Production and characterization of anti-porcine CXCL10 monoclonal antibodies.

Research on C-X-C motif chemokine ligand 10 (CXCL10) has been widely reported for humans and select animal species, yet immune reagents are limited for pig chemokines. Our goal is to provide veterinary immunologists and the biomedical community with new commercial immune reagents and standardized assays. Recombinant porcine CXCL10 (rPoCXCL10) protein was produced by yeast expression and used to generate a panel of α CXCL10 monoclonal antibodies (mAbs). All mAbs were assessed for cross-inhibition and reactivity to orthologous yeast expressed CXCL10 proteins. Characterization of a panel of nine α PoCXCL10 mAbs identified six distinct antigenic determinants. A sensitive quantitative sandwich ELISA was developed with anti-PoCXCL10-1.6 and -1.9 mAb; reactivity was verified with both rPoCXCL10 and native PoCXCL10, detected in supernatants of peripheral blood mononuclear cells stimulated with rPoIFNγ or PMA/Ionomycin. Immunostaining of in vitro rPoIFNγ stimulated pig spleen and blood cells verified CXCL10 + cells as CD3-CD4-CD172+, with occasional CD3-CD4 + CD172 + subsets. Comparison studies determined that α PoCXCL10-1.4 mAb was the ideal mAb clone for intracellular staining, whereas with α PoCXCL10-1.1 and -1.2 mAbs were best for immunohistochemistry analyses. These techniques and tools will be useful for evaluating swine immune development, responses to infectious diseases and vaccines, as well as for improving utility of pigs as an important biomedical model.

Genome wide association study of thyroid hormone levels following challenge with porcine reproductive and respiratory syndrome virus.

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory disease in piglets and reproductive disease in sows. Piglet and fetal serum thyroid hormone (i.e., T3 and T4) levels decrease rapidly in response to Porcine reproductive and respiratory syndrome virus infection. However, the genetic control of T3 and T4 levels during infection is not completely understood. Our objective was to estimate genetic parameters and identify quantitative trait loci (QTL) for absolute T3 and/or T4 levels of piglets and fetuses challenged with Porcine reproductive and respiratory syndrome virus. Methods: Sera from 5-week-old pigs (N = 1792) at 11 days post inoculation (DPI) with Porcine reproductive and respiratory syndrome virus were assayed for T3 levels (piglet_T3). Sera from fetuses (N = 1,267) at 12 or 21 days post maternal inoculation (DPMI) with Porcine reproductive and respiratory syndrome virus of sows (N = 145) in late gestation were assayed for T3 (fetal_T3) and T4 (fetal_T4) levels. Animals were genotyped using 60 K Illumina or 650 K Affymetrix single nucleotide polymorphism (SNP) panels. Heritabilities, phenotypic correlations, and genetic correlations were estimated using ASREML; genome wide association studies were performed for each trait separately using Julia for Whole-genome Analysis Software (JWAS). Results: All three traits were low to moderately heritable (10%-16%). Phenotypic and genetic correlations of piglet_T3 levels with weight gain (0-42 DPI) were 0.26 ± 0.03 and 0.67 ± 0.14, respectively. Nine significant quantitative trait loci were identified for piglet_T3, on Sus scrofa chromosomes (SSC) 3, 4, 5, 6, 7, 14, 15, and 17, and collectively explaining 30% of the genetic variation (GV), with the largest quantitative trait loci identified on SSC5, explaining 15% of the genetic variation. Three significant quantitative trait loci were identified for fetal_T3 on SSC1 and SSC4, which collectively explained 10% of the genetic variation. Five significant quantitative trait loci were identified for fetal_T4 on SSC1, 6, 10, 13, and 15, which collectively explained 14% of the genetic variation. Several putative immune-related candidate genes were identified, including CD247, IRF8, and MAPK8. Discussion: Thyroid hormone levels following Porcine reproductive and respiratory syndrome virus infection were heritable and had positive genetic correlations with growth rate. Multiple quantitative trait loci with moderate effects were identified for T3 and T4 levels during challenge with Porcine reproductive and respiratory syndrome virus and candidate genes were identified, including several immune-related genes. These results advance our understanding of growth effects of both piglet and fetal response to Porcine reproductive and respiratory syndrome virus infection, revealing factors associated with genomic control of host resilience.

