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In late 2011, the outbreak of pseudorabies (PR) occurred in Bartha-K61-vaccinated pig farms and spread rapidly to many provinces of China, causing substantial economic losses to the swine industry. A total of 4708 pig serum samples from Henan province during 2018-2019 were collected to screen for the presence of pseudorabies virus (PRV) gE-specific antibodies, and phylogenetic analysis based on the gE gene of PRV was performed. Of the 4708 serum samples tested, 30.14% (1419/4708) were seropositive for PRV antibodies, based on PRV gE-coated enzyme-linked immunosorbent assay (ELISA), with slaughterhouses having the highest seroprevalence. The seropositive rates of PRV also varied with the region and the season. Phylogenetic analysis showed that three PRV isolates from this study were clustered in an independent branch together with the Chinese variant PRV strains (after 2012), and had a closer genetic relationship with the Chinese variant PRV strains, but differed genetically from the 4 early Chinese PRV strains and 4 European-American strains. This study suggests that three PRV isolates may belong to PRV variants, and the development of a novel vaccine against PRV variants is particularly urgent., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Natural killer (NK) cells are cells of the innate immunity and play an important role in the defense against viral infections and cancer, but also contribute to shaping adaptive immune responses. Long-lived tissue-resident NK cells have been described in man and mouse, particularly in the liver, contributing to the idea that the functional palette of NK cells may be broader than originally thought, and may include memory-like responses and maintaining tissue homeostasis. Remarkably, liver resident (lr)NK cells in man and mouse show substantial species-specific differences, in particular reverse expression patterns of the T-box transcription factors Eomesodermin (Eomes) and T-bet (Eomes high T-bet low in man and vice versa in mouse). In pig, compared to blood NK cells which are CD3 - CD8α high cells, the porcine liver contains an abundant additional CD3 - CD8α dim NK cell subpopulation. In the current study, we show that this porcine CD3 - CD8α dim liver NK population is highly similar to its human lrNK counterpart and therefore different from mouse lrNK cells. Like human lrNK cells, this porcine NK cell population shows an Eomes high T-bet low expression pattern. In addition, like its human counterpart, the porcine liver NK population is CD49e - and CXCR6 + . Furthermore, the porcine Eomes high T-bet low liver NK cell population is able to produce IFN-γ upon IL-2/12/18 stimulation but lacks the ability to kill K562 or pseudorabies virus-infected target cells, although limited degranulation could be observed upon incubation with K562 cells or upon CD16 crosslinking. All together, these results show that porcine Eomes high T-bet low NK cells in the liver strongly resemble human lrNK cells, and therefore indicate that the pig may represent a unique model to study the function of these lrNK cells in health and disease., (Copyright © 2019 De Pelsmaeker, Denaeghel, Hermans and Favoreel.)

E74-like factor 4 (ELF4) is a novel transcription factor that initiates transcription of type I interferon (IFN) genes to control diverse pathogens. Here, porcine ELF4 (poELF4) was cloned and its role in type I IFN signaling was investigated in different porcine cell lines. Full-length cDNA of poELF4 encodes 663 amino acid residues and ectopic expression of poELF4 significantly induced IFN-β production. Interestingly, difference from the human ELF4 (huELF4), poELF4 mutants lacking the serine/threonine rich domain, which has been demonstrated to be responsible for the phosphorylation of huELF4, were still capable of activating IFN-β promoter. Using pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) as the models of DNA virus and RNA virus, respectively, we found that the replication of both PRV and PRRSV was reduced with poELF4 overexpression and enhanced with poELF4 knockdown. Taken together, these results suggested that poELF4 is an important antiviral host restriction factor., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aujeszky's disease (AD, pseudorabies) is a notifiable disease caused by Suid herpesvirus 1 (SuHV-1), also named pseudorabies virus (PrV). The study aimed at determining the occurrence and spatio-temporal trend of specific antibodies to AD virus (ADV) among wild boar of Saxony-Anhalt, a landlocked federal state situated in the western part of eastern Germany. To this end, a total of 7,209 blood samples were collected and tested from 2000 to 2011. An average seroprevalence of 6.8% was found for the entire observation period, whilst seroprevalence increased significantly between time periods January 2000-March 2009 (n = 3,605; prevalence = 4.5%) and March 2009-December 2011 (n = 3,604; prevalence = 9.1%). Spatial analysis revealed an extremely heterogenous distribution of seropositive samples with cluster formation [relative risk (RR) up to 41, P = 0.001] in the east. A comparison of spatial clusters between the aforementioned time intervals showed an expansion of the disease in the north to north-westerly direction. A test for spatial shift involving the entire territory of Saxony-Anhalt detected a spread of ADV infection in the latter direction (P = 0.079) at the average speed of 3.3 km/year. Detailed study of the distribution and spread of the disease among wild boar, including assessment of the speed of spatial spread, has not been done in the region before. Saxony-Anhalt has been officially recognised as being free of AD in domestic pigs since 1996. Despite increasing ADV seroprevalences in the wild boar population of Saxony-Anhalt and other federal states in the east of Germany, no spill-over to domestic pigs have been reported. The currently implemented monitoring will be continued in conjunction with surveillance of classical swine fever (CSF) to further trace ADV infections in the wild boar population of Saxony-Anhalt.

