<br/>PROJECT MODIFICATIONS: No major changes in approach.
IMPACT: The findings from this work broadly contributed to our understanding of the relationship between healthy cattle and the human pathogen Escherichia coli O157:H7. This science-based information will lead to interventions to eliminate or reduce the food-borne illness caused by these bacteria. Specific changes in knowledge occurred from our work and are summarized here. The major reservoir of STEC is healthy cattle and the frequency and numbers of STEC in feces of shedding animals varies strongly by season. The mechanism(s) underlying this variation is unknown. Higher summertime prevalence has been reported from diverse regions including the Netherlands, Italy, England, Canada, the United States, Turkey, and Korea. Seasonality of STEC shedding by cattle may be influenced by both exogenous factors such as ambient temperature-induced environmental proliferation or predation, and/or seasonal variation in feed components affecting STEC replication, as well as endogenous factors such as immune responses and possible endocrine effects. A two year study was undertaken to determine whether seasonal variation is due to factors intrinsic or extrinsic to cattle. Two cohorts of ten Holstein steers were housed in an open barn subject to seasonal temperature and humidity fluctuations. Feed was consistent for all seasons. Cattle were randomized into one of two groups by route of STEC oral or intra-rectal instillation and challenged in summer and winter. Each orally dosed animal received 109 cfu STEC O157 in saline and rectally dosed animals receive 107 cfu STEC O157 administered using a sponge. Inocula contained equal numbers of four strains belonging to the most common clinical- and bovine-biased genotypes: 1, 3, 5, and 6. Animals were sampled on days 1, 4, 7 and weekly thereafter for 60 days post-challenge and STEC measured by standard culture of the rectoanal junction mucosa. Presumptive STEC O57 were confirmed by O157-specific latex agglutination and genotypes were distinguished by PCR. Bedding, water and hair-coat were cultured weekly to determine the density of STEC O157. The results from the first year (two seasons) of this two year study showed that endogenous factors did not contriubte to carriage or shedding of E. coli O157:H7 and when exposure was equal, season did not contribute to quantity or duration of O157 passage by cattle. This finding will be re-tested during year two. In addition analysis of non-O157 EHEC were shown to adhere to bovine recto-anal junction epithelian cells in a manner distinct from O157. The previously developed PATS typing system was shown to identify divrese sets of bovine O157 isolates. An invasin-like protein of Yersinia pestis with homology to O157 intimin was shown to be required for virulence. Bovine-biased O157 strains were less virulent than human clinical isolates in a animal models of human disease.