Search

Sow immunity plays an important role in preventing viral infection and disease in newborn piglets. Vertical transmission of porcine circovirus type 2 (PCV2) may perpetuate porcine circovirus associated disease (PCVAD) in newborn and growing pigs. Hence, the immunological effects of maternal immunoglobulin transfer of PCV2-specific antibodies on PCV2 viremia and immune response in piglets in commercial swine herds were evaluated. Sow vaccination has been shown to reduce viral shedding and viremia, and increases the neutralizing antibody (NA) titers. Since NAs are important for control of PCVAD and mammary secretions may contain high anti-PCV2 NA levels, we examined the PCV2 NA levels in colostrum, milk, sow serum, and piglet serum over time to investigate an association between NA levels and protection against infection. NA titers were remarkably high (up to 10 -6 50% neutralizing titer) in sow serum and colostrum on all farms regardless of viremia levels. In piglets vaccinated at 3 weeks of age, NA titers peaked at 10 weeks of age and continued to maintain high viral neutralizing titers to slaughter. The impact of maternally derived neutralizing activity was most evident during the suckling period. Although PCV2 was transmitted from sows to piglets in colostrum, piglets were largely nonviremic at weaning. Thus, NAs appear to control or suppress initial infection even though they are unable to clear or prevent infection later in life.

In veterinary animal species, vaccines are the primary tool for disease prevention, a key tool for treatment of infection, and essential for helping maintain animal welfare and productivity. Traditional vaccine development by trial-and-error has achieved many successes. However, effective vaccines that provide solid cross-protective immunity with excellent safety are still needed for many diseases. The path to development of vaccines against difficult pathogens requires recognition of uniquely evolved immunological interactions of individual animal hosts and their specific pathogens. Here, general principles that currently guide veterinary immunology and vaccinology research are reviewed, with an emphasis on examples from swine. Advances in genomics and proteomics now provide the community with powerful tools for elucidation of regulatory and effector mechanisms of protective immunity that provide new opportunities for successful translation of immunological discoveries into safe and effective vaccines., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems., Results: The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome., Conclusions: This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig's adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

The TH1-TH2 paradigm provides an elegant model of directed response to infectious pathogens. Developed in the mouse, the model has provided a framework for systematic and mechanistic studies of immune regulation, protective immunity, and vaccine development in swine. Interleukin-4 (IL-4) plays a central role in the paradigm as a regulatory molecule directing development of the TH2 phenotype, as a developmental cytokine essential for antibody production, and as a soluble diagnostic marker of the TH2 cell type. In contrast, while characterizing the biological properties of porcine IL-4, we discovered that it was not a stimulatory factor for porcine B cells. Rather, it blocked antibody and IL-6 secretion and suppressed antigen-stimulated proliferation of B cells. Inhibition was not reversed by treatment with IL-2 and IL-6 treatment. IL-4 did not stimulate T lymphocyte proliferation, but induced cell growth in lymphoblasts in a dose-dependent fashion. These results suggest that IL-4 plays a different role in pigs than in mice and humans, in which it stimulates B cells and is essential for antibody production. Furthermore, the functions of IL-4 in swine cannot be inferred from results in model systems such as the mouse. General models of disease resistance show substantial variation between pigs and mice at the cellular and molecular level. Advances in somatic cell technologies and animal engineering to enable gene knockouts in pigs, in combination with a continuously expanding immunological toolkit, promise an exciting future for pig immunology, detailed mechanistic elucidation of the TH1-TH2 paradigm, and an improved understanding of the role of IL-4 in porcine immunity to infectious disease.

Cell-based diabetes therapy requires an abundant cell source. Here, we report reversal of diabetes for more than 100 d in cynomolgus macaques after intraportal transplantation of cultured islets from genetically unmodified pigs without Gal-specific antibody manipulation. Immunotherapy with CD25-specific and CD154-specific monoclonal antibodies, FTY720 (or tacrolimus), everolimus and leflunomide suppressed indirect activation of T cells, elicitation of non-Gal pig-specific IgG antibody, intragraft expression of proinflammatory cytokines and invasion of infiltrating mononuclear cells into islets.

Peyer's patches are organized lymphoid tissues of the small intestine that play a critical role in disease resistance and oral tolerance. Peyer's patches in the jejunum contain lymphocytes, dendritic cells, macrophages, villous epithelium, and specialized follicle-associated epithelium. Little is known about the mechanisms and processes by which cells of the Peyer's patches discriminate food nutrients and commensal microflora from pathogenic microbiota. We hypothesize that the jejunal Peyer's patches express genes that mediate and regulate its essential functions. Expression patterns of approximately 2600 cDNAs from a porcine Peyer's patch subtracted library were examined by microarray profiling. Individual mRNAs of interest were further examined by quantitative RT-PCR. Innate immunity-associated genes, including complement 3 and lysozyme, and the genes for epithelial chloride channel and trappin 1 were highly expressed by jejunal Peyer's patch in both juvenile and adult pigs. The growth- and apoptosis-associated genes CIDE-B, GW112, and PSP/Reg I (pancreatic stone protein or regenerating gene) were differentially expressed in juvenile pig Peyer's patches. Many sequences which were highly expressed in jejunal Peyer's patches have previously been described with functions in epithelial cells. Animal-to-animal variation in basal jejunal Peyer's patch gene expression was considerable and reflects the dynamic physiological environment of the gut in addition to genetic, epigenetic, and microbiological variation in the small intestine.

