Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication

Summary.  Experimental infection of colostrum-deprived (CD) pigs with a combined inoculum of porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiated the replication and distribution of PCV2 virus, when compared with pigs inoculated with PCV2 alone. The replication and distribution of PRRSV in dually infected pigs was not enhanced, when compared to pigs inoculated with PRRSV alone. The mechanisms involved in the potentiation of PCV2 replication in PCV2/PRRSV and PCV2/porcine parvovirus (PPV) dually infected pigs may relate to the fact that monocyte/macrophage cell types are common targets of these 3 viruses. Received May 2, 2000/Accepted May 24, 2000     

A multiplex PCR for rapid and simultaneous detection of porcine circovirus type 2, porcine parvovirus,porcine pseudorabies virus,and porcine reproductive and respiratory syndrome virus in clinical specimens

A multiplex PCR (mPCR) assay was developed and evaluated for its ability to simultaneously detect multiple viral infections of swine. Specific primers were designed for each of the following four DNA or RNA viruses: porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each target produced a specific amplicon with a size of 353 bp (PCV2), 271 bp (PPV), 194 bp (PRV), or 434 bp (PRRSV). The assay was sensitive and specific in detecting each target agent in composite cell cultures and clinical specimens. Results from mPCR were confirmed by PCR for individual viruses and by virus isolation. In conclusion, the mPCR has the potential to be useful for routine molecular diagnosis and epidemiology.     

Porcine reproductive and respiratory syndrome virus infection at the time of porcine circovirus type 2 vaccination has no impact on vaccine efficacy

Several porcine circovirus type 2 (PCV2) vaccines are now commercially available and have been shown to be effective at decreasing the occurrence of porcine circovirus-associated disease (PCVAD). Many herds are coinfected with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV). Some producers and veterinarians are concerned that if pigs are vaccinated for PCV2 at or near the time that they are typically infected with PRRSV, the efficacy of the PCV2 vaccine will be compromised. The impact of PRRSV on PCV2 vaccination is unclear and has not been investigated under controlled conditions. The objective of the present study was to determine whether the presence of PRRSV viremia has an effect on the efficacy of commercial PCV2 vaccinations. Three-week-old PCV2-negative conventional pigs with passively derived anti-PCV2 antibodies were either vaccinated with one of three commercial PCV2 vaccines or left nonvaccinated. A portion of the pigs were infected with PRRSV 1 week prior to PCV2 vaccination. To determine vaccine efficacy, a PCV2 challenge was conducted at 8 weeks of age. PCV2 vaccination, regardless of PRRSV infection status at the time of vaccination, was similarly effective in inducing an anti-PCV2 IgG response in the presence of maternally derived immunity and in protecting the pigs from PCV2 challenge, as determined by a reduction in the level of PCV2 viremia and a reduction in the prevalence and amount of PCV2 antigen in lymphoid tissues in vaccinated pigs compared to nonvaccinated pigs. The results indicate that acute PRRSV infection at the time of PCV2 vaccination has no adverse effect on PCV2 vaccine efficacy.     

Development of a Plexor real-time PCR assay for the detection of porcine circovirus type 2

A novel, real-time PCR system for the detection of porcine circovirus type 2 (PCV2) was developed. The system employed Plexor technology and detected 10⁸-10¹ copies per reaction of PCV2 DNA within a recombinant plasmid. The examination of clinical material showed consistent diagnostic sensitivity when samples contained more than 10³ viral copies per reaction. Specificity of Plexor real-time PCR was confirmed using the porcine viruses PCV1, PRRSV, CSFV, TTSuV1 and TTSuV2 employing the melting curve analysis of PCR products. The low values of coefficient of variation in the intra- (1.74%) and inter-assay (2.41%) analysis suggested that the assay was a highly reproducible. The Plexor real-time PCR was compared with three other real-time PCR systems (SYBR Green, TaqMan, LUX) with conclusion that it can be used as a method of choice for the detection and quantitation of PCV2.     

