Porcine Circovirus Type 2 Coinfection Research Articles

Glaesserella parasuis serotype 4 exploits fibronectin via RlpA for tracheal colonization following porcine circovirus type 2 infection.

Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality, but the role played by PCV2 and bacterial and host factors contributing to this process have not been defined. Bacterial attachment is assumed to occur via specific receptor-ligand interactions between adhesins on the bacterial cell and host proteins adsorbed to the implant surface. Mass spectrometry (MS) analysis of PCV2-infected swine tracheal epithelial cells (STEC) revealed that the expression of Extracellular matrix protein (ECM) Fibronectin (Fn) increased significantly on the infected cells surface. Importantly, efficient G. parasuis serotype 4 (GPS4) adherence to STECs was imparted by interactions with Fn. Furthermore, abrogation of adherence was gained by genetic knockout of Fn, Fn and Integrin β1 antibody blocking. Fn is frequently exploited as a receptor for bacterial pathogens. To explore the GPS4 adhesin that interacts with Fn, recombinant Fn N-terminal type I and type II domains were incubated with GPS4, and the interacting proteins were pulled down for MS analysis. Here, we show that rare lipoprotein A (RlpA) directly interacts with host Fibronectin mediating GPS4 adhesion. Finally, we found that PCV2-induced Fibronectin expression and adherence of GPS4 were prevented significantly by TGF-β signaling pathway inhibitor SB431542. Our data suggest the RlpA-Fn interaction to be a potentially promising novel therapeutic target to combat PCV2 and GPS4 coinfection.

A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide.We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap anddeveloped a multiplex crystal digital PCR (cdPCR) method after optimizing the primerconcentration, probe concentration, and annealing temperature.The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

Porcine circovirus type 2 and Glaesserella parasuis serotype 4 co-infection activates Snail1 to disrupt the intercellular junctions and facilitate bacteria translocation across the tracheal epithelium

Clinically, Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality. However, the mechanism of PCV2 and G. parasuis serotype 4 (GPS4) co-infection is still not fully understood. In this study, swine tracheal epithelial cells (STEC) were used as a barrier model, and our results showed that PCV2 infection increased the adhesion of GPS4 to STEC, while decreasing the levels of ZO-1, Occludin and increasing tracheal epithelial permeability, and ultimately facilitated GPS4 translocation. Snail1 is a transcriptional repressor, and has been known to induce epithelial-to-mesenchymal transition (EMT) during development or in cancer metastasis. Importantly, we found that Snail1, as a transcriptional repressor, was crucial in destroying the tracheal epithelial barrier induced by PCV2, GPS4, PCV2 and GPS4 coinfection. For the first time, we found that PCV2, GPS4, PCV2 and GPS4 coinfection cross-activates TGF-β and p38/MAPK signaling pathways to upregulate the expression of Snail1, down-regulate the levels of ZO-1 and Occludin, and thus disrupt the integrity of tracheal epithelial barrier then promoting GPS4 translocation. Finally, PCV2 and GPS4 co-infection also can activate TGF-β and p38/MAPK signaling pathways in vivo and upregulate Snail1, ultimately down-regulating the expression of ZO-1 and Occludin. Our study elucidates how PCV2 infection promotes GPS4 to breach the tracheal epithelial barrier and aggravate clinical manifestations.

Development of Duplex LAMP Technique for Detection of Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Circovirus Type 2 (PCV 2).

Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.

Development of multiplex PCR assay for simultaneous detection of African swine fever, porcine circo and porcine parvo viral infection from clinical samples

