Porcine Circovirus Type 2 Antigen Research Articles

Comparative immunogenic and immunoprotective activities of PCV2d Cap and Rep antigens delivered by an efficient eukaryotic expression system engineered into a Salmonella vaccine vector

Porcine circovirus type 2 (PCV2) stands as a predominant etiological agent in porcine circovirus-associated diseases. To manage the spread of the disease, it is necessary to develop a next-generation vaccine expressing PCV2 antigens that target the prevailing genotype such as PCV2d. A bacterial-mediated vaccine delivery by live-attenuated Salmonella has attracted interest for its low-cost production and highly effective vaccine delivery. Thus, in this study, we utilized the advantages of the Salmonella-mediated vaccine delivery by cloning PCV2d cap and rep into a eukaryotic expression plasmid pJHL204 and electroporation into an engineered live-attenuated Salmonella Typhimurium JOL2500 (Δlon, ΔcpxR, ΔsifA, Δasd). The eukaryotic antigen expression by JOL2995 (p204:cap) and JOL2996 (p204:rep) was confirmed in vitro and in vivo which showed efficient antigen delivery. Furthermore, vaccination of mice model with the vaccine candidates elicited humoral and cell-mediated immune responses as depicted by high levels of PCV2-specific antibodies, CD4+ and CD8+ T cells, and neutralizing antibodies, especially by JOL2995 (p204:cap) which correlated with the significant decrease in the viral load in PCV2d-challenged mice. Interestingly, JOL2996 (p204:rep) may not have elicited high levels of neutralizing antibodies and protective efficacy, but it elicited considerably higher cell-mediated immune responses. This study demonstrated Salmonella-mediated vaccine delivery system coupled with the eukaryotic expression vector can efficiently deliver and express the target PCV2d antigens for strong induction of immune response and protective efficacy in mice model, further supporting the potential application of the Salmonella-mediated vaccine delivery system as an effective novel approach in vaccine strategies for PCV2d.

Comparison of immune effects of porcine circovirus type 2d (PCV2d) capsid protein expressed by Escherichia coli and baculovirus-insect cells

Porcine circovirus type 2 (PCV2) is an important pathogen harmful to global pig production, which causes immunosuppression and serious economic losses. PCV2 capsid (Cap) protein expressed by E. coli or baculovirus-insect cells are often used in preparation of PCV2 subunit vaccines, but the latter is expensive to produce. It is therefore crucial to comparison of the immune effects of Cap protein expressed by the above two expression systems for reducing the production cost and guaranteeing PCV2 vaccine quality. In this study, the PCV2d-Cap protein lacking nuclear localization signal (NLS), designated as E. coli-Cap and Bac-Cap, was expressed by E. coli and baculovirus-Spodoptera frugiperda Sf9 (Bac-Sf9) cells, respectively. The expressed Cap proteins could self-assemble into virus-like particles (VLPs), but the Bac-Cap-assembled VLPs were more regular. The two system-expressed Cap proteins induced similar specific IgG responses in mice, but the neutralizing antibody levels of Bac-Cap-immunized mice was higher than those of E. coli-Cap. After PCV2 challenge, IL-10 in Bac-Cap immunized mice decreased significantly than that in E. coli-Cap. The lesions and PCV2 antigen positive cells in tissues of mice immunized with E. coli-Cap and Bac-Cap were significantly reduced, and Bac-Cap appeared mild lesions and fewer PCV2 antigen-positive cells compared with E. coli-Cap immunized mice. The study indicated that Cap proteins expressed by E. coli and Bac-Sf9 cells could induce specific protective immunity, but the latter induced more effective immunity, which provides valuable information for the research and development of PCV2 vaccine.