A live-attenuated chimeric porcine circovirus type 2 (PCV2) vaccine is transmitted to contact pigs but is not upregulated by concurrent infection with porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV) and is efficacious in a PCV2b-PRRSV-PPV challenge model

The live chimeric porcine circovirus type 2 (PCV2) vaccine with the capsid gene of the emerging subtype 2b cloned in the genomic backbone of the nonpathogenic PCV1 is attenuated in vivo and induces protective immunity against PCV2. To further determine the safety and efficacy of this experimental vaccine, we tested for evidence of pig-to-pig transmission by commingling nonvaccinated and vaccinated pigs, determined potential upregulation by simultaneous vaccination and infection with porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV), and determined vaccine efficacy by challenging pigs 4 weeks after vaccination with PCV2b, PRRSV, and PPV. Forty-six 21-day-old, PCV2-naïve pigs were randomly assigned to one of six groups. Twenty-nine of 46 pigs were challenged with PCV2b, PRRSV, and PPV at day 28, 8/46 remained nonvaccinated and nonchallenged and served as negative controls, and 9/46 remained nonchallenged and served as vaccination controls. All animals were necropsied at day 49. PCV1-PCV2 viremia was detected in nonvaccinated contact pigs commingled with vaccinated pigs, indicating pig-to-pig transmission; however, PCV1-PCV2 DNA levels remained low in all vaccinated and contact pigs regardless of concurrent infection. Finally, vaccination 28 days before challenge resulted in significantly (P < 0.05) decreased amounts of PCV2 in tissues and sera and significantly (P < 0.05) reduced macroscopic and microscopic lesions. The results of this study indicate that the experimental live-attenuated chimeric PCV2 vaccine, although transmissible to contact pigs, remains attenuated in pigs concurrently infected with PRRSV and PPV and induces protective immunity against PCV2b when it is administered 28 days before PCV2 exposure.     

Multiplex PCR and multiplex RT-PCR for inclusive detection of major swine DNA and RNA viruses in pigs with multiple infections

Multiplex PCR and multiplex RT-PCR were developed to identify nine viruses in pigs with multiple infections. These viruses are: porcine circovirus type 2 (PCV2), suid herpesvirus 1, porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus, porcine rotavirus A (PoRV-A), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and Getah virus. These methods were shown to be high specificity and sensitivity. In the clinical application, a total of 75 field samples were examined by our methods and previously reported methods for PCV2, PRRSV, TGEV, and PEDV. As a result, the detection rates of our multiplex PCR and multiplex RT-PCR were higher than those of the previously reported methods. Furthermore, it was confirmed that 24 PCV2 positive samples were co-infected with other viruses, 11 with PRRSV, 10 with PPV, 2 with PoRV-A, and 1 with TGEV by a combination of multiplex PCR and multiplex RT-PCR. PPV and PoRV-A were newly detected by multiplex PCR and multiplex RT-PCR. These results suggest that the combination of our multiplex PCR and multiplex RT-PCR is useful for rapid and accurate identification of nine major pathogenic viruses in pigs with multiple infections.     

Concurrent porcine circovirus type 2a (PCV2a) or PCV2b infection increases the rate of amino acid mutations of porcine reproductive and respiratory syndrome virus (PRRSV) during serial passages in pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) has a high degree of genetic and antigenic variability. The purpose of this study was to determine if porcine circovirus type 2 (PCV2) infection increases genetic variability of PRRSV during serial passages in pigs and to determine if there is a difference in the PRRSV mutation rate between pigs concurrently infected with PCV2a or PCV2b. After 8 consecutive passages of PRRSV alone (group 1), PRRSV with PCV2a (group 2), or PCV2b (group 3) in pigs, the sequences of PRRSV structural genes for open reading frame (ORF) 5, ORF6, ORF7 and the partial non-structural protein gene (Nsp) 2 were determined. The total number of identified amino acid mutations in ORF5, ORF6, ORF7 and Nsp2 sequences was 30 for PRRSV infection only, 63 for PRRSV/PCV2a concurrent infection, and 77 for PRRSV/PCV2b concurrent infection when compared with the original VR2385 virus used to infect the passage 1 pigs. Compared to what occurred in pigs infected with PRRSV only, the mutation rates in ORF5 and ORF6 were significantly higher for concurrent PRRSV/PCV2b infected pigs. The PRRSV/PCV2a pigs had a significantly higher mutation rate in ORF7. The results from this study indicated that, besides ORF5 and Nsp2, the PRRSV structural genes ORF6 and ORF7 were shown to mutate at various degrees when the PRRSV was passaged over time in vivo. Furthermore, a significantly higher mutation rate of PRRSV was observed when pigs were co-infected with PCV2 highlighting the importance of concurrent infections on PRRSV evolution and control.     

