Analyzing factors involved in genetic variation of porcine response to Salmonella (2012)

Asymptomatic Salmonella-carrier pigs present a major problem in pre-harvest food safety, with a recent survey indicating >50% of swine herds in the U.S. have Salmonella-positive animals. Salmonella can be shed from colonized swine and contaminate a) neighboring pigs; b) slaughter plants and pork products; c) edible crops when swine manure is used as a fertilizer; and d) water supplies if manure used as crop fertilizer runs off into streams and waterways. A potentially powerful method of addressing pre-harvest food safety at the farm level is through genetic improvement of disease resistance in animals.\nThe purpose of the research presented in this dissertation was to contribute to the understanding of factors underlying disease resistance in swine by investigating genetic variation associated with swine response to Salmonella. The main objectives of the research were: 1) using integrated global gene expression and bioinformatics analysis approach to discover candidate genes with single nucleotide polymorphisms (SNPs) that can be associated with Salmonella-related phenotypes and potentially affect Salmonella shedding and/or the carrier status in swine; 2) analyzing expression of a set of genes regulated by IFN-gamma, a cytokine whose serum levels are correlated with Salmonella shedding in swine, to determine potential biomarkers for assessing the quality of swine immune response that could be used to predict the shedding status of swine.\nTo identify SNPs in functional candidate genes to test for associations with swine response to Salmonella, integrative gene selection approach was employed. Recent global gene expression analysis of the porcine response to Salmonella combined with literature information on important candidate genes as well as sequence alignment-based predictions of SNPs resulted in successful identification of SNPs in functional candidate genes. The selected SNPs were genotyped by Sequenom technology using four porcine populations that total 750 swine. Genotyping and statistical association analysis resulted in discovery of several novel SNPs in 12 functional candidate genes associated with Salmonella-related phenotypes, such as tissue colonization and/or fecal Salmonella shedding. The associated SNPs were identified in such genes as: TAP1, GNG3, CCR1, CD163, AMT, ACP2, VCL, CCT7, HP, EMP1, NCF2 and PGD. Thus, the SNPs analysis study suggest several potential candidate genes to be further evaluated as markers for selection of swine with improved immune function and reduced Salmonella shedding. Additionally, the novel genetic associations provide useful information for the pig industry regarding strategies for selecting swine with improved disease resistance.\nOne of the major immune response mediators, IFN-gamma, has been shown to be important in swine response to Salmonella by transcriptomic and pathway analysis. Thus, this research focused on a postulated role of IFN-gamma-regulated genes in the differential swine response to Salmonella. Whole blood transcriptome analysis revealed many differentially expressed genes between pigs classified as persistent (PS) or low (LS) Salmonella shedders. Pathway analysis of differentially expressed genes showed that IFN-gamma-responsive genes represent the largest response network in the PS pigs. Using gene expression data and literature evidence, 15 candidate genes representing different aspects of the IFN-gamma-regulated response were selected. qPCR analysis confirmed in vivo patterns of the 15 genes. Further investigations verified that differential patterns observed in vivo in PS and LS pigs could be recapitulated in vitro. Whole blood from 3 healthy pigs was stimulated in vitro with 1, 10 or 100 ng/ml IFN-gamma with or without S. Typhimurium endotoxin (STE). The qPCR analysis revealed that the in vitro response to IFN-gamma alone for five genes (CXCL10, IL10, MMP8, PSMB9, TMEM176) was dose-dependent. Simultaneous stimulation of whole blood with STE and IFN-gamma induced IFN-gamma dose-dependent expression for these genes plus CASP4, CYBA, IRF1, JAK2, NCF1, SOD2, and TAP1. Cluster analysis revealed that in vivo gene expression patterns across all genes in PS swine clustered most closely with patterns in whole blood stimulated with the two highest IFN-gamma levels plus STE. This research indicates that quantitative differences in IFN-gamma levels explains the expression of most tested genes, and that the IFN-gamma regulon is a source of genes whose expression levels two days post-infection can predict shedding outcomes. Such genes can now be further evaluated as candidates for development of predictive assays for shedding outcome in swine.\nIn general, the research presented in this dissertation contributed new information for better understanding of swine genetic variation in response to Salmonella. Several novel SNPs in functional candidate genes were described that could be further analyzed as biomarkers for selection of swine with improved immune responses. Furthermore, several clusters of IFN-gamma-regulated genes suggest that expression analysis of the specific set of biomarkers could be useful for developing in vitro tests for prediction of swine Salmonella shedding phenotypes. IFNgamma-regulated genes are good candidates for further analysis of potential classifier sets to better understand and confirm the ability of small sets of genes to classify swine according to Salmonella shedding phenotypes and to develop predictive screening assays for swine with improved immune function and reduced bacterial shedding.