Effect of porcine circovirus type 2a or 2b on infection kinetics and pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model

To determine differences in infection kinetics of two temporally and genetically different type 2 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in vivo with and without concurrent porcine circovirus (PCV) type 2a or 2b infection, 62 pigs were randomly assigned to one of seven groups: negative controls (n=8); pigs coinfected with a 1992 PRRSV strain (VR-2385) and PCV2a (CoI-92-2a; n=9), pigs coinfected with VR-2385 and PCV2b (CoI-92-2b; n=9), pigs coinfected with a 2006 PRRSV strain (NC16845b) and PCV2a (CoI-06-2a; n=9), pigs coinfected with NC16845b and PCV2b (CoI-06-2b; n=9), pigs infected with VR-2385 (n=9), and pigs infected with NC16845b (n=9). Blood samples were collected before inoculation and at day post-inoculation (dpi) 3, 6, 9 and 12 and tested for the presence of PRRSV antibody and RNA, PCV2 antibody and DNA, complete blood counts, and interferon gamma (IFN-γ) levels. Regardless of concurrent PCV2 infection, VR-2385 initially replicated at higher levels and reached peak replication levels at dpi 6. Pigs infected with VR-2385 had significantly higher amounts of viral RNA in serum on both dpi 3 and dpi 6, compared to pigs infected with NC16845b. The peak of NC16845b virus replication occurred between dpi 9 and dpi 12 and was associated with a delayed anti-PRRSV antibody response in these pigs. PCV2 coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-PRRSV IgG response compared to pigs infected with PRRSV alone. This work further emphasizes in vivo replication differences among PRRSV strains and the importance of coinfecting pathogens.     

Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus

Three-week-old cesarean-derived colostrum-deprived (CD/CD) pigs were inoculated with porcine circovirus type 2 (PCV2, n = 19), porcine reproductive and respiratory syndrome virus (PRRSV, n = 13), concurrent PCV2 and PRRSV (PCV2/PRRSV, n = 17), or a sham inoculum (n = 12) to compare the independent and combined effects of these agents. Necropsies were performed at 7, 10, 14, 21, 35, and 49 days postinoculation (dpi) or when pigs became moribund. By 10 dpi, PCV2/PRRSV-inoculated pigs had severe dyspnea, lethargy, and occasional icterus; after 10 dpi, mortality in this group was 10/11 (91%), and all PCV2/ PRRSV-inoculated pigs were dead by 20 dpi. PCV2-inoculated pigs developed lethargy and sporadic icterus, and 8/19 (42%) developed exudative epidermitis; mortality was 5/19 (26%). PRRSV-inoculated pigs developed dyspnea and mild lethargy that resolved by 28 dpi. Microscopic lesions consistent with postweaning multisystemic wasting syndrome (PMWS) were present in both PCV2- and PCV2/PRRSV-inoculated pigs and included lymphoid depletion, necrotizing hepatitis, mild necrotizing bronchiolitis, and infiltrates of macrophages that occasionally contained basophilic intracytoplasmic inclusion bodies in lymphoid and other tissues. PCV2/ PRRSV-inoculated pigs also had severe proliferative interstitial pneumonia and more consistent hepatic lesions. The most severe lesions contained the greatest number of PCV2 antigen-containing cells. PRRSV-inoculated pigs had moderate proliferative interstitial pneumonia but did not develop bronchiolar or hepatic lesions or lymphoid depletion. All groups remained seronegative to porcine parvovirus. The results indicate that 1) PCV2 coinfection increases the severity of PRRSV-induced interstitial pneumonia in CD/CD pigs and 2) PCV2 but not PRRSV induces the lymphoid depletion, granulomatous inflammation, and necrotizing hepatitis characteristic of PMWS.     