Background: We have previously shown that pig-to-primate intraportal islet xenografts reverse diabetes, escape hyperacute rejection, and undergo acute cellular rejection in non-immunosuppressed recipients. To gain a better understanding of mechanisms contributing to xenoislet rejection in non-human primates we examined gene expression in livers bearing islet xenografts in the first 72 h after transplantation., Methods: Liver specimens were collected at sacrifice from seven non-immunosuppressed rhesus macaques at 12, 24, 48 and 72 h after intraportal porcine islet transplantation. Following total RNA extraction, mRNA was quantified using SYBR green real-time reverse transcription polymerase chain reaction (RT-PCR) for species-specific immune response genes. Data were analyzed using comparative cycle threshold (Ct) analysis, adjusted for specific primer-efficiencies and normalized to cyclophilin expression., Results: Porcine insulin mRNA was detected in all liver samples. Cluster analysis revealed differential gene expression patterns at 12 and 24 h (early) compared with at 48 and 72 h (late) post-transplant. Gene expression patterns were associated with histological findings of predominantly neutrophils and only a few lymphocytes at 12 and 24 h and an increasing number of lymphocytes and macrophages at 48 and 72 h. Transcript levels of CXCR3 and its ligands, interferon-inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig), significantly increased between early and late time points together with expression of MIP-1alpha, regulated on activation normal T expressed and secreted protein (RANTES) and MCP-1. CCR5 showed only a marginal, non-significant increase. Fas ligand, perforin and granzyme B transcripts were all elevated at 48 and 72 h post-transplant., Conclusions: Our data suggest that CXCR3, with ligands IP-10 and Mig, is involved in T cell recruitment in acute islet xenograft rejection in non-human primates. Upregulation of RANTES and MIP-1alpha transcripts in the absence of a significant CCR5 increase suggests a possible involvement of other chemokine receptors. MCP-1 expression is associated with T cell and macrophage infiltration. Elevated cytotoxic effector molecule expression (Fas ligand, perforin, granzyme B) indicates T-cell mediated graft destruction by cytotoxic and cytolytic mechanisms within 48 to 72 h after transplantation. These results identify the CXCR3-mediated chemoattractant pathway as an immunosuppressive target in pig-to-primate islet xenotransplantation.

Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) in semen and reproductive disease in pregnant swine might be reduced by vaccines that induce mucosal immunity in the reproductive tract. Cholera toxin (CT), when delivered orally, is a potent mucosal adjuvant and immunogen in swine. To determine if oral immunization additionally elicits immunity at distant mucosal surfaces, we examined antibody responses to CT-B subunit in the reproductive tract and oral cavity. Orally administered CT induced distant mucosal immunity, as measured by antibodies to CT-B subunit in saliva and vaginal secretions. Presentation of PRRSV nucleocapsid as a genetic fusion with CT resulted in local mucosal antibody production, but no response was observed in vaginal secretions. The results demonstrate the feasibility of using orally administered CT for the induction of immunity to reproductive pathogens in swine. However, effective induction of PRRSV-specific immune responses in the reproductive tract requires a better understanding of the mechanisms of antigenicity and adjuvanticity at distant mucosal sites.

Beta-glucan has been shown to enhance anti-tumor and anti-infection functions in animals. Pigs at 4 months of age were infected with porcine reproductive and respiratory syndrome virus (PRRSV), and peripheral blood monocytes (PBMC) were isolated for the detection of interferon gamma (IFNgamma)-producing cells. We found that soluble high molecular weight beta-glucan could increase IFNgamma-producing cell frequency in a dose-dependent manner in the enzyme-linked immunospot assay (ELISPOT) in the absence of antigenic restimulation. A concentration as low as 1.6 microg/ml gave a significant increase and a similarly high enhancement was achieved at concentrations from 3.2 to 100 microg/ml. In PRRSV-specific IFNgamma ELISPOT, soluble beta-glucan elicited increased PRRSV-specific responses at concentrations from 3.2 to 50 microg/ml, but not at 100 microg/ml, whereas insoluble beta-glucan had no effect. Soluble beta-glucan augmented the porcine cellular immune response in an antigen-independent fashion, whereas insoluble beta-glucan had no activity. This finding suggests that soluble beta-glucan may enhance innate antiviral immunity against PRRSV.

Although much research has been performed on porcine reproductive and respiratory syndrome virus (PRRSV), little quantitative information is available on the relationships between virulence and in vivo virus replication, among isolates recovered at different times in the history of PRRS, or the relative levels of virulence associated with individual virus isolates. In this study, the in vivo growth properties of virulent field isolates and attenuated PRRSV isolates were compared. The results show that virulent PRRSV isolates exhibit longer and more elevated levels of viremia, induce faster and more intense humoral immune responses, negatively affect body weight gain, induce higher death rates, and cause more severe clinical signs in a respiratory disease model. We found that the more virulent field isolates grew to significantly higher levels in pigs than did cell-culture adapted isolates. We concluded that the pathogenic consequences and immunological responses of pigs to PRRSV are directly related to viral load in acute infection as reflected in viral titers in blood.

Dendritic cells (DC) are potent inducers of acquired immunity due to their ability to present antigens in the context of a costimulatory environment and consequently serve an essential role in vaccine efficacy. Strategies to enhance their function, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 treatment to induce DC differentiation from peripheral blood monocytes, may therefore be useful as vaccine adjuvants. We now have evaluated the effect of recombinant GM-CSF on the differentiation of DC in swine. GM-CSF mRNA was readily detected in porcine splenocytes, with increased levels following treatment of the cells with ConA and LPS. Porcine GM-CSF was cloned and expressed in the methylotrophic yeast, Pichia pastoris, as a glycosylated protein that induced proliferation of porcine bone marrow cells. P. pastoris-derived GM-CSF induced expression of antigen presenting (MHC class II) and costimulatory (CD80-CD86) molecules and enhanced antigen presenting cell (APC) function consistent with the induction of functional DC. Thus, recombinant GM-CSF produced by P. pastoris may be a potent adjuvant for swine vaccines., (Copyright 2003 Elsevier Science B.V.)