A diagnostic method for simultaneously detecting and distinguishing African Swine Fever (ASF), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV) in clinical specimens is critical for differential diagnosis, monitoring, and control in the field. Three primer pairs were designed and used to create a multiplex PCR assay. In addition, 356 porcine post mortem tissue samples from various parts of India's North Eastern region were tested by the developed multiplex PCR assay to demonstrate its accuracy. Using the designed primers, each of the ASF, PCV2 and PPV target genes was amplified, but no other porcine virus genes were detected. The assay's limit of detection was 102 copies/µl of PCV2, PPV, or ASFV. The detection of PCV2, PPV, and ASF in postmortem tissue samples revealed that they are co-circulating in India's North-Eastern region. The percentage positivity (PP) for PCV2, PPV and ASF single infection were 7.02% (25/356), 3.93% (14/356), and 3.37% (12/356), respectively, while the PP for PCV2& PPV co-infection was 2.80% (10/356), ASF & PCV2 co infection was 1.4% (5/356) and the ASF, PPV& PCV2 co-infection was1.40% (5/356). The results also indicate that the ASF can infect pigs alongside PCV and PPV.

Prevalence and genetic analysis of porcine circovirus type 2 (PCV2) and type 3 (PCV3) between 2018 and 2020 in central China.

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVAD), causing substantial economic losses to the swine industry worldwide. PCV3, as a recently discovered virus, is associated with porcine dermatitis, nephropathy syndrome, reproductive failure, congenital tremors, and other clinical symptoms. To further investigate the epidemic profile and genetic diversity of the two viruses, a total of 198 samples from swine at various growth stages suspected for PCVAD on 55 different pig farms between 2018 and 2020 were analyzed for presence of PCV2 and PCV3 by using a multiplex real-time PCR assay. Among the 198 samples, 113 (57.07%) and 72 (36.36%) were positive for PCV2 and PCV3 respectively, and 39 (19.7%) were positive for PCV2 and PCV3 co-infection. Subsequently, whole genome sequences of 34 PCV2 and 19 PCV3 strains were obtained from 30 and 19 clinical samples, respectively. Of these, 8 PCV2 strains belonged to PCV2a, 10 belonged to PCV2b and 16 belonged to PCV2d, indicating PCV2d was the predominant PCV2 genotype circulating in central China. Furthermore, co-infection of different PCV2 genotype strains was identified in three samples (JZ-4, KF-2 and JY-1), and a cross-recombination was found in the ORF2 region of the sequenced 13 PCV2d strains whose putative parental strains were LN6/1999 (MF278777) and MEX/41238/2014 (KT795287) strains. The phylogenetic analysis of PCV3 showed high nucleotide identity (>98%) among sequences obtained in this study and reference sequences. These data will aid our understanding of the molecular epidemiology and evolution of PCV2 and PCV3.

Coinfection with Porcine Circovirus Type 2 (PCV2) and Streptococcus suis Serotype 2 (SS2) Enhances the Survival of SS2 in Swine Tracheal Epithelial Cells by Decreasing Reactive Oxygen Species Production.

Porcine circovirus type 2 (PCV2) and Streptococcus suis serotype 2 (SS2) clinical coinfection cases have been frequently detected. The respiratory epithelium plays a crucial role in host defense against a variety of inhaled pathogens. Reactive oxygen species (ROS) are involved in killing of bacteria and host immune response. The aim of this study is to assess whether PCV2 and SS2 coinfection in swine tracheal epithelial cells (STEC) affects ROS production and investigate the roles of ROS in bacterial survival and the inflammatory response. Compared to SS2 infection, PCV2/SS2 coinfection inhibited the activity of NADPH oxidase, resulting in lower ROS levels. Bacterial intracellular survival experiments showed that coinfection with PCV2 and SS2 enhanced SS2 survival in STEC. Pretreatment of STEC with N-acetylcysteine (NAC) also helps SS2 intracellular survival, indicating that PCV2/SS2 coinfection enhances the survival of SS2 in STEC through a decrease in ROS production. In addition, compared to SS2-infected STEC, PCV2/SS2 coinfection and pretreatment of STEC with NAC prior to SS2 infection both downregulated the expression of the inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β. Further research found that activation of p38/MAPK promoted the expression of inflammatory cytokines in SS2-infected STEC; however, PCV2/SS2 coinfection or NAC pretreatment of STEC inhibited p38 phosphorylation, suggesting that coinfection of STEC with PCV2 and SS2 weakens the inflammatory response to SS2 infection through reduced ROS production. Collectively, coinfection of STEC with PCV2 and SS2 enhances the intracellular survival of SS2 and weakens the inflammatory response through decreased ROS production, which might exacerbate SS2 infection in the host.