Development and Characterization of New Monoclonal Antibodies Against Porcine Interleukin-17A and Interferon-Gamma.

Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.

The Natural Cytotoxicity Receptor NKp44 (NCR2, CD336) Is Expressed on the Majority of Porcine NK Cells Ex Vivo Without Stimulation.

Natural killer (NK) cells have been studied extensively in humans and mice for their vital role in the vertebrate innate immune system. They are known to rapidly eliminate tumors or virus infected cells in an immune response utilizing their lytic properties. The natural cytotoxicity receptors (NCRs) NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1) are important mediators of NK-cell cytotoxicity. NKp44 expression was reported for NK cells in humans as well as in some non-human primates and found exclusively on activated NK cells. Previously, no information was available on NKp44 protein expression and its role in porcine lymphocytes due to the lack of species-specific monoclonal antibodies (mAbs). For this study, porcine-specific anti-NKp44 mAbs were generated and their reactivity was tested on blood and tissue derived NK cells in pigs of different age classes. Interestingly, NKp44 expression was detected ex vivo already on resting NK cells; moreover, the frequency of NKp44+ NK cells was higher than that of NKp46+ NK cells in most animals analyzed. Upon in vitro stimulation with IL-2 or IL-15, the frequency of NKp44+ NK cells, as well as the intensity of NKp44 expression at the single cell level, were increased. Since little is known about swine NK cells, the generation of a mAb (clone 54-1) against NKp44 will greatly aid in elucidating the mechanisms underlying the differentiation, functionality, and activation of porcine NK cells.

Future of biomedical, agricultural, and biological systems research using domesticated animals.

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.

Importance of the pig as a human biomedical model.

Pigs have substantial potential as biomedical models for studying human developmental processes, congenital diseases, and pathogen response mechanisms in addition to utility as xenotransplant organ donors and tools for vaccine and drug design. The similarity of pigs to humans in anatomical size and structure, physiology, immunology, and genome enhances their potential as models for humans. Hence, it is imperative that research is relevant and reproducible in animal models that more closely resemble humans, such as the pig. This review summarizes the current status of pigs as an investigative model for humans and highlights their future applications.

Thyroid hormone suppression in feeder pigs following polymicrobial or porcine reproductive and respiratory syndrome virus-2 challenge.

Thyroid hormones are powerful regulators of growth, development, and basal metabolic rate and can be dysregulated under conditions of severe stress or illness. To understand the role of these hormones in porcine disease response, serum samples were obtained from three batches of nursery-aged pigs (n = 208) exposed to a natural polymicrobial disease challenge with an array of bacterial and viral pathogens. Levels of total thyroxin (T4) and triiodothyronine (T3) assessed in sera by radioimmunoassay, decreased significantly by 14 days post-exposure (DPE). Levels of T3 partially rebounded by 48 DPE, while T4 levels remain depressed. Post-exposure T3 and T4 levels were positively correlated with acute and long-term average daily gain (ADG). Cross-sectional sampling of animals maintained at the high health source farms, showed no equivalent change in either hormone when managed under standard industrial conditions. To further elucidate the effect of porcine reproductive and respiratory syndrome virus (PRRSV)-infection on thyroid hormone levels, archived sera over 42 days post inoculation (DPI) from nursery pigs (N = 190) challenged with one of two PRRSV2 strains by the PRRS Host Genetics Consortium were similarly assessed, with animals selected in a two-by-two design, to investigate biological extremes in ADG and viral load (VL). All animals showed a similar decrease in both thyroid hormones reaching a minimum at 7 DPI and returning to near pre-challenge levels by 42 DPI. Post-challenge T3 and T4 levels were significantly greater in high ADG groups, with no significant association with VL or strain. The results of this study demonstrate porcine susceptibility to thyroid disruption in response to disease challenge and demonstrate a relationship between this response and growth performance.