Immune responses are stimulated in response to threats against health. In animals, defense against infectious agents, particularly rapidly growing viruses and bacteria, requires an immediate response to limit growth and dissemination, and then stimulation of a more prolonged, specific immunity to prevent re-infection. The process by which animals meet the dual needs of an immediate response to danger and initiation of long-term protection is substantially influenced by inflammatory cytokines produced primarily by macrophages and professional antigen presenting cells (APCs). Inflammatory cytokines mobilize the immune system in response to danger and increase the efficiency of an immune response as effectors of APC function. Here we review the evidence for the involvement of inflammatory cytokines in immune induction and as mediators of APC activity, with a particular emphasis on swine and on the induction of immunity at mucosal surfaces. The vast majority of infections occur at mucosal surfaces of the enteric, respiratory and reproductive tracts, and induction of protective immunity at these sites is particularly challenging. Induction of immunity at mucosal surfaces of the small intestine is greatly facilitated by the oral adjuvant, cholera toxin (CT). CT potentiates inflammatory cytokine and costimulatory molecule expression in macrophages, and stimulates humoral and cell-mediated immune responses both locally and systemically. These observations are consistent with the hypothesis that activation of APCs is a key step in the induction of antigen-specific immunity, and that inflammatory cytokine expression is a hallmark of activated APC function. The efficacy of vaccine adjuvants, particularly in the context of mucosal immunity, may be determined by their ability to induce a controlled inflammatory response in gut-associated lymphoid tissue, characterized by the expression of various costimulatory molecules and inflammatory cytokines. Thus, elucidation of the patterns of inflammatory cytokine expression and features of APC activation will help to facilitate the rational development of more efficacious vaccines.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine health and well-being worldwide largely due to an insufficient understanding of the adaptive immune response to infection leading to ineffective PRRSV control. The memory and anamnestic response to infection are critical gaps in knowledge in PRRSV immunity. The lack of effective tools for the evaluation of the memory response previously hindered the ability to effectively characterize the porcine memory response to infection. However, the creation and validation of a PRRSV nsp7-specific B cell tetramer now facilitates the ability to detect very rare memory B cells and thus define the memory response of the pig. Here, we describe the PRRSV nsp7-specific B cell response following vaccination and challenge in six key secondary lymphoid organs including the identification of PBMCs as the tissue of interest for the memory immune response in pigs. Following live virus challenge of immune animals, an anamnestic response of nsp7-specific memory B cells and neutralizing antibodies was observed. This characterization of the functional humoral immune response to PRRSV answers key questions involved in regional specialization of the immune response following intramuscular inoculation of PRRSV MLV. [ABSTRACT FROM AUTHOR]

Rotavirus B (RVB) is an important swine pathogen, but control and prevention strategies are limited without an available vaccine. To develop a subunit RVB vaccine with maximal effect, we characterized the amino acid sequence variability and predicted antigenicity of RVB viral protein 7 (VP7), a major neutralizing antibody target, from clinically infected pigs in the United States and Canada. We identified genotype-specific antigenic sites that may be antibody neutralization targets. While some antigenic sites had high amino acid functional group diversity, nine antigenic sites were completely conserved. Analysis of nucleotide substitution rates at amino acid sites (dN/dS) suggested that negative selection appeared to be playing a larger role in the evolution of the identified antigenic sites when compared to positive selection, and was identified in six of the nine conserved antigenic sites. These results identified important characteristics of RVB VP7 variability and evolution and suggest antigenic residues on RVB VP7 that are negatively selected and highly conserved may be good candidate regions to include in a subunit vaccine design due to their tendency to remain stable. [ABSTRACT FROM AUTHOR]

Background: Senecavirus A (SVA), a member of the family Picornaviridae, genus Senecavirus, is a recently identified single-stranded RNA virus closely related to members of the Cardiovirus genus. SVA was originally identified as a cell culture contaminant and was not associated with disease until 2007 when it was first observed in pigs with Idiopathic Vesicular Disease (IVD). Vesicular disease is sporadically observed in swine, is not debilitating, but is significant due to its resemblance to foreign animal diseases, such as foot-and-mouth disease (FMD), whose presence would be economically devastating to the United States. IVD disrupts swine production until foreign animal diseases can be ruled out. Identification and characterization of SVA as a cause of IVD will help to quickly rule out infection by foreign animal diseases. Methods: We have developed and characterized an indirect ELISA assay to specifically identify serum antibodies to SVA. Viral protein 1, 2 and 3 (VP1, VP2, VP3) were expressed, isolated, and purified from E. coli and used to coat plates for an indirect ELISA. Sera from pigs with and without IVD symptoms as well as a time course following animals from an infected farm, were analyzed to determine the antibody responses to VP1, VP2, and VP3. Results: Antibody responses to VP2 were higher than VP1 and VP3 and showed high affinity binding on an avidity ELISA. ROC analysis of the SVA VP2 ELISA showed a sensitivity of 94.2% and a specificity of 89.7%. Compared to IFA, the quantitative ELISA showed an 89% agreement in negative samples and positive samples from 4-60 days after appearance of clinical signs. Immune sera positive for FMDV, encephalomyocarditis virus, and porcine epidemic diarrhea virus antibodies did not cross-react. Conclusions: A simple ELISA based on detection of antibodies to SVA VP2 will help to differentially diagnose IVD due to SVA and rule out the presence of economically devastating foreign animal diseases. [ABSTRACT FROM AUTHOR]

Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is the most economically significant pathogen in the US swine industry. Aerosol transmission among herds is a major concern in pig dense regions and filtration of incoming air, in combination with standard biosecurity procedures, has been demonstrated to prevent transmission of PRRSV into susceptible herds. To quantify the impact of air filtration on reducing risk of PRRSV outbreaks, we compared the incidence rate of new PRRSV introductions in 20 filtered and 17 non-filtered control sow herds in a swine dense region of North America during a 7year study period. Events of novel virus introduction were ascertained by phylogenetic analysis of PRRSV ORF5 gene sequences. Putative new viruses were defined as exogenous (introduced) based on ORF5 nucleotide sequence differences compared to previous farm isolates. The influence of sequence difference cut-off values ranging from 2 to 10% on case definition and relative risk were evaluated. Non-filtered farms incurred about 0.5 outbreaks per year, with a seasonal increase in risk in cooler periods. Baseline risk, prior to filtration, in treatment farms was approximately 0.75 per year, approximately 50% higher than in control farms. Air filtration significantly reduced risk of PRRSV introduction events to 0.06–0.22 outbreaks per year, depending on the cut-off values used to classify a virus isolate as new to the herd. Overall, air filtration led to an approximately 80% reduction in risk of introduction of novel PRRSV, indicating that on large sow farms with good biosecurity in swine-dense regions, approximately four-fifths of PRRSV outbreaks may be attributable to aerosol transmission. [Copyright &y& Elsevier]