Development of a Gold Nanoparticle-Based Immunochromatographic Strip for Rapid Detection of Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is an important swine infectious pathogen that seriously threatens the global swine industry. PCV2 Cap protein is the only structural and the main immunogenic protein constituting the viral capsid. In this study, a gold nanoparticle-based immunochromatographic strip with high sensitivity and specificity was developed which could be used for rapid detection of PCV2 virions or Cap protein in research. The visual detection limit of the strip was 103.18 50% tissue culture infective does (TCID50)/mL for PCV2, and 2.03 μg/mL for PCV2 Cap protein. No cross-reactivity was observed with the PCV1 and PCV3 Cap proteins and other common swine pathogens such as porcine reproductive and respiratory syndrome virus, classical swine fever virus, pseudorabies virus, porcine epidemic diarrhea virus, porcine parvovirus, and swine influenza virus. The repeatability of the strip was good. The stability of the strip was perfect for 12 months in a dry state at room temperature. Visual results could be obtained within 5 min by simply inserting the strip into the diluted sample. The strip is a time-saving, labor-saving, and reliable tool for testing of PCV2 virions or Cap protein in research. The idea of this study might open a new perspective for the application of the strip. IMPORTANCE Porcine circovirus type 2 (PCV2) Cap protein is the only structural and the main immunogenic protein constituting the viral capsid. Although many methods can be used to identify PCV2 or PCV2 Cap protein in vaccine research, they usually require high workload and time. The developed strip can specifically detect PCV2 virions or Cap protein, and visual qualitative results can be obtained within 5 min by simply diluting the sample and inserting the strip into the sample. The final value of the strip is providing a simple and time-saving method for real-time monitoring of PCV2 antigen in vaccine research with reliable results, such as the different stages of PCV2 Cap protein expression and purification, as well as the different stages of PCV2 reproduction and purification.

Evaluation of cross-immunity among major porcine circovirus type 2 genotypes by infection with PCV2b and PCV2d circulating strains

There are three main genotypes of porcine circovirus type 2 (PCV2), namely PCV2a, PCV2b and PCV2d, of which PCV2b and PCV2d are currently the most common. There are antigenic differences between these different genotypes. To explore the effect of PCV2 antigen differences on the immune protection provided by vaccines, a cross-immune protection test was carried out in pigs. Three genotype strains, PCV2a-CL, PCV2b-MDJ and PCV2d-LNHC, were inactivated and emulsified to prepare inactivated vaccines to immunize pigs, who were then challenged with the circulating strains PCV2b-BY and PCV2d-LNHC. Immunoperoxidase monolayer assays (IPMAs) and micro-neutralization assays were used to detect antibodies against the three different genotypes of PCV2. The results showed that the three genotype vaccines induced pigs to produce antibodies against the same and different genotypes of PCV2, but the levels of IPMA and neutralizing antibodies against the same genotype were higher than those against different genotypes. Quantitative Polymerase Chain Reaction (qPCR), virus titration and immunohistochemistry were used to detect PCV2 genomic DNA, live virus and antigen, respectively, in inguinal lymph nodes of experimental pigs. Following challenge with the PCV2b-BY strain, the viral DNA load in the inguinal lymph nodes of pigs immunized with the three genotype vaccines was reduced by more than 99 % compared to the unimmunized group. Following challenge with the PCV2d-LNHC strain, the viral DNA loads in the inguinal lymph nodes of pigs immunized with PCV2a, PCV2b and PCV2d genotype vaccines were reduced by 93.8 %, 99.8 % and 98.3 %, respectively, compared to unimmunized controls. In addition, neither live PCV2 virus nor antigen were detected in the inguinal lymph nodes of pigs immunized with any of the genotype vaccines (0/18), but both were detected in the lymph nodes of experimental pigs in the unimmunized control group (6/6). These findings suggest that, although the antigenic differences of the three genotype strains induce significant differences in antibody levels, they seem to have little effect on cross-protection between different genotypes.

