PRRSV-2 viral load in critical non-lymphoid tissues is associated with late gestation fetal compromise.

The impact of late gestation PRRSV-2 infection is highly variable within a litter, with a subset of fetuses displaying varying degrees of compromise following infection while others remain viable despite significant systemic viral load. To understand the underlying cause of this variation, we examined the susceptibility, distribution and impact of viral infection within non-lymphoid tissues. Samples of brain, heart, kidney, liver, lung, and skeletal muscle were obtained from fetuses of pregnant gilts at gestation day 86, and the presence and distribution of CD163+ cells within each tissue evaluated via immunohistofluorescence. Equivalent samples were collected from phenotypic extremes representing resistant, resilient and susceptible fetuses at 21 days following infection of pregnant gilts with PRRSV-2 at day 86 of gestation. Viral load and its impact in each tissue was evaluated by a combination of qPCR, in vitro viral recovery, and local expression of IFNG and CD163. Resting populations of CD163+ cells were observed in all six non-lymphoid tissues from healthy day 86 fetuses, though the apparent density and the morphology of positive cells varied between tissue. Viral RNA was detected in all six tissues derived from fetuses previously classified as highly infected, and infectious viral particles successfully recovered. Significantly more viral RNA was detected in heart, brain, lung and skeletal muscle of susceptible fetuses, relative to their viable counterparts. Infection was associated with an increase in the expression of CD163 in brain, kidney and lung. In addition, the presence of virus in each tissue coincided with a significant upregulation in the expression of IFNG, but the scale of this response was not associated with fetal susceptibility. Thus, PRRSV-2 is widely distributed across these susceptible non-lymphoid fetal tissues, and fetal outcome is associated with local viral load in critical fetal organs.

GWAS and genetic and phenotypic correlations of plasma metabolites with complete blood count traits in healthy young pigs reveal implications for pig immune response.

Introduction: In this study estimated genetic and phenotypic correlations between fifteen complete blood count (CBC) traits and thirty-three heritable plasma metabolites in young healthy nursery pigs. In addition, it provided an opportunity to identify candidate genes associated with variation in metabolite concentration and their potential association with immune response, disease resilience, and production traits. Methods: The blood samples were collected from healthy young pigs and Nuclear Magnetic Resonance (NMR) was used to quantify plasma metabolites. CBC was determined using the ADVIA® 2120i Hematology System. Genetic correlations of metabolite with CBC traits and single step genome-wide association study (ssGWAS) were estimated using the BLUPF90 programs. Results: Results showed low phenotypic correlation estimates between plasma metabolites and CBC traits. The highest phenotypic correlation was observed between lactic acid and plasma basophil concentration (0.36 ± 0.04; p < 0.05). Several significant genetic correlations were found between metabolites and CBC traits. The plasma concentration of proline was genetically positively correlated with hemoglobin concentration (0.94 ± 0.03; p < 0.05) and L-tyrosine was negatively correlated with mean corpuscular hemoglobin (MCH; -0.92 ± 0.74; p < 0.05). The genomic regions identified in this study only explained a small percentage of the genetic variance of metabolites levels that were genetically correlated with CBC, resilience, and production traits. Discussion: The results of this systems approach suggest that several plasma metabolite phenotypes are phenotypically and genetically correlated with CBC traits, suggesting that they may be potential genetic indicators of immune response following disease challenge. Genomic analysis revealed genes and pathways that might interact to modulate CBC, resilience, and production traits.

Heritability and genetic correlations of plasma metabolites of pigs with production, resilience and carcass traits under natural polymicrobial disease challenge.

