2011 Annual Report 1a.Objectives (from AD-416) 1)Identify molecular mechanisms in Salmonella that impact virulence and antimicrobial resistance in the gastrointestinal environment of cattle and swine. 2)Characterize the genetic pathways in swine that respond to Salmonella infection, their relationship to carrier status, and the ability to predict and control Salmonella prevalence and ultimately food safety. 3)Investigate interactions between Salmonella and host microbiota that influence Salmonella colonization and persistence, alter the commensal composition in the host, and provide novel intervention strategies to decrease Salmonella in swine populations. 1b.Approach (from AD-416) 1) Use phenotypic assays, recombineering, and global transcriptional analysis to identify the genes and genetic pathways involved in regulating Salmonella DT104 hyper-invasion in response to a) rumen protozoa and b) sub-inhibitory concentrations of antibiotics.. 2)Use an attenuated, LPS/flagella-deficient S. Typhimurium strain to re-focus the pig’s immune response from serovar-specific antigens to conserved antigens during vaccination against Salmonella, thereby providing cross-protection against multiple Salmonella serovars.. 3)Classify the porcine gene expression differences between low and high shedders of Salmonella during colonization.. 4)Compare the gastrointestinal microbial communities of pigs before, during and after Salmonella colonization to assess correlations with Salmonella shedding status in the pigs. 3.Progress Report Within a host, salmonellae are exposed to a variety of different environmental conditions; however, it is not known how antibiotics affect the virulence of drug-resistant Salmonella Typhimurium DT104 under these various conditions. In order to test the effect each antibiotic has on invasion under a wide range of different growth conditions, we developed a high-throughput screening tool. A fluorescent reporter plasmid was constructed to determine the relative level of salmonella invasiveness by measuring the key invasion-regulating protein HilA. This allows us to quickly screen a multitude of different combinations of antibiotics, antibiotic concentrations, antibiotic exposure durations, and growth conditions, thereby determining which combination of antibiotics and environmental conditions affect Salmonella Typhimurium DT104 invasion. Since over 2,500 serovars of Salmonella exist, a live Salmonella vaccine was designed to improve vaccination strategies by providing broad protection against Salmonella. The Salmonella vaccine was tested in pigs and provided protection against subsequent exposure to a pathogenic strain of Salmonella. The goal is to provide pig producers with a vaccine that will improve both animal health and food safety. 4.Accomplishments 1. Identification of a novel survival and virulence mechanism in Salmonella. Salmonella Typhimurium is a human foodborne pathogen and is one of the most prominent Salmonella serovars isolated from swine production farms. Unfortunately, Salmonella Typhimurium can undetectably reside in pigs without causing noticeable infection. These Salmonella-carrier pigs are a food safety problem for humans through contamination of penmates, the environment and slaughter plants that process pork for consumption. In searching for improved intervention strategies against Salmonella on the farm, ARS researchers in Ames, IA, have identified a gene (poxA) in Salmonella Typhimurium that, when mutated, dramatically reduces the ability of the bacterium to survive numerous stress conditions as well as antibiotic and chemical exposures. Furthermore, the gene mutation decreased the ability of Salmonella Typhimurium to colonize the pig. As this genetic system is critical for the ability of the Salmonella to cause disease and resist antibiotics, it offers a novel target mechanism for intervention development against Salmonella. Review Publications Bearson, B.L., Bearson, S.M. 2011. Host specific differences alter the requirement for certain Salmonella genes during swine colonization. Veterinary Microbiology. 150:215-219. Brunelle, B.W., Bearson, S.M., Bearson, B.L. 2011. Salmonella enterica serovar Typhimurium DT104 invasion is not enhanced by sub-inhibitory concentrations of the antibiotic florfenicol. Journal of Veterinary Science and Technology. 2:104. Available: http://omicsonline.org/2157-7579/2157-7579-2-104.php. Bearson, S.M., Bearson, B.L., Brunelle, B.W., Sharma, V.K., Lee, I. 2011. A mutation in the poxA gene of Salmonella enterica serovar Typhimurium results in altered protein production, elevated susceptibility to environmental challenges, and decreased swine colonization. Foodborne Pathogens and Disease. 8(6):725-732. Tuggle, C.K., Bearson, S.M., Uthe, J.J., Huang, T.H., Qu, L., Couture, O.P., Wang, Y., Kuhar, D.J., Lunney, J.K., Nettleton, D., Honavar, V., Dekkers, J.C. 2010. Methods for transcriptomic analyses of the porcine host immune response: application to Salmonella infection using microarrays. Veterinary Immunology and Immunopathology. 138(4):280-291.