Efficacy evaluation of a bivalent subunit vaccine against classical swine fever virus and porcine circovirus type 2.

Classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2) are two of the most devastating and economically significant pathogens affecting pig populations worldwide. Administration of a combination of vaccines against swine pathogens has been demonstrated to be as efficacious as the administration of single vaccines. In this study, we developed and tested a novel bivalent subunit vaccine against CSFV and PCV2. The safety and efficacy of this vaccine were demonstrated in mice and specific pathogen-free (SPF) piglets. In addition to investigating the serological responses after immunization, challenge studies with both viruses were also conducted. The results showed that this CSFV/PCV2 bivalent vaccine elicited a high level of neutralizing antibodies against both viruses and provided protection in challenge studies. In conclusion, the CSFV/PCV2 bivalent vaccine is safe and effective against CSFV or PCV2 challenge.

Toll-like Receptor-Mediated Immunomodulation of Th1-Type Response Stimulated by Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2).

PRRSV infects CD163-positive macrophages and skews their polarization toward an M2 phenotype, followed by T-cell inactivation. In our previous study, we found that recombinant protein A1 antigen derived from PRRSV-2 was a potential vaccine or adjuvant for immunization against PRRSV-2 infection due to its ability to repolarize macrophages into M1 subtype, thereby reducing CD163 expression for viral entry and promoting immunomodulation for Th1-type responses, except for stimulating Toll-like receptor (TLR) activation. The aim of our current study was to evaluate the effects of another two recombinant antigens, A3 (ORF6L5) and A4 (NLNsp10L11), for their ability to trigger innate immune responses including TLR activation. We isolated pulmonary alveolar macrophages (PAMs) from 8- to 12-week-old specific pathogen free (SPF) piglets and stimulated them with PRRSV (0.01 MOI and 0.05 MOI) or antigens. We also investigated the T-cell differentiation by immunological synapse activation of PAMs and CD4+ T-cells in the cocultured system. To confirm the infection of PRRSV in PAMs, we checked the expression of TLR3, 7, 8, and 9. Our results showed that the expression of TLR3, 7, and 9 were significantly upregulated in PAMs by A3 antigen induction, similar to the extent of PRRSV infection. Gene profile results showed that A3 repolarizes macrophages into the M1 subtype potently, in parallel with A1, as indicated by significant upregulation of proinflammatory genes (TNF-α, IL-6, IL-1ß and IL-12). Upon immunological synapse activation, A3 potentially differentiated CD4 T cells into Th1 cells, determined by the expression of IL-12 and IFN-γ secretion. On the contrary, antigen A4 promoted regulatory T cell (T-reg) differentiation by significant upregulation of IL-10 expression. Finally, we concluded that the PRRSV-2 recombinant protein A3 provided better protection against PRRSV infection, suggested by its capability to reeducate immunosuppressive M2 macrophages into proinflammatory M1 cells. As M1 macrophages are prone to be functional antigen-presenting cells (APCs), they can call for TLR activation and Th1-type immune response within the immunological synapse.

Antibody Evaluation and Mutations of Antigenic Epitopes in the Spike Protein of the Porcine Epidemic Diarrhea Virus from Pig Farms with Repeated Intentional Exposure (Feedback).

The feedback strategy, or controlled exposure of pig herd to the porcine epidemic diarrhea virus (PEDV), significantly decreased losses during a severe outbreak in late 2013 in Taiwan. However, some pig farms still suffered from recurrent outbreaks. To evaluate the association between antibody titers and clinical manifestations, sera and colostra were analyzed from one pig farm that employed the feedback strategy. Furthermore, spike (S) gene full sequences from six positive samples of two farms with and without using feedback were compared to investigate the evolution of PEDV variants circulating in pig herds. The results in this study showed that high PEDV antibody titers do not correlate with the high rate of protection from PEDV infection. In addition, repeated feedback generated the emergence of PEDV variants with unique substitutions of N537S and Y561H in the COE domain and S769F in the SS6 epitopes. These mutations indicated the pathogenetic evolution of PEDV strains existing in the cycle of the feedback method. A very strict biosecurity practice to block the routes of pathogen transfer should be followed to achieve successful control of PEDV infections in pig herds.

Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) Promotes M1 Repolarization of Porcine Alveolar Macrophages and Th1 Type Response.