Metabolites in plasma of healthy nursery pigs were quantified using nuclear magnetic resonance. Heritabilities of metabolite concentration were estimated along with their phenotypic and genetic correlations with performance, resilience, and carcass traits in growing pigs exposed to a natural polymicrobial disease challenge. Variance components were estimated by GBLUP. Heritability estimates were low to moderate (0.11 ± 0.08 to 0.19 ± 0.08) for 14 metabolites, moderate to high (0.22 ± 0.09 to 0.39 ± 0.08) for 17 metabolites, and highest for L-glutamic acid (0.41 ± 0.09) and hypoxanthine (0.42 ± 0.08). Phenotypic correlation estimates of plasma metabolites with performance and carcass traits were generally very low. Significant genetic correlation estimates with performance and carcass traits were found for several measures of growth and feed intake. Interestingly the plasma concentration of oxoglutarate was genetically negatively correlated with treatments received across the challenge nursery and finisher (- 0.49 ± 0.28; P < 0.05) and creatinine was positively correlated with mortality in the challenge nursery (0.85 ± 0.76; P < 0.05). These results suggest that some plasma metabolite phenotypes collected from healthy nursery pigs are moderately heritable and genetic correlations with measures of performance and resilience after disease challenge suggest they may be potential genetic indicators of disease resilience.

The porcine trophoblast cell line PTr2 is susceptible to porcine reproductive and respiratory syndrome virus-2 infection.

INTRODUCTION: Porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) breaches the maternal-fetal interface (MFI) to infect porcine fetuses, yet the exact mechanism(s) of transmission is not understood. The objective of this study was to determine the susceptibility of porcine trophoblast cell line (PTr2) to PRRSV-2 infection to understand the potential role of the trophoblast in viral transmission to fetuses in vivo. METHODS: PTr2 cells were exposed in vitro to PRRSV-2 and then subjected to immunofluorescence analysis (IF), flow cytometry (FCM), real-time quantitative PCR (RT-qPCR), transmission electron microscopy (TEM) and immunogold electron microscopy (IEM) to assess viral infection. The effects of PRRSV-2 on PTr2 cell cycle progression and apoptosis, as well as the ability of PTr2 cells to produce infectious viral particles were also examined. RESULTS: PRRSV-2 was readily detected in PTr2 cells by IF, FCM, RT-qPCR, TEM and IEM techniques. RT-qPCR and FCM results of a time course of infection of PTr2 cells indicated PRRSV-2 load decreased over time after initial infection up to 72 h. PRRSV-2 infection altered PTr2 cell cycle with a selective increase of cells within the G2/M phase and also induced apoptosis. TEM and IEM demonstrated PRRSV-2 within and on the surface of PTr2 cells and PRRSV-2 virions released from PTr2 cells infected naïve MARC-145 cells inducing cytopathic effects. DISCUSSION: Trophoblast cells are susceptible to PRRSV-2 infection and release live virions capable of inducing cytopathic effects in naïve cells. This suggests a possible mechanism by which PRRSV-2 can breach the MFI resulting in fetal infection and death.

Spatiotemporal immunofluorescent evaluation of porcine reproductive and respiratory syndrome virus transmission across the maternal-fetal interface.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes severe reproductive failure characterized by high fetal morbidity and mortality leading to substantial economic losses to the swine industry. Evaluation of spatiotemporal transmission of PRRSV at the maternal-fetal interface (MFI) is critical for understanding fetal infection. Localization of PRRSV-2 strain NVSL 97-7895 at different regions of the MFI in 20 pregnant gilts at 2, 5, 8, 12 and 14 days post-inoculation (dpi) were analyzed by immunofluorescence (IF). Samples of MFI were collected from 15 inoculated and 5 control gilts and transplacental PRRSV transmission assessed in randomly selected fetuses from each litter. Localization of NVSL 97-7895 antigen immunoreactivity in the MFI was focused in three major areas: endometrial connective tissues (ENDO), the feto-maternal junction (FMJ) and fetal placenta (PLC). NVSL 97-7895 was detected at the FMJ by 2 dpi. At 2, 5 and 8 dpi, NVSL 97-7895 was localized within the ENDO and FMJ, whereas at 12 and 14 dpi, it was mainly localized in the PLC. Using a novel IF strategy for counting and size sorting NVSL 97-7895 viral antigen in situ, results of this study indicate that non-cell-associated mechanisms are involved in PRRSV transmission across the MFI.

