Comparison of different prime-boost regimes with DNA and recombinant Orf virus based vaccines expressing glycoprotein D of pseudorabies virus in pigs.

Both DNA and Orf virus (ORFV; Parapox virus) based vaccines have shown promise as alternatives for conventional vaccines in pigs against pseudorabies virus (PRV) infection causing Aujeszky's disease. In the present study we evaluated the efficacy of different prime-boost regimes in pigs in terms of immunogenicity and protection against challenge infection with PRV. The different prime-boost regimes consisted of the homologous prime-boost regimes (DNA followed by DNA or ORFV followed by ORFV) and the heterologous prime-boost regimes (DNA followed by ORFV and ORFV followed by DNA), all based on glycoprotein D (gD) of PRV. Moreover, we compared the efficacy of the different prime-boost regimes with the efficacy of a conventional modified live vaccine (MLV). The different prime-boost regimes resulted in different levels of immunity and protection against challenge infection. Most effective was the regime of priming with DNA vaccine followed by boosting with the ORFV based vaccine. This regime resulted in strong antibody responses, comparable to the antibody responses obtained after prime-boost vaccination with a conventional MLV vaccine. Also with regard to protection, the prime DNA-boost ORFV regime performed better than the other prime-boost regimes. This study demonstrates the potential of a heterologous prime-boost vaccination strategy against PRV based on a single antigen, and that in the natural host, the pig.

A DNA vaccine coding for gB and gD of pseudorabies virus (suid herpes type 1) primes the immune system in the presence of maternal immunity more efficiently than conventional vaccines.

DNA vaccines are capable of priming the immune system of neonates in the presence of maternal antibodies. However, it is still not clear whether the extent of priming and protection against challenge infections induced by a DNA vaccine in maternally immune newborns is better than that induced by conventional vaccines. To study this, we used the pseudorabies virus (PRV) infection model in the natural host, the pig. We compared the efficacy of a DNA vaccine with the efficacy of a conventional modified live vaccine (MLV) and an inactivated vaccine (IV) in maternally immune newborn piglets. We measured the priming of the immune response and the degree of protection against challenge infection for all vaccine types. We vaccinated piglets with or without maternal immunity twice, at the age of 5 and 9 weeks, and we assessed protection by challenge infection with virulent PRV at the age of 15 weeks. Vaccination with DNA or conventional vaccines induced both humoral and cell-mediated immune responses in maternally immune animals. DNA vaccination seemed not to suffer from suppression by maternal immunity and resulted in similar or stronger immune responses in maternally immune piglets as compared in naïve piglets. In contrast, vaccination with conventional vaccines resulted in weaker immune responses in maternally immune piglets than in naïve piglets. Moreover, DNA vaccination provided better protection against challenge infection in maternally immune piglets than in naive piglets, whereas vaccination with conventional vaccines did not.

Vaccine-induced T cell-mediated immunity plays a critical role in early protection against pseudorabies virus (suid herpes virus type 1) infection in pigs.

The aim of our study was to evaluate the relative importance of antibody and T cell-mediated immunity in protection against pseudorabies virus (suid herpes virus type 1) infection in pigs. We induced different levels of immune responses by using: (1) a modified live vaccine; (2) the same modified live vaccine with an oil-in-water (o/w) adjuvant; (3) an inactivated vaccine; and (4) the same inactivated vaccine with an o/w adjuvant. Subsequently, we challenged pigs with virulent pseudorabies virus (PRV). We demonstrated that best-protected pigs stood out by maintaining strong T cell-mediated immune (CMI) responses after challenge. Of the immune parameters tested, protection against virus shedding was correlated best with the magnitude of the IFN-gamma response of in vitro re-stimulated peripheral blood mononuclear cells (PBMC) with an additional role for PRV-specific IgG2 antibodies. The use of an o/w adjuvant resulted in higher antibody and CMI responses, in particular with an increased frequency of memory T helper blast cells of in vitro re-stimulated PBMC. However, this adjuvant-induced enhancement of the immune response had a limited additional effect on the efficacy of inactivated vaccines. This study suggests a major contribution of the CMI response in early protection against PRV infection and that PRV-induced IFN-gamma responses may serve as a suitable indicator for assessing the immune status of vaccinated pigs.