A novel magnetic electrochemical immunosensor has been developed for the detection of pseudorabies virus antibody in swine serum. The magnetic glass carbon electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and gold nanoparticles were chosen as electroactive labels for the electrochemical detection. The parameters concerning the assay strategy were carefully investigated. Under the optimal conditions, the linear response range of pseudorabies virus antibody dilution ratio (standard positive serum) was 1:250 to 1:1000 with a detection limit of 1:1000. Finally, this developed immunoassay method was successfully applied in the detection of pseudorabies virus antibody in swine serum, and had a good diagnostic accordance in comparison with ELISA., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The stimulator of interferon genes (STING) of human/mouse has been identified recently as an adaptor that links virus-sensing receptors to interferon regulatory factor 3 (IRF3) activation. Here we report the cloning and characterization of porcine STING (poSTING). The full-length poSTING cDNA sequence encodes 378 amino acids and contains one endoplasmic reticulum (ER) retention motif, RAR. Phylogenetic analysis revealed that poSTING, together with bovine STING, is more closely related to the human than to mouse STING. poSTING mRNA expression was mainly detected in the spleen, lymph node and lung. Enhanced green fluorescence protein (EGFP)-labeled poSTING was found to reside predominantly in the ER, and also in the mitochondrial membrane in PK-15 cells. Over-expression of poSTING activated both IRF3 and nuclear factor kappaB (NF-kappaB) transcription factors to induce IFN-beta production, while knockdown of poSTING significantly inhibited poly(I:C)- and poly(dAT:dAT)-induced IFN-beta promoter activation and IFN-beta mRNA production. Furthermore, the pseudorabies virus (PRV), a dsDNA virus, has been shown to activate the IFN-beta promoter in a poSTING-dependent way in porcine cell lines. Altogether, these results indicate that STING is an important regulator of porcine innate immune signaling. The results will help better understand the biological role(s) of STING in innate immunity during evolution., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Plausible representatives of plasmacytoid dendritic cells (pDCs) in pigs have been characterized as being CD4(hi)CD172(lo). Due to their paucity in blood, we utilized novel fluorescent-activated cell sorting procedures to isolate them from PBMC. The resultant subset was greater than 98% homogeneous in regards to the selected phenotype and contained the preponderance of individuals secreting IFN-alpha after exposure to a known stimulant, transmissible gastroenteritis virus (TGEV). In addition to being a potent source of IFN-alpha, other properties of these porcine CD4(hi)CD172(lo) cells including their morphological transition from a plasma cell-like shape during quiescence to one resembling a dendritic cell (DC) after activation by TGEV and their relatively strong constitutive expression of interferon regulatory factor-7 (IRF-7) conformed to the expectations of genuine pDCs. While a substantial IFN-alpha response was also elicited from the porcine pDCs by pseudorabies virus (PrV), swine influenza virus (SIV), and TLR7 and 9 agonists, there was an agent-dependent induction of varying amounts of IL-2, IL-6, IL-8, IL-12, IFN-gamma, and TNF-alpha. Notably, porcine reproductive and respiratory syndrome virus (PRRSV) failed to provoke the pDCs to secrete any of the measured cytokines except IL-2. Moreover, whereas pDCs exposed to TGEV or the TLR9 agonist rapidly increased IRF-7 production and morphed into DCs with enhanced CD80/86 expression, similar alterations were not observed during incubation with PRRSV. This atypical response of pDCs to PRRSV may contribute to its pathogenesis, which unlike that associated with PrV, SIV or TGEV includes persistent infection and limited development of protective immunity., (Copyright 2009 Elsevier B.V. All rights reserved.)

This study aimed to map quantitative trait loci (QTL) for traits related to humoral innate immune defence. Therefore, haemolytic complement activity in the alternative and the classical pathway, serum concentration of C3c and of haptoglobin (HP) were measured in blood samples obtained from F2 piglets (n = 457) of a porcine F2 resource population before and after Mycoplasma hyopneumoniae, Aujeszky's disease virus (Suid herpesvirus I, SuHVI) and porcine reproductive and respiratory syndrome virus (PRRSV) vaccination at 6, 14 and 16 weeks of age. Animals were genotyped at 88 autosomal markers. QTL analysis was performed under the line cross and the half sib. Phenotypic data were adjusted for systematic effects by mixed models with and without repeated measures statement. In total, 46 and 21 estimated QTL positions were detected with genome-wide significance at the 0.05 and 0.01 level, respectively. The proximal region of SSC2 (orthologous to HSA11 0-70 Mb), the distal region of SSC4 (HSA1 95-155 Mb), and the intermediate region of SSC16 (HSA5 0-73 Mb and 150-174 Mb) showed a clustering of estimated QTL positions for complement activity based on the different models. A common genetic background, i.e. a single true QTL, might underlie these QTL positions for related traits. In addition, QTL for antibody titres were detected on SSC1, 2, 6 and 7. With regard to number and magnitude of their impact, QTL for humoral innate immune traits behave like those for other quantitative traits. Discovery of such QTL facilitates the identification of candidate genes for disease resistance and immune competence that are applicable in selective breeding and further research towards improving therapeutic and prophylactic measures.

The purpose of this study was to test fermentation, for its products of a Chinese medicinal mushroom, Ganoderma lucidum, cultured by submerged fermentation for its effect on growth performance and immunocompetence in weanling piglets. In Experiment 1, 72 weanling piglets were allotted to one of four treatments receiving these fermentation products (GLF, expressed as amount of beta-glucans) at 0 (control), 50, 100, and 150 mg/kg feed for 4 weeks. The results showed that at a supplementation level of 50 mg/kg feed, GLF caused the best growth performance, the highest pseudorabies antibody titre, and a decrease of blood glucose level. It was also demonstrated that GLF up-regulated the cell-mediated immune response related cytokines (IL-2, IFN-gamma, and TNF-alpha) expression in different lymphoid tissues. After challenging with porcine circovirus (PCV) type 2 (Experiment 2), a supplementation with 50 mg GLF per kg feed also inhibited PCV-2 virus amplification, and ameliorated lymphocyte depletion in different lymphoid tissues. Conclusively, feed supplemented with GLF at 50 mg/kg could be beneficial to counteract the physiological stress in weanling piglets.