Low Altitude Solar Magnetic Reconnection, Type III Solar Radio Bursts, and X-ray Emissions.

Type III solar radio bursts are the Sun's most intense and frequent nonthermal radio emissions. They involve two critical problems in astrophysics, plasma physics, and space physics: how collective processes produce nonthermal radiation and how magnetic reconnection occurs and changes magnetic energy into kinetic energy. Here magnetic reconnection events are identified definitively in Solar Dynamics Observatory UV-EUV data, with strong upward and downward pairs of jets, current sheets, and cusp-like geometries on top of time-varying magnetic loops, and strong outflows along pairs of open magnetic field lines. Type III bursts imaged by the Murchison Widefield Array and detected by the Learmonth radiospectrograph and STEREO B spacecraft are demonstrated to be in very good temporal and spatial coincidence with specific reconnection events and with bursts of X-rays detected by the RHESSI spacecraft. The reconnection sites are low, near heights of 5-10 Mm. These images and event timings provide the long-desired direct evidence that semi-relativistic electrons energized in magnetic reconnection regions produce type III radio bursts. Not all the observed reconnection events produce X-ray events or coronal or interplanetary type III bursts; thus different special conditions exist for electrons leaving reconnection regions to produce observable radio, EUV, UV, and X-ray bursts.

Genomic investigation of piglet resilience following porcine epidemic diarrhea outbreaks.

Porcine epidemic diarrhea virus (PEDV) belongs to the Coronaviridae family and causes malabsorptive watery diarrhea, vomiting, dehydration and imbalanced blood electrolytes in pigs. Since the 1970s, PED outbreaks have become a source of problems in pig producing countries all over the world, causing large economic losses for pig producers. Although the infection in adults is not fatal, in naïve suckling piglets mortality is close to 100%. In this study, we investigated genome-wide differences between dead and recovered suckling piglets from commercial farms after PED outbreaks. Samples from 262 animals (156 dead and 106 recovered) belonging to several commercial lines were collected from five different farms in three different countries (USA, Canada and Germany) and genotyped with the porcine 80K SNP chip. Mean Fst value was calculated in 1-Mb non-overlapping windows between dead and recovered individuals, and the results were normalized to find differences within the comparison. Seven windows with high divergence between dead and recovered were detected-five on chromosome 2, one on chromosome 4 and one on chromosome 15-in total encompassing 152 genes. Several of these genes are either under- or overexpressed in many virus infections, including Coronaviridae (such as SARS-CoV). A total of 32 genes are included in one or more Gene Ontology terms that can be related to PED development, such as Golgi apparatus, as well as mechanisms generally linked to resilience or diarrhea development (cell proliferation, ion transport, ATPase activity). Taken together this information provides a first genomic picture of PEDV resilience in suckling piglets.

Pathological features and proposed diagnostic criteria of porcine periweaning failure-to-thrive syndrome.

Porcine periweaning failure-to-thrive syndrome (PFTS) is a clinical syndrome characterized by anorexia and progressive debilitation of newly weaned pigs. The objectives of the current case-control study were to describe the histopathologic features of PFTS in North America and test for selected pathogens in case and control pigs on 8 farms allegedly fulfilling the clinical definition of PFTS. Based on observations during farm visits, 5 farms fully met the case definition (PFTS farms), whereas 3 farms only partially fulfilled the definition (NON-PFTS farms). Necropsy and histopathologic examination were performed on case (n = 8 or 9) and control (n = 4) pigs from each farm. Superficial gastritis, which was mainly localized in the fundus and characterized by attenuation of superficial foveolar cells, was significantly more frequent in case pigs from PFTS farms compared with all the other pigs (odds ratio [OR], 16.7). The same was found for thymic atrophy (OR, 30.1) and small intestinal (SI) villous atrophy in the duodenum (OR, 28.7), jejunum (OR, 67.4), and ileum (OR, 56.3). All pigs with PFTS had at least 2 of these 3 lesions: gastritis, thymic atrophy, and SI villous atrophy. PFTS was not associated with any relevant porcine pathogen tested. We propose the diagnosis of PFTS be based on the fulfillment of the clinical case definition, the presence of the above lesions, and exclusion of other common swine diseases and pathogens. However, PFTS can be ruled out if debilitated pigs do not have at least 2 of the above 3 lesions.