Virological kinetics and immunological responses to a porcine reproductive and respiratory syndrome virus infection of pigs at different ages.

The objective of this study was to measure the effect of two variables (pig age and virus strain) on selected responses (clinical signs, viraemia, virus excretion and seroconversion) of pigs following exposure to porcine reproductive and respiratory syndrome (PRRS) virus. Therefore, young (6 till 8 weeks old) and old (6 months old) pigs were infected with 3 different PRRSV strains, i.e. LV ter Huurne (LVTH), LV4.2.1 and SDSU#73. Regardless of the strain used for exposure, young pigs were more susceptible to infection as shown by a higher number of viraemic and virus excreting pigs. Strain differences were also evident. LV ter Huurne induced virus excretion in a higher number of pigs and with a higher virus titre, whereas SDSU#73 induced most severe clinical signs. LV4.2.1 induced viraemia and virus excretion in a low number of pigs. The kinetics of the antibody response differed per virus strain. The results presented here are useful in developing a less expensive standardised infection model, consisting of young pigs intranasally infected with a virulent PRRSV strain, to study the efficacy of new vaccine strains.

Effect of maternally derived antibodies on the clinical signs and immune response in pigs after primary and secondary infection with an influenza H1N1 virus.

The aim of this study was to determine the role of maternally derived antibodies (MDA) against an influenza H1N1 virus in the clinical protection of piglets and especially their effect on the development of the active immunity after an infection with a homologous influenza H1N1 virus. Twenty piglets with MDA and 10 piglets without MDA were housed together and inoculated twice with influenza H1N1 virus, at 7 and 15 weeks of age. Nine piglets without MDA were added to these groups at 12 weeks of age to be inoculated at 15 weeks of age only. Clinical signs, body temperature, growth performance, virus excretion, antibody responses, and influenza-specific T-cell response were monitored. It was shown that MDA protect piglets against the clinical consequences of a primary influenza infection, but that this protection is not complete. A short but significant rise in body temperature was observed and growth seemed to be inhibited due to the infection. Piglets with MDA shed virus for a longer period after an infection than piglets without MDA. Piglets with and without MDA were protected against the clinical consequences of a secondary infection. However, both after primary and secondary infection significant differences in immune responses were observed that indicated that pigs with MDA developed a weaker immunity than pigs without MDA. Furthermore, overall growth performances from weaning to slaughter show a trend in favour of pigs without maternal antibodies, compared to pigs with maternal antibodies, mainly caused by a significant better performance in the second half of the finishing period. The results of this study provide us insight in the role of MDA in clinical protection and their influence on active immunity after an influenza virus infection of pigs. Furthermore, it leads us to the discussion about the profitability of massive sow herd vaccinations in an attempt to increase MDA levels in piglets, taking into account the overall performance of these piglets and the possible effects on antigenic drift.

Analysis of the quality of protection induced by a porcine influenza A vaccine to challenge with an H3N2 virus.