Pseudorabies virus (PRV) propagated in rabbit kidney-derived RK-13 cells (PRV-RK) was neutralized by serum obtained from specific pathogen-free pigs through the activation of complement. The virus-neutralizing activity of swine serum was lost after treatment with ethylene glycol-bis-aminoethylether-N,N,N',N'-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA). Anti-C1q and anti-IgM antibodies also inhibited virus-neutralizing activity. Though IgG-depleted swine serum neutralized PRV, IgM and IgG-free swine serum lost virus-neutralizing activity. Pre-incubation of swine serum with RK-13 cells, but not with swine kidney-derived CPK cells, at 4 degrees C eliminated the virus-neutralizing activity to PRV-RK. Results indicated that swine serum contained natural IgM against an antigen(s) on the RK-13 cell surface and that this surface antigen was integrated into the PRV envelope during the budding process. Thus the natural IgM in swine serum reacted with the RK-13 antigen on the viral envelope, activated the complement cascade and neutralized the PRV-RK.

Industrial swine production is affected by several serious viral diseases, such as pseudorabies, hog cholera, porcine reproductive and respiratory syndrome, which are frequently complicated with the increased incidence of bacterial complications such as Actinobacillus pleuropneumoniae (APP). This clinical observation is suggestive of a virus-bacteria synergism on the pathogenesis. One hypothesis is that viruses induce polymorphonuclear cell (PMNs, primarily neutrophils) dysfunction resulting in defective antibacterial resistance. The purpose of this study was to use the pseudorabies virus (PrV) as a model to explore the possibility of virus-induced PMN dysfunctions in pigs. The goals were to evaluate, in ex vivo settings, the oxidative burst (OB) function of pig PMNs, and to evaluate whether PrV could affect these responses to APP. We found that PrV served as a mild OB stimulant (2-fold) to pig PMNs, which also launched a significant burst to phorbol 12-myristate 13-diacetate (PMA; 61-fold), to non-opsonized, heat-killed and formaldehyde-fixed APP (8-fold), and to normal pig serum-opsonized APP (34-fold). Interestingly, the PMA-induced OB could be reduced 50-70% by preincubating PMNs with PrV, and the critical target was not likely the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase itself. Inactivated PrV was as efficient as viable PrV at exerting the inhibitory effect. On the other hand, PrV exerted a primarily additive effect on APP-induced OB, when the cytotoxic effect of APP on PMNs was avoided. The current finding suggests the possibility that activated PMNs are susceptible to PrV-induced dysfunction, and that the PrV-APP synergism may require upstream stimuli of PMNs to be initiated.

In order to examine the loci factor B (BF), C3, corticotropin releasing hormone (CRH), DQB, DRB, haptoglobin (HP) and transforming growth factor beta 1 (TGFB1) for association with traits of humoral, specific and unspecific defence F2-animals of a porcine resource family were genotyped at single nucleotide polymorphisms within these loci. Haemolytic complement activity in the alternative and classical pathway, C3c and haptoglobin serum concentration and antibody titres were determined immediately prior and at days 4 and 10 after vaccinations against Mycoplasma hyopneumoniae (Mh), Aujeszky's disease virus, and porcine reproductive and respiratory syndrome virus at 6, 14 and 16 weeks of age, respectively. Analysis of variance revealed association of BF, HP and DRB with C3c serum concentration. The trend of haemolytic complement activity and C3c serum concentration during the experiment was affected by the interaction of DQB genotype and time of measurement. Association with antibody titres were found for BF, DQB and DRB. Results of the mixed model analyses were confirmed by quantitative transmission disequilibrium test that showed linkage and association with antibody titres, complement activity and acute phase reaction at certain times of measurement. The findings promote the importance of the candidate genes for humoral mechanisms of unspecific and specific defence that provide natural resistance against many pathogens., (Copyright 2004 Elsevier B.V.)

Pseudorabies virus (PRV) may cause abortion, even in the presence of vaccination-induced immunity. Blood monocytes are essential to transport the virus in these immune animals, including transport to the pregnant uterus. Infected monocytes express viral proteins on their cell surface. Specific antibodies recognize these proteins and should activate antibody-dependent cell lysis. Previous work showed that addition of PRV-specific polyclonal antibodies to PRV-infected monocytes induced internalization of viral cell surface proteins, protecting the cells from efficient antibody-dependent lysis in vitro (immune-masked monocytes). As a first step to reach the pregnant uterus, PRV has to cross the endothelial cell barrier of the maternal blood vessels. The current aim was to investigate in vitro whether immune-masked PRV-infected monocytes can transmit PRV in the presence of virus-neutralizing antibodies via adhesion and fusion of these monocytes with endothelial cells. Porcine blood monocytes, infected with a lacZ-carrying PRV strain, were incubated with PRV-specific antibodies to induce internalization. Then, cells were co-cultivated with endothelial cells for different periods of time. Only PRV-infected monocytes with internalized viral cell surface proteins adhered efficiently to endothelial cells. LacZ transmission to endothelial cells, as a measure for monocyte-endothelial cell fusion, could be detected after co-cultivation from 30 min onwards. Virus transmission was confirmed by the appearance of plaques. Adhesion of immune-masked PRV-infected monocytes to endothelial cells was mediated by cellular adhesion complex CD11b-CD18 and subsequent fusion was mediated by the virus. In conclusion, immune-masked PRV-infected monocytes can adhere and subsequently transmit virus to endothelial cells in the presence of PRV-neutralizing antibodies.

The influence of interferon (IFN)-alpha on the in vitro differentiation of myeloid porcine dendritic cells (DC) was evaluated as the ability of the DC to stimulate to cell proliferation in a mixed leukocyte reaction (MLR), and as their ability to produce cytokines at exposure to bacterial and viral preparations. Porcine monocytes were enriched from purified peripheral blood mononuclear cells (PBMC) by plastic adherence and cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 or in GM-CSF, IL-4 and IFN-alpha. After 5 days of culture, the cells developed a dendritic morphology and the proportion of cells expressing MHC class II and B7 molecules was increased as determined by flow cytometry. Dendritic cells, differentiated for 5 days in GM-CSF, IL-4 and IFN-alpha, were able to stimulate both allogeneic and syngeneic PBMC to proliferation in an MLR. The DC produced the Th1 associated cytokines IFN-alpha at Sendai virus stimulation, and IL-12 at stimulation with plasmid DNA (pre-incubated in the presence of lipofectin), heat-inactivated Actinobacillus pleuropneumoniae, UV-inactivated Aujeszky's disease virus and live Sendai virus. The heat-inactivated bacteria and Sendai virus also induced production of the Th2 associated cytokines IL-10 and IL-6. The addition of IFN-alpha during differentiation of DC in GM-CSF and IL-4 enhanced their ability to stimulate allogeneic and syngeneic MLR, but did not alter their ability to produce cytokines., (Copyright 2003 Elsevier Science B.V.)

