2011 Annual Report 1a.Objectives (from AD-416) (1) Using optimized sampling strategies, enumeration, and molecular diagnostic, identify management practices resulting in high and low Salmonella and Campylobacter prevalence turkey farms and monitor the efficacy of on-farm intervention strategies targeting specific risk factors. (2) Identify key virulence attributes to differentiate Salmonella and Campylobacter avirulent commensals from those pathogenic strains that pose a public health threat in humans. (3) Develop molecular methods to assess the dynamics of the microbial intestinal flora throughout hog and turkey production. Identify microbes associated with gut colonization by, and population shifts of, foodborne pathogens. (4) Determine prevalence and quantities of recognized foodborne pathogens, principally Salmonella but also Campylobacter and Yersinia, in hog carcasses and organs. 1b.Approach (from AD-416) Time of entry of Salmonella and Campylobacter will be monitored in turkeys. The study will document flock management practices which affect the prevalence of these foodborne pathogens initially in the brooder period and ultimately throughout live production. Key virulence attributes of C. jejuni and C. coli strains recovered from turkeys will be characterized in vitro (cell invasion assays)and in vivo (day of hatch poult model.) Ultimately, differential gene expression formats will be used to differentiate a virulent from virulent isolates of C. coli and C. jejuni. Bacterial and fungal communities of the ceca of domestic and wild turkeys will be described. This initial survey will provide a measurement of diversity between wild and domestic birds. Analysis of total community flux over time will focus on kinetics of Campylobacter community development and stability in the turkey ceca. Organisms that correlate with Campylobacter colonization or exclusion will be identified. Second generation PCR assays will be developed to detect and quantify Salmonella and Campylobacter on hog and turkey carcasses. Monitoring viscera will serve as an indicator of on-farm versus in-plant sources of contamination. These studies will assist in determining the critical control points of contamination during slaughter. 3.Progress Report This is the final report for project 3625-32000-080-00D terminated in December 2010 and replaced with 3625-31320-003-00D. Research progress was made to uncover the ecology of foodborne pathogens, Campylobacter, Salmonella, and Yersinia either in poultry or in swine. Investigations were made both on external factors affecting colonization of the animals by these foodborne pathogens and internal (intestinal) factors affecting colonization. Ecological factors within poultry intestines affected the ability of the Campylobacter to colonize that environment. Different Campylobacter species and subspecies were shown to correlate to intestinal bacteria fluctuations and host signals, such as maturation age. Campylobacter coli was found to be a commensal colonizer while Campylobacter jejuni behaved as an opportunistic colonizer implying different intervention strategies will be required to exclude the two species from the poultry intestine. Different Campylobacter species and subspecies were shown to correlate to intestinal bacteria fluctuations and host signals. Megamonas hypermegale (M. hypermegale) type II levels correlated with the exclusion of Campylobacter coli from the turkey intestine. Exclusion of the pathogen from the animal host is the best method to ensure safety of the national food supply. Consequently, the potential of M. hypermegale as an inhibitor of Campylobacter will be examined in the new project. Swine are the major animal reservoir for Yersinia enterocolitica (Y. enterocolitica), a major human foodborne pathogen. We conducted the first attempt to identify risk factors for Y. enterocolitica in the United States hog population. By analyzing feces and tonsilar swabs of hogs from 122 locations, we found three positive risk factors for Y. enterocolitica infections: vaccination for Escherichia coli, percentage of deaths due to scours, and presence of meat/bone meal in grower-finisher diet. Locations in a U.S. midwestern state had reduced risk. We determined that the frequency of detection of Campylobacter on harvested turkeys was three-fold greater when the turkey carcass was swabbed than when neck skin was sampled. These findings indicate that the USDA Food Safety and Inspection Service carcass swab protocol appears a better indicator of Campylobacter status than European neck skin sampling technique. Turkeys are reservoirs for Campylobacter, a major human foodborne pathogen. Transport from farm to abbatoir resulted in a significant increase in Campylobacter numbers in the gall bladders and crops of transported birds. Turkey poults were found to be free of Campylobacter at one day and nine days after hatch. At slaughter (138 days after hatch), Campylobacter prevalence in sampled birds was 92%. These results indicate that this foodborne pathogen is acquired from the environment and colonizes ceca of birds during their growth. Review Publications Scupham, A.J., Jones, J., Rettedal, E.A., Weber, T.E. 2010. Antibiotic Manipulation of Intestinal Microbiota to Identify Microbes Associated with Campylobacter Exclusion in Poultry. Applied and Environmental Microbiology. 76(24):8026-8032. Solis-Soto, L., Garcia, S., Wesley, I.V., Heredia, N. 2011. A charcoal- and blood-free enrichment broth for isolation and PCR detection of Campylobacter jejuni and Campylobacter coli. Journal of Food Protection. 74(2):221-227. Muraoka, W.T., Zhang, Q. 2011. Phenotypic and genotypic evidence for L-fucose utilization by Campylobacter jejuni. Journal of Bacteriology. 193(5):1065-1075.