Antigenic drift of swine influenza A (H3N2) viruses away from the human A/Port Chalmers/1/73 (H3N2) strain, used in current commercial swine influenza vaccines, has been demonstrated in The Netherlands and Belgium. Therefore, replacement of this human strain by a more recent swine H3N2 isolate has to be considered. In this study, the efficacy of a current commercial swine influenza vaccine to protect pigs against a recent Dutch field strain (A/Sw/Oedenrode/96) was assessed. To evaluate the level of protection induced by the vaccine it was compared with the optimal protection induced by a previous homologous infection. Development of fever, virus excretion, and viral transmission to unchallenged group mates were determined to evaluate protection. The vaccine appeared efficacious in the experiment because it was able to prevent fever and virus transmission to the unchallenged group mates. Nevertheless, the protection conferred by the vaccine was sub-optimal because vaccinated pigs excreted influenza virus for a short period of time after challenge, whereas naturally immune pigs appeared completely protected. The immune response was monitored, to investigate why the vaccine conferred a sub-optimal protection. The haemagglutination inhibiting and virus neutralising antibody responses in sera, the nucleoprotein-specific IgM, IgG, and IgA antibody responses in sera and nasal secretions and the influenza-specific lymphoproliferation responses in the blood were studied. Vaccinated pigs developed the same or higher serum haemagglutination inhibiting, virus neutralising, and nucleoprotein-specific IgG antibody titres as infected pigs but lower nasal IgA titres and lymphoproliferation responses. The lower mucosal and cell-mediated immune responses may explain why protection after vaccination was sub-optimal.

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

Summary of workshop findings for porcine B-cell markers.

Based on cluster groups from the first-round analyses of the Third International Swine CD Workshop, 38 monoclonal antibodies (MAbs) including eight internal controls were analysed by flow cytometry (FCM) and immunohistochemistry (IH) in the second-round analysis of the B-cell section of this workshop. Targets in this section included peripheral blood lymphocytes and cells isolated from ileal Peyer's patches (PP), mesenteric lymph nodes (MLN) of adult animals, bone marrow cells from newborn piglets and thymus cells isolated from foetuses at day 105 of gestation. Immunohistochemistry of these 38 MAbs identified four sets, whose ligands were co-expressed with CD21, which showed a tissue distribution compatible with specificity for cells including those of the B-cell lineage. Another group of miscellaneous antibodies appeared to identify other cells, several antibodies were negative. Two-colour flow cytometry (2C-FCM) was carried out by pairing each antibody of interest with antibodies to SWC7, CD21, sIgM and a polyclonal rabbit anti-swine immunoglobulin antiserum (RaSwIg). The anti-CD21 MAb BB6-11C9 (no. 20) and IAH-CC51 (no. 19), established in previous workshops, as well as the cross-reactive anti-human CD21 B-1y4 (no. 146), clustered together in FCM analyses of the first round and showed similar cellular distribution in IH. A further cluster was formed by the standard CC55 (no. 55) and 2A10/8 (no. 102) submitted as SWC7 specific. The second SWC7 standard 2F6/8 (no. 100) clustered separately, but IH showed an identical pattern of reactivity to the other SWC7 MAb.Unfortunately, this work could not identify any other novel clusters with specificity for B-cells, as the statistical clustering of other MAbs could not be substantiated by IH or subsequent two-colour-FCM work. However, we could identify MAb with similar cellular distribution. The ligands for the cross-reactive anti-human CD40 G28.5 (no. 25) and STH224 (no. 153) were expressed on very similar targets, similarly the ligands for the MAb pair JM1H1 (no. 139) with BB6-10A10 (no. 142) and the MAb pair 3F7/11 (no. 115) with 1C2F10 (no. 187).

Effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus.

