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Neutrophils are vital components of the immune system for limiting the invasion and proliferation of pathogens in the body. Surprisingly, the functional annotation of porcine neutrophils is still limited. The transcriptomic and epigenetic assessment of porcine neutrophils from healthy pigs was performed by bulk RNA sequencing and transposase accessible chromatin sequencing (ATAC-seq). First, we sequenced and compared the transcriptome of porcine neutrophils with eight other immune cell transcriptomes to identify a neutrophil-enriched gene listwithin a detected neutrophil co-expressionmodule. Second, we used ATAC-seq analysis to report for the first time the genome-wide chromatin accessible regions of porcine neutrophils. A combined analysis using both transcriptomic and chromatin accessibility data further defined the neutrophil co-expression network controlled by transcription factors likely important for neutrophil lineage commitment and function. We identified chromatin accessible regions around promoters of neutrophil-specific genes that were predicted to be bound by neutrophil-specific transcription factors. Additionally, published DNA methylation data from porcine immune cells including neutrophils were used to link low DNA methylation patterns to accessible chromatin regions and genes with highly enriched expression in porcine neutrophils. In summary, our data provides the first integrative analysis of the accessible chromatin regions and transcriptional status of porcine neutrophils, contributing to the Functional Annotation of Animal Genomes (FAANG) project, and demonstrates the utility of chromatin accessible regions to identify and enrich our understanding of transcriptional networks in a cell type such as neutrophils. [ABSTRACT FROM AUTHOR]

Understanding regional distribution and specialization of small intestinal epithelial cells is crucial for developing methods to control appetite, stress, and nutrient uptake in swine. To establish a better understanding of specific epithelial cells found across different regions of the small intestine in pigs, we utilized single-cell RNA sequencing (scRNA-seq) to recover and analyze epithelial cells from duodenum, jejunum, and ileum. Cells identified included crypt cells, enterocytes, BEST4 enterocytes, goblet cells, and enteroendocrine (EE) cells. EE cells were divided into two subsets based on the level of expression of the EE lineage commitment gene, NEUROD1. NEUROD1 hi EE cells had minimal expression of hormone-encoding genes and were dissimilar to EE cells in humans and mice, indicating a subset of EE cells unique to pigs. Recently discovered BEST4 enterocytes were detected in both crypts and villi throughout the small intestine via in situ staining, unlike in humans, where BEST4 enterocytes are found only in small intestinal villi. Proximal-to-distal gradients of expression were noted for hormone-encoding genes in EE cells and nutrient transport genes in enterocytes via scRNA-seq, demonstrating regional specialization. Regional gene expression in EE cells and enterocytes was validated via quantitative PCR (qPCR) analysis of RNA isolated from epithelial cells of different small intestinal locations. Though many genes had similar patterns of regional expression when assessed by qPCR of total epithelial cells, some regional expression was only detected via scRNA-seq, highlighting advantages of scRNA-seq to deconvolute cell type-specific regional gene expression when compared to analysis of bulk samples. Overall, results provide new information on regional localization and transcriptional profiles of epithelial cells in the pig small intestine. [ABSTRACT FROM AUTHOR]

Intraepithelial lymphocytes (IELs) include T cells and innate lymphoid cells that are important mediators of intestinal immunity and barrier defense, yet most knowledge of IELs is derived from the study of humans and rodent models. Pigs are an important global food source and promising biomedical model, yet relatively little is known about IELs in the porcine intestine, especially during formative ages of intestinal development. Due to the biological significance of IELs, global importance of pig health, and potential of early life events to influence IELs, we collate current knowledge of porcine IEL functional and phenotypic maturation in the context of the developing intestinal tract and outline areas where further research is needed. Based on available findings, we formulate probable implications of IELs on intestinal and overall health outcomes and highlight key findings in relation to human IELs to emphasize potential applicability of pigs as a biomedical model for intestinal IEL research. Review of current literature suggests the study of porcine intestinal IELs as an exciting research frontier with dual application for betterment of animal and human health. [ABSTRACT FROM AUTHOR]