The polarization status of porcine alveolar macrophages (PAMs) determines the infectivity of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV infection skews macrophage polarization toward an M2 phenotype, followed by T-cells inactivation. CD163, one of the scavenger receptors of M2 macrophages, has been described as a putative receptor for PRRSV. In this study, we examined two types of PRRSV-2-derived recombinant antigens, A1 (g6Ld10T) and A2 (lipo-M5Nt), for their ability to mediate PAM polarization and T helper (Th1) response. A1 and A2 were composed of different combination of ORF5, ORF6, and ORF7 in full or partial length. To enhance the adaptive immunity, they were conjugated with T cells epitopes or lipidated elements, respectively. Our results showed that CD163+ expression on PAMs significantly decreased after being challenged with A1 but not A2, followed by a significant increase in pro-inflammatory genes (TNF-α, IL-6, and IL-12). In addition, next generation sequencing (NGS) data show an increase in T-cell receptor signaling in PAMs challenged with A1. Using a co-culture system, PAMs challenged with A1 can induce Th1 activation by boosting IFN-γ and IL-12 secretion and TNF-α expression. In terms of innate and T-cell-mediated immunity, we conclude that A1 is regarded as a potential vaccine for immunization against PRRSV infection due to its ability to reverse the polarization status of PAMs toward pro-inflammatory phenotypes, which in turn reduces CD163 expression for viral entry and increases immunomodulation for Th1-type response.

Transcutaneous immunization of Streptococcus suis bacterin using dissolving microneedles.

Vaccine delivery using microneedle (MN) patches is an easy, safe and painless alternative to traditional needle injections. In this study, we examined whether MN patches can enhance the efficacy of a Streptococcus suis serotype 2 (S. suis 2) vaccine in a mouse model. Results showed that MNs can reach 200-250µm into the skin, a depth beneficial for targeted delivery of antigens to antigen-presenting cells in the epidermis and dermis. Vaccination with prime-boost of MN induced higher levels of IgG2a antibody titer, T cell proliferation, and TH1 cytokines (IFN-γ and IL-12) as compared to intramuscular (IM) injection. In addition, single dose MN vaccination better protected mice against lethal challenge than IM vaccination. MN vaccination also conferred long-term IgG2a antibody against S. suis 2 bacteria presence for up to 7 months. Taken together, these data showed that vaccine delivery by MNs results in superior immune response and protection rate when compared to IM injections.

Immunization with Streptococcus suis bacterin plus recombinant Sao protein in sows conveys passive immunity to their piglets.

BACKGROUND: Streptococcus suis (S. suis) causes arthritis, meningitis, septicemia, and sudden death in pigs and is also an zoonotic agent for humans. The present study demonstrated that immunization with recombinant Sao-L (surface antigen one-L, rSao-L) protein from a strain of S. suis serotype 2 in pigs was able to increase cross-serotype protection against S. suis serotype 1 and 2 challenge. Since weaning piglets are more susceptible to S. suis infections due to the stresses associated with weaning, prepartum immunization in sows may convey passive immunity to piglets and provide protection. RESULTS: Pregnant sows were immunized with a vaccine containing inactivated S. suis serotype 2 plus rSao as the antigens. Blood samples were collected from their piglets after birth for analysis of antigen-specific antibody titers and levels of various cytokines. Results demonstrated that the titers of S. suis and rSao-specific antibodies were significantly (p < 0.05) higher in the vaccinated piglets in comparison with that of piglets in the control group. The serum levels of interferon (IFN)-γ, interleukin (IL)-4, IL-6, and IL-12 were significantly (p < 0.05) increased in piglets born from vaccinated sows when compared to piglets from unvaccinated sows. In addition, piglets were challenged by heterologous and homologous S. suis. All piglets from unvaccinated sows developed severe symptoms of bacteremia, fever, anorexia, depression, and arthritis. On the other hand, piglets from vaccinated sows had significantly (p < 0.05) reduced clinical symptoms and lesion score (by 75 and 81%). CONCLUSIONS: Our results revealed that immunizing pregnant sows with the vaccine containing inactivated S. suis bacterin plus rSao as the antigens is able to enhance passive immunity against heterologous and homologous S. suis challenge in their piglets.