The aim of this study was to investigate the effects of a porcine reproductive and respiratory syndrome virus (PRRSV) infection on the development of the immune response after pseudorabies virus (PRV) vaccination in pigs. Pigs were intranasally inoculated with the European PRRSV strain, Lelystad virus ter Huurne, and were vaccinated intramuscularly with PRV 2 weeks later (LV-PRV group). Control pigs were vaccinated with PRV only (PRV group). Eight weeks after PRV vaccination, pigs from both groups were challenged intranasally with wild-type PRV. We measured the lymphoproliferative, and the cytolytic responses to PRV of peripheral blood mononuclear cells (PBMC), isolated from blood samples. In addition, serum samples were examined for antibodies against PRV and LV. One week after PRV vaccination, PBMC proliferated abundantly to PRV in both groups. However, in the LV-PRV group the lymphoproliferative response declined after 1 week, whereas, in the PRV group, the lymphoproliferative response was high for 3 weeks and declined thereafter (P<0.05). After challenge, the lymphoproliferative response was 1 week earlier and was consistently and significantly higher in the PRV group than in the LV-PRV group. The PRV-specific killing was higher at 3 weeks after PRV vaccination and 5 weeks after PRV challenge 19+/-3 and 24+/-6%, respectively, in the PRV group, compared to 7+/-4 and 6+/-9%, respectively, in the LV-PRV group (P<0.05). However, later after vaccination and challenge the cytolytic response was identical in both groups. The antibody titre against PRV developed equally in both groups. After challenge, no PRV virus was isolated from both groups. From these results we conclude that, although PRRSV infection did cause changes in the time course of the T-lymphocyte response after PRV vaccination, PRRSV infection did not inhibit the development of vaccine-induced protection after PRV.

Systemic and mucosal isotype-specific antibody responses in pigs to experimental influenza virus infection.

The immunoglobulin isotype-specific responses in serum and at the respiratory mucosa of pigs after a primary infection with influenza virus were studied. To do this, we developed an aerosol challenge model for influenza in specified pathogen-free (SPF) pigs and isotype-specific enzyme-linked immunosorbent assays (ELISAs). Ten-week-old pigs were inoculated without anesthesia in the nostrils with an aerosol of the field isolate influenza A/swine/Neth/St. Oedenrode/96 (H3N2). The infection caused acute respiratory disease that closely resembled the disease observed in some outbreaks of influenza among finishing pigs, which were not complicated by bacterial infections. Pigs showed clinical signs characterized by fever, dyspnea, and anorexia. At necropsy on postinfection days 1 and 2, an exudative endobronchitis was observed throughout the lung. Viral antigen was present in the epithelial cells of the bronchi and bronchioli and virus was isolated from bronchioalveolar and nasal lavage fluids and from pharyngeal swabs until 5 days after infection. With the isotype-specific ELISAs, viral nucleoprotein specific immunoglobulin (Ig) M, IgG1, and IgA antibody responses were measured in serum and bronchioalveolar and nasal lavage fluids. To determine whether the antibodies were produced and secreted at the respiratory mucosa or were serum-derived, the specific activity (ie, the ratio of antibody titer to Ig concentration) was calculated for each isotype. The IgA and interestingly also a substantial part of the IgG1 antibody response in pigs upon infection with influenza virus was shown to be a mucosal response. Local production of specific IgA in the nasal mucosa, and of specific IgA and IgG1 in the lung was demonstrated. These results indicate that protective efficacy of vaccination can be improved by an immunization procedure that preferentially stimulates a mucosal immune response. The aerosol challenge model in SPF pigs and the isotype-specific ELISAs that we developed can be useful for evaluating various strategies to improve efficacy of porcine influenza vaccines and to study the immune mechanisms underlying the observed protection.

Discrimination of different subsets of cytolytic cells in pseudorabies virus-immune and naive pigs.