Porcine reproductive and respiratory syndrome virus (PRRSV) influences infection dynamics of porcine circovirus type 2 (PCV2) subtypes PCV2a and PCV2b by prolonging PCV2 viremia and shedding

The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on porcine circovirus type 2 (PCV2) subtypes a (PCV2a) or b (PCV2b) viremia and shedding characteristics in oral, nasal and fecal samples in experimentally infected pigs. Twenty-three, 2- to 6-week-old pigs were randomly divided into five groups: negative control (n=3), PCV2a-I (n=5), PCV2a-PRRSV-CoI (n=5), PCV2b-I (n=5), and PCV2b-PRRSV-CoI (n=5). Blood, oral, nasal and fecal swabs were collected in regular intervals from day post inoculation (dpi) 0 until dpi 70 and tested by quantitative real-time PCR for the presence and amount of PCV2 DNA and by ELISA for the presence of PCV2-specific antibodies. The results indicate that there were significantly (P<0.05) higher loads of PCV2a and PCV2b DNA in serum, oral swabs, nasal swabs and fecal swabs and a higher prevalence of detectable PCV2 antigen in tissues of pigs concurrently infected with PCV2 and PRRSV compared to pigs singularly infected with PCV2 further confirming that PRRSV enhances replication of PCV2. Moreover, PRRSV infection significantly prolonged the presence of PCV2 DNA in serum and increased the amount of PCV2 DNA in oral and nasal secretions and fecal excretions in the later stages of infection between dpi 28 and 70. Shedding patterns were similar between groups infected with PCV2a and PCV2b, indicating that there was no subtype-specific interaction with the PRRSV isolate used in this study. The results from this study highlight the interaction between PRRSV and PCV2 and the importance of controlling PRRSV infection in order to reduce PCV2 virus loads in pig populations.     

Porcine circovirus type 2 (PCV2)-infection and re-inoculation with homologous or heterologous strains: virological, serological, pathological and clinical effects in growing pigs

Long-term PCV2 infection and/or concurrent infection with genotypes PCV2a and PCV2b may play a role in the development of clinical porcine circovirus-associated disease (PCVAD). To evaluate this premise, 24 11-week-old specific pathogen-free (SPF) pigs were randomly assigned to 1 of 4 treatments: negative controls, a single inoculation with PCV2a, single inoculation followed by re-inoculation with a homologous PCV2a strain, or repeated inoculations with heterologous strains (PCV2a, PCV2b). Pigs were evaluated for clinical signs daily through 140 days post inoculation (dpi). Serum samples were collected every other day from dpi 0 through 14 and weekly thereafter. PCV2-inoculated pigs were viremic by dpi 2 and 13 of 18 pigs remained viremic at 140 dpi. No statistical differences in the onset, level, or duration of PCV2 viremia were detected among treatment groups. Anti-PCV2 antibodies were detected between 14 and 28 dpi and were present through 140 dpi without statistical differences in antibody response among treatment groups. In the current study, pigs had extended viremia combined with detectable tissue PCV2 antigen levels despite the presence of high levels of anti-PCV2 antibody; however, no clinical disease was observed.     

Viremia and effect of fetal infection with porcine viruses with special reference to porcine circovirus 2 infection

This publication reviews some pathogenetic features of the transplacental infection with porcine viruses in sows. Viremia either with virus freely circulating or associated to peripheral blood mononuclear cells (PBMC) is an essential part of such pathogenesis. Virus replication occurs either in fetal tissues only or both in fetal and maternal tissues and the outcome may be different.Since porcine circovirus 2 (PCV2) has been associated with reproductive failure in sows, the question was asked what type of viremia PCV2 causes and what the effect of PCV2 is on the pregnant uterus. Seronegative gilts were oronasally inoculated and plasma and PBMC were monitored for infectious virus and for quantity of viral DNA copies. Infectious virus was found in plasma only at 21 days post-inoculation (DPI). Virus associated to PBMC was detected between 14 and 49 DPI. Viral DNA was found in plasma between 14 and 49 DPI and associated to PBMC between 7 and 63 DPI (end of experiment). Direct intra-fetal inoculation at 57, 75 and 92 days of gestation and collection of fetuses 21 days later showed that the virus replicates highly in fetal tissues, particularly in the heart. Fetal death occurred in the 57 days sows while virus and antibodies were observed in the 75- and 92-day inoculated sows. Inoculation at 57 and 75 days of gestation and collection of the piglets at the end of pregnancy showed that intrauterine spread had occurred to fetuses adjacent to the inoculated ones and that fetal death occurred also in the presence of antibodies. The pregnancy was not interrupted.This study shows that PCV2 causes viremia which is largely cell-associated and that virus replication in fetuses causes fetal death with mummification. Whether such transplacental infection occurs in the immune sow population is questionable.     