Abstract: Glycoprotein 5 (GP5) and membrane (M) protein are the major proteins in the envelope of porcine reproductive and respiratory syndrome virus (PRRSV). Although viral neutralization epitopes are reported in GP5 and M of type 2 PRRSV, their significance as targets of porcine humoral immunity is not well described. Thus, we constructed recombinant polypeptides containing ectodomain neutralization epitopes to examine their involvement in porcine antibody neutralization and antiviral immunity. PRRSV infection elicited ectodomain-specific antibodies, whose titers did not correlate with the neutralizing antibody (NA) response. Ectodomain-specific antibodies from PRRSV-neutralizing serum bound virus but did not neutralize infectivity. Furthermore, immunization of pigs with ectodomain polypeptides raised specific antibodies and provided partial protection without a detectable NA response. Finally the polypeptides did not block infection of porcine macrophages. These results suggest that the GP5/M ectodomain peptide epitopes are accessible for host antibody recognition, but are not associated with antibody-mediated virus neutralization. [Copyright &y& Elsevier]

Abstract: Porcine reproductive and respiratory syndrome (PRRS) virus appeared 20 years ago as the cause of a new disease in swine. Today PRRS is the most significant swine disease worldwide in spite of intensive immunological interventions. The virus showed remarkable genetic variation with two geographically distinct genotypes at the time of its discovery, indicating the possibility of prolonged evolutionary divergence prior to its appearance as a swine pathogen. Since then, both type 1 and type 2 have spread geographically, radiated genetically, and acquired new phenotypic characteristics, especially increased virulence. Here, we explore various hypotheses that might account for rapid expansion and diversification of PRRSV, including mechanisms specific to PRRSV and other arteriviruses, cellular modification processes, and immunological selection. Phylogenetic analysis of PRRSV has provided a broadly applicable means to relate diverse isolates, but it does not explain biological variation in virulence or immunological cross-protection. We present other methods of classification and review their limitations. Major questions about PRRSV remain unanswered despite intensive investigation, suggesting that the interaction of PRRSV with pigs involves novel biological processes that may be relevant to other RNA virus and host interactions. [ABSTRACT FROM AUTHOR]

Abstract: The TH1–TH2 paradigm provides an elegant model of directed response to infectious pathogens. Developed in the mouse, the model has provided a framework for systematic and mechanistic studies of immune regulation, protective immunity, and vaccine development in swine. Interleukin-4 (IL-4) plays a central role in the paradigm as a regulatory molecule directing development of the TH2 phenotype, as a developmental cytokine essential for antibody production, and as a soluble diagnostic marker of the TH2 cell type. In contrast, while characterizing the biological properties of porcine IL-4, we discovered that it was not a stimulatory factor for porcine B cells. Rather, it blocked antibody and IL-6 secretion and suppressed antigen-stimulated proliferation of B cells. Inhibition was not reversed by treatment with IL-2 and IL-6 treatment. IL-4 did not stimulate T lymphocyte proliferation, but induced cell growth in lymphoblasts in a dose-dependent fashion. These results suggest that IL-4 plays a different role in pigs than in mice and humans, in which it stimulates B cells and is essential for antibody production. Furthermore, the functions of IL-4 in swine cannot be inferred from results in model systems such as the mouse. General models of disease resistance show substantial variation between pigs and mice at the cellular and molecular level. Advances in somatic cell technologies and animal engineering to enable gene knockouts in pigs, in combination with a continuously expanding immunological toolkit, promise an exciting future for pig immunology, detailed mechanistic elucidation of the TH1–TH2 paradigm, and an improved understanding of the role of IL-4 in porcine immunity to infectious disease. [Copyright &y& Elsevier]

Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus 2 (PCV2), is characterized by a highly variable pathogenesis that is manifested by various disease syndromes and includes immune evasion. Hence, even though PCVAD is effectively controlled by vaccination, pigs and farms remain infected so that continued vaccination is necessary to control disease. We investigated the molecular interactions of PCV2 and its permissive VR1BL host cell for gene expression signatures that could provide insight into mechanisms leading towards disease. Molecular pathways involved in the innate immune response to PCV2 infection were examined to identify changes in gene expression associated with productive infection of VR1BL cells. RNA profiling from infected and uninfected cells showed that 139 genes were induced by infection and 43 genes were down-regulated, using a p value <0.05 and an absolute fold-change difference>2. A strong type 1 interferon response, including an increase in genes involved in the RIG-I/MDA5 pathway and downstream interferon induced genes, was observed. Key regulators involved in PCV2 infection were identified as IFNβ, DDX58 (RIG-I), and IRF7. PCV2 infection induces a strong interferon response which unexpectedly facilitates viral gene expression, perhaps due to the presence of an interferon-sensitive response element in the viral promoter. The findings suggest that PCV2 interventions that attenuate type 1 interferon responses at the cellular level might enhance immunity and eliminate persistent infection. [ABSTRACT FROM AUTHOR]

Highlights: [•] A porcine cell line, VR1BL, was characterized for high titer growth of PCV2. [•] PCV2 grows to higher titers in VR1BL cells, than in PK-15 cells. [•] This cell line provides an in vitro model of acute versus persistent infection that replicates in vivo behavior of PCV2. [•] High MOI infection induces apoptosis-mediated cell death. [Copyright &y& Elsevier]