Genetic analysis of reproductive traits and antibody response in a PRRS outbreak herd.

Porcine reproductive and respiratory syndrome (PRRS) is the most economically significant disease impacting pig production in North America, Europe, and Asia, causing reproductive losses such as increased rates of stillbirth and mummified piglets. The objective of this study was to explore the genetic basis of host response to the PRRS virus (PRRSV) in a commercial multiplier sow herd before and after a PRRS outbreak, using antibody response and reproductive traits. Reproductive data comprising number born alive (NBA), number alive at 24 h (NA24), number stillborn (NSB), number born mummified (NBM), proportion born dead (PBD), number born dead (NBD), number weaned (NW), and number of mortalities through weaning (MW) of 5,227 litters from 1,967 purebred Landrace sows were used along with a pedigree comprising 2,995 pigs. The PRRS outbreak date was estimated from rolling averages of farrowing traits and was used to split the data into a pre-PRRS phase and a PRRS phase. All 641 sows in the herd during the outbreak were blood sampled 46 d after the estimated outbreak date and were tested for anti-PRRSV IgG using ELISA (sample-to-positive [S/P] ratio). Genetic parameters of traits were estimated separately for the pre-PRRS and PRRS phase data sets. Sows were genotyped using the PorcineSNP60 BeadChip, and genome-wide association studies (GWAS) were performed using method Bayes B. Heritability estimates for reproductive traits ranged from 0.01 (NBM) to 0.12 (NSB) and from 0.01 (MW) to 0.12 (NBD) for the pre-PRRS and PRRS phases, respectively. S/P ratio had heritability (0.45) and strong genetic correlations with most traits, ranging from -0.72 (NBM) to 0.73 (NBA). In the pre-PRRS phase, regions associated with NSB and PBD explained 1.6% and 3% of the genetic variance, respectively. In the PRRS phase, regions associated with NBD, NSB, and S/P ratio explained 0.8%, 11%, and 50.6% of the genetic variance, respectively. For S/P ratio, 2 regions on SSC 7 (SSC7) separated by 100 Mb explained 40% of the genetic variation, including a region encompassing the major histocompatibility complex, which explained 25% of the genetic variance. These results indicate a significant genomic component associated with PRRSV antibody response and NSB in this data set. Also, the high heritability and genetic correlation estimates for S/P ratio during the PRRS phase suggest that S/P ratio could be used as an indicator of the impact of PRRS on reproductive traits.

Transcriptomic analysis identifies candidate genes and functional networks controlling the response of porcine peripheral blood mononuclear cells to mitogenic stimulation.

It is difficult to obtain phenotypic data on disease susceptibility directly from swine in an industry setting. The magnitude of the proliferative response of peripheral blood mononuclear cells (PBMC) to the T cell mitogen concanavalin A (Con A) has long been used as an indirect measure of the responsiveness of the immune system to antigenic stimulation. This trait is known to exhibit moderate heritability in swine, but little is known about the identity of the genes that control the response. In this study, we carried out a time-course microarray experiment to measure gene expression at 3 different stages (3, 20, and 68 h) poststimulation of PBMC with Con A. A total of 46, 452, and 418 differentially expressed (DifEx) genes were identified at each time point, respectively. Expression changes for a subset of these genes were subsequently confirmed by real-time PCR. Functional annotation analyses of the microarray results successfully identified sets of genes involved in processes associated with multiple aspects of cell division, such as DNA and protein synthesis, and control of mitosis. However, the discovery of genes that controlled the response of PBMC to mitogen was limited with this approach, because the drastic changes in the transcriptional program necessitated by cells undergoing division masked changes in smaller immune response gene sets. Pathway and network analyses that focused on immune cells proved to be a more effective strategy for the identification of genes that coordinate aspects of the mitogenic response that are specific to PBMC. The cytokine gene IL15 was shown to be central to the highest scoring network at 20 h and affect the expression of 16 other DifEx genes, including some genes known to regulate T cell activation, such as IL7R, JUN, TNFRSF9, and ZAP70. The IL15 gene maps to a previously identified QTL interval for immune responsiveness to Con A on SSC 8, which also contains the related IL2 gene. At 68 h, a distinct downregulation of major histocompatibility complex class II antigen presentation genes was observed. Overall, the gene expression profile of the Con A-stimulated porcine PBMC points to a Th(1) bias in immune activation. Further work is required to determine whether polymorphisms linked to genes identified in this study affect this immune response trait in pig populations and whether the trait itself correlates with decreased susceptibility to intracellular pathogens in swine.