The complement system catalyzes direct lysis of micro-organisms and modulates phagocytosis, inflammation, humoral and cellular immune responses. Since the complement protein C3 is the central component within all pathways of complement activation, C3 is a candidate gene for complement activity and also for improved protection against many pathogens. The pig C3 gene was sequenced, screened for polymorphisms, and analyzed for association with hemolytic complement activity of the alternative and classical pathway (AH(50), CH(50)). C3c serum levels and haptoglobin (HP) serum concentrations were measured before and after vaccination against Mycoplasma hyopneumoniae, Aujeszky virus, and porcine reproductive and respiratory syndrome virus in F2 animals of a pig resource population based on crossbreeding of Duroc and Berlin Miniature Pig. The genomic C3 sequence covers 444 bp of promoter region, 41 exons and 40 introns, as well as 881 bp of the 3'-flanking region. The cDNA codes for a 1,661-amino acid precursor C3. Five polymorphic sites were detected in the 5'-UTR, intron 13, exon 15, exon 30, and the 3'-UTR. Within the resource population two haplotypes were found to segregate. Analysis of variance applying a repeated measures model revealed a significant effect of the interaction of C3 genotype and time of measurement relative to immunization on CH(50), AH(50,)and C3c that is likely to be due to variation of C3 expression. In contrast, the time course of the HP acute-phase reaction is not associated with C3 genomic variation. The association of C3 with complement activity indicates the importance of C3 as a candidate gene for natural resistance to micro-organisms, although the causative polymorphism modulating the expression of C3 remains to be delineated.

Only few data are available on the effect of fumonisins on the immune response. The aim of the present study was to examine whether dietary fumonisin B1 (FB1) has any effect on the humoral and cellular immune response in weaned pigs, depending on the dose and the time of toxin exposure. Fusarium moniliforme fungal culture was added to the experimental animals' diet to ensure an FB1 intake of 1, 5 and 10 ppm (first experiment) or 100 mg per animal per day (second experiment). The control animals were fed a toxin-free diet. In order to determine the immune response, the animals were vaccinated against Aujeszky's disease with inactivated vaccine (Aujesping K, Phylaxia-Sanofi, Budapest, Hungary). Specific and nonspecific in vitro cellular immune response was measured by the lymphocyte stimulation test (LST) induced by PHA-P, Con A, LPS and inactivated suspension of the Aujeszky's disease virus. Humoral immune response, e.g. specific antibody titre, was measured by the virus neutralisation (VN) test. None of the immunological parameters examined showed significant differences between groups. It could be concluded that fumonisin B1 had no significant effect on the humoral and cellular specific and nonspecific immune response when fed in a high dose (100 mg/animal/day for 8 days) or in a low concentration even for a longer period (1, 5 and 10 ppm for 3-4 months).

A nonclinical study was conducted to characterize the replication behavior of a modified live gE-deleted pseudorabies virus (PRV MS+1) in swine and potential for reversion to virulence after animal passages. Two to 3 week-old weaned pigs, negative for PRV, were maintained in isolation and challenged by intranasal instillation. For the first passage, 6 pigs were given 1 mL of PRV MS+1 (10(7.3)TCID(50)/mL) and 2 were necropsied at 3, 4 and 5 days post-inoculation (PI). Brain and secondary lymphoid tissues were collected, homogenized and the supernatants individually pooled for virus isolation, and PRV was recovered from each sample. No clinical signs of PRV infection were observed, but each pig had a nasal swab suspect or positive for PRV. For the second passage, 5 pigs were given 1 mL of the homogenate of mixed tissues from 1 animal in the previous passage (PRV at 10(1.9) TCID(50)/mL). At 5 days PI, all pigs were necropsied, and PRV was not recovered from their tissue homogenates or nasal swabs, and no clinical signs were observed. During a second attempt at a second passage, tissue homogenates from all pigs in the first passage (PRV at approximately 10(1.7)TCID50(50)/mL) were pooled and used to inoculate 15 pigs with 2 mL for 3 consecutive days. Ten pigs were monitored for clinical signs and seroconversion through 21 days PI, and 5 pigs were necropsied at 5 days PI. No clinical signs or PRV antibodies were detected in the 10 monitored pigs, and no PRV was recovered from the homogenates or nasal swabs of the 5 necropsied pigs. Thus, no evidence of reversion to virulence was demonstrated in pigs given the attenuated PRV.

Swine neutrophils were quantitatively examined for the direct and indirect migratory responses to Actinobacillus pleuropneumoniae (APP) in vitro and the effects of pseudorabies virus (PrV), frequently co-infecting with APP, were also observed. About 30% of swine neutrophils responded to viable APP, while 3.2% of the neutrophils responded to 0.1% casein which served as the control. The migration of APP was not affected by preincubation of neutrophils with PrV, which inhibited the random migration. When the random migration was normalized to 1, the chemotactic indices for APP, opsonized-APP and casein were 64, 70 and 8.5, respectively. Heat-killed APP or E. coli lipopolysaccharide stimulated the production of interleukin-8 activity by adherent peripheral blood mononuclear cells (PBMC). Preincubation of PBMC with PrV inhibited the production of neutrophil attractant activity when stimulated with heat-killed APP. The results suggested that the direct chemotaxis of neutrophils to viable APP might contribute to early infiltration in Actinobacillus pleuropneumonia, and that PrV might inhibit indirect recruitment of neutrophils to infected lungs by compromising the functions of PBMC.