Abstract: The objectives of this study were to evaluate the effects of a therapeutic vaccine intervention with a modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine on the dynamics of a heterologous viral infection in a population of pigs, and to determine the clinical and virological response of previously exposed and vaccinated pigs against a second virulent heterologous challenge. A population of 320 pigs were infected with a field isolate, PRRSV MN-30100, alone or followed by Ingelvac® PRRS MLV vaccine administered one to three times at 30 days intervals beginning 1 week after infection. Vaccine intervention reduced the duration of viral shedding, but did not reduce the viral load in tissues or the proportion of persistently infected pigs. A different and highly virulent field isolate, MN-184, was then given as a heterologous viral challenge at 97 days after first exposure. Previously infected and vaccinated pigs showed a significant reduction in clinical signs and enhanced weight gain after the highly virulent challenge with PRRSV MN-184, but infection with and shedding of the challenge isolate were not prevented. [Copyright &y& Elsevier]

Abstract: Salmonella enterica serovar Choleraesuis is an enteric pathogen of swine, producing septicemia, enterocolitis, pneumonia, and hepatitis. The initial molecular events at the site of Salmonella infection are hypothesized to be critical in the initiation of innate and adaptive immune responses; however, the acute immune response elicited by porcine intestinal tissues is not well understood. To address this need, we employed explants of jejunal Peyer''s patch (JPP) mucosa from pigs to examine Salmonella-induced immune responses under controlled conditions as well as to overcome limitations of whole animal approaches. JPP explants mounted in Ussing chambers maintained normal histological structure for 2h and stable short-circuit current and electrical conductance for 2.5h. After ex vivo luminal exposure to Salmonella serovar Choleraesuis, JPP responded with an increase in mRNA expression of IL-1β and IL-8, but not TNFα. Increased IL-1β and IL-8 expression were dependent on efficient Salmonella adhesion and internalization, whereas mutant Salmonella did not induce inflammatory cytokine expression. Commensal enteric bacteria, present in some experiments, also did not induce inflammatory cytokine expression. These findings indicate that Salmonella uptake by Peyer''s patch is important in the induction of an innate response involving expression of IL-1β and IL-8, and that ex vivo intestinal immune tissue explants provide an intact tissue model that will facilitate investigation of mucosal immunity in swine. [Copyright &y& Elsevier]

β-Glucan has been shown to enhance anti-tumor and anti-infection functions in animals. Pigs at 4 months of age were infected with porcine reproductive and respiratory syndrome virus (PRRSV), and peripheral blood monocytes (PBMC) were isolated for the detection of interferon γ (IFNγ)-producing cells. We found that soluble high molecular weight β-glucan could increase IFNγ-producing cell frequency in a dose-dependent manner in the enzyme-linked immunospot assay (ELISPOT) in the absence of antigenic restimulation. A concentration as low as 1.6μg/ml gave a significant increase and a similarly high enhancement was achieved at concentrations from 3.2 to 100μg/ml. In PRRSV-specific IFNγ ELISPOT, soluble β-glucan elicited increased PRRSV-specific responses at concentrations from 3.2 to 50μg/ml, but not at 100μg/ml, whereas insoluble β-glucan had no effect. Soluble β-glucan augmented the porcine cellular immune response in an antigen-independent fashion, whereas insoluble β-glucan had no activity. This finding suggests that soluble β-glucan may enhance innate antiviral immunity against PRRSV. [Copyright &y& Elsevier]

Highlights • PAMs from young pigs are more susceptible to PRRSV infection than adult PAMs. • PRRSV receptor expression was similar between PAMs from young and adult pigs. • Age-dependent resistance to PRRSV infection is not due to receptor-mediated entry. Abstract Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the most economically important disease affecting swine production worldwide. The severity and susceptibility of PRRSV infection varies with age. Nursery pigs have been shown to be more susceptible to PRRSV infection and a more severe and prolonged infection is observed as compared to growing or adult pigs. However, antibody responses to PRRSV are observed independent of age. Swine are the only known hosts of PRRSV, infection is restricted to cells of monocytic lineage, and fully differentiated porcine alveolar macrophages are the primary target of natural infection. Pulmonary intravascular macrophages from young pigs have been shown to be more susceptible to infection than those from adult pigs. A better understanding of why young pigs and macrophages from young pigs are more susceptible to PRRSV infection is critical to identify mechanisms of infection that can be explored for enhanced treatment or prevention of disease. This study examined PRRSV susceptibility of porcine alveolar macrophages isolated from the lungs of pigs of different age groups, and the presence of cell surface receptors to determine if differences correlated with infection level. The younger the pigs were, the more susceptible the macrophage were to PRRSV infection, but no differences in cellular receptor expression were observed between pigs of different ages. Resistance to infection is likely related to intracellular innate immune mechanisms rather than receptor-mediated entry. [ABSTRACT FROM AUTHOR]

