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The objective of this cooperative research project is to characterize the transcriptional profile of the carrier state of Salmonella-infected pigs to identify differentially expressed genes with sequence variants in pig populations.
Approach: Pigs will be experimentally infected (n=40) with S. Typhimuriuim and fecal shedding will be quantitatively measured at regular intervals (0 hrs, 48 hrs, 7 days, 14 days, 21 days). Transcriptional profiling will be performed on animals with the lowest level of shedding at 21 days post inoculation (n=4, animals that showed infection but were culture negative for Salmonella after day 7) compared to animals with the highest shedding (n=6, animals that cultured positive for Salmonella at all time points) using DNA microarray analysis (Affymetrix porcine chip) followed by quantitative real-time PCR. Several methods (direct sequencing, PCR Restriction Fragment Length Polymorphism analysis, and similar analyses) will be used to identify, genotype and determine the frequency of DNA sequence variants (polymorphisms) in the challenge populations as well as commercial pig breeds. Such variants,termed DNA markers, may identify specific versions of genes (alleles) that are superior to other alleles of the gene in resisting disease. Data from our current agreement has revealed a positive correlation between Salmonella shedding and differential expression of immune-related genes as well as immune cells present in the blood. Extension of the SCA for the requested 5 months will allow expansion of the project to include additional samples from the infection that were not included in the original agreement, thereby providing additional parameters to assay for association to Salmonella shedding. Furthermore, the current cooperative agreement has identified single nucleotide polymorphisms (SNPs) in 10 porcine genes identified as differentially-expressed during infection with Salmonella (thereby meeting Objective II of the proposal). One of the identified SNPs in the CCT7 gene associates with Salmonella shedding at 7 days post-inoculation. Extension of the SCA will allow us to expand the association testing to a larger Salmonella challenge group (204 pigs); this set of DNA is property of the Tuggle laboratory.