Porcine dermatitis and nephropathy syndrome on natural infection of Porcine Circovirus Type-2 in pigs in Kerala, southern India

Porcine circovirus type-2 (PCV-2), is a widely studied pathogen in the swine population during the last decade. Even though, PCV-2 has many disease manifestations, the objective of the current study was to investigate the cutaneous and renal lesions in PCV-2 infected cases. Swine carcasses presented for post-mortem examination with a history of skin lesions, inappetance, dyspnoea and diarrhoea, during September 2021 to April 2022 from different parts of Kerala were utilised in this study. The samples were collected and screened with Polymerase chain reaction (PCR) after which, histological and immunohistochemical examination were performed. Three samples were found positive for PCV-2 out of 25 samples using PCR. Microscopically, skin revealed congestion, haemorrhages and infiltration of mononuclear infiltration cells and neutrophils in dermis and spongiosis in epidermis. Kidney revealed haemorrhages, congestion, tubular necrosis and degenerative alterations, interstitial mononuclear cell infiltration with occasional glomerulitis and vasculitis in the renal pelvis. Localisation of PCV-2 antigen was observed in renal tissue and lymph node with immunohistochemistry. Altogether, these findings were suggestive of porcine dermatitis and nephropathy syndrome (PDNS) in pigs. These results indicate the importance of including PCV-2 in the differential diagnosis of cutaneous lesions caused by bacterial and viral etiologies in pigs.

Development of gold Immunochromatographic assay strip based on specific polyclonal antibodies against capsid protein for rapid detection of porcine circovirus 2 in Zhejiang province, China

BackgroundThe existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host’s immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed.ResultsThe truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%.ConclusionsThis study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.

Pathomorphology, immunohistochemical, and molecular detection of an atypical porcine dermatitis and nephropathy syndrome (PDNS) due to PCV-2d-2 in naturally affected grower pigs of India

Porcine circovirus type 2 (PCV-2) is of great economic significance to porcine industry worldwide. PCV-2 variants and genotypes, alternating world over, are the etiological agent of several clinical syndromes such as porcine dermatitis and nephropathy syndrome (PDNS), post-weaning multi-systemic wasting syndrome (PMWS) and others in pigs. This study is reporting an atypical manifestation of PDNS in twelve grower pigs, 3- to - 4.5 months age and either sex, died of the disease, with predominant lesions of nephropathy and no obvious clinical lesions in skin. Necropsy examination of pigs showed lesions of petechial -to- ecchymotic hemorrhages in the kidneys and in the right auricular musculature of the hearts. Microscopic lesions in H & E sections of the kidneys showed acute glomerulonephritis, interstitial nephritis, and vasculitis, but the skin morphology and architecture remained unaltered in contrast to the pathognomonic lesions of PDNS described in the literature. Other syndromic associations of PDNS in these cases included-perimyocarditis, interstitial pneumonia, depleted lymphoid tissues, tonsillitis, enteritis, and meningo-encephalitis. The lesional sites in duplicate paraffin tissue sections of kidneys, heart, lungs, spleen, lymph nodes, intestine, and brain demonstrated PCV-2 antigen in the cytoplasm of cells as highlighted by the intense immunolabeling on IHC staining. The PCV-2 positive organs reconfirmed by PCR, targeting ORF2 gene, which yielded 481bp size of products. The sequencing results of 481bp products on phylogenetic analysis showed 94% similarity with that of PCV-2 sequences in the database that grouped into PCV2d-2 genotype. The present report confirms, probably for the first time, the atypical PDNS cases due to PCV2d-2 genotype in naturally affected grower pigs of India.

Characterization of the antigens in inactivated porcine circovirus type 2 vaccines and virus-like particle vaccines by high-performance size-exclusion chromatography coupled with multi-angle laser light scattering

This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×106 (±4.34%) Da and 2.40×106 (±2.51%) Da, respectively, as calculated by MALLS. The antigen peaks of the two VLP vaccines also appeared at 13.3 min and the molecular weight was 2.09×106 (±2.94%) Da and 2.88×106 (±11.85%) Da, respectively, which was close to the theoretical molecular weight of PCV2. Moreover, an antigen peak of VLP vaccine c was observed at 11.4 min and the molecular weight was 4.37×106 (±0.42%) Da. The antigen was verified to be the dimer of VLP by TEM. Vaccine d and purified Cap VLP antigens were tested repeatedly, and the RSD of the peak area (n=3) was all < 1.5%, indicating that the method was repeatable. The purified VLP were diluted in serial and tested for linearity. The result suggested good linear relationship between the peak area of VLP or VLP aggregates and the protein concentration of the sample with R2 of 0.999 and 0.997, respectively. Thus, the method met the requirement for quantification and aggregate analysis. This method is accurate and efficient in in vitro quality evaluation and improvement of PCV2 vaccine.