Vaccine control and prevention of porcine reproductive and respiratory syndrome (PRRS), the most important disease of swine, is difficult to achieve. However, the discovery of broadly neutralizing antibody activity against porcine reproductive and respiratory syndrome virus (PRRSV) under typical field conditions opens the door to new immunologic approaches for robust protection. We show here that passive administration of purified immunoglobulins with neutralizing antibodies reduced PRRSV2 infection by up to 96%, and PRRSV1 infection by up to 87%, whereas immune immunoglobulins lacking neutralizing activity had no effect on viral infection. Hence, immune competence of passive immunoglobulin transfer was associated specifically with antibody neutralizing activity. Current models of PRRSV infection implicate a minor envelope glycoprotein (GP) complex including GP2, GP3, and GP4, as critical to permissive cell infection. However, conserved peptides comprising the putative cell attachment structure did not attenuate neutralization or viral infection. The results show that immunological approaches aimed at induction of broadly neutralizing antibodies may substantially enhance immune protection against PRRSV. The findings further show that naturally occurring viral isolates are able to induce protective humoral immunity against unrelated PRRSV challenge, thus removing a major conceptual barrier to vaccine development. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, dehydration in pigs, and high mortality rates in piglets <3 weeks of age. Maternal immunity protects piglets, but information on vaccination before or after natural infection in endemically exposed sow herds is limited. Currently, the recovery goal in sow units infected with PEDV is to become fully naive again or use natural virus infection to develop immune gilts through a feedback program before introduction into the sow herd. Since neutralizing antibodies in the gut are critical for protection against enteric viral infections such as PEDV, we evaluated the effect of a conditionally licensed, adjuvanted inactivated PEDV vaccine on neutralizing antibody levels in milk and colostrum in both naive and previously naturally exposed sow herds. The results illustrate that intramuscular vaccination increased neutralizing antibody titers, and anti-PEDV IgA and IgG in milk and colostrum of sows that were previously infected. Thus, inactivated PEDV vaccines may provide increased protection to piglets nursing on previously infected sows against exposure to PEDV through increased delivery of lactogenic neutralizing antibodies to the enteric site of infection. [ABSTRACT FROM AUTHOR]

Porcine circovirus type 2 (PCV2), a small, single-stranded circular DNA virus and the causative agent of porcine circovirus associated disease (PCVAD), was first observed in the mid-1990s in pigs with a post-weaning wasting disease. In 2006 the number of PCVAD cases greatly increased, marking it as an important viral pathogen for the United States (US) swine industry. PCV2 vaccines were introduced to the US in 2006 in response to widespread outbreaks of PCVAD. These vaccines were effective in preventing disease, but did not eliminate virus from the animals. In 2006, prior to vaccine use, a study of PCV2 prevalence in pig herds across the US was performed in conjunction with the US National Animal Health Monitoring System. In 2012, 6 years after widespread PCV2 vaccination, this study was repeated. Since the introduction of PCV2 vaccines in 2006, viral presence and viral loads have greatly decreased, and a genotypic shift dominated by PCV2b has occurred. Antibody levels have decreased in the pig population, but approximately 95% of sites continue to be antibody-positive. Widespread vaccination has controlled PCVAD and decreased PCV2 prevalence to the point that viremia is not detected on many sites. Thus, continued vaccination may lead to PCV2 elimination in the national herd over time. [ABSTRACT FROM AUTHOR]

Porcine circovirus 2 (PCV2) was prevalent in swine in the United States before PCV2-associated disease (PCVAD) appeared in 2006. Limited nucleotide sequencing of open reading frame 2 (ORF2) encoding capsid, the only structural protein, revealed the presence of two genotypes, PCV2a and PCV2b. Later, PCV2c and mutant PCV2b, or PCV2d, were also described. However, extensive PCV2 ORF2 sequence databases in veterinary diagnostic laboratories have not been analyzed systematically to determine the genetic diversity of field isolates. Here, we interrogated >1100 PCV2 ORF2 nucleotide sequences to assess population diversity and genetic variation. We detected a novel PCV2 genotype that is substantially different, primarily in ORF2, from all known PCV2. Notably, ORF2 contains a unique carboxyl terminal amino acid insertion resulting in a 238 amino acid ORF2. All other PCV2 ORF2 proteins are 233 or 234 aa in length. Phylogenetic analysis indicates that it is more ancient than other PCV2 genotypes. The findings demonstrate the value of analyzing routine diagnostic laboratory sequence databases in population genetic analyses of animal pathogens. [ABSTRACT FROM AUTHOR]

Neutralizing antibodies are a critical part of the immune armory for defense against viruses, and the mechanism by which many effective vaccines work to protect against viral infections. However, infections by rapidly evolving and genetically diverse viruses are often characterized by ineffective neutralizing antibody responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly genetically diverse RNA virus that causes PRRS, the most significant disease of pigs worldwide. The prevailing view of immunity to PRRSV is characterized by delayed and ineffectual production of neutralizing antibodies lacking cross-reactivity that is necessary for vaccine efficacy. Using an ELISA-based neutralizing assay developed to analyze PRRSV growth in porcine alveolar macrophages, the naturally permissive cell of PRRSV, we showed that sera from previously infected commercial sows had high levels of neutralizing activity against diverse PRRSV strains, including across distinct genotypes of PRRSV. Fifty percent cross-neutralization titers in excess of 1/1024 were observed. Neutralizing activity was dose-dependent and was maintained in the immunoglobulin fraction. Presence of high-titer, anti-PRRSV antibody activity that cross-neutralizes diverse strains of virus has prompted reevaluation of the role of neutralizing antibodies for cross-protection against PRRSV under field conditions. Understanding conditions that favor development of cross-neutralizing activity will be crucial for improved strategies to enhance cross-protection against PRRSV. More detailed studies are expected to elucidate mechanisms of neutralizing antibody production and maturation and to investigate conserved epitope targets of cross-neutralization in this rapidly evolving virus. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widespread pathogen of pigs causing significant economic losses to the swine industry. The expanding diversity of PRRSV strains makes the diagnosis, control and eradication of the disease more and more difficult. In the present study, the authors report the full genome sequencing of a type 2 PRRSV strain isolated from piglet carcasses in Hungary. Next generation sequencing was used to determine the complete genome sequence of the isolate (PRRSV-2/Hungary/102/2012). Recombination analysis performed with the available full-length genome sequences showed no evidence of such event with other known PRRSV. Unique deletions and an insertion were found in the nsp2 region of PRRSV-2/Hungary/102/2012 when it was compared to the highly virulent VR2332 and JXA-1 prototype strains. The majority of amino acid alterations in GP4 and GP5 of the virus were in the known antigenic regions suggesting an important role for immunological pressure in PRRSV-2/Hungary/102/2012 evolution. Phylogenetic analysis revealed that it belongs to lineage 1 or 2 of type 2 PRRSV. Considering the lack of related PRRSV in Europe, except for a partial sequence from Slovakia, the ancestor of PRRSV-2/Hungary/102/2012 was most probably transported from North-America. It is the first documented type 2 PRRSV isolated in Europe that is not related to the Ingelvac MLV. [ABSTRACT FROM AUTHOR]