An adjuvant effect of invertebrate DNA has been attributed to its relative high frequency of unmethylated CpG dinucleotides. Here we describe the interferon-alpha (IFN-alpha) and interleukin-6 (IL-6) inducing properties of a commonly used eukaryotic expression vector, pcDNA3, in porcine leukocytes. The magnitude of the cytokine response was compared to that induced by the synthetic ds RNA analogue poly(I):poly(C), inactivated preparations of Aujeszky's disease virus (ADV) and the Gram-negative bacteria Actinobacillus pleuropneumoniae. The plasmid, as well as poly(I):poly(C), required lipofectin to induce IFN-alpha production whereas both preparations induced IL-6 irrespective of preincubation with lipofectin. However, the nucleic acid-induced levels of IL-6 were low compared to those induced by A. pleuropneumoniae. The IFN-alpha response elicited by pcDNA3 in the presence of lipofectin was as high as, or higher than that induced by ADV. Interestingly, also A. pleuropneumoniae induced a substantial production of IFN-alpha when preincubated with lipofectin. Plasmid expression was not necessary for induction of IFN-alpha. Furthermore, the IFN-alpha inducing capacity of pcDNA3 was not reduced when the two predicted immunostimulatory sequences 5'AACGTT3' were deleted. Nor did the ability of the plasmid to induce IFN-alpha production decrease when the ampicillin resistance (ampR) gene was replaced with the kanamycin resistance (kanR) gene. However, methylation of all cytidines in CpG dinucleotides of pcDNA3 abolished the IFN-alpha inducing capacity. These in vitro results indicate an immunomodulatory role of bacterial DNA also in the pig. Unmethylated CpG dinucleotides are crucial for induction of IFN-alpha by the plasmid, but other CpG motifs than those within the 5'AACGTT3' sequences of the ampR gene contribute to this induction in porcine cells.

For in vivo determination of innate and memory immune reactions we have implanted sterile gelatin sponges subcutaneously in swine for the monitoring of the following immunological parameters: 1. Analysis of local cell population phenotypes after vascularization of the gelatin sponges using flow cytometry. 2. Comparative analysis of sponge-infiltrating cells after loading with viral antigen in primed as well as naive animals. 3. Performance of reverse transcription quantitative competitive PCR (RT-qcPCR) for the detection of porcine cytokine mRNA indicative for IFN-gamma, IL-2, IL-4, IL-8 and IL-10. The in vitro analysis, e.g. by re-exposure to viral antigens, allows the determination of cytokine reaction patterns of sponge derived cells, draining lymph node cells as well as PBMC of the same individual. Studies of innate reactions and modulation of cellular infiltration in transplanted gelatin sponges are possible. Functional assays, e.g. cell-mediated cytotoxicity, antigen specific cell proliferation, using sponge-derived cells will provide additional information about the suitability of the model for example in vaccine potency tests.

Immune capacity traits show considerable genetic variation in outbred populations. To identify quantitative trait loci (QTLs) for immune capacity in the pig, various measures of immune function (total and differential leukocyte counts, neutrophil phagocytosis, mitogen-induced proliferation, IL-2 production, and virus induced IFN-alpha production in whole blood cultures, and Ab responses to two Escherichia coli antigens) were determined in 200 F2 animals from a wild pig-Swedish Yorkshire intercross. The pedigree has been typed for 236 genetic markers covering all autosomes, the X chromosome and the X/Y pseudoautosomal region. Through interval mapping using a least-squares method, four QTLs with significant effects were identified; one for total leukocyte counts, one for mitogen-induced proliferation, one for prevaccination levels of Abs to E. coli Ag K88, and one for Ab response to the O149 Ag. In addition, several putative QTLs were indicated. The results from the present study conclusively show that it is possible to identify QTLs for immune capacity traits in outbred pig populations by genome analysis.

Cell-mediated immunity is a major component of the host defense system against viral infections. Since interleukin (IL)-12 has been shown to be a potent stimulus for the in vivo generation of interferon-gamma (IFN-gamma)-producing T cells (i.e. Th-1 cells) in laboratory animals, we evaluated the effect of IL-12 on the cellular immune response of pigs to vaccination against pseudorabies virus (PrV), a herpesvirus of swine. The magnitude of the cellular immune response was measured by IFN-gamma ELISPOT analysis of peripheral blood mononuclear cells (PBMC) from pigs which had been immunized twice, at 2-week intervals, with either, modified live virus (MLV) alone or with a commercial inactivated PrV vaccine with or without the coadministration of human recombinant IL-12 (HrIL-12). No significant differences in the titer of virus-neutralizing antibodies or in the intensity of the virus-specific lymphoproliferative response among the different treatment groups was observed. However, the number of virus-specific IFN-gamma-producing cells among PBMC isolated from animals receiving the MLV vaccine was on average 3.5 times more than animals immunized with the inactivated vaccine (P = 0.01). Administration of the inactivated vaccine and IL-12 induced a two-fold higher frequency of virus-specific IFN-gamma-producing cells from that induced by the inactivated vaccine alone (P < 0.05). Despite this enhancement, the level of protection from lethal PrV challenge provided by the inactivated vaccine in combination with IL-12 was the same as that induced by the inactivated vaccine alone. Both of these vaccination regimes provided significantly lower levels of protection than those afforded by the MLV vaccine. This study demonstrates that an inactivated PrV vaccine is a poor inducer of virus-specific IFN-gamma-producing cells and that this response can be enhanced by administration of exogenous IL-12. The data provides evidence of a dichotomy in the humoral and cellular immune responses of pigs to a viral antigen and implies the existence of a Th-1/Th-2 type regulation of the anti-viral immune response in pigs.

A NYVAC vaccinia vector containing genes for pseudorables virus glycoproteins gII and gp50 was administered to pigs to determine if it would have a greater protective effect than a vector containing the gene for gp50 alone. Both NYVAC vectors protected pigs similarly from virulent pseudorabies virus challenge.