Combined Antibody Tagged HRP Gold Nanoparticle Probe for Effective PCV2 Screening in Pig Farms.

IntroductionPorcine circovirus type 2 (PCV2) causes immune repression and intercurrent infections in pigs, resulting in a huge economic loss to the pig breeding industry. Additionally, the spread of PCV2 in pig farms can pollute the living environment of the residents in the farm’s vicinity, which increases the rate of infections. Therefore, rapid and sensitive detection methods are needed for disease prevention and timely environmental cleaning.MethodsThis research describes a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) that utilizes gold nanoparticles (AuNPs) in a functional, specific antibody labeled probe for the detection of PCV2. Due to their high specific surface area and histocompatibility, AuNPs were used as carriers of HRP labeled anti-PCV2 antibodies to amplify the detection signal.ResultsCompared to conventional sandwich ELISA procedures, this method resulted in higher sensitivity (51-fold) and a shorter assay time with a limit of detection of 195 TCID50/mL. The cross-reactivity assay demonstrated that this assay was PCV2 specific.ConclusionThe amplified Ab (HRP) labeled AuNPs probe provides a sensitive analytical approach for the determination of the traces of the PCV2 antigen in early diagnosis.

A Comparison of Virulence of Three Porcine Circovirus Type 2 (PCV2) Genotypes (a, b, and d) in Pigs Singularly Inoculated with PCV2 and Dually Inoculated with PCV2 and Porcine Reproductive and Respiratory Syndrome Virus.

The aim of this study was to compare the virulence of porcine circovirus type 2 (PCV2) genotypes in dually inoculated pigs with both three genotypes (a, b, and d) of PCV2 and porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) versus pigs singularly inoculated with the same three PCV2 genotypes (a, b, and d). Differences in this comparison were found in PCV2 viremia levels, lung and lymphoid lesion severity, and the amount of PCV2 antigen within the lymphoid lesions. Regardless of PCV2 genotypes, pigs that were dually inoculated with PCV2/PRRSV had significantly higher clinical scores, less average daily weight gain, higher levels of PCV2 viremia, and more severe lug and lymphoid lesions compared to pigs singularly inoculated with PCV2. Among the dually infected pig groups, pigs infected with PCV2d/PRRSV-2 had significantly higher levels of PCV2 viremia, more severe lung and lymphoid lesions, and more PCV2-positive cells within lymphoid lesions compared to pigs dually inoculated with PCV2a/PRRSV-2 and PCV2b/PRRSV-2. The results of this study demonstrated significant differences in the virulence among dual inoculation of PCV2a/PRRSV-2, PCV2b/PRRSV-2, and PCV2d/PRRSV-2. A significant difference in the virulence among PCV2a, PCV2b, and PCV2d single-inoculated pig groups was not found with respect to the levels of PCV2 viremia and production of PCV2-associated lymphoid lesions.

Detection of porcine circovirus type 2 (PCV2) in the Philippines and the complexity of PCV2-associated disease diagnosis.

