C. K. Tuggle

Comparative Analysis of Monocyte Subsets in the Pig

Human and mouse monocyte can be divided into two different subpopulations based on surface marker expression: CD14/16 and Ly6C/CX3CR1, respectively. Monocyte subpopulations in the pig were identified based on reciprocal expression of CD14 and the scavenger receptor CD163. The two populations, CD14hi-CD163low and CD14low-CD163hi, show approximately equal abundance in the steady-state. Culture of pig PBMCs in CSF1 indicates that the two populations are a maturation series controlled by this growth factor. Gene expression in pig monocyte subpopulations was profiled using the newly developed and annotated pig whole genome snowball microarray. Previous studies have suggested a functional equivalence between human and mouse subsets, but certain genes such as CD36, CLEC4E, or TREM-1 showed human-specific expression. The same genes were expressed selectively in pig monocyte subsets. However, the profiles suggest that the pig CD14low-CD163high cells are actually equivalent to intermediate human monocytes, and there is no CD14− CD16+ “nonclassical” population. The results are discussed in terms of the relevance of the pig as a model for understanding human monocyte function.

Correlating blood immune parameters and a CCT7 genetic variant with the shedding of Salmonella enterica serovar Typhimurium in swine.

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.

Gene expression in intestinal mucosal biopsy specimens obtained from dogs with chronic enteropathy

OBJECTIVEnTo characterize mucosal gene expression in dogs with chronic enteropathy (CE).nnnANIMALSn18 dogs with CE and 6 healthy control dogs.nnnPROCEDURESnSmall intestinal mucosal biopsy specimens were endoscopically obtained from dogs. Disease severity in dogs with CE was determined via inflammatory bowel index scores and histologic grading of biopsy specimens. Total RNA was extracted from biopsy specimens and microchip array analysis (approx 43,000 probe sets) and quantitative reverse transcriptase PCR assays were performed.nnnRESULTSn1,875 genes were differentially expressed between dogs with CE and healthy control dogs; 1,582 (85%) genes were downregulated in dogs with CE, including neurotensin, fatty acid-binding protein 6, fatty acid synthase, aldehyde dehydrogenase 1 family member B1, metallothionein, and claudin 8, whereas few genes were upregulated in dogs with CE, including genes encoding products involved in extracellular matrix degradation (matrix metallopeptidases 1, 3, and 13), inflammation (tumor necrosis factor, interleukin-8, peroxisome proliferator-activated receptor γ, and S100 calcium-binding protein G), iron transport (solute carrier family 40 member 1), and immunity (CD96 and carcinoembryonic antigen-related cell adhesion molecule [CEACAM] 18). Dogs with CE and protein-losing enteropathy had the greatest number of differentially expressed genes. Results of quantitative reverse transcriptase PCR assay for select genes were similar to those for microchip array analysis.nnnCONCLUSIONS AND CLINICAL RELEVANCEnExpression of genes encoding products regulating mucosal inflammation was altered in dogs with CE and varied with disease severity. Impact for Human Medicine-Molecular pathogenesis of CE in dogs may be similar to that in humans with inflammatory bowel disease.

Integrating Comparative Expression Profiling Data and Association of SNPs with Salmonella Shedding for Improved Food Safety and Porcine Disease Resistance

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.

Not All SCID Pigs Are Created Equally: Two Independent Mutations in the Artemis Gene Cause SCID in Pigs

Mutations in >30 genes are known to result in impairment of the adaptive immune system, causing a group of disorders collectively known as SCID. SCID disorders are split into groups based on their presence and/or functionality of B, T, and NK cells. Piglets from a line of Yorkshire pigs at Iowa State University were shown to be affected by T−B−NK+ SCID, representing, to our knowledge, the first example of naturally occurring SCID in pigs. In this study, we present evidence for two spontaneous mutations as the molecular basis for this SCID phenotype. Flow cytometry analysis of thymocytes showed an increased frequency of immature T cells in SCID pigs. Fibroblasts from these pigs were more sensitive to ionizing radiation than non-SCID piglets, eliminating the RAG1 and RAG2 genes. Genetic and molecular analyses showed that two mutations were present in the Artemis gene, which in the homozygous or compound heterozygous state cause the immunodeficient phenotype. Rescue of SCID fibroblast radiosensitivity by human Artemis protein demonstrated that the identified Artemis mutations are the direct cause of this cellular phenotype. The work presented in the present study reveals two mutations in the Artemis gene that cause T−B−NK+ SCID in pigs. The SCID pig can be an important biomedical model, but these mutations would be undesirable in commercial pig populations. The identified mutations and associated genetic tests can be used to address both of these issues.