An epidemiological investigation of porcine circovirus type 2 and porcine circovirus type 3 infections in Tianjin, North China.

Novel porcine circovirus type 3 (PCV3), first identified in the United States, has been detected in many other countries. Porcine circovirus is associated with postweaning multisystemic wasting syndrome, reproductive failure, congenital tremors, and other clinical symptoms. In this study, we established a double polymerase chain reaction assay for detecting both porcine circovirus type 2 (PCV2) and PCV3. This is the first study to detect and characterize the PCV3 genome in the Tianjin region of North China. We collected a total of 169 tissue samples from seven farms between 2016 and 2018. The PCV3-positive rate of all tissue samples was 37.3% (63/169) and the rate of PCV2 and PCV3 coinfection was 14.8% (25/169). PCV2 and PCV3 coinfections with more serious clinical symptoms were found in only three farms. We sequenced three PCV3 strains selected from tissue samples that were positively identified. The complete genome sequences of the three strains shared 97.6–99.4% nucleotide identities with the PCV3 strains in GenBank. Our results showed the extent of PCV3’s spread in Tianjin, and the need to further study PCV3’s pathobiology, epidemiology, isolation, and coinfection.

Reduction of Postweaning Multisystemic Wasting Syndrome-Associated Clinical Symptoms by Virus-Like Particle Vaccine Against Porcine Parvovirus and Porcine Circovirus Type 2.

The porcine circovirus type 2 (PCV2) capsid (Cap) protein and porcine parvovirus (PPV) VP2 protein have been studied in vaccines to control postweaning multisystemic wasting syndrome (PMWS). Virus-like particle (VLP) vaccines are nonreplicative vectors that deliver epitopes and induce immune responses. However, most VLP vaccines are recombinant proteins expressed in eukaryotic systems and are expensive and complex. In this study, the full-length PCV2-Cap and PPV-VP2 proteins were expressed in Escherichia coli, which self-assembled into VLPs. The highly soluble proteins were purified using Ni-chelating affinity chromatography. The proteins self-assembled into VLPs of ∼20 nm (Cap VLP) and 25 nm (VP2 VLP) in diameter. The immunogenicities of Cap VLP and VP2 VLP were determined in piglets coinfected with PPV and PCV2 postimmunization. The results suggested that Cap VLP and VP2 VLP did not antagonize each other. The combined vaccine induced stronger humoral and cellular immune responses and provided the best protection against PPV and PCV2 coinfection. On a farm containing PMWS-infected pigs, the combined Cap VLP and VP2 VLP vaccine significantly improved piglet growth indices; the average daily weight gains were significantly higher than those of the Cap VLP vaccine and nonimmunized groups. Thus, Cap and VP2 protein expression in E. coli is feasible for large-scale VLP vaccine production. The combined vaccine may be a promising candidate vaccine for better preventing PMWS-associated diseases coinfected with PCV2 and PPV.

Porcine circovirus type 2 exploits JNK-mediated disruption of tight junctions to facilitate Streptococcus suis translocation across the tracheal epithelium

Porcine circovirus type 2 (PCV2) is considered as the primary pathogen of porcine circovirus-associated disease (PCVAD), which results in significant economic losses worldwide. Clinically, PCV2 often causes disease through coinfection with other bacterial pathogens, including Streptococcus suis (S. suis), and especially the highly prevalent S. suis serotype 2 (SS2). The present study determined that continuous PCV2 infection in piglets down-regulates tight junction proteins (TJ) ZO-1 and occludin in the lungs. Swine tracheal epithelial cells (STEC) were used to explore the mechanisms and consequences of disruption of TJ, and an in vitro tracheal epithelial barrier model was established. Our results show that PCV2 infection in STEC decreases the expression levels of ZO-1 and occludin and increases the permeability of the tracheal epithelial barrier, resulting in easier translocation of SS2. Moreover, Western blot analysis indicates that PCV2 infection activates the JNK/MAPK pathway. The disruption of TJ in SETC and increased permeability of the epithelial barrier induced by PCV2 could be alleviated by inhibition of JNK phosphorylation, which indicates that the JNK/MAPK pathway regulates the expression of ZO-1 and occludin during PCV2 infection. This study allows us to better understand the mechanisms of PCV2 coinfection with bacterial pathogens and provides new insight into controlling the occurrence of PCVAD.

