RESUMEN
Salmonella in swine is a major food safety problem, as the majority of US swine herds are Salmonella-positive. Salmonella can be shed from colonized swine and contaminate (i) neighbouring pigs; (ii) slaughter plants and pork products; (iii) edible crops when swine manure is used as a fertilizer; and (iv) 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. In this research, we describe a successful strategy for discovering genetic variation at candidate genes associated with disease resistance in pigs. This involves integrating our recent global gene expression analysis of the porcine response to Salmonella with information from the literature about important candidate genes. We identified single-nucleotide polymorphisms (SNPs) in these functional candidate genes and genotyped three independent pig populations that had data on Salmonella faecal shedding or internal burden (total n = 377) at these loci. Of 31 SNPs genotyped, 21 SNPs segregated in at least two populations with a minor allele frequency of 15% or greater. Statistical analysis revealed thirteen SNPs associated with Salmonella faecal shedding or tissue colonization, with an estimated proportion of false positives (PFP) ≤0.2. The genes with associated SNPs included GNG3, NCF2, TAP1, VCL, AMT, CCR1, CD163, CCT7, EMP1 and ACP2. These associations provide new information about the mechanisms of porcine host response to Salmonella and may be useful in improving genetic resistance to this bacterium.
Asunto(s)
Derrame de Bacterias , Carne/microbiología , Polimorfismo de Nucleótido Simple , Salmonelosis Animal/inmunología , Sus scrofa , Enfermedades de los Porcinos/inmunología , Animales , Inocuidad de los Alimentos , Perfilación de la Expresión Génica , Inmunidad InnataRESUMEN
Asymptomatic Salmonella-carrier pigs present a major problem in preharvest food safety, with a recent survey indicating >50% of swine herds in the United States have Salmonella-positive animals. Salmonella-carrier pigs serve as a reservoir for contamination of neighbouring pigs, abattoir pens and pork products. In addition, fresh produce as well as water can be contaminated with Salmonella from manure used as fertilizer. Control of Salmonella at the farm level could be through genetic improvement of porcine disease resistance, a potentially powerful method of addressing preharvest pork safety. In this research, we integrate gene expression profiling data and sequence alignment-based prediction of single nucleotide polymorphisms (SNPs) to successfully identify SNPs in functional candidate genes to test for the associations with swine response to Salmonella. A list of 2527 genes that were differentially regulated in porcine whole blood in response to infection with Salmonella enterica serovar Typhimurium were selected. In those genes, SNPs were predicted using ANEXdb alignments based on stringent clustering of all publically available porcine cDNA and expressed sequence tag (EST) sequences. A set of 30 mostly non-synonymous SNPs were selected for genotype analysis of four independent populations (n = 750) with Salmonella faecal shedding or tissue colonization phenotypes. Nine SNPs segregated with minor allele frequency ≥15% in at least two populations. Statistical analysis revealed SNPs associated with Salmonella shedding, such as haptoglobin (HP, p = 0.001, q = 0.01), neutrophil cytosolic factor 2 (NCF2 #2, p = 0.04, q = 0.21) and phosphogluconate dehydrogenase (p = 0.066, q = 0.21). These associations may be useful in identifying and selecting pigs with improved resistance to this bacterium.
Asunto(s)
Biología Computacional , Regulación de la Expresión Génica , Polimorfismo de Nucleótido Simple , Salmonelosis Animal/genética , Enfermedades de los Porcinos/genética , Animales , Genotipo , PorcinosRESUMEN
Technological developments in both the collection and analysis of molecular genetic data over the past few years have provided new opportunities for an improved understanding of the global response to pathogen exposure. Such developments are particularly dramatic for scientists studying the pig, where tools to measure the expression of tens of thousands of transcripts, as well as unprecedented data on the porcine genome sequence, have combined to expand our abilities to elucidate the porcine immune system. In this review, we describe these recent developments in the context of our work using primarily microarrays to explore gene expression changes during infection of pigs by Salmonella. Thus while the focus is not a comprehensive review of all possible approaches, we provide links and information on both the tools we use as well as alternatives commonly available for transcriptomic data collection and analysis of porcine immune responses. Through this review, we expect readers will gain an appreciation for the necessary steps to plan, conduct, analyze and interpret the data from transcriptomic analyses directly applicable to their research interests.