Abstract: Porcine circovirus 2 (PCV2), the cause of porcine circovirus-associated disease (PCVAD), is widespread in swine farms throughout the United States with vaccine controlling disease, but not eliminating infection. We examined the PCV2 virological and immunological status of sows, pre-suckling piglets, and the farrowing environment of sow farms to determine PCV2 exposure risks, transmission dynamics, and immunological impacts at the time of farrowing. PCV2 was widely distributed in animals and the farrowing environment of 6 midwestern US sow farms irrespective of sow vaccination status. High levels of PCV2 capsid-specific antibodies were observed in sow serum and colostrum and had no apparent effect on PCV2 transmission to and infection in piglets. In 281 pre-suckling piglets from 59 sows, PCV2 DNA was detected in 63% of serum samples and on 93% of axillary skin swabs. PCV2 was present in one or more samples from 58 of 59 sows and in the farrowing environment. Isolated infectious virus samples from sows, presuckling piglets, and the environment were shown by sequencing to be genetically similar from all farms. In conclusion, piglets are readily infected with PCV2 in utero and are under constant challenge by PCV2 through contact with infected sows and a contaminated farrowing environment. However, maternal immunity did not affect PCV2 transmission to piglets or the viral load in sows. These findings illustrate the importance of maternal infection, despite robust anti-PCV2 immunity, in early infection of newborn piglets, and the need to develop appropriate infection models for elucidation of mechanisms of protective immunity. [Copyright &y& Elsevier]

Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus responsible for PRRS in swine, a disease with globally significant animal welfare and economic concerns. There is no specific treatment and variably effective immune protection. Molecular mechanisms responsible for virulence, pathogenesis and protective immune response remain poorly understood. These factors limit progress toward development of effective measures for prevention and treatment of PRRS. A novel PRRSV ORF5a protein, encoded in an open reading frame (ORF) that overlaps the major envelope glycoprotein GP5 ORF, was recently identified. Because ORF5a is highly conserved in diverse PRRSV isolates, is a structural protein in the virion, and elicits a specific antibody response in infected pigs, we investigated its potential role in immune protection against PRRSV infection. Pigs immunized with ORF5a protein had robust serologic responses. However, the antibodies did not neutralize virus, and immunity did not protect against challenge infection. We conclude from these findings that the ORF5a antibody response is neither neutralizing nor protective. [Copyright &y& Elsevier]

Abstract: Porcine circovirus 2 (PCV2) is believed to be a necessary but not sufficient underlying cause of porcine circovirus associated disease (PCVAD) in swine (). Since the potential threat of PCVAD is dependent on the prevalence of PCV2 in swine populations, accurate diagnostic tests are important for epidemiologic surveillance. Therefore, we evaluated the diagnostic sensitivity (Se) and specificity (Sp) of a new indirect ELISA and two quantitative PCR tests for PCV2 in a series of latent class models that used Bayesian estimation procedures. A total of 4140 samples from finisher pigs were tested for evidence of PCV2 by the ELISA and a TaqMan (TM) quantitative PCR, 995 by the ELISA and a SYBR Green (SG) dye-binding PCR, 998 by both PCRs and 993 by all three tests. Overall, the median (95% probability interval) ELISA Se and Sp was 0.85 (0.83–0.87) and 0.74 (0.68–0.79), respectively, when all three tests were analyzed together at an ELISA absorbance (optical density or OD) cutoff of ≥0.3. The TM PCR Se and Sp was 0.86 (0.84–0.88) and 0.94 (0.87–0.97), respectively, and the SG PCR Se and Sp was 0.83 (0.81–0.85) and 0.98 (0.94–1.00), respectively when all three tests were analyzed together at an ELISA OD cutoff of ≥0.3. Sensitivity analysis revealed that Sp estimates in general had less stability than Se estimates, but the SG PCRSp was the most stable. Limited conditional dependence between the two PCR tests was detected. We conclude that the ELISA had the highest diagnostic Se at an absorbance cutoff of ≥0.3, while the SG PCR had the highest diagnostic Sp. The prevalence levels for exposure to PCV2 in finishing swine populations across all analyses ranged from 58 to 100%. [Copyright &y& Elsevier]

Abstract: Porcine circovirus (PCV) appeared in 1974 as an unidentified, innocuous viral inhabitant of cell cultures and pigs. Today PCV1 is a contaminant of some human vaccines, and PCV2 is a major pathogen of swine. PCV1 is reportedly ubiquitous in swine but nonpathogenic. Since the interplay of PCV1 and PCV2 in swine might explain variable disease results and shed light on the potential for human exposure, we analyzed in depth the prevalence of PCV1 and PCV2 infection and exposure in the U.S. finishing swine herd. Over 82% of sera from 185 farms were positive for PCV2 by PCR, whereas only 2.4% were positive for PCV1. More than 80% of PCV2 DNA-positive swine were also positive for anti-PCV2 antibodies. PCV1 was only rarely present. Exposure of swine, and therefore humans via pigs, to PCV1 is negligible. We conclude that PCV2 causes a persistent infection in pigs and that PCV1 is absent or rare in swine. [Copyright &y& Elsevier]