Associations of natural variation in the CD163 and other candidate genes on host response of nursery pigs to porcine reproductive and respiratory syndrome virus infection.

Pigs with complete resistance to porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) have been produced by genetically knocking out the CD163 gene that encodes a receptor of the PRRSV for entry into macrophages. The objectives of this study were to evaluate associations of naturally occurring single nucleotide polymorphisms (SNPs) in the CD163 gene and in three other candidate genes (CD169, RGS16, and TRAF1) with host response to PRRSV-only infection and to PRRS vaccination and PRRSV/porcine circovirus 2b (PCV2b) coinfection. SNPs in the CD163 gene were not included on SNP genotyping panels that were used for previous genome-wide association analyses of these data. An additional objective was to identify the potential genetic interaction of variants at these four candidate genes with a mutation in the GBP5 gene that was previously identified to be associated with host response to PRRSV infection. Finally, the association of SNPs with expression level of the nearby gene was tested. Several SNPs in the CD163, CD169, and RGS16 genes were significantly associated with host response under PRRSV-only and/or PRRSV/PCV2b coinfection. The effects of all SNPs that were significant in the PRRSV-only infection trials depend on genetic background. The effects of some SNPs in the CD163, CD169, and RGS16 genes depend on genotype at the putative causative mutation in the GBP5 gene, which indicates a potential biological interaction of these genes with GBP5. In addition, genome-wide association results for the PRRSV-only infection trials revealed that SNPs located in the CDK5RAP2 or MEGF9 genes, near the TRAF1 gene, had suggestive effects on PRRS viral load, which indicates that these SNPs might contribute to PRRSV neuropathogenesis. In conclusion, natural genetic variants in the CD163, CD169, and RGS16 genes are associated with resistance to PRRSV and/or PCV2b infection and appear to interact with the resistance quantitative trait locus in the GBP5 gene. The identified SNPs can be used to select for increased natural resistance to PRRSV and/or PRRSV-PCV2b coinfection.

Reference Transcriptomes of Porcine Peripheral Immune Cells Created Through Bulk and Single-Cell RNA Sequencing.

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 αß 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.

Gene expression in tonsils in swine following infection with porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a threat to pig production worldwide. Our objective was to understand mechanisms of persistence of PRRS virus (PRRSV) in tonsil. Transcriptome data from tonsil samples collected at 42 days post infection (dpi) were generated by RNA-seq and NanoString on 51 pigs that were selected to contrast the two PRRSV isolates used, NVSL and KS06, high and low tonsil viral level at 42 dpi, and the favorable and unfavorable genotypes at a genetic marker (WUR) for the putative PRRSV resistance gene GBP5. RESULTS: The number of differentially expressed genes (DEGs) differed markedly between models with and without accounting for cell-type enrichments (CE) in the samples that were predicted from the RNA-seq data. This indicates that differences in cell composition in tissues that consist of multiple cell types, such as tonsil, can have a large impact on observed differences in gene expression. Based on both the NanoString and the RNA-seq data, KS06-infected pigs showed greater activation, or less inhibition, of immune response in tonsils at 42 dpi than NVSL-infected pigs, with and without accounting for CE. This suggests that the NVSL virus may be better than the KS06 virus at evading host immune response and persists in tonsils by weakening, or preventing, host immune responses. Pigs with high viral levels showed larger CE of immune cells than low viral level pigs, potentially to trigger stronger immune responses. Presence of high tonsil virus was associated with a stronger immune response, especially innate immune response through interferon signaling, but these differences were not significant when accounting for CE. Genotype at WUR was associated with different effects on immune response in tonsils of pigs during the persistence stage, depending on viral isolate and tonsil viral level. CONCLUSIONS: Results of this study provide insights into the effects of PRRSV isolate, tonsil viral level, and WUR genotype on host immune response and into potential mechanisms of PRRSV persistence in tonsils that could be targeted to improve strategies to reduce viral rebreaks. Finally, to understand transcriptome responses in tissues that consist of multiple cell types, it is important to consider differences in cell composition.