Weaned pigs exposed daily to either unpredictable draught (experiment 1) or intermittent unpredictable draught (experiment 2) showed different lymphocyte blastogenic responses after mitogenic stimulation with phytohaemagglutinin (PHA) and concanavalin A (ConA). In both experiments PHA skin test responses were lower for draught exposed pigs than for control pigs and leucocyte numbers or profiles were altered compared to those of control pigs. Superoxide production and chemiluminescence of porcine granulocytes were similar for draught exposed and control animals. Furthermore, serum globulin content did not differ significantly between pigs in the experimental and control room. The strong increase in serum gamma-globulin after the Aujeszky Disease Virus (ADV)-challenge was the same for draught exposed and control pigs. The same held for the lymphocyte blastogenic response with ADV protein as antigenic stimulus. The present study shows the effects of climatic stress on immunological reactivity, which may reflect a homeostatic disturbance of the pig's immune system elicited by exposure to unpredictable draught.

Research was undertaken to study the effect of a 'three times a year vaccination schedule in sows' against Aujeszky's disease (AD) on the maternal immunity of their offspring at a large pig farm. Only gI-negative sows and piglets were induced in the study. The maternal immunity was measured in the 8-week-old offspring of 6 gilts and 13 sows, vaccinated in early pregnancy and in 8-week-old piglets born from 12 gilts and 21 sows vaccinated 3 weeks prior to farrowing. At an age of 8 weeks only 5 percent of the piglets, born from sows vaccinated during early pregnancy showed antibody titres against AD of > 1:40 in their serum. In piglets born from sows vaccinated 3 weeks prior to farrowing more than 50 (54.4) percent of the piglets showed antibody titres > 1:40 in their serum. It was concluded that using a 'three times a year vaccination schedule' in a gI negative herd may lead to groups of seronegative piglets on breeding and multiplier farms. The implications are discussed.

We examined primary and memory isotype-specific antibody responses directed against pseudorabies virus in serum and mucosal fluids of pigs with and without passively acquired maternal antibody, and we studied the relationship between these responses and protection against virus challenge. Pigs were inoculated intranasally with the virulent NIA-3 strain or the avirulent Bartha strain, or they were inoculated IM with an inactivated vaccine containing the Phylaxia strain. Ten weeks later, all pigs were challenge-exposed intranasally with strain NIA-3. Only pigs that were without passively acquired antibody at the time they were inoculated with virulent virus appeared to have complete protective immunity against challenge exposure, as evidenced by lack of clinical signs of pseudorabies and lack of virus excretion. In contrast, pigs inoculated with strain Bartha or with the inactivated vaccine developed fever, had a period of growth arrest, and excreted virus after challenge exposure. In pigs without passively acquired antibody, intranasal inoculation with strains NIA-3 or Bartha was followed by primary IgM and IgA responses in serum and in oropharyngeal fluid as well as primary IgG1 and IgG2 responses in serum. Intramuscular inoculation with the inactivated vaccine induced primary serum IgM, IgG1, and IgG2 responses, but no mucosal responses. Challenge exposure of pigs that had been inoculated with the Bartha strain or the inactivated vaccine was followed by clear memory responses in serum and in oropharyngeal fluid. In contrast, challenge exposure of pigs that had been inoculated by the virulent NIA-3 strain was not followed by memory responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody titres against Aujeszky's disease virus (ADV) in the plasma of slaughter pigs were compared with respect to the site of sampling, namely on the farm (one day before delivery to the slaughterhouse) and in the slaughterhouse during slaughter. No significant differences were noted in the antibody titres against ADV with respect to the different sampling sites. Blood samples collected at the slaughterhouse can be used for monitoring and surveillance systems within the framework of organised pig health care.

In a cohort study, 40 pig finishing herds were selected: twenty pig herds with a low and twenty pig herds with a high prevalence of several pathological lesions recorded at slaughter in a six-month period before the start of the study. Blood samples were taken from 20 pigs per herd at the end of the finishing period to investigate haematological and clinicochemical profiles. There was only a significant difference in serum albumin concentration between the low and high lesion prevalence groups. There were distinct differences in blood profiles between pig herds, but also between batches of pigs within a herd, housed in different compartments. Differences between castrated males and gilts were also demonstrated with respect to mean values of the blood variables haemoglobin, iron, copper, beta-globulin, eosinophils and segmented neutrophils. However, the differences were not of a biologically important magnitude. The mean values of the blood variables pepsinogen and lymphocytes differed significantly between pig herds from the two participating integration groups. Pigs with a higher albumin concentration, a lower gamma-globulin concentration, a higher copper concentration and a higher creatine kinase activity in serum showed a higher daily weight gain.

The infectivity and potential immunosuppressive effects of Pseudorabies virus (PRV) was evaluated in swine peripheral blood mononuclear cells (PBMC). Virus progeny titers and viral DNA synthesis at various intervals post-inoculation revealed the replication of PRV in both peripheral blood monocytes and lymphocytes; however, replication in lymphocytes was restricted compared with monocytes. PRV infection resulted in the damage and death of monocytes. Although PRV did not appear to affect the viability of the lymphocytes, PRV infection suppressed lymphocyte functions such as proliferation and interleukin-2 (IL-2) synthesis in response to Concanavalin A. This immunosuppression was dependent upon the multiplicity of infection (MOI) of infectious PRV. UV-inactivated PRV was not immunosuppressive. There was no effect of PRV on natural killer (NK) cell activity. The reduction of lymphocyte proliferation by PRV was not reversible by the addition of supernatant containing porcine IL-2 and non-infected monocytes to the infected cultures. The results from these in vitro studies demonstrate that PRV can infect and cause immunosuppressive effects on swine PBMC. These effects may explain the potential role of PRV in predisposing infected pigs to secondary infection and support the hypothesis that PRV can spread systemically by infected PBMC in blood and lymph.