We previously observed that pseudorabies virus (PRV)-induced, cell-mediated cytolysis in pigs includes killing by natural killer (NK) cells. We also observed that IL-2 stimulation in vitro of naive PBMC expands porcine NK cells. The purpose of this study was to compare the phenotypes of the cytolytic subsets stimulated in vitro by PRV and by IL-2. PBMC were isolated from blood of PRV-immune and naive pigs and stimulated in vitro with PRV or IL-2. After 6 days, the frequency of various lymphocyte subsets in these cultured PBMC was determined by flow cytometry: the cells were separated with a magnet-activated cell sorter and the cytolytic activity of the separated populations was determined. When lymphocytes were separated and analysed with FACScan, the following lymphocyte subsets were discriminated: CD6(+) CD8(bright+) CD4(-) (CTL phenotype), CD6(+) CD8(dull+) CD4(+) (the fraction containing memory T helper cells), CD6(+) CD8(-) CD4(+) (T helper cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(+) ( gammadelta-T cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(-) (NK phenotype) and CD6(-) CD8(-) CD4(-) gammadelta-T(-) or gammadelta-T(+). Flow cytometry analysis demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of more CD6(+) CD8(+) and CD4(+) CD8(+) and fewer CD6(-) CD8(+) and gammadelta-T(+) CD8(+) lymphocytes than IL-2 stimulation of naive PBMC (P<0.05). It was demonstrated further that killing by PRV-stimulated PBMC was mediated mainly by CD6(+) CD8(+) T lymphocytes. Killing by IL-2-stimulated PBMC was mediated mainly by CD6(-) CD8(+) T lymphocytes. These results demonstrate that both natural killing and killing by classical PRV-specific CTL were detected in PRV-immune pigs, whereas IL-2 stimulation of PBMC isolated from naive pigs mainly induced natural killing.

A DNA vaccine coding for glycoprotein B of pseudorabies virus induces cell-mediated immunity in pigs and reduces virus excretion early after infection.

Glycoproteins B (gB), gC and gD of pseudorabies virus (PRV) have been implicated as important antigens in protective immunity against PRV infection. As cell-mediated immunity plays a major role in this protective immunity, we determined the significance of these glycoproteins in the actual induction of cell-mediated immunity. We vaccinated pigs with plasmid DNA constructs coding for gB, gC or gD and challenged them with the virulent NIA-3 strain of pseudorabies virus. Vaccination with plasmid DNA coding for gB induced the strongest cell-mediated immune responses including cytotoxic T cell responses, whereas plasmid DNA coding for gD induced the strongest virus neutralising antibody responses. Interestingly, vaccination with gB-DNA reduced virus excretion early after challenge infection while vaccination with gC-DNA or gD-DNA did not.This is the first study to demonstrate that DNA vaccination induces cytotoxic T cell responses in pigs and that cell-mediated immunity induced by vaccination with gB-DNA is important for the reduction of virus excretion early after challenge infection.

Changes of leukocyte phenotype and function in the broncho-alveolar lavage fluid of pigs infected with porcine reproductive and respiratory syndrome virus: a role for CD8(+) cells.

Porcine reproductive and respiratory virus (PRRSV) primarily infects and destroys alveolar macrophages of the pig. The aim of the present study was to characterize the changes of leukocyte populations in the broncho-alveolar lavage fluid (BALF) of PRRSV-infected pigs. Piglets were inoculated intranasally with PRRSV strain LV ter Huurne. On various days post-infection the piglets were sacrificed and the lungs removed, washed semi-quantitatively and analysed by flow cytometry. The total number of recovered BALF cells increased approximately 10 times between day 10 and day 21 of infection and decreased thereafter. The number of small low-autofluorescent cells (SLAC), i.e. lymphocytic and monocytic cells, increased very strongly from day 2 until day 21 of infection; in contrast, the number of large highly autofluorescent cells (LHAC), i.e. mostly macrophages, remained constant until day 14 of infection, increased slightly on day 21 and then decreased. On day 21 of infection in specific-pathogen-free piglets approximately 60% of the SLAC consisted of CD2(+)CD8(+)CD4(-)gammadeltaTCR(-) cells, which were partly CD8(+)CD6(+) and partly CD8(+)CD6(-). These phenotypes correspond to that of cytotoxic T-cells and natural killer cells respectively. From these results we can conclude that during a PRRSV infection the total number of BALF cells increases mainly due to an influx of lymphocytic cells with a cytolytic phenotype.

Cytolytic function for pseudorabies virus-stimulated porcine CD4+ CD8dull+ lymphocytes.