Three experiments (EXP) were conducted to determine the effect of feed additives on performance, intestinal integrity, gastrointestinal volatile fatty acids (VFA), and energy and nutrient digestion in nonchallenged nursery pigs. In EXP 1, 480 pigs (6.36-kg body weight, BW) were placed into 96 pens with 5 pigs/pen, and allotted to 1 of 10 dietary treatments: 1) negative control containing no feed additive (NC), 2) NC + 44 mg chlortetracycline and 38.5 mg tiamulin/kg diet (CTsb), 3) NC + 5% resistant potato starch (RSpo), 4) NC + 5% soluble corn fiber (SCF), 5) NC + 5% sugar beet pulp (SBP), 6) NC + 0.30% fatty acid mix (FAM), 7) NC + 0.10% phytogenic blend of essential oils and flavoring compounds (PHY), 8) NC + 50 mg Cu and 1,600 mg zinc oxide/kg diet (CuZn), 9) NC + 5% resistant corn starch (RScn), and 10) NC + 0.05% β-glucan (BG) for 28 d. There was no impact of dietary treatment on BW gain or feed intake (P ≥ 0.22). Pigs fed diets containing SCF, CTsb, and RSpo resulted in microbial community differences compared to pigs fed the NC (P < 0.05). In EXP 2, 48 barrows (12.8 kg BW) were selected at the end of EXP 1 and fed the same dietary treatments they had previously received: 1) NC, 2) NC + 5% RScn, 3) NC + 5% SCF, and 4) NC + FAM for 8 d. There was no effect of feeding diets containing RScn, SCF, or FAM on in vivo intestinal permeability (P ≤ 0.21). Ileal or colon pH, concentrations of VFA did not differ due to dietary treatment (P ≥ 0.36), but pigs fed diets containing FAM resulted in a greater butyric acid concentration in the cecum compared to pigs fed the NC (P ≤ 0.05). In EXP 3, 156 pigs (6.11 kg BW) were placed into 52 pens with 3 pigs/pen and allotted to 1 of 4 dietary treatments arranged in a factorial manner: 1) NC, 2) NC + 5% RSpo, 3) NC + 0.30% FAM, and 4) NC + 5% RSpo + 0.30% FAM for 24 d. Feeding pigs diets containing RSpo did not affect BW gain (P = 0.91) while pigs fed diets containing FAM grew improved BW gain (P = 0.09). Colonic butyric acid concentrations were greater in pigs fed diets containing RSpo (P = 0.03), while pigs fed diets containing FAM exhibited reduced total VFA concentrations (P = 0.11). The results indicate that supplementing diets with digestively resistant but fermentable fibers, short- and medium-chain fatty acids, or antibiotics do not have a consistent effect, positive or negative, on markers of intestinal integrity or barrier function, intestinal VFA patterns, ATTD of energy and nutrients, or on pig performance. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis is a swine pathogen of significant industry concern, but little is known about how the organism causes disease. A related human pathogen, Haemophilus influenzae, has been better studied, and many of its virulence factors have been identified. Two of these, outer membrane proteins P2 and P5, are known to have important virulence properties. The goals of this study were to identify, analyze, and compare the genetic relatedness of orthoiogous genes encoding P2 and P5 proteins in a diverse group of 35 H. parasuis strains. Genes encoding P2 and P5 proteins were detected in all H. parasuis strains evaluated. The predicted amino acid sequences for both P2 and P5 proteins exhibit considerable heterogeneity, particularly in regions corresponding to predicted extracellular loops. Twenty-five variants of P2 and 17 variants of P5 were identified. The P2 proteins of seven strains were predicted to contain a highly conserved additional extracellular loop compared to the remaining strains and to H. influenzae P2. Antigenic-site predictions coincided with predicted extracellular loop regions of both P2 and P5. Neighbor-joining trees constructed using P2 and P5 sequences predicted divergent evolutionary histories distinct from those predicted by a multilocus sequence typing phylogeny based on partial sequencing of seven housekeeping genes. Real-time reverse transcription-PCR indicated that both genes are expressed in all of the strains. doi: 10.1128/JB.00469-09

Innate immunomodulation via induction of innate memory is one mechanism to alter the host's innate immune response to reduce or prevent disease. Microbial products modulate innate responses with immediate and lasting effects. Innate memory is characterized by enhanced (training) or depressed (tolerance) innate immune responses, including pro-inflammatory cytokine production, to secondary exposure following a priming event. To investigate the ability of β-glucans and bacillus Calmette-Guerin to induce innate training or tolerance in pig cells, porcine monocytes were cultured with priming agonist (β-glucans or bacillus Calmette-Guerin) then re-stimulated 5 d later with a heterologous microbial agonist to determine induction of innate memory. Priming with β-glucan from Saccharomyces cerevisiae depressed IL-1β and TNF-α cytokine responses to re-stimulation with LPS, indicative of a tolerized state. However, bacillus Calmette-Guerin priming induced a trained state in porcine monocytes, as LPS re-stimulation enhanced IL-1β and TNF-α gene expression and protein production. We present the first evidence of innate memory in pig monocytes, with bacillus Calmette-Guerin (training) or Saccharomyces cerevisiae β-glucan (tolerance). Induction of a trained or tolerized state in vitro is a first step to identify agonists to alter the innate immune system at the animal level with the intent of enhancing disease resistance. [ABSTRACT FROM AUTHOR]

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B-cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B-cell, conventional CD4 and CD8 αβ T-cells, NK cells, and γδ T-cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T-cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology. [ABSTRACT FROM AUTHOR]

Changes in chromatin structure, especially in histone modifications (HMs), linked with chromatin accessibility for transcription machinery, are considered to play significant roles in transcriptional regulation. Alveolar macrophages (AM) are important immune cells for protection against pulmonary pathogens, and must readily respond to bacteria and viruses that enter the airways. Mechanism(s) controlling AM innate response to different pathogen-associated molecular patterns (PAMPs) are not well defined in pigs. By combining RNA sequencing (RNA-seq) with chromatin immunoprecipitation and sequencing (ChIP-seq) for four histone marks (H3K4me3, H3K4me1, H3K27ac and H3K27me3), we established a chromatin state map for AM stimulated with two different PAMPs, lipopolysaccharide (LPS) and Poly(I:C), and investigated the potential effect of identified histone modifications on transcription factor binding motif (TFBM) prediction and RNA abundance changes in these AM. The integrative analysis suggests that the differential gene expression between non-stimulated and stimulated AM is significantly associated with changes in the H3K27ac level at active regulatory regions. Although global changes in chromatin states were minor after stimulation, we detected chromatin state changes for differentially expressed genes involved in the TLR4, TLR3 and RIG-I signaling pathways. We found that regions marked by H3K27ac genome-wide were enriched for TFBMs of TF that are involved in the inflammatory response. We further documented that TF whose expression was induced by these stimuli had TFBMs enriched within H3K27ac-marked regions whose chromatin state changed by these same stimuli. Given that the dramatic transcriptomic changes and minor chromatin state changes occurred in response to both stimuli, we conclude that regulatory elements (i.e. active promoters) that contain transcription factor binding motifs were already active/poised in AM for immediate inflammatory response to PAMPs. In summary, our data provides the first chromatin state map of porcine AM in response to bacterial and viral PAMPs, contributing to the Functional Annotation of Animal Genomes (FAANG) project, and demonstrates the role of HMs, especially H3K27ac, in regulating transcription in AM in response to LPS and Poly(I:C). [ABSTRACT FROM AUTHOR]