Development and validation of a duplex real-time PCR assay for the simultaneous detection and quantification of porcine circovirus type 2 and an internal control on porcine semen samples

A duplex real-time quantitative PCR (qPCR) method for the simultaneous detection of porcine circovirus type 2 (PCV2) and an exogenous internal positive control (IPC) in porcine semen samples was developed. The IPC was included to monitor DNA extraction and PCR inhibition and consisted of a mutated PCV2 plasmid clone which differed from the target PCV2 in the probe binding region and thus was detected by the use of a second probe with different end-labeling. The sensitivity, specificity and repeatability of the assay were validated by testing semen samples from 12 boars inoculated experimentally with PCV2, 10 boars infected naturally with PCV2, and 3 PCV2 negative control boars. The duplex qPCR assay was found to be more sensitive, specific, rapid, and repeatable than nested PCR (nPCR) methods for the detection of PCV2 DNA in semen. Analysis of separated semen fractions by the duplex qPCR assay showed PCV2 DNA to be present mainly in the cell fraction as opposed to the seminal plasma fraction which is in contrast to previous reports. The duplex qPCR assay was found to be a valuable tool for accurate and quantitative detection of PCV2 DNA in boar semen.     

Comparison of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs

Postweaning multisystemic wasting syndrome (PMWS) diagnosis is based on the presence of characteristic histopathological lymphoid lesions and porcine circovirus type 2 (PCV2) within these lesions. Previous studies indicate that PCV2 load is higher in PMWS affected than in PCV2 infected, healthy pigs. On the other hand, PCV2 has been suggested to play a role in porcine dermatitis and nephropathy syndrome (PDNS) pathogenesis. This study describes a new TaqMan real time PCR assay and its use to quantify viral load in serum samples. Serum viral loads were related with different degrees of PMWS characteristic lesions and PDNS cases. DNA extracted from serum samples from 75 animals with mild, moderate and severe PMWS lesions and 12 animals with PDNS was used as template. PCV2 DNA was quantified in 69 of 75 PMWS cases and in 11 of 12 PDNS cases. Significant differences in PCV2 load were observed between animals with severe, moderate and mild PMWS lesions, although variability within each group was high, probably due to heterogeneity in disease progression. These results suggest that high viral load is a major feature of PMWS affected pigs. PDNS affected animals had lower PCV2 loads. No significant differences in viral load were found between animals suffering from PDNS and animals with mild PMWS characteristic lesions, which were unaffected clinically.     

Cardiovascular lesions in pigs naturally or experimentally infected with porcine circovirus type 2

Abundant intracytoplasmic porcine circovirus type 2 (PCV2) was associated with myocardiocyte swelling or necrosis, or myocardial fibrosis (or both) in three naturally infected pigs aged 4-7 weeks from three different farms. One 6 week old pig from a fourth farm had severe diffuse segmental to circumferential lymphohistiocytic and plasmacytic periarteritis and endarteritis in several organs, PCV2 antigen was demonstrated in endothelial cells, and inflammatory cells in the arterial walls. In three pigs experimentally infected with PCV2, viral antigen was also associated with obliterated blood vessels in areas of granulomatous and necrotizing lymphadenitis. Together these findings suggest that the cardiovascular system in general and endothelial cells in particular play an important role in the pathogenesis of PCV2-associated diseases.     

Multiplex PCR for the simultaneous detection of pseudorabies virus, porcine cytomegalovirus, and porcine circovirus in pigs

The use of porcine organs is being developed as a means to alleviate the shortage of human organs for transplantation. Recommendations have been published for the microbiological specifications of organ-source pigs to reduce the possibility of a microorganism from pigs being inadvertently transferred to the recipient of the xenograft. The pseudorabies virus (PRV), porcine cytomegalovirus (PCMV), and porcine circovirus (PCV) are infectious agents in pigs that are considered to be of significance for the microbiological safety of xenotransplantation. A multiplex polymerase chain reaction (mPCR) was developed to detect and differentiate among PRV, PCMV, and PCV. The sensitivities of the multiplex PCR were 10(2.5) TCID(50)/ml for PRV, 10(1.8) TCID(50)/ml for PCMV, and 10(1.8) TCID(50)/ml for PCV. The lowest viral concentrations detected by single PCR were 10(1.5) TCID(50)/ml for PRV, 10(1.0) TCID(50)/ml for PCMV, and 10(1.4) TCID(50)/ml for PCV2. Non-specific reactions were not observed when other viruses, bacteria, and Vero cells were used to assess the multiplex PCR. The multiplex PCR was effective in detecting various combinations of one or more of these viruses in pig specimens collected for xenotransplantation.     