Commercial spray-dried porcine plasma does not transmit porcine circovirus type 2 in weaned pigs challenged with porcine reproductive and respiratory syndrome virus

The objective of this study was to evaluate if spray dried porcine plasma (SDPP) containing porcine circovirus type 2 (PCV2) genome supplemented in feed could transmit PCV2 to pigs challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Twenty-three PRRSV-free pigs, non-viraemic for PCV2, were housed in bio-safety level 3 facilities and assigned to four groups in a 2×2 factorial design consisting of PRRSV challenge and a negative control. The diet contained 0 or 8kg SDPP per 100kg of feed. PRRSV challenge groups were inoculated intranasally with 2mL of a suspension containing 10(6) TCID(50)/mL PRRSV. The SDPP used in the study contained 7.56×10(5) PCV2 genome copies per gram. Dietary treatments were fed from 4days prior to PRRSV inoculation until 28days post-inoculation (PI). All challenged pigs developed PRRSV viraemia by day 3PI and PRRSV antibodies were detected in sera by day 14PI, with no difference between diet treatments. Neither PRRSV viraemia nor seroconversion was observed in non-challenged pigs. PCV2 was not detected in the serum of any pigs throughout the experimental period. SDPP containing the PCV2 genome supplemented in feed did not result in PCV2 transmission to either healthy or PRRSV-infected pigs under these experimental conditions.     

Effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus

The objective of this study was to determine whether vaccination with bacterins commonly used in the USA, when administered at a time typical of US protocol, enhances porcine circovirus type 2 (PCV2) replication and the incidence and severity of clinical signs and lesions characteristic of postweaning multisystemic wasting syndrome (PMWS) in conventional pigs. Sixty-one pigs free of PCV2 were randomly assigned to four groups. Groups 1 (n = 15) and 2 (n = 15) pigs served as sham-inoculated negative controls. Groups 3 (n = 14) and 4 (n = 17) pigs were inoculated intralymphoid with PCV2 field isolate ISU-40895. Pigs in groups 2 and 4 were vaccinated with Actinobacillus pleuropneumoniae (APP) and Mycoplasma hyopneumoniae (M. hyopneumoniae) bacterins 21 days before and again 1 day before inoculation with PCV2. Mild transient respiratory disease and diarrhea were observed from 13 to 34 days postinoculation (DPI) in pigs in groups 3 and 4. Half the pigs from each group were necropsied at 22 and 34 DPI, respectively. Moderately enlarged, tan-colored lymph nodes were observed in the majority of pigs in groups 3 and 4. There was a significantly (P < 0.05) longer length of viremia (2.14 +/- 0.26 versus 4.44 +/- 0.23 weeks), a higher copy number of the PCV2 genome in serum, a wider range of tissue distribution of PCV2 antigen, and an increased severity of lymphoid depletion in pigs vaccinated with commercial APP and M. hyopneumoniae vaccines and inoculated with PCV2 compared with PCV2-inoculated unvaccinated pigs. Swine producers and veterinarians may need to consider changes in vaccination protocols in herds with recurrent PCV2-associated PMWS.     

Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3⁺CD8^high cells starting from 14 dpi. Although CD3⁺CD8^high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3⁺CD8^high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages.     

Experimental infection of 3-week-old conventional colostrum-fed pigs with porcine circovirus type 2 and porcine parvovirus

This report describes an experimental infection with porcine circovirus type 2 (PCV2) in combination with porcine parvovirus (PPV) in 3-week-old conventional colostrum-fed pigs with maternal antibodies to both viruses. Two groups of four pigs each were inoculated with PCV2 and PPV. One of the groups received also a commercial inactivated vaccine against porcine pleuropneumonia to evaluate possible effects of the stimulation of the immune system of pigs on the infection. Another group of four pigs was kept as uninfected control. Clinical signs, rectal temperatures and body weights were recorded. Serum antibody titers to PCV2 and PPV were determined at weekly intervals. Pigs were killed 42 days after inoculation and tissue samples were examined for the presence of gross and microscopic lesions. Tissues were also analyzed for the presence of PCV2 and PPV DNA by PCR, and for the presence of PCV2 antigen by immunohistochemistry (IHC). All the pigs had serum antibodies to PCV2 and PPV at the beginning of the trial. None of them developed clinical symptoms or pathological lesions typical of post-weaning multisystemic wasting syndrome (PMWS), a disease associated to PCV2 infection. However, IHC and/or PCR analyses showed that clinically silent PCV2 infection developed in five of the eight inoculated pigs, regardless of the administration of the vaccine. In particular, PCV2 DNA and/or antigen were detected in most of the tissues examined in the two pigs with the lowest titer of maternal PCV2 antibodies at the beginning of the trial. PPV DNA was not detected in any of the samples examined. The five pigs with PCR and/or IHC evidence of PCV2 infection had a mean weight gain during the experiment lower than that of the inoculated PCR-negative pigs considered together and that of the control pigs. In conclusion, it would appear that passive immunity against PCV2 can play a role in preventing the development of PMWS, but is not able to prevent the establishing of clinically silent PCV2 infections. The dissemination and persistence of the virus in the tissues may depend on the level of PCV2 antibodies at the time of inoculation.     