Impact of Mated Female Nonproductive Days in Breeding Herd after Porcine Epidemic Diarrhea Virus Outbreak.

Porcine epidemic diarrhea virus (PEDV) is an important pathogen that has a significant economic impact on the swine industry by imposing a high rate of mortality in suckling piglets. However, limited information on the productivity values of gilts and sows infected with PEDV is available. Here, we evaluate the productivity index in gilts and sows during the 1-year period before (19 January 2013 to 18 January 2014) and after (19 January 2014 to 18 January 2015) a PEDV outbreak from a 2000-sow breeding herd in Taiwan. The farrowing rate (FR), return rate (RR), total pigs born per litter (TB), pigs born alive per litter (BA), weaning pigs per litter (WPL), pre-weaning mortality, percentage of sows mated by 7 days after weaning, weaning to first service interval (WFSI), mated female nonproductive days (NPDs), replacement rate of sows and sow culling rate were compared using productive records. The FR (-9.6%), RR (+9.8%), TB (-1.6), BA (-1.1), WPL (-1.1), sows mated by 7 days after weaning (-6.9%), WFSI (+0.8 days), NPDs (+6.9 days) and sow culling rate (+7.2%) were significantly different between the 1-year pre-PEDV outbreak period and the post-PEDV outbreak period. Impacts of the PEDV infection on the reproductive performance were more severe in pregnant gilts than in sows. In conclusion, these findings indicate that the outbreak of PEDV caused an increase in the rate of NPDs in breeding herds.

Expression of Toll-like receptor signaling-related genes in pigs co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2.

Pigs co-infected with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) have been shown to develop more severe diseases than pigs infected with PRRSV or PCV2 only. The underlying interaction mechanisms between the two viruses in developing the disease are unclear. The present study investigates the mRNA expression of Toll-like receptor (TLR) signaling-related molecules in peripheral blood mononuclear cells from pigs infected with PRRSV or PCV2 or both. The mRNA expression levels were determined by quantitative real-time RT-PCR. Co-infection of pigs with PRRSV and PCV2 resulted in a negatively synergistic effect on the mRNA expression of the negative regulators of TLR, including A20, Bcl-3, IRAK-M, MKP-1, SARM1 and SIGIRR, as well as the TLR downstream transcription factors IRF-1 and IRF-3. A positively synergistic effect of a combined infection of PRRSV and PCV2 on the CD14 mRNA expression was also observed.

Expression profile of Toll-like receptor mRNA in pigs co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2.

Field and experimental studies have shown that co-infection of pigs with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) increases the severity of the disease. The present study investigates the mRNA expression profile of Toll-like receptors (TLRs) in pigs co-infected with PRRSV and PCV2. SPF pigs were infected with PRRSV, PCV2 or in a combination of both. The mRNA expression levels of TLRs and related cytokines in peripheral blood mononuclear cells (PBMCs) of pigs were determined by quantitative real-time RT-PCR. The mRNA expression profiles of TLRs by PBMCs from pigs co-infected with PRRSV and PCV2 displayed two distinct patterns: an increased expression profile for TLRs2, 4 and 8, and a decreased expression profile for TLRs3, 7 and 9. An up-regulated expression of IL-1ß and IL-10 mRNA and a down-regulated expression of INF-α and TNF-α mRNA in PBMCs of co-infected pigs were also observed.

US-like strain of porcine epidemic diarrhea virus outbreaks in Taiwan, 2013-2014.

Since late 2013, several outbreaks of porcine epidemic diarrhea virus (PEDV) infection have emerged in Taiwan. Suckling piglets under 2 weeks of age showed severe vomiting and watery yellowish diarrhea with morbidity and mortality ranging from 80 to 100% and 90 to 100%, respectively. A total of 68 samples from 25 pig farms were confirmed as positive for PEDV and negative for rotavirus and transmissible gastroenteritis virus by reverse transcription PCR, and the partial S gene of PEDV was analyzed. Phylogenetic analysis places all 18 Taiwanese PEDV isolates collected during this outbreak in the same clade as the US strains of PEDV. This novel PEDV is prevailing and currently causing severe outbreaks in Taiwan.

Identification of cytotoxic T lymphocyte epitopes on swine viruses: multi-epitope design for universal T cell vaccine.