Differential responses in placenta and fetal thymus at 12 days post infection elucidate mechanisms of viral level and fetal compromise following PRRSV2 infection.

BACKGROUND: A pregnant gilt infected with porcine reproductive and respiratory syndrome virus (PRRSV) can transmit the virus to her fetuses across the maternal-fetal-interface resulting in varying disease outcomes. However, the mechanisms leading to variation in fetal outcome in response to PRRSV infection are not fully understood. Our objective was to assess targeted immune-related gene expression patterns and pathways in the placenta and fetal thymus to elucidate the molecular mechanisms involved in the resistance/tolerance and susceptibility of fetuses to PRRSV2 infection. Fetuses were grouped by preservation status and PRRS viral load (VL): mock infected control (CTRL), no virus detected (UNINF), virus detected in the placenta only with viable (PLCO-VIA) or meconium-stained fetus (PLCO-MEC), low VL with viable (LVL-VIA) or meconium-stained fetus (LVL-MEC), and high VL with viable (HVL-VIA) or meconium-stained fetus (HVL-MEC). RESULTS: The host immune response was initiated only in fetuses with detectable levels of PRRSV. No differentially expressed genes (DEG) in either the placenta or thymus were identified in UNINF, PLCO-VIA, and PLCO-MEC when compared to CTRL fetuses. Upon fetal infection, a set of core responsive IFN-inducible genes (CXCL10, IFIH1, IFIT1, IFIT3, ISG15, and MX1) were strongly upregulated in both tissues. Gene expression in the thymus is a better differentiator of fetal VL; the strong downregulation of several innate and adaptive immune pathways (e.g., B Cell Development) are indicative of HVL. Gene expression in the placenta may be a better differentiator of fetal demise than the thymus, based-on principle component analysis clustering, gene expression patterns, and dysregulation of the Apoptosis and Ubiquitination pathways. CONCLUSION: Our data supports the concept that fetal outcome in response to PRRSV2 infection is determined by fetal, and more significantly placental response, which is initiated only after fetal infection. This conceptual model represents a significant step forward in understanding the mechanisms underpinning fetal susceptibility to the virus.

Neonatal and infant immunity for tuberculosis vaccine development: importance of age-matched animal models.

Neonatal and infant immunity differs from that of adults in both the innate and adaptive arms, which are critical contributors to immune-mediated clearance of infection and memory responses elicited during vaccination. The tuberculosis (TB) research community has openly admitted to a vacuum of knowledge about neonatal and infant immune responses to Mycobacterium tuberculosis (Mtb) infection, especially in the functional and phenotypic attributes of memory T cell responses elicited by the only available vaccine for TB, the Bacillus Calmette-Guérin (BCG) vaccine. Although BCG vaccination has variable efficacy in preventing pulmonary TB during adolescence and adulthood, 80% of endemic TB countries still administer BCG at birth because it has a good safety profile and protects children from severe forms of TB. As such, new vaccines must work in conjunction with BCG at birth and, thus, it is essential to understand how BCG shapes the immune system during the first months of life. However, many aspects of the neonatal and infant immune response elicited by vaccination with BCG remain unknown, as only a handful of studies have followed BCG responses in infants. Furthermore, most animal models currently used to study TB vaccine candidates rely on adult-aged animals. This presents unique challenges when transitioning to human trials in neonates or infants. In this Review, we focus on vaccine development in the field of TB and compare the relative utility of animal models used thus far to study neonatal and infant immunity. We encourage the development of neonatal animal models for TB, especially the use of pigs.

The Veterinary Immunological Toolbox: Past, Present, and Future.