Use of Bioinformatic SNP Predictions in Differentially Expressed Genes to find SNPs Associated with Salmonella Colonization in Swine

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.

Genomewide association of piglet responses to infection with one of two porcine reproductive and respiratory syndrome virus isolates.

Porcine reproductive and respiratory syndrome (PRRS) is a devastating disease in the swine industry. Identification of host genetic factors that enable selection for improved performance during PRRS virus (PRRSV) infection would reduce the impact of this disease on animal welfare and production efficiency. We conducted genomewide association study (GWAS) analyses of data from 13 trials of approximately 200 commercial crossbred nursery-age piglets that were experimentally infected with 1 of 2 type 2 isolates of PRRSV (NVSL 97-7985 [NVSL] and KS2006-72109 [KS06]). Phenotypes analyzed were viral load (VL) in blood during the first 21 d after infection (dpi) and weight gain (WG) from 0 to 42 dpi. We accounted for the previously identified QTL in the region on SSC4 in our models to increase power to identify additional regions. Many regions identified by single-SNP analyses were not identified using Bayes-B, but both analyses identified the same regions on SSC3 and SSC5 to be associated with VL in the KS06 trials and on SSC6 in the NVSL trials ( < 5 × 10); for WG, regions on SSC5 and SSC17 were associated in the NVSL trials ( < 3 × 10). No regions were identified with either method for WG in the KS06 trials. Except for the region on SSC4, which was associated with VL for both isolates (but only with WG for NVSL), identified regions did not overlap between the 2 PRRSV isolate data sets, despite high estimates of the genetic correlation between isolates for traits based on these data. We also identified genomic regions whose associations with VL or WG interacted with either PRRSV isolate or with genotype at the SSC4 QTL. Gene ontology (GO) annotation terms for genes located near moderately associated SNP ( < 0.003) were enriched for multiple immunologically (VL) and metabolism- (WG) related GO terms. The biological relevance of these regions suggests that, although it may increase the number of false positives, the use of single-SNP analyses and a relaxed threshold also increased the identification of true positives. In conclusion, although only the SSC4 QTL was associated with response to both PRRSV isolates, genes near associated SNP were enriched for the same GO terms across PRRSV isolates, suggesting that host responses to these 2 isolates are affected by the actions of many genes that function together in similar biological processes.

Identification of potential serum biomarkers to predict feed efficiency in young pigs.

Identification of biomarkers for feed efficiency in livestock will aid in the efficient production of high-quality protein to meet the demands of a growing population. The overall objective of this research was to identify biomarkers in serum for swine feed efficiency and to discover pathways affected by divergent selection for residual feed intake (RFI). Serum was collected from young pigs (between 35 and 42 d of age) from 2 lines of pigs that have been genetically selected to be either more efficient (low-RFI) or less efficient (high-RFI). After blood collection, during finishing, pigs from each line were placed on either a low-energy/high-fiber diet or a traditional high-energy/low-fiber diet to test for any diet effects on RFI. Subsets of 6 pigs per line within each diet were used in 3 independent experiments. Pigs with extreme RFI phenotypes from the low-energy/high-fiber diet were used to confirm the results from the first 2 comparisons. Two-dimensional difference in gel electrophoresis and mass spectrometry were used to identify proteins with different abundances between RFI line or finishing diet. Three proteins had consistent and significant ( < 0.05) RFI line differences for both diets: gelsolin, vitronectin, and serine protease inhibitor A3 (serpinA3). Abundance of gelsolin, a protein with roles in actin filament assembly and immune response, was greater in the more efficient low-RFI pigs (9 to 39%). Vitronectin was also more abundant in the low-RFI pigs (39 to 56%) and has known roles in blood homeostasis and may regulate adiposity. SerpinA3 is a member of a very large family of proteins referred to as serine protease inhibitors. A total of 14 spots that were more abundant in the low-RFI line, some at least twice as abundant, were identified as serpinA3. Multiple isoforms of serpinA3 have been reported (serpinA3-1 to serpinA3-4 in pigs and serpinA3-1 to serpinA3-8 in cattle) with serpinA3 having many different functions dependent on isoform. Gelsolin, vitronectin, and serpinA3 are 3 proteins that may play direct and important biological roles in the pathways that control RFI and, ultimately, feed efficiency through energy utilization and homeostasis. These data demonstrate that serum proteins can be a useful source of potential biomarkers for feed efficiency and provide information on pathways with distinct expression patterns between animals that differ in feed efficiency.