T cells resident within the intestinal epithelium play a central role in barrier integrity and provide a first line of immune defense. Intraepithelial T cells (IETs) are among the earliest immune cells to populate and protect intestinal tissues, thereby giving them an important role in shaping gut health early in life. In pigs, IETs are poorly defined, and their maturation in young pigs has not been well-studied. Given the importance of IETs in contributing to early life and long-term intestinal health through interactions with epithelial cells, the microbiota, and additional environmental factors, a deeper characterization of IETs in pigs is warranted. The objective of this study was to analyze age- and intestinal location-dependent changes in IETs across multiple sites of the small and large intestine in pigs between 4- and 8-weeks of age. IETs increased in abundance over time and belonged to both γδ and αβ T cell lineages. Similar compositions of IETs were identified across intestinal sites in 4-week-old pigs, but compositions diverged between intestinal sites as pigs aged. CD2+CD8α+ γδ T cells and CD4−CD8α+ αβ T cells comprised >78% of total IETs at all intestinal locations and ages examined. Greater percentages of γδ IETs were present in large intestine compared to small intestine in older pigs. Small intestinal tissues had greater percentages of CD2+CD8α− γδ IETs, while CD2+CD8α+ γδ IET percentages were greater in the large intestine. Percentages of CD4−CD8α+ αβ IETs increased over time across all intestinal sites. Moreover, percentages of CD27+ cells decreased in ileum and large intestine over time, indicating increased IET activation as pigs aged. Percentages of CD27+ cells were also higher in small intestine compared to large intestine at later timepoints. Results herein emphasize 4- to 8-weeks of age as a critical window of IET maturation and suggest strong associations between intestinal location and age with IET heterogeneity in pigs. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is a significant problem in health care, animal health, and food safety. To limit AMR, there is a need for alternatives to antibiotics to enhance disease resistance and support judicious antibiotic usage in animals and humans. Immunomodulation is a promising strategy to enhance disease resistance without antibiotics in food animals. One rapidly evolving field of immunomodulation is innate memory in which innate immune cells undergo epigenetic changes of chromatin remodeling and metabolic reprogramming upon a priming event that results in either enhanced or suppressed responsiveness to secondary stimuli (training or tolerance, respectively). Exposure to live agents such as bacille Calmette-Guerin (BCG) or microbe-derived products such as LPS or yeast cell wall ß-glucans can reprogram or "train" the innate immune system. Over the last decade, significant advancements increased our understanding of innate training in humans and rodent models, and strategies are being developed to specifically target or regulate innate memory. In veterinary species, the concept of enhancing the innate immune system is not new; however, there are few available studies which have purposefully investigated innate training as it has been defined in human literature. The development of targeted approaches to engage innate training in food animals, with the practical goal of enhancing the capacity to limit disease without the use of antibiotics, is an area which deserves attention. In this review, we provide an overview of innate immunomodulation and memory, and the mechanisms which regulate this long-term functional reprogramming in other animals (e.g., humans, rodents). We focus on studies describing innate training, or similar phenomenon (often referred to as heterologous or non-specific protection), in cattle, sheep, goats, swine, poultry, and fish species; and discuss the potential benefits and shortcomings of engaging innate training for enhancing disease resistance. [ABSTRACT FROM AUTHOR]

Background: Glaesserella parasuis, the causative agent of Glӓsser's disease, is widespread in swine globally resulting in significant economic losses to the swine industry. Prevention of Glӓsser's disease in pigs has been plagued with an inability to design broadly protective vaccines, as many bacterin based platforms generate serovar or strain specific immunity. Subunit vaccines are of interest to provide protective immunity to multiple strains of G. parasuis. Selected proteins for subunit vaccination should be widespread, highly conserved, and surface exposed. Results: Two candidate proteins for subunit vaccination (RlpB and VacJ) against G. parasuis were identified using random mutagenesis and an in vitro organ culture system. Pigs were vaccinated with recombinant RlpB and VacJ, outer membrane proteins with important contributions to cellular function and viability. Though high antibody titers to the recombinant proteins and increased interferon-γ producing cells were found in subunit vaccinated animals, the pigs were not protected from developing systemic disease. Conclusions: It appears there may be insufficient RlpB and VacJ exposed on the bacterial surface for antibody to bind, preventing high RlpB and VacJ specific antibody titers from protecting animals from G. parasuis. Additionally, this work confirms the importance of utilizing the natural host species when assessing the efficacy of vaccine candidates. [ABSTRACT FROM AUTHOR]