Porcine circovirus type 2 (PCV2), an important pig viral pathogen, can cause porcine circovirus-associated disease (PCVAD), resulting in economic losses associated with decreased growth and mortalities. The diagnosis of PCVAD is complex requiring clinical, pathological and virological approaches. This study assessed PCV2 infection using histopathology and immunohistochemistry (IHC) on tissue samples and quantitative polymerase chain reaction (qPCR) on serum samples from 47 grower-finisher pigs allocated in three clinical groups in the Philippines. Typical PCV2 histopathological lesions were observed in mediastinal lymph nodes (MLN) of eight of 47 pigs. Lymphoid depletion was seen in all eight pigs and granulomatous inflammation in one of these pigs. Four of these eight pigs were PCV2 positive by both IHC and qPCR. IHC revealed PCV2 antigen in 8 pigs in at least one of the following tissues: MLN (5/8), spleen (3/8), tonsils (4/8) and lungs (5/8). PCV2 antigen was observed in 3/8 MLN with lymphoid depletion and in one MLN with depletion and granulomatous inflammation. The qPCR test showed that 33 sera had a non-detectable level, twelve had < 106 and two had > 106 PCV2 DNA copies/ml serum. One pig with lymphoid depletion had > 106 PCV2 DNA copies/ml serum, and another pig without MLN lesions also had > 106 PCV2 DNA copies/ml serum. These findings suggest that PCVAD is present in the Philippines and confirm the challenges of PCVAD diagnosis as different patterns of results were obtained from the different tests.

A broad spectrum monoclonal antibody against porcine circovirus type 2 for antigen and antibody detection.

This study described the production, characterization, and application of monoclonal antibodies (mAbs) against porcine circovirus type 2 (PCV2). Twelve stable hybridomas were produced by immunization with purified PCV2a/LG strain and characterized by immunoperoxidase monolayer assay (IPMA), Western blotting, and neutralization assays. All mAbs could react with the PCV2 Cap protein and neutralize PCV2a/LG strain. One of them, mAb 3A5, reacted to all PCV2 strains from PCV2a, PCV2b, and PCV2d and it could be applied to detect PCV2 antigen and antibodies. It was shown that the mAb 3A5 could be used to locate PCV2 antigen in PK15 cells and the inguinal lymph nodes of PCV2b/YJ stain-infected piglets. Furthermore, this mAb could immunoprecipitate the Cap protein in PCV2-infected PK15 cells. Meanwhile, a capture ELISA based on mAb 3A5 was developed and used to specifically test PCV2 antigen from cultures; a linear relationship was observed between the optical density at 405nm of the ELISA and viral titers (200-12,800 TCID50/mL), with a correlation coefficient of 0.9999. Finally, a competitive ELISA based on mAb 3A5 was developed to specifically detect antibodies in PCV2-infected and immunized pigs, and its sensitivity was higher than that of the blocking ELISA. This study suggested that the mAb 3A5 could be used in several convenient and efficient methods for PCV2 clinical and pathological studies, as well as surveillance in pigs and seroconversion monitoring in the vaccinated pigs.

Genetic variation and phylogenetic analysis of Porcine circovirus type 2 in China from 2016 to 2018.

Infection with Porcine circovirus type 2 (PCV2), the essential cause of porcine circovirus (PCV) associated diseases, is a growing problem in swine industry around the world. High nucleotide alteration leads to the constant evolution of PCV2 and outbreak of disease caused by PCV2. In this study, 48 PCV2 strains were isolated in China between 2016 and 2018 and the genetic diversity of these PCV2 isolates was determined. Results showed that these PCV2 isolates were classified into genotypes PCV2a (4 of 48), PCV2b (13 of 48) and PCV2d (31 of 48). Among them, 54.5% isolated in 2016, 65.2% isolated in 2017 and 71.4% isolated in 2018 belonged to PCV2d. It indicated that the prevalence of PCV2d genotype increased. All strains shared 93.4%-100% nucleotide sequence identity for the whole genome. Results of the analysis using RDP 4.0 molecular recombination software showed there were no recombinant events among the 48 PCV2 isolates in this study. Further analysis indicated the presence of higher amino acid residues diversity in important epitopes (43D/G, 115D/G, 134N/T, 165P/T, 169G/R/S, and 210E/G/D) in the predominant genotype PCV2d. Animal tests showed the viral titer of the PCV2d strain LN-3 in sera was higher than that of PCV2b strain HeB-1 at 14 and 21 days post-challenge, however, the differences were not statistically significant. There were also no obvious differences between PCV2d strain LN-3 and PCV2b strain HeB-1 in the amount of PCV2 antigen in lymphoid tissues. On the account of the increasing prevalence of PCV2d genotype, it is necessary to find the cause of PCV2 genotype change, to evaluate the effect of existing commercial vaccines and to develop new vaccines based on PCV2d genotype, if necessary. Keywords: epitope; PCV2d; phylogeny; Porcine circovirus type 2; recombination; genetic diversity.