Potentiation of porcine circovirus 2-induced postweaning multisystemic wasting syndrome by porcine parvovirus is associated with excessive production of tumor necrosis factor-alpha

This study investigated the potentiation of porcine circovirus 2 (PCV2)-induced postweaning multisystemic wasting syndrome by porcine parvovirus (PPV) and found it was associated with excessive production of tumor necrosis factor-alpha (TNF-alpha). Colostrum-deprived conventional pigs were inoculated intranasally with PCV2 or PPV alone or in combination (PCV2 and PPV). In vitro assay of TNF-alpha, obtained from alveolar macrophages coinfected with PCV2 and PPV, showed a significant increase in TNF-alpha compared to single infection of macrophages with either PCV2 or PPV alone (P < 0.05). All pigs inoculated with PCV2 and PPV developed severe postweaning wasting syndrome, whereas clinical signs (e.g., weight loss) were present but perhaps less severe in either PCV2- or PPV-inoculated pigs. Compared to the pigs inoculated with PCV2 or PPV alone, pigs inoculated dually with PCV2 and PPV showed significantly (P < 0.05) increased levels of TNF-alpha. Levels of TNF-alpha in the sera were reversely correlated with the body weight in pigs experimentally infected with dual inoculation of PCV2 and PPV (r(s) = -0.92, P < 0.001). These data suggest that a potentiation of PPV in PCV2-induced PMWS is associated with the excessive production of TNF-alpha.     

Effect of porcine parvovirus vaccination on the development of PMWS in segregated early weaned pigs coinfected with type 2 porcine circovirus and porcine parvovirus

The objectives of this study were to determine if coinfection of segregated early weaned (SEW) pigs with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) induces an increase in the incidence of post-weaning multisystemic wasting syndrome (PMWS) compared to singular PCV2 infection, and to determine if vaccination against PPV protects pigs against PMWS associated with PCV2/PPV coinfection in SEW pigs. Seventy, 3-week-old, SEW pigs were randomly assigned to one of the five groups. Pigs in group 1 (n = 14) served as the negative controls, group 2 pigs (n = 14) were inoculated with PCV2, group 3 pigs (n = 12) were inoculated with PPV, groups 4 (n = 16) and 5 (n = 14) pigs were inoculated with both PCV2 and PPV. Pigs in groups 1-3 and 5 were vaccinated with two doses of a killed parvovirus-leptospira-erysipelothrix (PLE) vaccine prior to inoculation. The PCV2/PPV-coinfected pigs (groups 4 and 5) had significantly (P < 0.05) higher and more persistent fevers than the singular PCV2-infected pigs. One pig in each of the coinfected groups developed clinical disease (fever, respiratory disease, jaundice, weight loss) consistent with PMWS. Lymphoid depletion was significantly (P < 0.05) more severe in the dually-infected pigs at 42 days post-inoculation (DPI). Vaccinated, coinfected pigs (group 5) remained viremic significantly (P < 0.05) longer and had higher copy numbers of genomic PCV2 DNA in sera at 28, 35, and 42 DPI compared to the unvaccinated coinfected pigs (group 4). PPV-viremia was detected only in the unvaccinated group 4 pigs. PLE-vaccination prevented PPV-viremia but did not prevent clinical PMWS or reduce the severity of lymphoid depletion in PCV2/PPV-coinfected pigs. Evidence of increased incidence of clinical PMWS due to vaccination was not observed in this model.     

Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2

The objectives of this study were to investigate the interactions between Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV2) and to establish a model for studying the pathogenesis of and testing intervention strategies for the control of PCV2-associated porcine respiratory disease complex (PRDC). Sixty-seven pigs were randomly assigned to four groups. Group 1 (n=17) pigs served as controls, group 2 (n=17) pigs were inoculated with M. hyopneumoniae, group 3 (n=17) pigs were dual infected with M. hyopneumoniae and PCV2, and group 4 (n=16) pigs were inoculated with PCV2. Pigs were inoculated intratracheally with M. hyopneumoniae at 4 weeks of age followed by intranasal inoculation with PCV2 at 6 weeks of age. Dual-infected pigs had moderate dyspnea, lethargy, and reduced weight gain. The overall severity of macroscopic lung lesions, PCV2-associated microscopic lesions in lung and lymphoid tissues, and the amount of PCV2-antigen associated with these lesions were significantly (P <0.05) higher in dual-infected pigs compared with all other groups. Four of 17 (23.5%) dual-infected pigs had decreased growth rate and severe lymphoid depletion and granulomatous lymphadenitis associated with high amounts of PCV2-antigen consistent with postweaning multisystemic wasting syndrome (PMWS). PCV2-antigen in lung tissue was most often associated with M. hyopneumoniae-induced peribronchial lymphoid hyperplasia, suggesting that this is an important site for PCV2 replication in the lung. This study indicates that M. hyopneumoniae potentiates the severity of PCV2-associated lung and lymphoid lesions, increases the amount and prolongs the presence of PCV2-antigen, and increases the incidence of PMWS in pigs.     

Bacterial lipopolysaccharide induces porcine circovirus type 2 replication in swine alveolar macrophages

Monocyte/macrophage lineage cells are major target cells of porcine circovirus type 2 (PCV2). From tissues of field pigs suffering from postweaning multisystemic wasting syndrome (PMWS), both intracytoplasmic and intranuclear PCV2 signals, including antigens and nucleic acid, were easily detected in the monocyte/macrophage lineage cells. However, there was a high incidence of intracytoplasmic PCV2-positive signals, but lack of intranuclear signals and PCV2 replication in these cells in vitro. Concurrent infection with bacteria and activation of immune system are suggested to promote viral replication. In this study, lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate (PMA) were used to stimulate PCV2-inoculated alveolar macrophages (AMs). A decrease in intracytoplasmic but increase in intranuclear PCV2-positive signals, including antigens and nucleic acid, were detected in LPS-treated PCV2-inoculated AMs, but not in PMA-treated cells. Additionally, the replication product corresponding to PCV2 spliced major capsid protein (Cap) mRNA and a significant elevation in PCV2 titer were demonstrated in the LPS-treated PCV2-inoculated AMs. The results imply that Gram-negative bacterial co-infection in PCV2-infected pigs may be an important factor in promoting PCV2 replication and contributing, at least partially, to the full development of PMWS.     

Porcine circovirus 2 infection in swine foetuses inoculated at different stages of gestation

The ability of porcine circovirus 2 (PCV2) to replicate and cause pathologic abnormalities in foetuses at selected time points of gestation was examined in this study. Two foetuses were inoculated in utero in each of two sows at 57, 75 and 92 days of gestation, respectively, with PCV2 (1121). The remaining foetuses were left uninoculated to assess whether intra-uterine spread occurred. Twenty-one days after inoculation, the foetuses were collected and examined for gross lesions and for virus and infected cells in different organs. Serum samples from all foetuses were tested for PCV2 antibodies. Virus replication was detected in all inoculated foetuses. Spread to non-inoculated foetuses did not occur. Virus replication was significantly higher in foetuses inoculated at 57 days compared to that inoculated at 75 and 92 days. The heart contained the highest virus titre and highest number of viral antigen positive cells. Gross lesions were observed only in foetuses inoculated at 57 days of age. PCV2 antibodies were detected only in foetuses inoculated at 75 and 92 days. This study shows the ability of PCV2 to replicate in foetuses at different stages of gestation and to cause pathologic abnormalities in foetuses inoculated at 57 gestational days.     