It is well-recognized that research capability in veterinary species is restricted by a lack of immunological reagents relative to the extensive toolboxes for small rodent biomedical model species and humans. This creates a barrier to the strategic development of disease control solutions for livestock, companion animals and wildlife that not only affects animal health but can affect human health by increasing the risk of transmission of zoonotic pathogens. There have been a number of projects aimed at reducing the capability gaps in the veterinary immunological toolbox, the majority of these focusing on livestock species. Various approaches have been taken to veterinary immunological reagent development across the globe and technological advances in molecular biology and protein biochemistry have accelerated toolbox development. While short-term funding initiatives can address specific gaps in capability, they do not account for long-term sustainability of reagents and databases that requires a different funding model. We review the past, present and future of the veterinary immunological toolbox with specific reference to recent developments discussed at the International Union of Immunological Societies (IUIS) Veterinary Immunology Committee (VIC) Immune Toolkit Workshop at the 12th International Veterinary Immunology Symposium (IVIS) in Seattle, USA, 16-19 August 2019. The future availability of these reagents is critical to research for improving animal health, responses to infectious pathogens and vaccine design as well as for important analyses of zoonotic pathogens and the animal /human interface for One Health initiatives.

Porcine cytokines, chemokines and growth factors: 2019 update.

Pigs are a major food source worldwide as well as major biomedical models for human physiology and therapeutics. A thorough understanding of porcine immunity is essential to prevent and treat infectious diseases, and develop effective vaccines and therapeutics. The use of pigs as biomedical models is dependent on the growing molecular and immune toolbox. This paper summarizes current knowledge of swine cytokines, chemokines and growth factors, identifying 289 pig proteins, characterizing knowledge of their gene structures and families. It identifies areas in the current swine genome build that need to be clarified. A broad-based literature and vendor search was conducted to identify defined sets of monoclonal and polyclonal antibodies reacting with porcine cytokines, chemokines, growth factors along with availability of cloned recombinant proteins and assays for their quantitation. This process identified numerous reagents that are reportedly reactive with 170 pig cytokines, chemokines, growth factors: 118 have at least one commercial antibody reagent, 66 a cloned recombinant peptide, and 97 with quantitative assays. This affirms the great need to develop and characterize additional reagents. There are panels of reagents for numerous high priority targets that have been essential reagents for characterizing porcine immunity, disease and vaccine responses, and factors regulating development of innate immune responses, polarized macrophages and lymphoid cells including T regulatory cells. Yet there are many areas requiring investment of efforts to more effectively explore the pig immune system. The development of more reagents to understand the complex of cytokines, chemokines, and growth factors will clearly advance these initiatives.

The NC229 multi-station research consortium on emerging viral diseases of swine: Solving stakeholder problems through innovative science and research.

The NC229 research consortium was created in 1999 in response to the emergence of porcine reproductive and respiratory syndrome virus (PRRSV), a viral agent responsible for devastating economic losses to the swine industry. The project follows the traditional "consortium" approach for Multistate Agricultural Research driven through the US State Agricultural Experiment Stations (SAES), wherein stakeholder-driven needs to combat swine infectious diseases are identified and scientific solutions pursued by combining funds from federal, state, commodity groups, and the animal health industry. The NC229 consortium was the main driving force in successfully competing for a USDA multi-station Coordinated Agricultural Project (PRRS CAP-I) in 2004-2008, immediately followed by a renewal for 2010-2014 (PRRS CAP-II)-, resulting in an overall record achievement of almost $10 million dollars. The CAP funding was not only useful for quality research, extension, and education in PRRS and related diseases, but also instrumental in enabling the group to leverage swine industry funding of more than $34 million dollars, distributed between creative research and extension on PRRS during the last 20 years. The North American/International PRRS Symposium, now recognized by the community as a highly effective platform for the exchange of basic research findings and fundamental translational technology, is directly derived from the NC229 consortium. Other significant offshoots from NC229 include the PHGC (PRRS Host Genomic Consortium), a platform for discoveries on the role of host genetics during PRRSV infection, since 2007. Since 2009, the NC229 consortium has expanded its collective research interests beyond PRRSV to include nine other emerging viral diseases of swine. In the current project (2019-2024), African Swine Fever Virus (ASFV) retains a central focus, with the goal of harnessing the group's expertise in promoting preparedness for the global control of ASFV.