Post-weaning diarrhea caused by enterotoxigenic E. coli (ETEC) causes significant economic losses for pig producers. This study was to test the hypotheses that an ETEC challenge disrupts intestinal microbial homeostasis and the inclusion of dietary soluble (10% sugar beet pulp) or insoluble fiber (15% corn distillers dried grains with solubles) with or without exogenous carbohydrases will protect or restore the gut microbial homeostasis in weaned pigs. Sixty crossbred piglets (6.9 ± 0.1 kg) were blocked by body weight and randomly assigned to one of six treatments (n = 10), including a non-challenged control (NC), ETEC F18-challenged positive control (PC), ETEC-challenged soluble fiber without (SF-) or with carbohydrases (SF+), and ETEC-challenged insoluble fiber without (IF-) or with carbohydrases (IF+). Pigs were housed individually and orally received either ETEC inoculum or PBS-sham inoculum on day 7 post-weaning. Intestinal contents were collected on day 14 or 15. The V4 region of the bacterial 16S rRNA was amplified and sequenced. High-quality reads (total 6,671,739) were selected and clustered into 3,330 OTUs. No differences were observed in α-diversity among treatments. The ileal microbiota in NC and PC had modest separation in the weighted PCoA plot; the microbial structures were slightly altered by SF+ and IF- compared with PC. The PC increased ileal Escherichia-Shigella (P < 0.01) and numerically decreased Lactobacillus compared to NC. Predicted functional pathways enriched in the ileal microbiota of PC pigs indicated enhanced activity of Gram-negative bacteria, in agreement with increased Escherichia-Shigella. The SF+ tended to decrease (P < 0.10) ileal Escherichia-Shigella compared to PC. Greater abundance of ileal Streptococcus , Turicibacter , and Roseburia and colonic Prevotella were observed in SF- and SF+ than PC (P < 0.05). Pigs fed IF + had greater Lactobacillus and Roseburia than PC pigs (P < 0.05). The ETEC challenge reduced total volatile fatty acid (VFA) compared with NC (P < 0.05). The SF+ tended to increase (P < 0.10) and SF- significantly increased (P < 0.05) colonic total VFA compared with PC. Collectively, ETEC challenge disrupted gut microbial homeostasis and impaired microbial fermentation capacity. Soluble fiber improved VFA production. Dietary fiber and carbohydrases altered microbiota composition to maintain or restore microbial homeostasis. [ABSTRACT FROM AUTHOR]

Severe combined immunodeficiency (SCID) is described as the lack of functional T and B cells. In some cases, mutant genes encoding proteins involved in the process of VDJ recombination retain partial activity and are classified as hypomorphs. Hypomorphic activity in the products from these genes can function in the development of T and B cells and is referred to as a leaky phenotype in patients and animals diagnosed with SCID. We previously described two natural, single nucleotide variants in ARTEMIS (DCLR1EC) in a line of Yorkshire pigs that resulted in SCID. One allele contains a splice site mutation within intron 8 of the ARTEMIS gene (ART16), while the other mutation is within exon 10 that results in a premature stop codon (ART12). While initially characterized as SCID and lacking normal levels of circulating lymphoid cells, low levels of CD3ε+ cells can be detected in most SCID animals. Upon further assessment, we found that ART16/16 , and ART12/12 SCID pigs had abnormally small populations of CD3ε+ cells, but not CD79α+ cells, in circulation and lymph nodes. Newborn pigs (0 days of age) had CD3ε+ cells within lymph nodes prior to any environmental exposure. CD3ε+ cells in SCID pigs appeared to have a skewed CD4α+CD8α+CD8β− T helper memory phenotype. Additionally, in some pigs, rearranged VDJ joints were detected in lymph node cells as probed by PCR amplification of TCRδ V5 and J1 genomic loci, as well as TCRβ V20 and J1.1, providing molecular evidence of residual Artemis activity. We additionally confirmed that TCRα and TCRδ constant region transcripts were expressed in the thymic and lymph node tissues of SCID pigs; although the expression pattern was abnormal compared to carrier animals. The leaky phenotype is important to characterize, as SCID pigs are an important tool for biomedical research and this additional phenotype may need to be considered. The pig model also provides a relevant model for hypomorphic human SCID patients. [ABSTRACT FROM AUTHOR]

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T− B− NK− SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS) −/− genetic background. We confirmed ART −/− IL2RG −/ Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART −/− IL2RG −/ Y male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal ART −/− IL2RG −/ Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model. [ABSTRACT FROM AUTHOR]