Assessing peripheral blood cell profile of Yorkshire pigs divergently selected for residual feed intake

The cost of feed is a serious issue in the pork industry, contributing about 65 to 75% of the total production cost. To prevent economic losses and decreased productivity of the herd, it is important to select for animals that eat less for the same lean gain, or more efficient animals. Residual feed intake (RFI) is the difference between observed feed intake and expected feed intake based on estimated maintenance and production requirements. Selection for decreased RFI, or more efficient animals, is a potential solution to higher feed costs in pig production. However, animals that are highly selected for decreased RFI may have reduced energy input to the immune system and fail to withstand diseases and stressors after infection that negatively impact profitability. The objective of this study was to evaluate differences in circulating blood cell profiles at a young age between 2 lines of Yorkshire pigs that were divergently selected for RFI as well as the heritability of these traits, to investigate effects of selection for RFI on immune system parameters, and to identify potential biomarkers for feed efficiency. Previous work has shown that the 2 lines had diverged for IGF-1 in serum in young pigs and, therefore, this stage was investigated for other potential physiological differences. Blood samples were drawn for a complete blood count (CBC) analysis from 517 gilts and barrows, ages 35 to 42 d, across the 2 lines. In general, the low-RFI line had lower numbers of specific types of white blood cells but higher hemoglobin concentration and red blood cell volume compared to the high-RFI line. No significant correlations were found between CBC traits and RFI across and within the lines (0.05 < < 0.1). Of the 15 CBC traits that were measured, 3 were highly heritable (0.56 < < 0.62), 9 were moderately heritable (0.12 < < 0.47), and 3 were lowly heritable ( < 0.12), suggesting a substantial genetic component for CBC traits and that selection for CBC traits could be effective. Our results also show that selection for RFI has significantly impacted the number of circulating blood cells. In this experiment, we studied only healthy animals that were not under known pathogen challenge; therefore, our results cannot be directly applied to a disease challenge situation. Future work will be to challenge the animals and determine the effect of challenge on CBC levels.

Transcript profiles in longissimus dorsi muscle and subcutaneous adipose tissue: A comparison of pigs with different postweaning growth rates

Although most pigs recover rapidly from stresses associated with the transition of weaning, a portion of the population lags behind their contemporaries in growth performance. The underlying biological and molecular mechanisms involved in postweaning differences in growth performance are poorly understood. The objective of this experiment was to use transcriptional profiling of skeletal muscle and adipose tissue to develop a better understanding of the metabolic basis for poor weaned-pig transition. A total of 1,054 pigs was reared in commercial conditions and weighed at birth, weaning, and 3 wk postweaning. Transition ADG (tADG) was calculated as the ADG for the 3-wk period postweaning. Nine pigs from both the lowest 10th percentile (low tADG) and the 60th to 70th percentile (high tADG) were harvested at 3 wk postweaning. Differential expression analysis was conducted in longissimus dorsi muscle (LM) and subcutaneous adipose tissue using RNA-Seq methodology. In LM, 768 transcripts were differentially expressed (DE), 327 with higher expression in low tADG and 441 with higher expression in high tADG pigs (q < 0.10). Expression patterns measured in LM by RNA-Seq were verified in 30 of 32 transcripts using quantitative PCR. No DE transcripts were identified in adipose tissue. To identify biological functions potentially underlying the effects of tADG on skeletal muscle metabolism and physiology, functional annotation analysis of the DE transcripts was conducted using DAVID and Pathway Studio analytic tools. The group of DE genes with lower expression in LM of low tADG pigs was enriched in genes with functions related to muscle contraction, glucose metabolism, cytoskeleton organization, muscle development, and response to hormone stimulus (enrichment score > 1.3). The list of DE genes with higher expression in low tADG LM was enriched in genes with functions related to protein catabolism (enrichment score > 1.3). Analysis of known gene-gene interactions identified possible regulators of these differences in gene expression in LM of high and low tADG pigs; these include forkhead box O1 (FOXO1), growth hormone (GH1), and the glucocorticoid receptor (NR3C1). Differences in gene expression between poor transitioning pigs and their contemporaries indicate a shift to decreased protein synthesis, increased protein degradation, and reduced glucose metabolism in the LM of low tADG pigs.