We previously observed that pseudorabies (PRV) virus-specific killing in vitro was mediated by CD6+ CD8+ lymphocytes. Also a high percentage of CD4+ lymphocytes, among these CD6+ CD8+ lymphocytes, was observed. The purpose of this study was, therefore, to further characterize the killing ability of PRV-stimulated CD4+ CD8+ lymphocytes. Peripheral blood mononuclear cells (PBMC) were isolated from blood of PRV-immune pigs and were stimulated in vitro with PRV. After 6 days, the frequency of CD4+ CD8+ lymphocytes in peripheral blood was determined by flow cytometry analyses. Lymphocytes were separated using a magnet-activated cell sorter or a FACSVantage SE, and the cytolytic activity of the isolated populations was determined. Flow cytometry analyses demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of 26% +/- 4% CD4+ CD8dull+ lymphocytes. We further demonstrated that killing by PRV-stimulated PBMC was mediated by CD4+ CD8dull+ T lymphocytes and CD4- CD8+ T lymphocytes (classic cytolytic T lymphocytes and natural killer cells). The CD4+ CD8dull+ T lymphocytes showed major histocompatibility complex (MHC) II-restricted PRV-specific killing. The CD4- CD8+ T lymphocytes showed both PRV-specific and natural killing. The CD4+ CD8dull+ lymphocytes, which are unique in the pig, seemed to have a more heterogeneous function than was earlier demonstrated. In conclusion, we demonstrated that PRV-specific CD4+ CD8dull+ lymphocytes are able to kill PRV-infected target cells in a MHC II-restricted manner.

Detection of pseudorabies virus DNA in individual single-reactor pigs found in certified pseudorabies-free herds.

During monitoring of certified pseudorabies (PRV)-free herds to confirm their PRV -free status, occasional individual gE-seropositive pigs are detected. These single-reactor pigs remain gE-seropositive when further serum samples are collected and tested. For the eradication programme to proceed, it is important to determine whether these pigs are only false positives or are; in fact, infected with field PRV. The purpose of this study was to determine whether the polymerase chain reaction (PCR) could detect field PRVDNA in single-reactor pigs and so confirm positive reactions in the serologic monitoring programme. First, DNA samples of various tissues from 15 single-reactor pigs all from different herds were examined for field PRV by PCR. Additionally, serum samples from these pigs were analyzed in a gE-confirmation enzyme linked immunosorbent assay (gE-confirmation ELISA). PCR detected PRVDNA in five of the 15 pigs, and these results were confirmed by the gE-confirmation ELISA. The remaining 10 pigs that tested negative in the PCR also tested negative in the gE- confirmation ELISA. We conclude that PCR can be used to discriminate between true and false serological positive single-reactor pigs and, moreover, that the gE-confirmation ELISA confirms these PCR results.

Development of the natural response of immunoglobulin secreting cells in the pig as a function of organ, age and housing.