Vaccination-induced Escherichia coli O157:H7-specific immune responses have been shown to reduce E. coli O157:H7 shedding in cattle. Although E. coli O157:H7 colonization is correlated with perturbations in intestinal microbial diversity, it is not yet known whether vaccination against E. coli O157:H7 could cause shifts in bovine intestinal microbiota. To understand the impact of E. coli O157:H7 vaccination and colonization on intestinal microbial diversity, cattle were vaccinated with two doses of different E. coli O157:H7 vaccine formulations. Six weeks post-vaccination, the two vaccinated groups (Vx-Ch) and one non-vaccinated group (NonVx-Ch) were orally challenged with E. coli O157:H7. Another group was neither vaccinated nor challenged (NonVx-NonCh). Fecal microbiota analysis over a 30-day period indicated a significant (FDR corrected, p <0.05) association of bacterial community structure with vaccination until E. coli O157:H7 challenge. Shannon diversity index and species richness were significantly lower in vaccinated compared to non-vaccinated groups after E. coli O157:H7 challenge (p < 0.05). The Firmicutes:Bacteroidetes ratio (p > 0.05) was not associated with vaccination but the relative abundance of Proteobacteria was significantly lower (p < 0.05) in vaccinated calves after E. coli O157:H7 challenge. Similarly, Vx-Ch calves had higher relative abundance of Paeniclostridium spp. and Christenellaceae R7 group while Campylobacter spp., and Sutterella spp. were more abundant in NonVx-Ch group post-E. coli O157:H7 challenge. Only Vx-Ch calves had significantly higher (p < 0.001) E. coli O157:H7-specific serum IgG but no detectable E. coli O157:H7-specific IgA. However, E. coli O157:H7-specific IL-10-producing T cells were detected in vaccinated animals prior to challenge, but IFN-γ-producing T cells were not detected. Neither E. coli O157:H7-specific IgG nor IgA were detected in blood or feces, respectively, of NonVx-Ch and NonVx-NonCh groups prior to or post vaccinations. Both Vx-Ch and NonVx-Ch animals shed detectable levels of challenge strain during the course of the study. Despite the lack of protection with the vaccine formulations there were detectable shifts in the microbiota of vaccinated animals before and after challenge with E. coli O157:H7. [ABSTRACT FROM AUTHOR]

Background: It is unclear whether improving feed efficiency by selection for low residual feed intake (RFI) compromises pigs’ immunocompetence. Here, we aimed at investigating whether pig lines divergently selected for RFI had different inflammatory responses to lipopolysaccharide (LPS) exposure, regarding to clinical presentations and transcriptomic changes in peripheral blood cells. Results: LPS injection induced acute systemic inflammation in both the low-RFI and high-RFI line (n = 8 per line). At 4 h post injection (hpi), the low-RFI line had a significantly lower (p = 0.0075) mean rectal temperature compared to the high-RFI line. However, no significant differences in complete blood count or levels of several plasma cytokines were detected between the two lines. Profiling blood transcriptomes at 0, 2, 6, and 24 hpi by RNA-sequencing revealed that LPS induced dramatic transcriptional changes, with 6296 genes differentially expressed at at least one time point post injection relative to baseline in at least one line (n = 4 per line) (|log2(fold change)| ≥ log2(1.2); q < 0.05). Furthermore, applying the same cutoffs, we detected 334 genes differentially expressed between the two lines at at least one time point, including 33 genes differentially expressed between the two lines at baseline. But no significant line-by-time interaction effects were detected. Genes involved in protein translation, defense response, immune response, and signaling were enriched in different co-expression clusters of genes responsive to LPS stimulation. The two lines were largely similar in their peripheral blood transcriptomic responses to LPS stimulation at the pathway level, although the low-RFI line had a slightly lower level of inflammatory response than the high-RFI line from 2 to 6 hpi and a slightly higher level of inflammatory response than the high-RFI line at 24 hpi. Conclusions: The pig lines divergently selected for RFI had a largely similar response to LPS stimulation. However, the low-RFI line had a relatively lower-level, but longer-lasting, inflammatory response compared to the high-RFI line. Our results suggest selection for feed efficient pigs does not significantly compromise a pig’s acute systemic inflammatory response to LPS, although slight differences in intensity and duration may occur. [ABSTRACT FROM AUTHOR]