Comparative Effects of Vaccination against Porcine Circovirus Type 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in a PCV2-PRRSV Challenge Model

The objective of the present study was to determine the effects of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) vaccinations in an experimental PCV2-PRRSV challenge model, based on virological (viremia), immunological (neutralizing antibodies [NAs], gamma interferon-secreting cells [IFN-γ-SCs], and CD4⁺ CD8⁺ double-positive cells), and pathological (lesions and antigens in lymph nodes and lungs) evaluations. A total of 72 pigs were randomly divided into 9 groups (8 pigs per group): 5 vaccinated and challenged groups, 3 nonvaccinated and challenged groups, and a negative-control group. Vaccination against PCV2 induced immunological responses (NAs and PCV2-specific IFN-γ-SCs) and reduced PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. However, vaccination against PCV2 did not affect the PRRSV immunological responses (NAs and PRRSV-specific IFN-γ-SCs), PRRSV viremia, PRRSV-induced lesions, or PRRSV antigens in the dually infected pigs. Vaccination against PRRSV did not induce immunological responses (PRRSV-specific IFN-γ-SCs) or reduce PRRSV viremia, PRRSV-induced lesions, or PRRSV antigens in the dually infected pigs. In addition, vaccination against PRRSV increased PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. In summary, vaccination against PCV2 reduced PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. However, vaccination against PRRSV increased PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. Therefore, the PCV2 vaccine decreased the potentiation of PCV2-induced lesions by PRRSV in dually infected pigs. In contrast, the PRRSV vaccine alone did not decrease the potentiation of PCV2-induced lesions by PRRSV in dually infected pigs.     

Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR

Porcine circovirus type 2 (PCV2) has been linked to several disease syndromes during the last decade. In this context, postweaning multisystemic wasting syndrome (PMWS) has emerged as a significant disease. As most pig herds are infected with PCV2, the determination of viral load in animals may be useful in discriminating between healthy and PMWS pigs. A TaqMan-based real-time PCR for quantitation of PCV2 in serum/plasma and tissue samples was established. A standard curve was created from serial dilutions of a plasmid encoding ORF2 (cap gene) of PCV2 and used to estimate the number of viral DNA copies in the analyzed samples. Comparison of viral load in mesenteric lymph nodes and serum/plasma from healthy animals and PMWS animals showed statistical significant difference between the two groups (p < 0.01). No healthy pigs had viral load greater than 10(6) PCV2 genomes per ml serum or 500 ng tissue sample, while all clinically sick PMWS pigs had PCV2 loads above 10(7) in both serum/plasma and in tissue samples. Furthermore, the estimated viral load in tissue samples from PMWS pigs was related to the immunohistochemical findings, with especially lymph nodes, ileum, and tonsil giving both high viral load, and a high degree of staining by immunohistochemistry.     

Distribution of porcine parvovirus in porcine circovirus 2-infected pigs with postweaning multisystemic wasting syndrome as shown by in-situ hybridization

In-situ hybridization with a nonradioactive digoxigenin-labelled probe was used to study the distribution of porcine parvovirus (PPV) in formalin-fixed paraffin wax-embedded tissues from 10 porcine circovirus 2 (PCV2)-infected weaned pigs with naturally occurring postweaning multisystemic wasting syndrome (PMWS). A 226 base pair DNA fragment from a VP2 structural gene was generated by polymerase chain reaction (PCR) and used as a probe. Lymph node, spleen, thymus and tonsil were positive by PCR, demonstrating the presence of PPV DNA in the tissue samples from four of 10 pigs tested. PPV nucleic acid was also detected consistently in lymph node, spleen, thymus and tonsil by in-situ hybridization. Detection of PPV DNA from PCV2-infected pigs with PMWS suggests that PPV also plays a role in the pathogenesis of PMWS.     

Porcine circovirus type 2 infection decreases the efficacy of a modified live porcine reproductive and respiratory syndrome virus vaccine

Porcine reproductive and respiratory syndrome virus (PRRSV)-induced pneumonia is a major problem, and vaccination is used to reduce losses associated with PRRSV. Porcine circovirus type 2 (PCV2) causes lymphoid depletion, and there is concern that this adversely affects the immune response. The objective of this study was to investigate the effect of PCV2 infection on the efficacy of modified live virus (MLV) PRRSV vaccine. Sixty-nine 2-week-old pigs were randomly assigned to one of seven groups of 9 to 10 pigs each. At 6 weeks of age, pigs in groups 4, 5, and 6 were inoculated intranasally with PCV2 ISU-40895. At 8 weeks of age, groups 3, 4, 6, and 7 were vaccinated with a PRRSV MLV vaccine. At 12 weeks of age, groups 2, 3, and 4 were challenged with PRRSV SDSU73. All pigs were necropsied 14 days after PRRSV challenge. PCV2-infected, PRRSV-vaccinated, and PRRSV-challenged pigs had significantly (P < 0.05) more-severe macroscopic lung lesions than did the PRRSV-vaccinated and PRRSV-challenged pigs that were not exposed to PCV2 prior to PRRSV vaccination. Nonvaccinated PRRSV-infected pigs had a significantly (P < 0.001) higher incidence of PRRSV antigen in lungs than did all other groups except the group infected with PCV2 prior to PRRSV vaccination and challenge. The nonvaccinated PRRSV-challenged group and the group challenged with PCV2 prior to PRRSV vaccination and challenge had significantly (P < 0.001) lower average daily weight gain than did the control and the vaccinated groups. This work suggests that PCV2 infection has an adverse effect on the development of protective immunity induced by PRRSV vaccine.     