Classical swine fever (CSF), foot-and-mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are the primary diseases affecting the pig industry globally. Vaccine induced CD8(+) T cell-mediated immune response might be long-lived and cross-serotype and thus deserve further attention. Although large panels of synthetic overlapping peptides spanning the entire length of the polyproteins of a virus facilitate the detection of cytotoxic T lymphocyte (CTL) epitopes, it is an exceedingly costly and cumbersome approach. Alternatively, computational predictions have been proven to be of satisfactory accuracy and are easily performed. Such a method enables the systematic identification of genome-wide CTL epitopes by incorporating epitope prediction tools in analyzing large numbers of viral sequences. In this study, we have implemented an integrated bioinformatics pipeline for the identification of CTL epitopes of swine viruses including the CSF virus (CSFV), FMD virus (FMDV) and PRRS virus (PRRSV) and assembled these epitopes on a web resource to facilitate vaccine design. Identification of epitopes for cross protections to different subtypes of virus are also reported in this study and may be useful for the development of a universal vaccine against such viral infections among the swine population. The CTL epitopes identified in this study have been evaluated in silico and possibly provide more and wider protection in compared to traditional single-reference vaccine design. The web resource is free and open to all users through http://sb.nhri.org.tw/ICES.

Differentiation of foot-and-mouth disease-infected pigs from vaccinated pigs using antibody-detecting sandwich ELISA.

The presence of serum antibodies for nonstructural proteins of the foot-and-mouth disease virus (FMDV) can differentiate FMDV-infected animals from vaccinated animals. In this study, a sandwich ELISA was developed for rapid detection of the foot-and-mouth disease (FMD) antibodies; it was based on an Escherichia coli-expressed, highly conserved region of the 3ABC nonstructural protein of the FMDV O/TW/99 strain and a monoclonal antibody derived from the expressed protein. The diagnostic sensitivity of the assay was 98.4%, and the diagnostic specificity was 100% for naïve and vaccinated pigs; the detection ability of the assay was comparable those of the PrioCHECK and UBI kits. There was 97.5, 93.4 and 66.6% agreement between the results obtained from our ELISA and those obtained from the PrioCHECK, UBI and CHEKIT kits, respectively. The kappa statistics were 0.95, 0.87 and 0.37, respectively. Moreover, antibodies for nonstructural proteins of the serotypes A, C, Asia 1, SAT 1, SAT 2 and SAT 3 were also detected in bovine sera. Furthermore, the absence of cross-reactions generated by different antibody titers against the swine vesicular disease virus and vesicular stomatitis virus (VSV) was also highlighted in this assay's specificity.

Investigation of beta-glucans binding to human/mouse dectin-1 and associated immunomodulatory effects on two monocyte/macrophage cell lines.

Dectin-1, a specific pattern recognition receptor for ß-1,3/ß-1,6-glucans, is expressed mainly on phagocytes. Human dectin-1 (hDectin-1) and mouse dectin-1 (mDectin-1) were separately expressed on HEK293 cell surfaces for examination of the binding abilities of a synthetic particulate ß-glucan (pßG), a product extracted from Saccharomyces cerevisiae, in this study. The binding of zymosan-FITC to hDectin-1 and mDectin-1 was inhibited by pßG at similar concentrations for 50% inhibition of binding (IC50). However, the kinetics of the time course and dose response to zymosan stimulation observed for U937 and J774A.1 differed. Superoxide anion production was increased in U937 but reduced in J774A.1 when cells were treated with pßG, zymosan, or laminarin, whereas ovalbumin endocytosis was enhanced in U937 and J774A.1 treated either with pßG, zymosan, laminarin, or barley-glucan. These results indicate that the binding affinity of pßG to hDectin-1 is similar to the binding affinity to mDectin-1, and that stimulation by pßG as well as various forms of ß-1,3-glucans on U937 and J774A.1 resulted in upregulation of cell activity and ovalbumin endocytosis. Additionally, other coreceptors on U937 and J774A.1 may be involved in directing different responses to superoxide anion production in these two types of cells. These results will likely contribute to further investigations on identifying the biological forms of ß-glucans capable of binding its specific receptor as the effective immunomodulators.