Asunto(s)
Perfilación de la Expresión Génica/veterinaria , Análisis de Secuencia por Matrices de Oligonucleótidos/veterinaria , Salmonelosis Animal/genética , Salmonelosis Animal/inmunología , Sus scrofa/genética , Sus scrofa/inmunología , Enfermedades de los Porcinos/genética , Enfermedades de los Porcinos/inmunología , Animales , Biología Computacional , Minería de Datos , Bases de Datos Genéticas , Perfilación de la Expresión Génica/métodos , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Bases del Conocimiento , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Regiones Promotoras Genéticas , Sitios de Carácter Cuantitativo , PorcinosRESUMEN
The porcine response to Salmonella infection is critical for control of Salmonella fecal shedding and the establishment of Salmonella carrier status. In this study, 40 crossbred pigs were intranasally inoculated with Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) and monitored for Salmonella fecal shedding and blood immune parameters at 2, 7, 14 and 20 days post-inoculation (dpi). Using a multivariate permutation test, a positive correlation was observed between Salmonella Typhimurium shedding levels at 2 and 7dpi and serum interferon-gamma (IFNgamma) levels at 2dpi (p<0.05), with Salmonella being shed in greater numbers from animals with higher IFNgamma levels. A positive correlation was also observed between IFNgamma levels and the number of banded neutrophils (2dpi), circulating neutrophils (7 and 14dpi), monocytes (7dpi), and white blood cells (WBCs) (7, 14 and 20dpi). We have further performed association studies on these immune response parameters as well as shedding status of the Salmonella-infected pigs with a single nucleotide polymorphism (SNP) in the porcine gene CCT7, previously shown by our group to be transcriptionally up-regulated in swine experimentally inoculated with Salmonella Typhimurium. Our analyses with the 40 pigs suggest a positive association (p=0.0012) of SNP genotype A/G at position AK240296.c1153G>A of the CCT7 gene with Salmonella shedding at 7dpi compared to the G/G homozygote genotype. Linking specific genes and genetic polymorphisms with the porcine immune response to Salmonella infection and shedding may identify potential markers for carrier pigs as well as targets for disease diagnosis, intervention and prevention.
Asunto(s)
Salmonella typhi/genética , Enfermedades de los Porcinos/microbiología , Fiebre Tifoidea/veterinaria , Esparcimiento de Virus/inmunología , Animales , Cartilla de ADN , ADN Viral/genética , ADN Viral/aislamiento & purificación , Heces/virología , Femenino , Interferón gamma/sangre , Interferón gamma/genética , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Salmonelosis Animal/sangre , Salmonelosis Animal/genética , Salmonelosis Animal/inmunología , Salmonella typhi/aislamiento & purificación , PorcinosRESUMEN
We are investigating the porcine gut immune response to infection through gene expression profiling. Porcine Affymetrix GeneChip data was obtained from RNA prepared from mesenteric lymph node of swine infected with either Salmonella enterica serovar Typhimurium (ST) or S. Choleraesuis (SC) for 0, 8, 24, 48 or 504 hours post-inoculation (hpi). In total, 2365 genes with statistical evidence for differential expression (DE; p < 0.01, q < 0.26, fold-change > 2) between at least two time-points were identified. Comparative Gene Ontology analyses revealed that a high proportion of annotated DE genes in both infections are involved in immune and defence responses. Hierarchical clustering of expression patterns and annotations showed that 22 of the 83 genes upregulated from 8-24 hpi in the SC infection are known NF-kappaB targets. The promoter sequences of human genes orthologous to the DE genes were collected and TFM-Explorer was used to identify a set of 72 gene promoters with significant over-representation of NF-kappaB DNA-binding motifs. All 22 known NF-kappaB target genes are in this list; we hypothesize that the remaining 51 genes are un-recognized NF-kappaB targets. Integration of these results and verification of putative target genes will increase our understanding of the porcine response pathways responding to bacterial infection.