The spread of Aujeszky's disease virus (ADV) from nasal mucosa via the olfactory pathway was studied in HPCD pigs. ADV antigen was detected in the epithelial cells, nasal gland cells, olfactory nerve cells and peripheral nerve fibres in the nasal cavity and in neuroglial cells in the olfactory bulb. Results indicate that the olfactory pathway is one of the most important neuronal pathways of ADV infection in pigs.

The possibility of using sow's milk to detect antibodies to the gI protein of Aujeszky's virus was studied by the present authors. Antibody titres in serum (collected less than 30 days ante partum) and colostrum from sixty-three sows were determined using a commercially available ELISA; twenty-four of thirty-seven animals vaccinated with gI-positive vaccines showed an antibody response in both colostrum and serum. The titre in colostrum was significantly higher than it was in serum. Antibodies could not be detected in serum or in colostrum of the twenty-six animals which were only vaccinated with gI-negative vaccines. Subsequently, anti-gI titres were determined in milk collected during lactation. Antibodies were also detectable in milk of animals showing high colostrum titres (greater than 128) for at least seventeen days. This period decreased as the initial colostrum titre became lower. Antibodies could still be detected in milk of animals vaccinated with gI-positive vaccine, which were sero-negative at the time of the experiment. Milk therefore seems to be a useful alternative to serum in detecting anti-gI antibodies in sows.

Two Aujeszky's disease virus glycoprotein genes, gX and g1, have been used to produce deletion mutants which have then been developed into vaccines. These deletions then allow differentiation between pigs infected with wild type virus and those given the vaccine. It is not clear whether the glycoproteins encoded for by these genes are needed to induce a full protective immune response, in which case deletion mutants would suffer from lack of potency. To test this, commercially available Aujeszky's virus vaccines which lacked either gX or g1 were compared and isogenic constructs were made which differed only in the absence or presence of gX and, or, g1. These constructs and vaccines were used to vaccinate the natural host of Aujeszky's disease, the pig, and potency was measured using challenge with wild type virus. In all cases vaccines which lacked g1 performed significantly less well than those in which g1 was present, whereas deletions of gX had no significant effect on vaccine performance.

The introduction of Aujeszky's disease virus into a herd of pigs usually results in a rapid spread of the virus and a high percentage of pigs become seropositive. However, herd monitoring for the virus occasionally reveals a single seropositive breeding pig, referred to as a single reactor. The seropositive status of single reactors may be due to previous vaccination against Aujeszky's disease, or to exposure to a field strain of the virus, or to a false positive reaction in the serological assay. During a monitoring programme in Minnesota, 30 pig herds with single serological reactors were detected. Twenty-seven of these single reactors from 19 herds were segregated from their herds immunosuppressed with dexamethasone. Aujeszky's disease virus was isolated from four of the 27 pigs. Three of the four herds subsequently had outbreaks of Aujeszky's disease, suggesting that some single reactors were infected with Aujeszky's disease virus and had the potential to spread the virus within and between herds.

Antibodies to Aujeszky's disease virus (ADV) glycoproteins gII, gIII, and gp50 were compared using four in vitro tests. Antibodies generated by vaccination with a modified-live vaccine (MLV) were also compared. The serological assays employed were: serum neutralization test (SNT), complement facilitated serum neutralization test (C'SNT), complement-mediated cytolysis and antibody dependent cellular cytotoxicity (ADCC). Pigs were immunized with single glycoproteins twice 14 days apart, or once with the modified-live vaccine. Fourteen days after the second immunization, sera were collected. Virus neutralizing activity (SNT) was demonstrated in the sera from all pigs immunized with gp50 and in one out of three immunized with gIII. Sera from the MLV group all had neutralization titers higher than animals immunized with single glycoproteins. Addition of guinea pig complement to the serum neutralization test (i.e., C'SNT) produced an enhancement of antibody titers in all groups except the pigs immunized with gIII. The complement-mediated cytolysis test rendered antibody titers similar in magnitude for all pigs immunized with single glycoproteins, but slightly lower than values for MLV vaccinated pigs. ADCC activity was clearly displayed in sera from pigs immunized with gIII or vaccinated with MLV, whereas sera from pigs immunized with gII or gp50 had a minimal response. The results indicate that the relative efficiency of antibodies against ADV glycoproteins in protection should be considered for selecting or producing gene-deleted strains for use in vaccine production.

Studies on the maternal state of immunity were done in 4-10 week-old piglets born of vaccinated sows free from Aujeszky's disease. The sows were inoculated with three different vaccines: two live vaccines (Aescovac Aujeszky) and the MK25 vaccine) and an inactivated vaccine (Geskyvac). The maternal state of immunity of the piglets was assessed from the titre of the virus-neutralizing (VN) antibodies in the blood. To do so, samples were taken from 300-400 4-10-week-old piglets per vaccine inoculated in the sow. At the same time, the VN titre of the blood of the sow was determined. The VN titres were highest in those sows which were vaccinated with Geskyvac. There was an obvious correlation between the VN titre of the sow and the maternal VN titres of the piglets. As a result, the maternal state of immunity in the sows vaccinated with Geskyvac also was highest. The value of the maternal VN titres in the assessment of maternal immunity was tested by exposing 28-37-day-old piglets showing various maternal VN titres to intracerebral challenge with 2000 TCID50 of Aujeszky disease virus (Van Doorn strain). Piglets showing VN titres higher than 1:8 were usually found to be immune against this challenge.

