The long-term goals of this project are to use genomics tools to identify genetic variants that are associated with decreased Salmonella shedding on farms; and to use these variants to select for animals that shed fewer bacteria and so are less likely to cause abattoir carcass contamination. Thus, the aim is to directly improve pork safety, as well as decrease other on-farm contamination by contaminated fecal matter that may be used as fertilizer. To achieve the long term goals, we worked on 4 specific objectives: 1) generate a large bank of Salmonella fecal shedding data and matched DNA samples using US industry pigs; 2) create DNA tests for genetic variation at candidate genes, selected based on our previous data on porcine gut lymph node and whole blood gene expression patterns in response to Salmonella infection; 3) perform genotyping using new and established DNA tests on US industry Salmonella-positive–control groups; and 4) test associations of genotypes with Salmonella incidence and integrate data from epidemiology and genomics projects to understand Salmonella contamination.
For objective 1, during a 2 year period a total of 462 belly flap tissue samples have been collected that include 163 pigs that shed Salmonella in their feces 7 days before marketing as well as 299 farm-mate negative controls matched for farm-visit cohort where a positive sample was found. Shedding of Salmonella was determined by qualitative fecal testing. Objective 1 was performed in collaboration with a large epidemiology study funded by the USDA-NRI Competitive Grants Program. We emphasize here the extraordinary contribution of this USDA-funded project; it would not have been possible to collect such a large number of Salmonella positive field samples within the current funding structure for standard NPB projects . In our laboratory, the DNA from Salmonella-positive and negative-control pigs belly flap tissue have been isolated and was used for genotyping. We also used other 3 porcine populations that we created using prior NPB funding or current funding from the USDA-NRI Functional Genomics program, such as the NADC-40 pig and NADC-77 pig Salmonella Typhimurium challenge populations; and the 228 pig IAH-Compton Salmonella Choleraesuis challenge population kindly provided by the Pig Improvement Company. In objective II we selected candidate genes for analysis of genetic variants (single nucleotide polymorphisms, SNPs). As candidates we selected genes that, based on our previous research, are involved in the porcine response to Salmonella infection. For objective III during the 1st-year period we genotyped 29-31 SNPs across the 4 populations and identified 18 SNPs with minor allele frequency (MAF) of 15% or higher in at least 2 populations. During the 2nd year period, we have genotyped a set of 31 additional SNPs across our pig populations and identified 10 new genetic variants with MAF of 15% or larger in at least 2 populations. In all, during the two year period we selected and genotyped across the 4 populations 62 SNPs in 53 genes that are involved in porcine response to Salmonella infection and confirmed 28 SNPs segregating in at least 2 populations. This output exceeded the original proposed goals of 10 segregating SNPs per year of funding. To genotype this larger number of SNPs, we adopted a Sequenom technology due to its increased throughput and reduced cost. In objective IV, we have newly applied a statistical approach to measuring Salmonella shedding/tissue colonization levels over the 3-week period after infection. Statistical analyses revealed several SNPs associated with Salmonella fecal shedding or tissue colonization in pigs. Fecal shedding associated SNPs were CCT7 #3 (p=0.041), GNG3 (p=0.029), PGD (p=0.047) and HP #2 (p=0.0002) in the field population, AMT (p=0.005) in the NADC-40 pigs population and PGD (p=0.001) in the NADC-77 pigs population. In the IAH-Compton population three SNPs in the ACP2 gene were associated with Salmonella burden in spleen (ACP2#1, p=0.013, ACP2#2, p=0.026, ACP2#3, p=0.033); and in the NADC-40 pig population, a SNP in the EMP1 (p=0.002) gene was associated with bacterial load in ileo-cecal lymph node. The genotyping and statistical analysis data identified genetic markers that are potentially useful for selecting animals that shed fewer bacteria and are less likely to cause pen-mate Salmonella contamination in a farm and on a slaughter plant. Additional validation of these SNPs should be performed, but this project has provided novel information for the pig industry to implement in strategies for selecting pigs with reduced shedding and/or disease susceptibility.
Contact: 515-294-4252, email: [email protected].