Importance of the Major Histocompatibility Complex (Swine Leukocyte Antigen) in Swine Health and Biomedical Research.

In pigs, the major histocompatibility complex (MHC), or swine leukocyte antigen (SLA) complex, maps to Sus scrofa chromosome 7. It consists of three regions, the class I and class III regions mapping to 7p1.1 and the class II region mapping to 7q1.1. The swine MHC is divided by the centromere, which is unique among mammals studied to date. The SLA complexspans between 2.4 and 2.7 Mb, depending on haplotype, and encodes approximately 150 loci, with at least 120 genes predicted to be functional. Here we update the whole SLA complex based on the Sscrofa11.1 build and annotate the organization for all recognized SLA genes and their allelic sequences. We present SLA nomenclature and typing methods and discuss the expression of SLA proteins, as well as their role in antigen presentation and immune, disease, and vaccine responses. Finally, we explore the role of SLA genes in transplantation and xenotransplantation and their importance in swine biomedical models.

The effects of a globin blocker on the resolution of 3'mRNA sequencing data in porcine blood.

BACKGROUND: Gene expression profiling in blood is a potential source of biomarkers to evaluate or predict phenotypic differences between pigs but is expensive and inefficient because of the high abundance of globin mRNA in porcine blood. These limitations can be overcome by the use of QuantSeq 3'mRNA sequencing (QuantSeq) combined with a method to deplete or block the processing of globin mRNA prior to or during library construction. Here, we validated the effectiveness of QuantSeq using a novel specific globin blocker (GB) that is included in the library preparation step of QuantSeq. RESULTS: In data set 1, four concentrations of the GB were applied to RNA samples from two pigs. The GB significantly reduced the proportion of globin reads compared to non-GB (NGB) samples (P = 0.005) and increased the number of detectable non-globin genes. The highest evaluated concentration (C1) of the GB resulted in the largest reduction of globin reads compared to the NGB (from 56.4 to 10.1%). The second highest concentration C2, which showed very similar globin depletion rates (12%) as C1 but a better correlation of the expression of non-globin genes between NGB and GB (r = 0.98), allowed the expression of an additional 1295 non-globin genes to be detected, although 40 genes that were detected in the NGB sample (at a low level) were not present in the GB library. Concentration C2 was applied in the rest of the study. In data set 2, the distribution of the percentage of globin reads for NGB (n = 184) and GB (n = 189) samples clearly showed the effects of the GB on reducing globin reads, in particular for HBB, similar to results from data set 1. Data set 3 (n = 84) revealed that the proportion of globin reads that remained in GB samples was significantly and positively correlated with the reticulocyte count in the original blood sample (P < 0.001). CONCLUSIONS: The effect of the GB on reducing the proportion of globin reads in porcine blood QuantSeq was demonstrated in three data sets. In addition to increasing the efficiency of sequencing non-globin mRNA, the GB for QuantSeq has an advantage that it does not require an additional step prior to or during library creation. Therefore, the GB is a useful tool in the quantification of whole gene expression profiles in porcine blood.

Minipigs as a neonatal animal model for tuberculosis vaccine efficacy testing.

Many vaccines against childhood diseases are administered early after birth, but vaccine development studies frequently test efficacy in adult rather than in neonatal animal models. In countries with endemic tuberculosis (TB), Bacillus Calmette-Guerin (BCG) is administered as part of the neonatal vaccine regimen because it prevents against the disseminated form of TB in children, although it has variable efficacy against pulmonary TB. Several promising new vaccines against TB are currently being tested in adult animal models. Here we evaluated neonatal piglets as an animal model to test vaccine efficacy. For this purpose, minipigs were vaccinated or not with BCG 48 h after birth and their immune response followed longitudinally until adolescence. We characterized the memory and activation phenotype of T cells, cytokine profile, and monocyte activation in response to BCG stimulation from 4 weeks of age into adolescence- age of 24 weeks. Immunological responses in vaccinated and non-vaccinated animals were further monitored upon infection with a low dose exposure to Mycobacterium tuberculosis strain HN878 via the aerosol route. Comparing the immunological response elicited by BCG vaccination in minipigs vs similar studies in infants, suggest that minipigs have the potential to serve as an effective neonatal animal model for vaccine development.