Abstract: The innate immune response is critical to enteric disease resistance and the induction of mucosal adaptive immunity. In mucosae of the small intestine, Peyer''s patches play a central role in immune surveillance and sampling of bacteria by specialized M cells. The innate immune response to Salmonella enterica serovar Choleraesuis, an enteric pathogen of swine, involves IL-1β and IL-8 mRNA induction but not that of IL-6 and TNFα, in contrast to Salmonella serovar Typhimurium infection of murine small intestine. We investigated in vivo responses to Salmonella and potential effects of animal variation since the gut environment is highly dynamic and constantly changing physiologically. Salmonella serovar Choleraesuis induced an early proinflammatory cytokine response at 6h after infection, which was characterized by a 4-fold increase in production of CXCL2 mRNA by jejunal Peyer''s patches (JPP), and a 12-fold increase in IL-1β and 4-fold increase in IL-8 (CXCL8) mRNAs by distal ileal Peyer''s patches (IPP). Levels of IL-1β and IL-8 mRNA were positively correlated with numbers of mucosal neutrophils in the distal IPP. Salmonella DNA was also detected in ileal tissues, including Peyer''s patches, absorptive epithelium and mesenteric lymph nodes, in 33–83% of infected animals, compared to the jejunal tissues, which were positive in 0–33% of infected pigs. Notwithstanding substantial animal-to-animal variation, IL-1β was increased in both proximal and distal IPP, IL-8 was increased in the distal IPP, and calprotectin was associated with both by cluster analysis. These data indicate that IL-1β and IL-8 expression in the IPP plays a key role early in the interaction between Salmonella serovar Choleraesuis and the small intestine. [Copyright &y& Elsevier]

Objective--To evaluate the influences of animal age, bacterial coinfection, and porcine reproductive and respiratory syndrome virus (PRRSV) isolate pathogenicity on virus concentration in pigs. Animals--Twenty-one 2-month-old pigs and eighteen 6-month-old pigs. Procedure--Pigs were grouped according to age and infected with mildly virulent or virulent isolates of PRRSV. The role of concurrent bacterial infection was assessed by infecting selected pigs with Mycoplasma hyopneumoniae 21 days prior to inoculation with PRRSV. On alternating days, blood and swab specimens of nasal secretions and oropharyngeal secretions were collected. On day 21 after inoculation with PRRSV, selected tissues were harvested. Concentrations of PRRSV were determined by use of quantitative real-time PCR and expressed in units of TCID50 per milliliter (sera and swab specimens) or TCID50 per gram (tissue specimens). Results--Concentrations of virus were higher in blood and tonsils of pigs infected with virulent PRRSV. Pigs infected with virulent PRRSV and M hyopneumoniae had significantly higher concentrations of viral RNA in lymphoid and tonsillar tissue. Coinfection with M hyopneumoniae resulted in a higher viral load in oropharyngeal swab specimens and blood samples, independent of virulence of the PRRSV isolate. Two-month-old pigs had significantly higher viral loads in lymph nodes, lungs, and tracheal swab specimens than did 6-month-old pigs, independent of virulence of the PRRSV isolate. Conclusions and Clinical Relevance--Multiple factors affect PRRSV concentration in pigs, including pathogenicity of the PRRSV isolate, age, and concurrent infection with M hyopneumoniae. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important infectious disease agent of pigs worldwide, causing reproductive failure in pregnant sows and respiratory problems in nursing and growing pigs. PRRSV infection is characterized by a prolonged viremia of 30 or more days and an extended persistent infection of lymphoid tissues. To better understand the immunological basis for prolonged acute and persistent PRRSV infection, we have examined the cell-mediated immune (CMI) response throughout the course of infection and compared the results to the local distribution and abundance of PRRSV in infected tissues. PRRSV-specific T cells, enumerated by gamma interferon enzyme-linked immunospot assay, did not appear until 2 weeks after PRRSV inoculation, and their abundance exhibited substantial variation over time and among animals. In all cases the T-cell response was transient. High levels of viral RNA were present in lymphoid tissues of all animals in the acute phase of infection. Viral loads were decreased 1,000-fold or more in persistent infections, with the primary sites of persistence being tonsil, sternal lymph node, and inguinal lymph node. The abundance of virus-specific T cells in either acutely or persistently infected animals was highly variable and showed no correlation to the level of virus in lymphoid tissues. No significant difference in antigen-specific T-cell abundance was observed in secondary lymphoid tissues in either acute or persistent infection except for tonsil, in which the number of responding cells was extremely low. CD4+- and CD8+-T-cell frequencies did not change after PRRSV infection, though a decrease in γδ T cells was observed. Macrophages, the permissive cell type for PRRSV, were present in various levels in all tissue preparations and were not in proportion to local virus load. These findings indicate that a weak CMI response contributes to prolonged PRRSV infection and suggests that PRRSV suppresses T-cell recognition of infected macrophages. Thus, the slow but eventual resolution of PRRSV infection may be dependent on limiting permissive macrophages and on innate immune factors. [ABSTRACT FROM AUTHOR]

The lack of available, well characterized, established, domestic porcine cell lines hinders the advancement of porcine cellular immunology. A case of multicentric lymphoma was diagnosed in a market weight pig at the time of slaughter. Affected lymph nodes and spleen were collected and used for single cell isolation and analysis. Cell lines were established by 3 rounds of limiting dilution from splenic and subiliac lymph node lymphomas. Surface marker staining identified the cells as CD21+, CD79a+, CD20+, PAX5+, and CD3− and cells were grown and easily passaged in cell culture. Transcriptome analysis was carried out to further characterize these rapidly proliferating cells validating the initial cytometric findings, confirming their identity as B cell lymphomas, and suggesting that they arose from germinal center centroblasts with aberrant control of BCL6 expression. Functional analysis identified the cells as being involved in cancer, cell movement, cell survival, and apoptosis. These new porcine B cell lymphoma cell lines will be a valuable resource for more in-depth cellular investigations into the porcine immune system and cancer, as well as providing a potential tool for the growth of lymphotropic viruses of pigs and humans. • Three porcine B cell lines were established from a pig with multicentric lymphoma. • Transcriptome sequencing identified the cell origin as germinal center centroblasts. • Lymphoma cells have decreased cell signaling pathways and increased survival. • This cell line is a valuable resource for cancer and viral infection investigations. [ABSTRACT FROM AUTHOR]