Porcine circovirus type 2 associated reproductive failure in a specific pathogen free (SPF) piglet producing herd in Norway: a case report

BackgroundSince 1999, scientists have published evidence of transplacental infection by porcine circovirus type 2 (PCV2) and reproductive failure in pigs. Affected herds have frequently been start-up herds, either naïve or with a high proportion of PCV2 susceptible gilts. Here, delayed farrowing in non-vaccinated gilts was observed in a commercial specific pathogen free (SPF) herd. Mummified fetuses and stillborn piglets recovered from these gilts were PCV2 positive.Case presentationThe case herd was a self-recruiting, piglet producing unit of 240 sows. After detecting livestock associated methicillin resistant Staphylococcus aureus (LA-MRSA, CC398), stamping out was imposed by the authorities. An SPF herd was re-established and all dams were vaccinated against PCV2 until the farmer decided to exclude this vaccine. The first non-vaccinated batch consisted of 76% gilts. Here, one gilt showed signs of impending farrowing. This gilt was slaughtered three to four weeks after the expected farrowing date without having expelled any uterine contents. In the subsequent batch consisting of 79% gilts, three gilts showed similar clinical signs. Delayed farrowing was observed in two of these gilts and the uterine contents from the third gilt were recovered at the abattoir. Mummified fetuses and stillborn piglets were recovered from all three gilts. High levels of PCV2 DNA (>107 viral genomic copies/ 500 ng tissue) were found in myocardial samples by real-time PCR analysis. One myocardial sample submitted for immunohistochemical (IHC) analysis showed moderate amounts of PCV2 antigen. In the subsequent batch consisting of 77% gilts, several weak-born piglets were seen across different litters.ConclusionsThis case report describes an apparent link between in utero PCV2 infection, pre partum nest-building behaviour, mammary development and delayed farrowing. To date, no reports have described imminent signs of farrowing and delayed farrowing as clinical signs in conjunction with transplacental PCV2 infection in Norway. Reinitiation of PCV2 vaccination was strongly advised in this herd due to recent depopulation and repopulation and the high proportion of gilts. Vaccination was effective because no further cases have occurred since.

Granulomatous Necrotizing Myositis in Swine Affected by Porcine Circovirus Disease.

Porcine circovirus type 2 (PCV2) is associated with multiple clinical syndromes in pigs, known as porcine circovirus diseases. This work describes an outbreak of porcine circovirus diseases with severe lesions affecting the skeletal muscle. Ninety-two pigs had apathy, weight loss, and diarrhea over a clinical course of 7 to 10 days. Approximately 30 of the pigs had stiff gait, muscle weakness, hind limb paresis, and recumbency. Twelve of the 92 pigs were necropsied, and 4 had pale discoloration of skeletal muscles with microscopic lesions of granulomatous necrotizing myositis. Immunohistochemistry of skeletal muscle showed that PCV2 antigen was located primarily in the cytoplasm and nuclei of macrophages, lymphocytes, and multinucleated giant cells, with a lower amount in the cytoplasm of endothelial cells, necrotic fibers, and satellite cells. Affected muscle samples were polymerase chain reaction-positive for PCV2 and the amplicon exhibited 99% identity with sequences belonging to the PCV2b genotype. Locomotor clinical signs and granulomatous necrotizing myositis should be considered as another expression of PCV2 infection in pigs.