Interactions between diet, the microbiota, and the host set the ecological conditions in the gut and have broad implications for health. Prebiotics are dietary compounds that may shift conditions toward health by promoting the growth of beneficial microbes that produce metabolites capable of modulating host cells. This study's objective was to assess how a dietary prebiotic could impact host tissues via modulation of the intestinal microbiota. Pigs fed a diet amended with 5% resistant potato starch (RPS) exhibited alterations associated with gut health relative to swine fed an unamended control diet (CON). RPS intake increased abundances of anaerobic Clostridia in feces and several tissues, as well as intestinal concentrations of butyrate. Functional gene amplicons suggested bacteria similar to Anaerostipes hadrus were stimulated by RPS intake. The CON treatment exhibited increased abundances of several genera of Proteobacteria (which utilize respiratory metabolisms) in several intestinal locations. RPS intake increased the abundance of regulatory T cells in the cecum, but not periphery, and cecal immune status alterations were indicative of enhanced mucosal defenses. A network analysis of host and microbial changes in the cecum revealed that regulatory T cells positively correlated with butyrate concentration, luminal IgA concentration, expression of IL-6 and DEF1B, and several mucosa-associated bacterial taxa. Thus, the administration of RPS modulated the microbiota and host immune status, altering markers of cecal barrier function and immunological tolerance, and suggesting a reduced niche for bacterial respiration. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effects of a source of dietary soluble (SF) and insoluble fiber (IF) without or with exogenous carbohydrases (xylanase, β-glucanase, and pectinase) on diarrhea incidence, selected immune responses, and growth performance in enterotoxigenic Escherichia coli (ETEC)-challenged pigs. Sixty weaned pigs (6.9 ± 0.1 kg BW, ~23 d of age) were blocked by initial BW and placed in individual pens. Pens were randomly assigned to one of six treatments (n = 10 per treatment), including a nonchallenged control (NC), a positive challenge control (PC), the PC + a soluble fiber diet (10% sugar beet pulp) without (SF−) or with carbohydrases (SF+), and PC + an IF diet (15% corn distillers dried grains with solubles) without (IF−) or with carbohydrases (IF+). The control diet was primarily based on corn and soybean meal with 13.5% whey powder. The two sources of fiber were added at the expense of cornstarch in the control diet. Pigs were orally inoculated with 6 mL hemolytic F18 ETEC (~3.5 × 109 cfu/mL) or sham infected with 6 mL phosphate-buffered saline on day 7 (0 d postinoculation, dpi) postweaning. All ETEC challenged pigs were confirmed to be genetically susceptible to F18 ETEC. Pigs had free access to feed and water throughout the 14-d trial. Pig BW and feed intake were recorded on dpi − 7, 0, and 7 or 8. Fecal swabs were collected on dpi − 7, 0, 1, 2, 3, 5, and 7 or 8 to evaluate hemolytic E. coli shedding. Fecal score was visually ranked daily postchallenge to evaluate diarrhea incidence. Blood samples were collected on dpi − 1, 3, and 7 or 8 at necropsy and intestinal tissues were collected at necropsy. Pigs on PC had lower dpi 1 to 7 ADG and ADFI than those on NC (P < 0.05). Compared with PC pigs, SF+ pigs had greater ADG during both pre- and postchallenge period (P < 0.05). The IF − increased postchallenge diarrhea incidence compared with PC (P < 0.05). Pigs on SF − had lower ileal E. coli attachment than PC (P < 0.05). The SF+ reduced haptoglobin and IF+ reduced C-reactive protein on dpi 3 compared with PC (P < 0.05). Compared with PC pigs, SF+ pigs tended to have lower ileal tumor necrosis factor alpha and greater ileal occludin (OCLN) mRNA (P < 0.10) and had greater (P < 0.05) colonic OCLN mRNA levels. Collectively, IF − increased incidence of diarrhea and fecal E. coli shedding compared with PC. The SF+ pigs had improved growth compared with PC pigs, likely due in part to a reduction in inflammatory intermediates. [ABSTRACT FROM AUTHOR]

Highlights • Delivery of G-CSF to pigs via an adenovirus vector induced a sustained increase in circulating neutrophil numbers. • There was a longer mean survival time after Streptococcus suis challenge in pigs treated with G-CSF. • G-CSF may be a useful adjunct to antibiotics for prevention of Streptococcal disease in swine. Abstract The use of immunomodulators is a promising alternative to the use of antibiotics for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease. Previously we demonstrated a replication-defective adenovirus vector that expresses porcine granulocyte colony-stimulating factor (G-CSF) elicited a sustained neutrophilia, lasting nearly 3 weeks, which may be beneficial to prevent bacterial diseases during times of peak incidence. In a pilot study using the vectored G-CSF with a Caesarian-derived, colostrum-deprived (CDCD) pig model of Streptococcus suis disease, only 1 of 4 pigs given G-CSF developed disease, while 3 of 4 non-treated pigs developed Streptococcal disease. In a subsequent study using a larger number of pigs, although there was no difference in overall survival, there was a longer mean survival time in G-CSF treated pigs. S. suis infection is more severe in CDCD pigs than conventionally raised pigs, consequently results in the field may be superior to the ones reported in this study. Although there were positive effects from the use of G-CSF in this study, further research is needed to determine if improved clinical outcomes could be achieved under field conditions and whether the use of G-CSF in pigs to induce a sustained increase in circulating neutrophil numbers may be useful as an adjunct to antibiotics to diminish the severity of Streptococcal disease, especially during times of stress and pathogen exposure such as post-weaning. [ABSTRACT FROM AUTHOR]