We analysed the development of the natural immunoglobulin-secreting cell (Ig-SC) response in systemic- and mucosal-lymphoid tissues of specified pathogen free pigs between 1 and 40 weeks of age. As antigen exposure may influence the development of the Ig-SC repertoire we also compared the frequencies of Ig-SC in various lymphoid tissues of 40 weeks old specified pathogen free pigs and conventional pigs. A procedure to isolate lamina propria cells from porcine intestine was adapted for this study. The frequencies of IgM-, IgG-, and IgA-secreting (spot forming) cells were determined with a reversed enzyme linked immunospot assay, which was also adapted for detection of Ig-SC in pigs. The Ig-SC frequencies were calculated as percentage of the mononuclear leukocytes isolated from the various organs. The observations till 40 weeks of age were as follows: Splenic IgM-SC predominated at all ages and reached a plateau of 0.1-0.2% of the mononuclear leukocytes already at 4 weeks of age. The IgM-SC of mesenteric lymph node (MLN) predominated up till 12 weeks of age and reached an optimum of 0.15% reached at 4 weeks of age. The frequencies of IgG-SC of spleen and MLN had dips around 4 weeks of age and increased thereafter till 40 weeks of age (spleen 0.025%, MLN 0.05% at 40 weeks of age). The frequencies of IgA-SC were low in the spleen (< or =0.003%) and moderate in the MLN (0.01-0.02%) at all ages tested. In peripheral lymph node (PLN) and bone marrow (BM), the frequencies of IgM-SC (0.03-0.05%) were much lower than in the spleen. The IgG-SC frequencies of BM and MLN also had dips around 4 weeks of age and increased thereafter. The IgG-SC frequency of BM reached a plateau at 12 weeks of age (0.15%) and for PLN the highest frequency was observed at 40 weeks of age (0.05%). The frequencies of IgA-SC were low in BM and PLN (<0.003%). High frequencies of IgA-SC were observed in mucosa associated tissue like Peyer's patches (PP) and intestinal lamina propria (till 20% of the mononuclear leukocytes in intestinal lamina propria of 12-40 weeks of age). IgM and IgA are both important isotypes in mucosal lymphoid organs in the pig. The shift from IgM to IgAas predominant, mucosal isotype was first observed in duodenum and jejunum (12 weeks) and later in ileum (40 weeks). The influence of ageing on the frequency of Ig-SC in PP was only observed in jejunal PP. whereas in ileal PP the frequencies of Ig-SC did not vary over time. We combined our data about the frequencies of IgM-, IgG-, and IgA-SC in various organs with data obtained by others about the distribution of lymphocytes over porcine lymphoid organs at about 12 weeks of age. Based on these calculations we concluded that the small intestine, with more than 80% of all Ig-SC, is fair most the major site of Ig production in the pig. We also concluded that the small intestine is the major site of IgA and IgM production cells in the pig. Although IgA becomes predominant along the intestine, the results demonstrated that in the pig IgM is more a mucosal isotype compared with other species. With 40% of all IgG-SC the porcine BM appeared to be the major site of IgG production. Unexpected results were obtained for IgG-SC in the systemic lymphoid organs. In these organs the frequencies of IgG-SC dropped firstly from 1 to 4 weeks of age and steadily increased thereafter till 40 weeks of age. This observation is discussed in relation to the possibility that systemic IgG-SC at one week of age were passively acquired from maternal colostrum. The influence of housing/antigenic load at 40 weeks of age was mainly expressed by an increase (2-8x) of the frequency of IgG-SC in spleen, PLN, BM, and intestinal lamina propria, whereas the typical mucosal IgA-SC frequencies in the lamina propria were hardly affected.

Vaccination of pigs against pseudorabies virus with plasmid DNA encoding glycoprotein D.

We analysed the ability of a plasmid carrying the gene encoding glycoprotein D (gD) of pseudorabies virus (PRV) to induce humoral and cell-mediated immune responses and assessed the protection provided by PRV-gD DNA vaccination against challenge infection with PRV. Immunization with plasmid PRV-gD induced neutralizing antibodies and lymphocyte proliferative responses both in mice and pigs. Moreover, when challenged with virulent PRV six weeks following the last immunization, PRV-gD DNA vaccinated pigs excreted virus for a significantly shorter period and showed less clinical symptoms than pigs vaccinated with a control plasmid. Thus, in the target animal, DNA vaccination with PRV-gD DNA induces protective immunity against challenge infection.

Effects of mild stress on the immune response against pseudorabies virus in mice.

Stress is a recognised problem in intensive pig husbandry, which might lead to changes in immune reactivity. To study the effect of stress on the development of an anti-viral immune response, we used a murine model in which mice were immunized with an attenuated strain of pseudorabies virus (PRV). The effect of two stress treatments, both relevant to intensive pig husbandry, on the development of the specific immune response against PRV was investigated. The stress treatments consisted of restraint, social isolation, and transport and they differed in predictability. The specific immune response against PRV, which developed in the draining lymph nodes, was measured by a lymphocyte proliferation assay and cytokine production assays. Our results showed that the unpredictable stress treatment had no effect on the development of the immune response against PRV in mice, whereas the predictable stress treatment actually hastened the immune response.