Dual heterologous porcine circovirus genogroup 2a/2b infection induces severe disease in germ-free pigs.

Our primary objectives were to determine: the relative virulence of porcine circovirus (PCV) 2a and PCV2b, if heterologous infection induces severe illness, and the relative concentration of PCV2a and PCV2b in tissues of heterologously infected pigs. In experiment 1, 18 germ-free piglets served as controls or were infected with PCV2a or PCV2b. Half were immune stimulated with keyhole limpet hemocyanin (KLH) emulsified in incomplete Freund's adjuvant (2aKLH, 2bKLH). No piglets demonstrated severe illness. Lesion severity did not differ, but PCV2 capsid staining was more intense in 2a- than 2b-infected pigs (P<.05). In experiment 2, 20 germ-free piglets were dual inoculated 7 days apart with PCV2a and PCV2b (2a2b, 2b2a), PCV2b twice (2b2b), or PCV2a (2a2a) twice. Five of 9 heterologous-infected pigs developed severe illness. All heterologously infected pigs demonstrated ascites or edema, and 8/9 developed thymic atrophy. By contrast, 1 of 5 2b2b-infected pigs developed bronchopneumonia and pleural effusion. No 2a2a-infected pig developed illness. Gross lesions were more severe in heterologously infected pigs than in 2b2b pigs (P<.05), and were more severe in 2b2b than 2a2a pigs (P<.05). PCV2 capsid staining intensity did not differ by group. In heterologously infected pigs, higher levels of PCV2 DNA reflective of the first inoculum compared to the second were found in mesenteric lymph node (P=.04), spleen (P=.004) and liver (P=.04). These results indicate that dual heterologous PCV2a/2b inoculation 7 days apart may induce severe clinical illness, but PCV2a and PCV2b when administered singularly or in combination with KLH appear to be of equivalent virulence.

Prenatal programming of postnatal development in the pig.

Studies of low birth weight offspring have a long history in pig science. These pigs have reduced growth potential and poor carcass quality compared to their higher birth weight littermates. In contemporary commercial sows with between 10 and 15 total pigs born/litter, between-litter differences in average birth weight appear to make the largest contribution to variation in postnatal growth performance, independent of numbers born. Low birth weight is a characteristic of a subpopulation of these sows, likely as a consequence of an imbalance between ovulation rate and uterine capacity due to ongoing selection for litter size. Based on experimental studies, we hypothesize that increased crowding at day 30 of gestation primarily affects placental development and persistent negative impacts on placental growth then affect fetal development. However, embryonic myogenic gene expression is already affected at day 30. Latent effects of metabolic state on oocyte quality and early embryonic development have also been reported. In contrast to effects of uterine crowding, the embryo is primarily affected by previous catabolism. The large body of literature on gene imprinting, and the interactions between metabolism, nutrition, and methylation state, suggest that classic imprinting mechanisms may be involved. However, the potential use of genomics, epigenomics, nutrigenomics, and proteomics to investigate these mechanisms brings new demands on experimental design and data management that present a considerable challenge to the effectiveness of future research on prenatal programming in the pig.