Identification of factors associated with virus level in tonsils of pigs experimentally infected with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important global swine diseases from both an economic and animal welfare standpoint. PRRS has plagued the US swine industry for over 25 yr, and containment of PRRS virus (PRRSV) has been unsuccessful to date. The primary phase of PRRS, tracked by serum viremia, typically clears between 21 and 42 d postinfection (dpi) but tonsils are a main site of PRRSV persistence and PRRSV can be detected in tonsils in excess of 150 dpi. Measuring tonsil virus (TV) levels at late stages of infection (6 to 7 wk postinfection) can be used to assess tonsil persistence, as levels of virus in tonsil at this time likely influence how long the virus will remain in the tissue. TV levels were measured on pigs experimentally infected with either the NVSL-97-7895 (NVSL; n = 524) or KS-2006-72109 (KS06; n = 328) PRRSV type 2 isolates across five trials. The objectives of this study were to (i) estimate the heritability of TV levels at 35 or 42 dpi; (ii) identify factors the affect TV level, including serum viremia; (iii) identify genomic regions associated with TV level; and (iv) compare results for the two PRRSV isolates. TV level was lowly heritable for both isolates (NVSL: 0.05 ± 0.06; KS06: 0.11 ± 0.10). Level of TV was phenotypically associated with traits related to viral clearance from serum: pigs with low TV levels had an earlier and faster rate of maximal serum viral clearance, lower total serum viral load, and lower viremia level at 35 or 42 dpi. Although no genomic regions with major effects on TV level were identified, several showed some association (>0.1% of total genetic variance in the NVSL-infected dataset, the KS06-infected dataset, and the combined dataset). These regions contained the genes CCL1, CCL2, CCL8, HS3ST3B1, GALNT10, TCF7, C1QA/B/C, HPSE, G0S2, and CD34, which are involved in viral infiltration or replication, immune cell migration, and viral clearance from tissue. Results were similar between the two PRRSV isolates. In conclusion, selection for viral clearance traits in serum may reduce PRRSV persistence in the tonsil across PRRSV isolates. However, genetic correlations need to be estimated to determine whether this will be successful.

A Vision for Development and Utilization of High-Throughput Phenotyping and Big Data Analytics in Livestock.

Automated high-throughput phenotyping with sensors, imaging, and other on-farm technologies has resulted in a flood of data that are largely under-utilized. Drastic cost reductions in sequencing and other omics technology have also facilitated the ability for deep phenotyping of livestock at the molecular level. These advances have brought the animal sciences to a cross-roads in data science where increased training is needed to manage, record, and analyze data to generate knowledge and advances in Agriscience related disciplines. This paper describes the opportunities and challenges in using high-throughput phenotyping, "big data," analytics, and related technologies in the livestock industry based on discussions at the Livestock High-Throughput Phenotyping and Big Data Analytics meeting, held in November 2017 (see: https://www.animalgenome.org/bioinfo/community/workshops/2017/). Critical needs for investments in infrastructure for people (e.g., "big data" training), data (e.g., data transfer, management, and analytics), and technology (e.g., development of low cost sensors) were defined by this group. Though some subgroups of animal science have extensive experience in predictive modeling, cross-training in computer science, statistics, and related disciplines are needed to use big data for diverse applications in the field. Extensive opportunities exist for public and private entities to harness big data to develop valuable research knowledge and products to the benefit of society under the increased demands for food in a rapidly growing population.