Although the impact of carbohydrases on performance and nutrient utilization has been well studied, their effects on immune status and intestinal microbiota are less known in pigs. This study aimed to evaluate the impact of xylanase (X) and a carbohydrase enzyme blend (EB ; cellulase, ß-glucanase, and xylanase) on the immune profile of the intestine and peripheral system as well as intestinal microbes and microbial metabolites of weaned pigs fed higher fiber diets. Pigs (n = 460; 6.43 ± 0.06 kg BW; F25 × 6.0 Genetiporc) were blocked by initial BW. Pens (n = 48; 12 per treatment; 9 or 10 pigs per pen) were randomly assigned to 1 of 4 dietary treatments, including a higher fiber control diet (CON) and the CON supplemented with 0.01% X, 0.01% EB, or both enzymes (X + EB), arranged in a 2 × 2 factorial. The diets were based on corn, soybean meal, corn distillers dried grains with solubles, and wheat middlings. After 7-d adaptation to the environment, pigs were fed experimental diets ad libitum for 28 d. Blood samples were collected from the same pig within each pen on days 0, 7, 14, and 28. Intestinal tissues and digesta were collected on day 28. Bacteria 16S rRNA gene copy numbers were quantified using qPCR. The mRNA levels of colonic IL-17 , occludin (OCLN), and claudin 3 (CLDN3) were greater in pigs fed diets with X + EB, but not X or EB, compared with those fed CON (P < 0.05). The EB in the diet reduced plasma IL-8 over the 28-d trial compared with diets without EB (P < 0.05). There was an X × EB interaction on plasma tumor necrosis factor α and IL-1ß (P < 0.05); their levels were decreased when X and EB were added together, but not individually, compared with CON. The EB decreased cecal propionate, butyrate, and total volatile fatty acids (P < 0.05). Pigs fed X had lower ileal Lactobacillus and greater ileal and cecal Enterobacteriaceae compared with those fed unsupplemented diets (P < 0.05). The EB decreased Lactobacillus (P < 0.05) and tended to decrease (P = 0.065) Enterobacteriaceae in the colon compared with diets without EB. In conclusion, the addition of X and EB together decreased systemic markers of immune activation, potentially diverting energy and nutrients towards growth. The EB reduced colonic Lactobacillus and cecal total volatile fatty acids, probably due to improved prececal fiber and starch degradation and thus reduced substrate availability in the large intestine. These data corroborated previously observed enhanced growth in pigs fed EB-supplemented diets. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effects of dietary xylanase (X) and a carbohydrase enzyme blend (EB: cellulase, β-glucanase, and xylanase) on nutrient digestibility, intestinal barrier integrity, inflammatory status, and growth performance in weaned piglets fed higher fiber diets. A total of 460 pigs (6.43 ± 0.06 kg BW; F25 × 6.0 Genetiporc) were blocked by initial BW and pens (n = 12 per treatment) were randomly assigned to 1 of 4 dietary treatments. The diets included a higher fiber unsupplemented control diet (CON) and the CON supplemented with 0.01% X, 0.01% EB, or both enzymes, arranged in a 2 × 2 factorial. The diets were based on corn, soybean meal, corn distillers dried grains with solubles (DDGS), and wheat middlings. Pigs had 7 d to adapt to the environment and consumed the same commercial diet. Pigs were fed the experimental diets for 28 d with free access to feed and water. Body weight and feed disappearance were recorded weekly. One pig with BW closest to the pen average from each pen was selected and moved to metabolism crates on day 16 and intragastric gavaged a solution of lactulose and mannitol on day 22 followed by 12-h urine collection. Feces were collected from day 23 to 25. Intestinal tissues and mucosal scrapings were collected on day 28. Data were analyzed using PROC MIXED of SAS (9.4). Xylanase, EB, and their interaction were fixed effects and block was a random effect. The EB, but not X, increased pig BW and improved ADG over 28 d (P < 0.05). Neither carbohydrase impacted ADFI, G:F, or apparent total tract digestibility (ATTD) of DM, GE, or CP. The EB improved ATTD of ADF (32.45 vs. 26.57%; P < 0.01), but had no effect on NDF. Unexpectedly, X reduced ATTD of NDF and ADF (P < 0.01). The EB reduced urinary lactulose:mannitol and increased ileal claudin-3 mRNA abundance (P < 0.05), indicating improved small intestinal barrier integrity. There was a X × EB interaction on ileal secretory immunoglobulin A (sIgA) concentration (P < 0.05); in the absence of X, EB decreased sIgA compared to CON, but this effect disappeared in the presence of X. The EB also reduced ileal IL-22 mRNA abundance (P < 0.05), probably indicating decreased immune activation. In conclusion, EB but not X enhanced growth rate of weaned pigs fed higher fiber diets, which may be partly explained by the improved small intestinal barrier integrity and reduced immune activation, rather than improvement in nutrient digestibility. [ABSTRACT FROM AUTHOR]

Within the last decade there have been several severe combined immunodeficient (SCID) pig models discovered or genetically engineered. The animals have mutations in ARTEMIS, IL2RG , or RAG1/2 genes, or combinations thereof, providing SCID pigs with NK cells, but deficient in T and B cells, or deficient in NK, T, and B cells for research studies. Biocontainment facilities and positive pressure isolators are developed to limit pathogen exposure and prolong the life of SCID pigs. Raising SCID pigs in such facilities allows for completion of long-term studies such as xenotransplantation of human cells. Ectopically injected human cancer cell lines develop into tumors in SCID pigs, thus providing a human-sized in vivo model for evaluating imaging methods to improve cancer detection and therapeutic research and development. Immunocompromised pigs have the potential to be immunologically humanized by xenotransplantation with human hematopoietic stem cells, peripheral blood leukocytes, or fetal tissue. These cells can be introduced through various routes including injection into fetal liver or the intraperitoneal (IP) space, or into piglets by intravenous, IP, and intraosseous administration. The development and maintenance of transplanted human immune cells would be initially (at least) dependent on immune signaling from swine cells. Compared to mice, swine share higher homology in immune related genes with humans. We hypothesize that the SCID pig may be able to support improved engraftment and differentiation of a wide range of human immune cells as compared to equivalent mouse models. Humanization of SCID pigs would thus provide a valuable model system for researchers to study interactions between human tumor and human immune cells. Additionally, as the SCID pig model is further developed, it may be possible to develop patient-derived xenograft models for individualized therapy and drug testing. We thus theorize that the individualized therapeutic approach would be significantly improved with a humanized SCID pig due to similarities in size, metabolism, and physiology. In all, porcine SCID models have significant potential as an excellent preclinical animal model for therapeutic testing. [ABSTRACT FROM AUTHOR]