Development of a multiplex PCR to detect and discriminate porcine circoviruses in clinical specimens

BackgroundA diagnostic method to simultaneously detect and discriminate porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) in clinical specimens is imperative for the differential diagnosis and monitoring and control of PCVs in the field.MethodsThree primer pairs were designed and used to develop a multiplex PCR assay. And 286 samples from 8 farms in Hubei province were tested by the developed multiplex PCR assay to demonstrate the accuracy.ResultsEach of target genes of PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine viruses genes were detected. The limit of detection of the assay was 10 copies/μL of PCV1, PCV2 OR PCV3. The results of the tissue samples detection showed that PCV1, PCV2 and PCV3 are co-circulating in central China. The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co-infection rate was 1.7% (5/286), respectively, which were 100% consistent with the sequencing method and real-time PCR methods.ConclusionsThe multiplex PCR assay could be used as a differential diagnostic tool for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection and their co-infection are severe in Hubei province, Central China.

Effect of porcine circovirus type 2 on the severity of lung and brain damage in piglets infected with porcine pseudorabies virus

Porcine circovirus type 2 (PCV2) is widespread throughout Chinese farms, and the infection rate of porcine pseudorabies virus (PRV) is very high. The emergence of mixed infection involving PCV2 and PRV has been difficult to prevent and control and has caused considerable economic loss. The present study investigated lung and brain damage caused by PRV in piglets with PCV2 infection. Twenty piglets were divided randomly into two experiment groups (PRV group and PRV + PCV2 group; n = 10 per group). The pigs were observed for clinical signs at specified times. At necropsy, lung and brain tissue samples were collected for histopathological examination, and tissue virus load was determined using quantitative polymerase chain reaction. Severe pathogenicity due to PRV was evident in two-month-old piglets. PCV2 and PRV co-infection led to more severe neurological and respiratory symptoms and a higher mortality rate in the piglets. In addition, the pathological damage to the lung and brain was also aggravated. The co-infection was associated with a significant increase in the content of PRV in the brain and lung tissue. In conclusion, PCV2 and PRV co-infection could cause severe and irreversible damage to piglets.

In Vitro Coinfection and Replication of Classical Swine Fever Virus and Porcine Circovirus Type 2 in PK15 Cells.

Increasing clinical lines of evidence have shown the coinfection/superinfection of porcine circovirus type 2 (PCV2) and classical swine fever virus (CSFV). Here, we investigated whether PCV2 and CSFV could infect the same cell productively by constructing an in vitro coinfection model. Our results indicated that PCV2-free PK15 cells but not ST cells were more sensitive to PCV2, and the PK15 cell line could stably harbor replicating CSFV (PK15-CSFV cells) with a high infection rate. Confocal and super-resolution microscopic analysis showed that PCV2 and CSFV colocalized in the same PK15-CSFV cell, and the CSFV E2 protein translocated from the cytoplasm to the nucleus in PK15-CSFV cells infected with PCV2. Moreover, PCV2-CSFV dual-positive cells increased gradually in PK15-CSFV cells in a PCV2 dose-dependent manner. In PK15-CSFV cells, PCV2 replicated well, and the production of PCV2 progeny was not influenced by CSFV infection. However, CSFV reproduction decreased in a PCV2 dose-dependent manner. In addition, cellular apoptosis was not strengthened in PK15-CSFV cells infected with PCV2 in comparison with PCV2-infected PK15 cells. Moreover, using this coinfection model we further demonstrated PCV2-induced apoptosis might contribute to the impairment of CSFV HCLV strain replication in coinfected cells. Taken together, our results demonstrate for the first time the coinfection/superinfection of PCV2 and CSFV within the same cell, providing an in vitro model to facilitate further investigation of the underlying mechanism of CSFV and PCV2 coinfection.