Pathogenicity of experimental infection with 'pneumotropic' porcine coronavirus

Virus localisation and lesions were studied in 14-one-week-old piglets following combined intranasal-oral inoculation with a British isolate of 'pneumotropic' porcine coronavirus (PCV) and were compared with the effects of transmissible gastroenteritis virus (TGEV) infection in five piglets. Unlike TGEV-infected piglets, all PCV-inoculated piglets remained clinically healthy. Seroconversion was detected at seven days after inoculation. Mild bronchointerstitial pneumonia involving terminal airways was consistently present at two days after infection and thereafter. Both PCV and TGEV infected bronchiolar epithelium and alveolar macrophages but, unlike TGEV, replication by PCV in villous enterocytes was limited and did not cause villous atrophy.     

The involvement of Fas/FasL interaction in porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus co-inoculation-associated lymphocyte apoptosis in vitro

Lymphoid depletion of various lymphoid organs is one of the major lesions in pigs suffering from postweaning multisystemic wasting syndrome (PMWS). The co-existence of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) in PMWS-affected pigs along with the more severe and wider range of lymphocyte depletion of lymphoid organs in PCV2 and PRRSV dually-inoculated pigs imply that PCV2 and PRRSV may interact in the pathogenesis of PMWS. The mechanism for the development of lymphoid depletion in PMWS-affected pigs remains controversial. The objective of the present study was to evaluate and compare the effects of inoculation of both viruses, singularly or in combination, on swine splenic macrophages (SMs) and co-cultured splenic (SLs) and peripheral blood (PBLs) lymphocytes in vitro. A significant reduction in the survival rate and increase in the apoptotic rate of the co-cultured SLs and PBLs and concurrent increase in the expression levels of Fas ligand (FasL) in SMs and Fas in co-cultured SLs and PBLs were demonstrated in PRRSV alone- and PCV2 and PRRSV dually-inoculated groups with the latter more prominent. The increased FasL was proven capable of inducing Fas/FasL-mediated apoptosis in co-cultured FasL-sensitive Jurkat T cells. The de novo expression and production of FasL in PCV2 and PRRSV dually-inoculated SMs and concurrently increased surface expression of Fas in co-cultured lymphocytes may contribute, at least partially, to lymphoid depletion in PMWS-affected pigs with PCV2 and PRRSV dual infection.     

Quantification of PCV2 capsid transcript in peripheral blood mononuclear cells (PBMCs) in vitro

The presence of PCV2 DNA or spliced capsid mRNA (Cap mRNA) for viral replication was assessed following addition of PCV2 to resting or concanavalin A (ConA) stimulated peripheral blood mononuclear cells (PBMCs). Real-time PCR or real-time RT-PCR assays were used to measure viral DNA or Cap mRNA, respectively. The study demonstrated that PCV2 replication increased in infected PBMCs over time. Replication within infected PBMCs was significantly (P<0.05) increased when PBMCs were stimulated with ConA, compared to unstimulated PBMCs. The data showed a strong correlation between the level of PCV2 Cap mRNA and the level of viral DNA in the ConA stimulated PBMCs. Replication of PCV2 was also assessed in T lymphocyte- and monocyte/macrophage-enriched or monocyte/macrophage-depleted PBMC populations which had been stimulated with ConA for 3 days. It was demonstrated that the enriched T lymphocytes and the monocyte/macrophage-depleted PBMCs had significantly higher Cap mRNA and viral DNA levels (P<0.05) compared to the monocyte/macrophage-enriched population, indicating that in addition to monocytes/macrophages, PCV2 replicates in lymphocytes, particularly T lymphocytes following stimulation. These results suggest that the presence of activated T lymphocytes may play an important role in PCV2 replication and potentially the development of clinical disease.     

Butyrate differentially regulates cytokines and proliferation in porcine peripheral blood mononuclear cells