Intranasally administered live-attenuated influenza virus (LAIV) vaccines provide significant protection against heterologous influenza A virus (IAV) challenge. However, LAIV administration can modify the bacterial microbiota in the upper respiratory tract, including alterations in species that cause pneumonia. We sought to evaluate the effect of Bordetella bronchiseptica colonization on LAIV immunogenicity and efficacy in swine, and the impact of LAIV and IAV challenge on B. bronchiseptica colonization and disease. LAIV immunogenicity was not significantly impacted by B. bronchiseptica colonization, but protective efficacy against heterologous IAV challenge in the upper respiratory tract was impaired. Titers of IAV in the nose and trachea of pigs that received LAIV were significantly reduced when compared to non-vaccinated, challenged controls, regardless of B. bronchiseptica infection. Pneumonia scores were higher in pigs colonized with B. bronchiseptica and challenged with IAV, but this was regardless of LAIV vaccination status. While LAIV vaccination provided significant protection against heterologous IAV challenge, the protection was not sterilizing and IAV replicated in the respiratory tract of all LAIV vaccinated pig. The interaction between IAV, B. bronchiseptica , and host led to development of acute-type B. bronchiseptica lesions in the lung. Thus, the data presented do not negate the efficacy of LAIV vaccination, but instead indicate that controlling B. bronchiseptica colonization in swine could limit the negative interaction between IAV and Bordetella on swine health. [ABSTRACT FROM AUTHOR]

Type I interferons, such as interferon alpha (IFN-α), contribute to innate antiviral immunity by promoting production of antiviral mediators and are also involved in promoting an adaptive immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating and costly viruses to the swine industry world-wide and has been shown to induce a meager IFN-α response. Previously we administered porcine IFN-α using a replication-defective adenovirus vector (Ad5-IFN-α) at the time of challenge with virulent PRRSV and demonstrated an increase in the number of virus-specific IFNγ secreting cells, indicating that the presence of IFN-α at the time of infection can alter the adaptive immune responses to PRRSV. In the current experiment, we explored the use of IFN-α as an adjuvant administered with live-attenuated PRRSV vaccine as a method to enhance immune response to the vaccine. Unlike the previous studies with fully virulent virus, one injection of the Ad5-IFN-α abolished replication of the vaccine virus and as a result there was no detectible adaptive immune response. Although IFN-α did not have the desired adjuvant effect, the results further highlight the use of IFN-α as a treatment for PRRSV infection. [ABSTRACT FROM AUTHOR]

Background Predicting vaccine efficacy against emerging pathogen strains is a significant problem in human and animal vaccine design. T-cell epitope cross-conservation may play an important role in cross-strain vaccine efficacy. While influenza A virus ( IAV) hemagglutination inhibition ( HI) antibody titers are widely used to predict protective efficacy of 1 IAV vaccine against new strains, no similar correlate of protection has been identified for T-cell epitopes. Objective We developed a computational method (Epi CC) that facilitates pairwise comparison of protein sequences based on an immunological property-T-cell epitope content-rather than sequence identity, and evaluated its ability to classify swine IAV strain relatedness to estimate cross-protective potential of a vaccine strain for circulating viruses. Methods T-cell epitope relatedness scores were assessed for 23 IAV HA sequences representing the major H1 swine IAV phylo-clusters circulating in North American swine and HA sequences in a commercial inactivated vaccine (FluSure XP®). Scores were compared to experimental data from previous efficacy studies. Results Higher Epi CC scores were associated with greater protection by the vaccine against strains for 23 field IAV strain vaccine comparisons. A threshold for Epi CC relatedness associated with full or partial protection in the absence of cross-reactive HI antibodies was identified. Epi CC scores for field strains for which FluSure protective efficacy is not yet available were also calculated. Conclusion Epi CC thresholds can be evaluated for predictive accuracy of protection in future efficacy studies. Epi CC may also complement HI cross-reactivity and phylogeny for selection of influenza strains in vaccine development. [ABSTRACT FROM AUTHOR]

Alternatives to antibiotics to improve animal performance, limit the negative impact of infectious disease, and/or reduce colonization with foodborne pathogens is a major focus of animal agricultural research. β-glucans, a generally-recognized-as-safe (GRAS) product derived from various sources, are used in swine and can serve as both a prebiotic and/or stimulant of the immune system given the expression of β-glucan receptors on immune cells. When supplied in the diet of nursery pigs, it is unclear how dietary additives, particularly those known to modulate immune status, impact immunogenicity and efficacy of mucosal-delivered vaccines. Salmonellosis is one of the most common bacterial foodborne infections in the United States, and consumption of contaminated pork is a major source of human infection. Reduction of foodborne Salmonella in pigs via vaccination is one strategy to reduce contamination risk and subsequently reduce human disease. We examined the ability of dietary β-glucan to modulate fecal microbial diversity, and immunogenicity and efficacy of a mucosally-delivered, live-attenuated Salmonella vaccine during the nursery period. While dietaryβ-glucan did modulate fecal alpha diversity, it did not alter the induction of peripheral Salmonella -specific IFN-γ secreting Tcells or Salmonella -specific IgA in oral fluids. In addition, vaccination reduced Salmonella enterica serovar Typhimurium fecal shedding and tissue colonization. Overall, addition of β-glucan to the nursery diet of pigs impacted the microbiota but did not alter mucosal vaccine immunogenicity and efficacy. • Dietary, yeast-derived β-glucan modulates intestinal microbial diversity. • Oral Salmonella vaccine induces minimal peripheral immune response. • Mucosal Salmonella vaccination limits Salmonella shedding upon exposure. • Vaccine response not impacted by inclusion of yeast β-glucan in diet. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structures has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria. [ABSTRACT FROM AUTHOR]