Markedly different immune responses and virus kinetics in littermates infected with porcine circovirus type 2 or porcine parvovirus type 1

Porcine parvovirus type 1 (PPV1) and porcine circovirus type 2 (PCV2) are small single-stranded DNA viruses with high prevalence in the global pig population. The aim of this study was to compare and contrast PCV2 and PPV1 infections in high-health status pigs and to describe PCV2 long-term infection dynamics. Six caesarian-derived colostrum-deprived pigs were randomly divided into two groups and were experimentally infected with PCV2 or PPV1 at 5 weeks of age. All pigs had detectable viremia by day (D) 3 post-infection. Pigs infected with PPV1 had a detectable INF-α response by D3 followed by a high IFN-γ response by D6. The PPV1 pigs developed antibodies against PPV1 by D6 resulting in decreasing virus titers until PPV1 DNA became undetectable from D28 until D42. In contrast, PCV2-infected pigs had no detectable INF-α or IFN-γ response after PCV2 infection. PCV2-infected pigs had no detectable anti-PCV2 humoral response until D49 and had a sustained high level of PCV2 DNA for the duration of the study. While PPV1-infected pigs were clinically normal, PCV2-infected pigs developed severe clinical illness including fatal systemic porcine circovirus associated disease (PCVAD) by D28, fatal enteric PCVAD by D56 and chronic PCVAD manifested as decreased weight gain and periods of diarrhea. Microscopically, all three PCV2-infected pigs had lymphoid lesions consistent with PCVAD and associated with low (chronic disease) to high (acute disease) levels of PCV2 antigen. Under the study conditions, there was a lack of early IFN-γ and INF-α activation followed by a delayed and low humoral immune response and persisting viremia with PCV2 infection. In contrast, PPV1-infected pigs had IFN-γ and INF-α activation and an effective immune response to the PPV1 infection.

The use of oral fluids to monitor key pathogens in porcine respiratory disease complex

BackgroundThe usefulness of oral fluid (OF) sampling for surveillance of infections in pig populations is already accepted but its value as a tool to support investigations of porcine respiratory disease complex (PRDC) has been less well studied. This study set out to describe detection patterns of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), swine influenza virus type A (SIV) and Mycoplasma hyopneumoniae (M. hyo) among farms showing differing severity of PRDC.The study included six wean-to-finish pig batches from farms with historical occurrence of respiratory disease. OF samples were collected from six pens every two weeks from the 5th to the 21st week of age and tested by real time PCR for presence of PRRSV, SIV and M. hyo and by quantitative real time PCR for PCV2. Data was evaluated alongside clinical and post-mortem observations, mortality rate, slaughter pathology, histopathology, and immunohistochemistry testing data for PCV2 antigen where available.ResultsPRRSV and M. hyo were detectable in OF but with inconsistency between pens at the same sampling time and within pens over sequential sampling times. Detection of SIV in clinical and subclinical cases showed good consistency between pens at the same sampling time point with detection possible for periods of 2–4 weeks. Quantitative testing of OF for PCV2 indicated different patterns and levels of detection between farms unaffected or affected by porcine circovirus diseases (PCVD). There was good correlation of PCR results for multiple samples collected from the same pen but no associations were found between prevalence of positive test results and pen location in the building or sex of pigs.ConclusionsDetection patterns for PRRSV, SIV and M. hyo supported the effectiveness of OF testing as an additional tool for diagnostic investigation of PRDC but emphasised the importance of sampling from multiple pens and on multiple occasions. Preliminary evidence supported the measurement of PCV2 load in pooled OF as a tool for prediction of clinical or subclinical PCVD at farm level.

Depletion of follicular dendritic cells in tonsils collected from PMWS-affected pigs.