Effect of vaccination route and composition of DNA vaccine on the induction of protective immunity against pseudorabies infection in pigs.

Vaccination with naked DNA may be an alternative to conventional vaccines because it combines the efficacy of attenuated vaccines with the biological safety of inactivated vaccines. We recently showed that the vaccination with naked DNA coding for the immunorelevant glycoprotein D (gD) of pseudorabies virus (PRV) induced both antibody and cell-mediated immunity in pigs and provided protection against challenge infection. To determine whether the efficacy of the naked DNA vaccination against PRV could be improved, we compared three sets of variables. First, the efficacy of the naked DNA vaccine coding only for the immunorelevant gD was compared with a cocktail vaccine containing additional plasmids coding for two other immunorelevant glycoproteins, gB and gC. Second, the intramuscular route of vaccination was compared with the intradermal route. Third, the commonly used needle method of inoculation was compared with the needleless Pigjet injector method. Five groups of five pigs were vaccinated three times at 4-weeks intervals and challenged with the virulent NIA-3 strain of PRV 6 weeks after the last vaccination. Results showed that although the cocktail vaccine induced stronger cell-mediated immune responses than the vaccine containing only gD plasmid, both vaccines protected pigs equally well against challenge infection. Intradermal inoculation with a needle induced significantly stronger antibody and cell-mediated immune responses and better protection against challenge infection than intramuscular inoculation. Our data show that the route of administering DNA vaccines in pigs is important for an optimal induction of protective immunity.

Time course of the porcine cellular and humoral immune responses in vivo against pseudorabies virus after inoculation and challenge: significance of in vitro antigenic restimulation.

We investigated the time course of porcine cellular and humoral immune responses against pseudorabies virus (PRV) after pigs were inoculated with PRV gE(-) mutant strain M141 and challenged with wild-type virus NIA-3. Peripheral blood mononuclear cells (PBMC) were isolated from blood samples; half were used directly and half were restimulated with PRV in vitro before use in a cytolytic assay. We determined time course and extent of PRV-specific lymphoproliferative and cytolytic response. In addition, serum samples were examined for neutralizing antibodies. After inoculation, the frequency of various lymphocyte subsets in peripheral blood was determined by FACScan. One week after inoculation, T-lymphocytes proliferated abundantly and a B-lymphocyte response was observed. When PBMC were used directly without restimulation, only 15% of the PRV-infected target cells were lysed, and about 15-20% of uninfected target cells were lysed. In contrast, when PBMC were restimulated with PRV, up to 50% of the PRV-infected target cells were lysed while only 30% of the uninfected target cells were lysed. The frequency of various T-lymphocyte subsets in the circulation did not change significantly after inoculation, which indicates that the number of PRV-specific lymphocytes in circulation was very small. After challenge, the T-lymphocyte response was enhanced, but the B-lymphocyte response was not. When PBMC were used directly, only 20% of the PRV-infected and uninfected target cells were lysed after challenge. In contrast, when PBMC were restimulated with PRV, they again lysed more PRV-infected target cells than uninfected target cells. Cytolytic cells were detected for a longer period after challenge than after inoculation. Since it was only possible to clearly detect cytolysis after lymphocytes were restimulated with PRV, it may be that they do not preferentially localize in blood or that they are too few in blood to be detected without further antigenic restimulation in vitro. These lymphocytes may instead localize in other tissues, such as mucosal tissues, tonsils and draining lymph nodes. Whether such a reservoir of PRV-specific cytolytic cells is important in clearing the virus is still unknown. In this study we demonstrated PRV-specific lymphocytes in circulation after they were restimulated in vitro with PRV.