Although butyrate modulates proliferation and cytokine production by PBMC in some species, the role of butyrate as a regulator of immunocyte function in the pig has not been studied. Therefore, the primary objective of this study was to determine whether butyrate influences peripheral blood mononuclear cell (PBMC) proliferation, cytokine secretion and mRNA expression in the pig in vitro. We also sought to determine whether alterations in cytokine production attributable to butyrate were associated with changes in the expression of suppressor of cytokine signaling-3 (SOCS3). Porcine PBMC were isolated from venous blood and stimulated with concanavalin A (ConA) in the presence or absence of sodium butyrate at 0.2 or 2.0 mM. Butyrate at 2.0 mM suppressed (P<0.05) ConA-induced PBMC proliferation and led to a paradoxical increase (P<0.05) in IL-2 mRNA expression. The secretion and mRNA expression of interferon-gamma (IFN-gamma) by ConA-activated PBMC was increased (P<0.05) by butyrate at 2.0 mM. Exposing activated PBMC to butyrate at 2.0 mM decreased (P<0.05) the secretion of interleukin-10 (IL-10). In contrast, butyrate at 0.2 mM increased (P<0.05) both IL-10 secretion and mRNA expression. Activation of porcine PBMC with ConA increased (P<0.05) the expression of SOCS3 mRNA, and butyrate treatment further augmented (P<0.05) SOCS3 mRNA expression in a dose-dependent manner. Mechanistically, pretreatment with the adenyl cyclase inhibitor 2,5-dideoxyadenosine abolished (P<0.05) the inhibitory effect of 2.0 mM butyrate on IL-10 secretion, and partially reversed (P<0.05) the increase in IFN-gamma secretion induced by 2.0mM butyrate. These data indicate that the effect of butyrate on cytokine production by porcine PBMC is dose-dependent, and that butyrate increases the expression of SOCS3 in activated PBMC. In addition, we provide evidence that the effects of butyrate on IFN-gamma and IL-10 production are mediated in part via a cAMP-dependent mechanism.     

Expression of human CD46 has no effect on porcine circovirus type 2 infection and shedding in the experimental pig model

Xenotransplantation of tissues from transgenic pigs with desired genetic modifications such as CD46 expression help minimize xenograft rejections. However, CD46 is a known receptor for some viruses. In this study, pigs transgenic for human CD46 (CD46-TG) and appropriate non-transgenic (non-TG) control pigs were utilized to determine possible differences in the level of replication and shedding of porcine circovirus type 2 (PCV2). Non-TG and CD46-TG were blocked by transgenic status and randomly divided into three groups: Non-TG negative controls (n?=?3), non-TG-PCV2 (n?=?10; PCV2a?=?5, PCV2b?=?5), and CD46-TG-PCV2 (n?=?6; PCV2a?=?3, PCV2b?=?3). Blood, oral, nasal and fecal swabs were collected at regular intervals from the day of arrival until 70 days post inoculation (DPI). All samples were tested by quantitative real-time PCR for the presence of PCV2 DNA and serum was tested for presence of PCV2 antibodies by ELISA. Overall, the main effects "transgenic status" and "PCV2 subtype" had no influence on degree of PCV2 viremia and shedding or the anti-PCV2 humoral immune response in CD46-TG-PCV2 pigs compared to non-TG-PCV2 pigs. Differences in PCV2 concentrations between non-TG-PCV2 and CD46-TG-PCV2 pigs were minimal and limited to DPI 35 in sera, DPI 7 in fecal swabs and DPI 5 in nasal swabs when CD46-TG-PCV2 pigs had significantly higher concentrations of PCV2 DNA. At DPI 1, CD46-TG-PCV2 pigs had significantly lower concentrations of PCV2 DNA in oral swabs. Under the study conditions, the presence of human CD46 in transgenic pigs had no effect on PCV2 infection in otherwise healthy pigs capable of a normal immune response.     

Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica

Porcine circovirus (PCV) was recently divided into 2 antigenically distinct types that differ (65% amino acid identity) in the protein encoded by open reading frame 2 (ORF2). Porcine circovirus 1 is apparently non-pathogenic and, in contrast, PCV2 is associated with porcine multisystemic wasting syndrome (PMWS). Our objective was to determine the extent of exposure of normal pigs in Canada and Costa Rica to PCV2. Recombinant DNA techniques were used to produce an antigen from ORF2 of PCV2 that was suitable for the detection of antibody in swine sera. The presence of PCV2 nucleotide sequences was detected using polymerase chain reaction (PCR) techniques. Using these tests, specific antibody and nucleotide sequences were demonstrated in sera from a cohort of pigs during a PMWS outbreak. Antibody was detected in normal, healthy hogs slaughtered in Canada (82.4% of 386) and in Costa Rica (14.6% of 322). This is the first report indicating the presence of PCV2 in Latin America. More than 50% of these sera also contained PCV2 nucleotide sequence. Although these hogs were healthy when slaughtered, they were infected with PCV2 and may have previously been ill. The widespread occurrence of PCV2 in swine suggests that this virus is adapted to replication in porcine tissue.