Toll-Like Receptor expressions in porcine alveolar macrophages and Dendritic Cells in responding to poly IC stimulation and porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Antigen-presenting cells play critical roles in recognizing, presenting and processing antigens and consequently induce adequate immune response for defending infections. The immature DCs (imDCs) and mature DCs (mDCs) were obtained from in vitro differentiation of bone marrow haematopoietic cells. Results showed that poly IC stimulation down-regulated the expressions of TLR7 and TLR8 in alveolar macrophages (AMs) and imDCs. The release of IL-12 was inhibited from imDCs and mDCs in response to poly IC. Porcine reproductive and respiratory syndrome virus (PRRSV)-infection inhibited TLR3 and TLR7 expressions in AMs and imDCs at 6h post-infection (PI); both of expressions were then restored at 24h PI in both types of cells while they exhibited up-regulated IL-10 and IL-12 expression at 24h PI. Hence, the differential TLR expression patterns in porcine AMs and DCs in discrimination of the imitated viral dsRNA or PRRSV infection may determine their cytokine expressions and thus affect the resulting immune responses.

Immunomodulatory effects of beta-glucans on porcine alveolar macrophages and bone marrow haematopoietic cell-derived dendritic cells.

The immunopharmacological activities of beta-glucans with a backbone of beta-1,3/beta-1,6-linkages associated with anti-tumor, anti-viral, bacterial and fungal infections have been well documented. Dectin-1, a specific pattern recognition receptor for beta-1,3/beta-1,6-glucans, is expressed mainly on phagocytes, especially macrophages and dendritic cells (DCs). In this study, the encoding nucleotide for the carbohydrate-recognition domain (CRD) of porcine dectin-1 was sequenced for the first time, and the immunomodulatory functions of a synthetic particulate beta-glucan (p-beta-glucan) were examined. Results showed that p-beta-glucan significantly enhanced cell activity and phagocytosis in porcine alveolar macrophages (AMs), immature DCs (imDCs) and mature DCs (mDCs), in a similar way to zymosan. Zymosan enhanced dectin-1/TLR2/TLR4 expression and TNF-alpha/IL-10 production in all of three types of cell, whereas p-beta-glucan increased dectin-1/TLR4 and TNF-alpha/IL-12 production in AMs but inhibited IL-10 in mDCs. These results indicate that the complex collaborating interactions between dectin-1 and TLRs in the recognition of beta-1,3/beta-1,6-glucans with different structural features may direct different cellular responses.

Modulations of phenotype and cytokine expression of porcine bone marrow-derived dendritic cells by porcine reproductive and respiratory syndrome virus.

Phenotypic and functional property changes of bone marrow-derived immature dendritic cells (BM-imDCs) after porcine reproductive and respiratory syndrome virus (PRRSV) infection have been detailed in a previous report. A down-regulated expression of MHC I molecules along with an up-regulated expression of CD80/86 were observed in BM-imDCs after the exposure to PRRSV. In this study, we further investigate the expression of surface phenotypes of BM-imDCs in relation to their infection status. Exposure of PRRSV to BM-imDCs resulted in a down-regulated expression of MHC I and an up-regulated expression of CD80/86 in infected cells, as demonstrated by significant alterations in both percentage of expressing cells and mean fluorescence intensity (MFI) in PRRSV-positive cells. A significant suppression in MFI of MHC I and an increase in percentage of cells expressing CD80/86 were observed in noninfected bystander cells. We also demonstrated that exposure of BM-imDCs to PRRSV resulted in a significantly increased secretion of IL-1, IL-6, IL-8, IL-10 and IFN-gamma but not IL-12 or TNF-alpha. In addition, the PRRSV infection modulates cytokine expressions of BM-imDCs through their response to microbial pathogen-associated molecular patterns. These results will prove helpful in clarification of the factors that mediate host defense against PRRSV, as well as the possible interaction mechanisms between PRRSV and other microbes in the pathogenesis of PRRSV infection in pigs.

Expression of Toll-like receptor mRNA and cytokines in pigs infected with porcine reproductive and respiratory syndrome virus.