Post-weaning multisystemic wasting syndrome (PMWS) is a relevant, worldwide disease caused by porcine circovirus type 2 (PCV2). Microscopically, PMWS is mainly characterized by lymphocytic depletion, macrophage infiltration and syncytia in lymphoid tissues. Some data suggest that follicular dendritic cells (FDCs) could be infected by PCV2, thus likely playing a role in the pathogenesis of PMWS. The present paper aims at assessing, qualitatively and quantitatively, the FDCs' network in the soft palate tonsils of clinically healthy and PMWS-affected pigs. Consecutive tissue sections were tested by immunohistochemistry to detect PCV2, FDCs and macrophages. FDCs and PCV2 antigens were quantitatively assessed by means of the Image J software and results submitted to statistical analysis. Our data demonstrated that FDCs are significantly reduced in PMWS-affected pigs compared with healthy pigs and that FDCs' depletion should be considered among microscopic features of PMWS. It is reasonable to hypothesize that depletion of FDCs further compromises the immune response and enhances the occurrence and the severity of secondary infections, which are relevant for the clinical manifestation of PMWS.

Fluorescence Microsphere Immunoassay for Detection of Antibodies to Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus type 2 using Protein A, Protein G, and Protein A/G

The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) specific IgG antibodies by incorporation of non-species-specific conjugates Protein A, G, and A/G in place of the secondary antibody. A total of 205 serum samples obtained from pigs experimentally infected with PRRSV and/or PCV2 were tested. For the production of recombinant antigens, PRRSV nucleoprotein (N) and PCV2 capsid protein (CP) were expressed in Escherichia coli and purified on a nickel affinity column. The purified proteins were covalently coupled to carboxylated microsphere beads. Four target antigens were assembled into a single multiplex and tested against sera from swine infected or co-infected with PRRSV or PCV2. Protein A, G, and A/G have been tested in place of secondary conjugate porcine IgG. All conjugates were capable of detecting antibody and the detection of IgG responses against PRRSV and PCV2 antigen targets were varied with A> A/G> G.In the absence of species specific reagents the incorporation of Protein A, G, and A/G provide a suitable substitutes.

A commercial porcine circovirus (PCV) type 2a-based vaccine reduces PCV2d viremia and shedding and prevents PCV2d transmission to naïve pigs under experimental conditions

Porcine circovirus type 2 (PCV2) vaccination has been effective in protecting pigs from clinical disease and today is used extensively. Recent studies in vaccinated populations indicate a major PCV2 genotype shift from the predominant PCV2 genotype 2b towards 2d. The aims of this study were to determine the ability of the commercial inactivated PCV2a vaccine Circovac® to protect pigs against experimental challenge with a 2013 PCV2d strain and prevent transmission. Thirty-eight pigs were randomly divided into four groups with 9–10 pigs per group: NEG (sham-vaccinated, sham-challenged), VAC (PCV2a-vaccinated, sham-challenged), VAC+CHAL (PCV2a-vaccinated and PCV2d-challenged), and CHAL (sham-vaccinated, PCV2d-challenged). Vaccination was done at 3weeks of age using Circovac® according to label instructions. The CHAL and VAC+CHAL groups were challenged with PCV2d at 7weeks of age and all pigs were necropsied 21days post-challenge (dpc). The VAC-CHAL pigs seroconverted to PCV2 by 21days post vaccination (dpv). At PCV2d challenge on 28dpv, 3/9 VAC and 1/9 VAC+CHAL pigs were seropositive. NEG pigs remained seronegative for the duration of the study. Vaccination significantly reduced PCV2d viremia (VAC+CHAL) at dpc 14 and 21, PCV2d fecal shedding at dpc 14 and 21 and PCV2d nasal shedding at dpc 7, 14 and 21 compared to CHAL pigs. Vaccination significantly reduced mean PCV2 antigen load in lymph nodes in VAC+CHAL pigs compared to CHAL pigs. When pooled serum or feces collected from VAC+CHAL and CHAL pigs at dpc 21 were used to expose single-housed PCV2 naïve pigs, a pooled fecal sample from CHAL pigs contained infectious PCV2 whereas this was not the case for VAC+CHAL pigs suggesting reduction of PCV2d transmission by vaccination. Under the study conditions, the PCV2a-based vaccine was effective in reducing PCV2d viremia, tissue loads, shedding and transmission indicating that PCV2a vaccination should be effective in PCV2d-infected herds.