This serologic study was done to gain information on the spread, maintenance, and effect upon performance of five porcine viruses. Blood samples were taken from two groups of 8- to 11-week-old pigs from a large number of Indiana swine herds in a performance-testing station 1 week after entry, 7 weeks after entry (one group only), and at slaughter. The sera were tested by indirect fluorescent antibody tests for antibodies to transmissible gastroenteritis virus (TGEV), swine influenza virus (SIV), hemagglutinating encephalomyelitis virus (HEV), porcine adenovirus (PAV), and pseudorabies virus (PRV). Seroconversions to TGEV, HEV, and PAV occurred in a group of pigs entered in May and slaughtered in August (group 1). In the group that was entered in October and slaughtered in January (group 2), pigs developed antibodies to SIV, HEV, and PAV, but not to TGEV. Only 1 of the 434 pigs tested had antibodies to PRV, and there were no seroconversions to this virus. The only statistically valid effect of infection on performance was found in group 1 pigs, which had seroconverted to TGEV during the first 7 weeks of their stay. These pigs gained 0.077 kg less per day than pigs that did not develop antibodies to TGEV during that period. The pattern of serologic reactions was indicative of a relatively slow spread of these viruses in the groups. We interpret this as supporting the concept that a relatively slow spread of these viruses through large groups of pigs kept under conditions that are less than optimum for virus spread may be an important means of their interepizootic survival.

In pseudorabies virus (PRV) infection of pigs, antibody-dependent cellular cytotoxicity (ADCC) may be an early defense mechanism. Peripheral blood leukocytes (PBL) and pulmonary macrophages mediate ADCC activity. Antibody-dependent cellular cytotoxicity against PRV-infected target cells was assessed, and the effect of infection of cells having an ADCC-effector function was determined. Although pulmonary lavage cells (PLC) had ADCC activity, in vitro infection of PLC led to PRV replication, loss of cell viability, and loss of ADCC activity. In contrast, infection of PBL did not lead to replication, decreased cell viability, or reduced ADCC activity, compared with those in non-infected controls. Measuring ADCC activity in a longitudinal study revealed that PBL from neonates had lower ADCC activity than did PBL from pigs greater than 3.5 months old. Peripheral blood leukocytes and not PLC may have a greater role in control of PRV dissemination in the pig. The difference in activity between cells from neonates and older pigs might explain, in part, the age dependency in the severity of the disease.

Twenty Aujeszky's disease (AD) virus antigens were demonstrated by crossed immunoelectrophoresis in a Triton-X-100 detergent extract of virus-infected PK-1a cells. Eight of these antigens were shown to be glycosylated based on their ability to be specifically bound by the lectin Ricinus communis agglutinin II. Pigs nasally infected with AD virus showed a significant serum antibody titer to seven of the known glycosylated antigens and to four additional antigens. The antibody titer to these antigens persisted for at least 116 days. Pigs which were vaccinated parenterally with the whole detergent extract survived a nasal challenge of 10(8 . 5) PFU of virulent AD virus. The antibody response of these vaccinated pigs on the day of challenge was essentially identical to the recovery response previously observed in non-vaccinated nasally infected pigs. These results indicate that the optimum components of future AD virus subunit vaccines and their complementary diagnostic reagents should be selected from these 11 antigens.

The effects of challenge exposure on the humoral and cellular immune responses in pseudorabies vaccinated swine were studied in 84 barrows. The pigs were divided into seven groups and challenge exposed to a virulent strain of pseudorabies virus on months 1, 3, 5, 8, 10, 12 and 14 after vaccination. The pigs were vaccinated with commercial attenuated and inactivated pseudorabies virus vaccines. The protection conferred by vaccination was equally effective with both types of vaccines. The levels of cellular and humoral immunity after challenge exposure in pigs vaccinated with either type of vaccine were similar. The cell-mediated immune response can be effectively used for the early detection of pigs exposed to pseudorabies virus. Virus isolation attempts from the brain and spleen in most of the vaccinated pigs were unsuccessful.

Embryo transfer was used to conserve genetic material from 2 swine herds seropositive for pseudorabies virus (PRV). Embryos (n = 805) were recovered from 38 PRV-seropositive Duroc sows in Iowa and, after 4 to 10 hours' culture and shipment to Illinois, were transferred to 34 recipients from a herd seronegative for PRV. All recipients remained seronegative for PRV, and 22 of the recipients farrowed 208 pigs (189 alive) that also were seronegative for PRV. There was no evidence of PRV in the embryo recovery medium or in the uterine and oviductal cells recovered with the embryos. Transfer of morulae resulted in higher (P less than 0.02) farrowing rates than did transfer of 4- to 8-cell embryos, but litter size was not affected.

A thymidine kinase deficient mutant of the Indiana-Funkhauser strain of pseudorabies virus was tested for its ability to stimulate protective immunity in mice and young pigs. Mice vaccinated intraperitoneally were protected from morbidity and mortality when challenged with 50 LD50 of virulent pseudorabies virus. Eight week old pigs were protected from serious morbidity and mortality when challenged with virulent pseudorabies virus. The thymidine kinase mutant was not shed from the nasal passages of pigs vaccinated intramuscularly, but did not prevent shedding of challenge virus.

An indirect solid-phase microradioimmunoassay (IRIA) was developed for detection and quantitation of antibodies to pseudorabies virus (PRV) in swine serum. Qualitative results of the IRIA compared closely with results of the serum neutralization test (NT) and the microimmunodiffusion test (MIDT). The IRIA was more sensitive than the NT for detection of antibodies to PRV in swine serum. The IRIA result is expressed numerically. With the IRIA and NT, antibody to PRV was first detectable in 3 experimentally infected pigs at 9 days after inoculation. With MIDT, antibody was detected in the 3 experimentally infected pigs at 9 days after inoculation. With the MIDT, antibody was detected in the 3 experimentally infected pigs at 7, 8, and 9 days after inoculation. The IRIA results are obtainable within a few hours; the NT and MIDT require 48 hours for completion.

A countercurrent immunoelectrophoresis test was developed for the detection of precipitating antibodies to pseudorabies virus in pig serum. The precipitation reaction occurred only between the pseudorables antigen and the homologous porcine antiserum. The sensitivity of the method was compared to that of the serum neutralization test. On the basis of its sensitivity, its specificity and the rapidity with which the results were obtained, the countercurrent immunoelectrophoresis may become a potentially valuable screening method to test large numbers of porcine serum.