Field observations have suggested that porcine reproductive and respiratory syndrome virus (PRRSV) predispose pigs to secondary infections. The interaction between PRRSV and the secondary invaders has not yet been well elucidated. In this study, we investigated the mRNA expression of Toll-like receptors (TLR) in lymphoid organs and cells, and cytokine secretions by alveolar macrophages (AMs) and peripheral blood mononuclear cells (PBMCs) in response to pathogen-associated molecular patterns (PAMPs) in PRRSV-challenged pigs. TLR mRNA expressions were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and cytokine concentrations were determined using commercially available ELISA kits. PRRSV infection led to significantly increased secretions of IL-1beta and IL-6 by AMs of PRRSV-infected pigs. Infection of pigs with PRRSV also resulted in an increased secretion of IL-1beta by AMs in response to lipoteichoic acid (LTA) stimulation, and IL-6 by PBMCs in response to lipopolysaccharide (LPS) and LTA stimulation. Infection of pigs with PRRSV tended to up-regulate the mRNA expression of TLR2, 3, 4, 7 and 8 in at least one of the lymphoid tissues and cells. Further research is required to demonstrate the association between the enhanced expressions of the specific TLRs and the increased susceptibility to secondary agents with more severe clinical outcomes in PRRSV-infected pigs.

Leukotriene C4 release and gene expressions of IL-8 and MCP-1 in porcine alveolar epithelial type II cells.

Leukotrienes (LT) and chemokines are important chemotactic compounds in regulating the recruitment and activation of immune cells during pulmonary inflammatory reactions. Results showed that LTC4 release by porcine alveolar epithelial type II cells (AEC IIs) is significantly enhanced by either LTB4 or LPS stimulation. The basal level of IL-8 gene expression in AEC IIs was only 1/3 of that observed in alveolar macrophages (AMs) while AEC IIs expressed a higher basal level of monocyte chemotactic peptide-1 (MCP-1) and also in response to LPS stimulation than do AMs. The increasing basal and LT-induced MCP-1 gene expressions after 8h of incubation were observed in AEC IIs but decreased in AMs. These findings suggest that AEC IIs play an important role in initial inflammatory reactions of the lung by releasing LTC4, and that they also modulate later inflammatory reactions, evidenced by consistent elevation of MCP-1 gene expression after and during exogenous challenge in pigs.

Phenotypic and functional modulation of bone marrow-derived dendritic cells by porcine reproductive and respiratory syndrome virus.

It is well documented that there is a delay in the development of effective immunity to porcine reproductive and respiratory syndrome virus (PRRSV) in infected and vaccinated pigs. This suggests that PRRSV might possess some inherent properties to evade host defense mechanisms during the early stage of infection. Dendritic cells (DCs) play a crucial role in the activation and control of T-cells in response to viral antigens. In this study, we investigated the phenotypic and functional property changes of bone marrow-derived immature DCs (BM-imDCs) that take place after infection by PRRSV. Results showed that BM-imDCs were permissive to PRRSV infection, as productive replication took place in these cells. A down-regulated expression of MHC I molecules along with an up-regulated expression of CD80/86 is observed at 48 h following infection. Also at 48h following PRRSV infection, a significant increase of IL-10 secretion by BM-imDCs was noticed. Results suggest that the inhibited expression of MHC I and the enhanced secretion of IL-10 by BM-imDCs after PRRSV infection might be among the strategies used by the virus to evade the host immune defenses.

Real-time PCR for quantitation of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in naturally-infected and challenged pigs.

Real-time PCR assays were developed for quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2). The established real-time PCR for the quantitation of PRRSV cDNA and PCV2 DNA were found to be in the 9-log(10) linear dynamic range with excellent linearity and reliable reproducibility. Using these techniques, the distribution and quantitation of PRRSV and PCV2 in naturally infected and challenged pigs were investigated. The viral concentrations were expressed as the mean log(10) viral DNA or cDNA copy numbers per mg or ml of tested samples. For pigs infected naturally with both viruses, the lung, spleen, tonsil and lymphoid organs had the highest viral burdens with ranges from 5.73 to 8.38 and 5.65 to 6.91 for PRRSV and PCV2, respectively. The injection of formalin-inactivated Salmonella choleraesuis emulsified in complete Freund's adjuvant 1 week before and after the inoculation of both viruses resulted in PRRSV replication enhancement 2 weeks post-challenge. However, this facilitated the clearance of PRRSV 4 weeks post-challenge. Results from this study show that the established quantitative PCR could be a useful tool when applied to vaccine development and pathogenesis studies in the future.