Experimental Reproduction of Porcine Circovirus Type 2 (PCV2)-Associated Enteritis in Pigs Infected with PCV2 Alone or Concurrently with Lawsonia intracellularis or Salmonella typhimurium

Porcine circovirus (PCV)-associated disease (PCVAD) has emerged to become one of the most economically important pig diseases globally. One of the less commonly recognized clinical manifestations of PCVAD is PCV2 type 2 (PCV2)-associated enteritis in growing pigs; however, experimental confirmation of the ability of PCV2 alone or PCV2 coinfection with other agent(s) to induce enteritis is lacking. In this study, 120 specific-pathogen-free (SPF) pigs were divided randomly into six groups: controls (negative control pigs), PCV2 (inoculated with PCV2), LAW (inoculated with Lawsonia intracellularis), SALM (inoculated with Salmonella typhimurium), PCV2-LAW (concurrently inoculated with PCV2 and Lawsonia intracellularis) and PCV2-SALM (concurrently inoculated with PCV2 and Salmonella typhimurium). One half of the pigs in each group were subject to necropsy examination 14 days postinoculation (dpi) and the remaining pigs were examined at 28 dpi. The average daily weight gain was not different (P>0.05) between groups. Individual pigs inoculated orally with PCV2 regardless of coinfection status (2/10 PCV2, 1/10 PCV2-LAW, 3/10 PCV2-SALM) developed PCVAD with diarrhoea and reduced weight gain or weight loss between 14 and 28 dpi. Those pigs had characteristic microscopic lesions in lymphoid and enteric tissues associated with abundant PCV2 antigen. Enteric lesions were characterized by necrosuppurative and proliferative enteritis with crypt elongation and epithelial hyperplasia in LAW and PCV2-LAW pigs by 14 dpi, ulcerative and necrosuppurative colitis in SALM and PCV2-SALM pigs by 14 dpi, and lymphohistiocytic enteritis with depletion of Peyer's patches in PCV2, PCV2-SALM and PCV2-LAW pigs by 28 dpi. To the authors' knowledge, this is the first report documenting that under experimental conditions, PCV2 can induce enteritis independently from other enteric pathogens and that oral challenge is a potentially important route and perhaps the natural route of PCV2 transmission in growing pigs.

Molecular detection of genotype 3 porcine hepatitis E virus in aborted fetuses and their sows

By reverse transcription (RT)-nested polymerase chain reaction (PCR), hepatitis E virus (HEV) RNA was simultaneously detected in the livers of aborted fetuses and in fecal and serum samples from their sows, obtained from two of 14 farms tested. Sequence and phylogenetic analyses showed that these HEVs belonged to genotype 3 HEV. HEV RNA was detected by real-time RT-PCR at 2.0 x 10(3)-2.8 x 10(4) viral copies/microl in the HEV-positive samples. All HEV-positive fetuses tested positive for porcine circovirus type 2 (PCV2). These results indicate that transplacental infection of HEV with PCV2 coinfection may occur in sows with reproductive failure, which is suggestive of similarities to HEV infection in women.

First report of porcine circovirus type 2 infections in Cuba

To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba and the probable association of PCV2 with other porcine viruses, tissue samples collected from ill pigs were evaluated using polymerase chain reaction (PCR). The PCR analysis showed that 67.7% of the samples (23/34) from seven swine herds of six different geographic regions were detected to be positive for PCV2. Ten of the 23 PCV2 positive samples (43.5%) shown a concurrent infection with porcine parvovirus (PPV) and 17 of 23 PCV2 positive samples (73.9%) exhibited a concomitant infection with classical swine fever virus (CSFV). This study is the first report of PCV2 infecting pigs with different clinical conditions in Cuban swine herds and provides evidence of PCV2 co-infection with PPV and CSFV in the field.