The contribution of NF-kappa B activity to spontaneous proliferation and resistance to apoptosis in human T-cell leukemia virus type 1 Tax-induced tumors.

Human T-cell leukemia virus type I is the etiologic agent of adult T-cell leukemia/lymphoma. The Tax protein of this virus is thought to contribute to cellular transformation and tumor development. In this report, we have used a Tax transgenic mouse model of tumorigenesis to study the contribution of nuclear factor (NF)-kappa B activity to spontaneous tumor cell proliferation and resistance to apoptosis. We have demonstrated elevated expression levels of NF-kappa B--inducible cytokines, including interleukin (IL)-6, IL-10, IL-15, and interferon (IFN)-gamma, in freshly isolated primary tumors from Tax transgenic mice. Inhibitors of NF-kappa B activity, sodium salicylate and cyclopentenone prostaglandins (prostaglandin A(1) and 15-deoxy-Delta(12,14)-prostaglandin J(2)), blocked spontaneous proliferation of Tax transgenic mouse spleen cells. In addition, Tax-induced tumor cells, which are resistant to irradiation-induced apoptosis, became sensitive to apoptosis in the presence of sodium salicylate and prostaglandins. These results strongly suggest that Tax-mediated induction of NF-kappa B activity contributes to tumorigenesis in vivo. (Blood. 2001;98:1200-1208)

Analysis of p53 inactivation in a human T-cell leukemia virus type 1 Tax transgenic mouse model.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). The HTLV-1 Tax protein has been strongly linked to oncogenesis and is considered to be the transforming protein of this virus. A Tax transgenic mouse model was utilized to study the contribution of p53 inactivation to Tax-mediated tumorigenesis. These mice develop primary, peripheral tumors consisting of large granular lymphocytic (LGL) cells, which also infiltrate the lymph nodes, bone marrow, spleen, liver, and lungs. Primary Tax-induced tumors and tumor-derived cell lines exhibited functional inactivation of the p53 apoptotic pathway; such tumors and tumor cell lines were resistant to an apoptosis-inducing stimulus. In contrast, p53 mutations in tumors were found to be associated with secondary organ infiltration. Three of four identified mutations inhibited transactivation and apoptosis induction activities in vitro. Furthermore, experiments which involved mating Tax transgenic mice with p53-deficient mice demonstrated minimal acceleration in initial tumor formation, but significantly accelerated disease progression and death in mice heterozygous for p53. These studies suggest that functional inactivation of p53 by HTLV-1 Tax, whether by mutation or another mechanism, is not critical for initial tumor formation, but contributes to late-stage tumor progression.

Association of tumour necrosis factor and acute phase reactant changes with post arrival disease in swine.

Severe weight loss in the absence of respiratory, enteric or systemic clinical disease or gross pathologic lesions is often observed when immunologically naive boars are placed in conventional health swine facilities. Affected animals develop this weight loss in spite of receiving pre-entry vaccinations against common swine pathogens, such as Haemophilus parasuis or Mycoplasma hyopneumoniae. In many cases, the weight loss is non-responsive to long term antibiotic therapy. In order to determine the relationships between the severity of post arrival weight loss and disease and its potential immunological or physiological indicators, tumour necrosis factor (TNF) and acute phase reactant levels were correlated with the clinical status in immunologically naive boars following their transfer to a conventional facility. Boars had higher TNF (P < 0.0001) and plasma protein (P = 0.0054) levels and decreased zinc (P = 0.0004) levels during periods of clinical sickness. Likewise, peak and average plasma TNF, serum haptoglobin, and serum zinc were correlated indicating a prolonged stress or pathogenic insult (r = 0.89, P < 0.0001 for TNF; r = 0.67, P = 0.01 for haptoglobin; r = 0.73, P = 0.005 for zinc). An acute phase response, a systemic TNF increase and the development of a lymphopenia were observed in post arrival disease in swine. This is the first time cytokines and acute phase reactants have been investigated in a field study involving immunologically naive or high health swine.