Rapid detection of porcine circovirus type 2 by loop-mediated isothermal amplification

A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). The amplification could be finished in 60 min under isothermal condition at 64 degrees C by employing a set of four primers targeting the cap gene of PCV2. The LAMP assay showed higher sensitivity than the conventional PCR, with a detection limit of five copies per tube of purified PCV2 genomic DNA. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1 (PCV1), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV). The detection rate of PCV2 LAMP for 86 clinical samples was 96.5% and appeared greater than that of the PCR method. The LAMP assay reported can provide a rapid yet simple test of PCV2 suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.     

Multiplex method for simultaneous serological detection of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are major contributors to the porcine respiratory disease complex (PRDC). Routine serological diagnosis and surveillance play an important role in the prevention of PRDC, as it is a leading cause of economic losses to the swine industry. We herein describe an advanced microsphere-based immunoassay that permits the simultaneous detection of antibodies to PCV2 and PRRSV, thereby reducing the time and effort involved in testing. Recombinant PRRSV nucleoprotein antigen and the PCV2 capsid antigen were coupled to fluorophore-dyed beads with distinct spectral addresses. Weekly serum samples from 72 pigs that were experimentally exposed to either PCV2, PRRSV, or both PCV2 and PRRSV were used to validate the microbead assay (MBA) in comparison with the "gold standard" enzyme-linked immunosorbent assays. The kinetics of the PCV2- and PRRSV-specific antibody responses measured by the microbead assay were comparable to those of the standard assays; Spearman's rank correlations were 0.72 (P < 0.001) for PRRSV and 0.80 (P < 0.001) for PCV2. Diagnostic sensitivity and specificity were determined using field sera whose positive or negative status was determined by the standard tests. The diagnostic sensitivity and specificity were both 98% for PCV2 and were 91% and 93%, respectively, for PRRSV (kappa coefficients, 0.85 and 0.67 for PCV2 and PRRSV, respectively). Multiplexing did not interfere with assay performance or diagnostic sensitivity. Therefore, the described study demonstrates proof of concept for the development of more versatile and economical microbead array-based multiplex serological test panels for veterinary use.     

Multiplex PCR for detection and typing of porcine circoviruses

Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogenic PK-15 strain of porcine circovirus (PCV) type 1 (PCV-1). By the PCR performed with the various primer sets, genomic DNA or RNA from other bacterial or viral pathogens of the respiratory tracts of pigs could not be amplified. A positive amplification reaction could be visualized with DNA extracted from a viral suspension containing as few as 10 viral particles per ml. No DNA fragment could be amplified from lysates of continuous porcine cell lines (PT, ST, and PFT cells) known to be negative for PCV. When tested with clinical samples from pigs, the results of the single PCR method showed nearly 93% (13 of 14 samples) correlation with histopathological and immunohistochemical findings. Interestingly, subclinical PCV infections could be detected by single PCR with clinical samples that have been submitted from animals with irrelevant cases of respiratory and/or enteric problems. On the basis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Quebec, Canada, pig farms, other primers were designed from the PCV-1 genome, and these primers failed to amplify genomic fragments specific to the ORF1 or ORF2 genes of clinical isolates associated with PWMS but amplified DNA from the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been developed to distinguish between both genotypes of PCV. By those two mPCR methods, (i) species-specific primer pairs were used to amplify a DNA fragment of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fragment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) species-specific primer pairs were used to amplify a DNA fragment of 646 bp specific for the ORF1 genes of both genotypes, whereas a 425-bp fragment was amplified from the ORF2 gene of the PCV-1 strain only. By both mPCR methods, a PCV-2 infection was demonstrated in tissues of 94.2% (33 of 35) of the sick pigs tested, in agreement with previous findings showing the close association of this new genotype of PCV with outbreaks of PMWS in Europe and North America. On the other hand, a PCV-1 infection was confirmed in only 5.7% (2 of 35) of the pigs, and confirmation of a mixed infection with PCV-2 was obtained by a single PCR with PCV-2-specific primers.