Effect of porcine circovirus type 2 (PCV2) vaccination on porcine reproductive and respiratory syndrome virus (PRRSV) and PCV2 coinfection

The objectives were to determine if PCV2 vaccination is effective in reducing disease and lesions associated with PRRSV and PCV2 coinfection and if there is a difference between intradermal (ID) and intramuscular (IM) route of PCV2 vaccination. Seventy-four, 21-day-old pigs were randomly allocated into one of six groups. On day 0, pigs were vaccinated with 2 ml Suvaxyn ® PCV2 One Dose (Fort Dodge Animal Health, Inc.) by intramuscular (VAC-M-COINF) or intradermal (VAC-D-COINF) routes. On day 28, pigs were either singularly (PRRSV-only, PCV2-only) or coinfected (COINF) with PRRSV and PCV2. All pigs in all groups were necropsied on day 42. All vaccinated pigs seroconverted (IgM, IgG, and neutralizing antibodies) to PCV2 between 14 and 28 days post-vaccination. After challenge, all groups inoculated with PRRSV had reduced average daily gain compared to CONTROLS and PCV2-only ( P < 0.001). COINF pigs had significantly ( P < 0.05) reduced anti-PCV2-IgG antibody levels and neutralizing antibody levels compared to both vaccinated groups. COINF pigs had more severe lung lesions compared to VAC-M-COINF ( P < 0.05). COINF pigs had higher amounts of PCV2 DNA in serum samples and feces ( P < 0.05) and increased amounts of PCV2 in lymphoid tissues ( P < 0.05) compared to both vaccinated groups. In summary, PCV2 vaccination was effective at inducing a neutralizing antibody response and significantly reducing PCV2-associated lesions and PCV2 viremia in pigs coinfected with PCV2 and PRRSV. Differences between intradermal and intramuscular routes of vaccine administration were not observed.

Changes in peripheral blood leukocyte subpopulations in piglets co-infected experimentally with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are pathogens, which can significantly affect the swine industry worldwide. Field surveys suggest that simultaneous PRRSV and PCV2 infection is common in pigs. The objective of this study was to measure the changes in peripheral blood leukocyte subpopulations in piglets co-infected experimentally with PRRSV and PCV2, in order to analyze the synergistic influence of co-infection on the immune system. Changes in peripheral blood leukocyte subpopulations were systematically measured by flow cytometry (FCM). The levels of antibodies to PRRSV and PCV2 were detected by indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) and the indirect fluorescent antibody test (IFA), respectively. Serum viral loads were measured using real-time PCR. The results showed that piglets co-infected with PRRSV and PCV2 exhibited slower generation and lower levels of antibodies to PRRSV and PCV2, and increased amounts and a prolonged presence of both PRRSV and PCV2 in serum, in comparison to the piglets infected with either virus alone. The major finding in our study was that the total and differential leukocyte counts, including white blood cells (WBCs), monocytes, granulocytes and lymphocytes (T, B and NK cells, as well as T-cell subpopulations), dramatically decreased early during co-infection with PRRSV and PCV2 for about two weeks, in contrast with animals singly infected with either PRRSV or PCV2. These results suggest that PRRSV and PCV2 co-infection results in a synergistic decrease in immune cells in the peripheral blood of piglets. These data contribute to the understanding of the immunosuppressive effects resulting from PRRSV and PCV2 co-infection in pigs.

Detection of Porcine Circovirus Type 2, Porcine Parvovirus and Porcine Pseudorabies Virus from Pigs with Postweaning Multisystemic Wasting Syndrome by Multiplex PCR

Multiplex PCR was established to detect porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV) and applied to samples from 137 piglets exhibiting clinical signs of postweaning multisystemic wasting syndrome (PMWS). PCV-2 DNA was detected from all samples. Moreover, 43 samples were positive for PPV but negative for PRV; 11 samples were positive for PRV but negative for PPV; and 35 samples were positive both for PPV and PRV. These results suggests that PCV-2 co-infection with PRV and PPV may play an important role in PMWS. Also, multiplex PCR is an appropriate candidate method for diagnosis of PCV-2, PRV and PPV simultaneously in field cases.