Porcine Circovirus Research Articles

Situational analysis on porcine diseases: general risk assessment and prioritization of epizootic threats to biosecurity systems of pig establishments in the Russian Federation

The results of the situational analysis on porcine diseases in the Russian Federation and the expert assessment prioritizing the list of porcine pathogens significant for the pig industry of the country are presented. The method applied to analyse the expert estimates in the situational analysis allows for rapid assessment and interpretation of the situation with identification of priority diseases to be further addressed. The calculations demonstrated the sufficient degree of agreement among the experts (coefficient of concordance W = 0.61), and Pearson’s chi-squared test statistic χ2 = 51.33 (≥ 21.02607) indicated that the concordance is not random and the results can be used in subsequent studies. The specific features of epizootiology of the agents of African swine fever, classical swine fever, porcine reproductive and respiratory syndrome that can impact the effectiveness of biosecurity systems of pig establishments, as well as further ways for improving biosecurity management measures are discussed. The overall risk for the pig industry in the Russian Federation that is associated with external sources is currently characterized as permanently high, requiring maintaining risk management measures at the pig establishments by both the managerial staff of the establishments and the State Veterinary Service. It is recommended that biosecurity measures against external threats should focus on diseases such as African swine fever (weight λ = 0.52), porcine reproductive and respiratory syndrome (λ = 0.071), classical swine fever (λ = 0.068) and infections considered emerging for the Russian Federation (λ = 0.05) according to the weights based on the expert estimation results. The biosecurity systems of the establishments should equally address other threats significant for the pig industry of the country: swine enzootic pneumonia, porcine pleuropneumonia (Actinobacillus pleuropneumoniae), Aujeszky’s disease, streptococcosis (Streptococcus suis), porcine circovirus infection, foot-and-mouth disease, leptospirosis, transmissible gastroenteritis, cysticercosis (λ = 0.02…0.05). The improvement of the governmental policy for eradication of African swine fever, porcine reproductive and respiratory syndrome, classical swine fever (including the substantial modification of the existing official pig turnover control, zoning, diagnosis and prevention quality, as well as the implementation of biosecurity standards) is the most significant factor, without which the disease eradication perspective is questionable.

Development of a RPA-CRISPR/Cas12a based rapid visual detection assay for Porcine Parvovirus 7.

Porcine Parvovirus (PPV) is a significant pathogen in the pig industry, with eight genotypes, including PPV7, identified since its emergence in 2016. Co-infections with viruses such as Porcine Circovirus 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) pose serious risks to swine health. Thus, there is an urgent need for rapid, sensitive, and specific detection methods suitable for use in field settings or laboratories with limited resources. We developed a CRISPR/Cas12a-based assay combined with recombinase polymerase amplification (RPA) for the rapid detection of PPV7. Specific RPA primers and five CRISPR RNAs (crRNAs) were designed to target a highly conserved region within the NS1 gene of PPV7. Optimization of crRNA and single-stranded DNA (ssDNA) concentrations was performed to enhance the assay's performance. CrRNA optimization identified crRNA-05 as the optimal candidate for Cas12a-based detection of PPV7, as all synthesized crRNAs demonstrated similar performance. The optimal crRNA concentration was determined to be 200 nM, yielding consistent results across tested concentrations. For ssDNA optimization, the strongest fluorescence signal was achieved with 500 nM of the FAM-BHQ ssDNA receptor. The assay showed a minimal detection limit of 100copies/μl for PPV7, confirmed through fluorescence and lateral flow detection methods. Specificity testing indicated that only PPV7 DNA samples returned positive results, confirming the assay's accuracy. In tests of 50 lung tissue samples from diseased pigs, the RPA-Cas12a assay identified 29 positive samples (58%), surpassing the 22 positive samples (44%) detected by conventional PCR. This highlights the RPA-Cas12a method's enhanced detection capability and its potential utility in clinical surveillance and management of PPV7 in swine populations. The RPA-Cas12a assay effectively detects PPV7 in clinical samples, enhancing disease surveillance and control in pigs. Its adaptability to resource-limited settings significantly improves PPV7 management and prevention strategies, thereby supporting the overall health and development of the pig industry.

Retrospective Analyses of Porcine Circovirus Type 3 (PCV-3) in Switzerland.

Porcine circovirus 3 (PCV-3) has emerged as a significant pathogen affecting global swine populations, yet its epidemiology and clinical implications remain incompletely understood. This retrospective study aimed to investigate the prevalence and histopathological features of PCV-3 infection in pigs from Switzerland, focusing on archival cases of suckling and weaner piglets presenting with suggestive lesions. An in-house qPCR assay was developed for detecting PCV-3 in frozen and formalin-fixed paraffin-embedded tissues, enhancing the national diagnostic capabilities. Histopathological reassessment identified PCV-3 systemic disease (PCV-3-SD) compatible lesions in 19 (6%) of archival cases, with 47% testing positive by qPCR across various organs. Notably, vascular lesions predominated, particularly in mesenteric arteries, heart, and kidneys. The study confirms the presence of PCV-3 in Switzerland since at least 2020, marking the first documented cases within the Swiss swine population. Despite challenges in in situ hybridization validation due to prolonged formalin fixation, the findings indicate viral systemic dissemination. These results contribute to the understanding of PCV-3 epidemiology in Swiss pigs, emphasizing the need for continued surveillance and further research on its clinical implications and interaction with host factors. Our study underscores the utility and limitations of molecular techniques in confirming PCV-3 infections.

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.

Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3.

The porcine circovirus type 3 (PCV3) infection is an emerging disease associated with clinical signs of porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs. Currently, there is a lack of effective vaccines and therapeutics against this disease. Therefore, rapid, effective, sensitive, and specific detection methods are crucial for the timely identification, prevention, and control of PCV3. In this study, we developed one- and two-pot visual detection methods for PCV3 using a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a detection system combined with recombinase polymerase amplification (RPA). These two methods demonstrated no cross-reactivity with eight other swine viruses and exhibited minimum detection limits of five and two copies of viral DNA, respectively, revealing their high specificity and sensitivity. During a clinical sample detection within 30 min, the coincidence rates between the one- and two-pot detection methods and real-time quantitative polymerase chain reaction (qPCR) were 100%. In conclusion, both one- and two-pot RPA-CRISPR/Cas12a detection methods have significant potential for the rapid, sensitive, and specific visual detection of PCV3.

Can immunocrit be used as a monitoring tool for swine vaccination and infection studies?

BackgroundThe immunocrit is a cost-effective and straightforward technique traditionally used to assess passive immunity transfer to newborn piglets. However, it has not been previously used for monitoring the effect of vaccination and/or infections. Therefore, this study aimed to evaluate the usefulness of the immunocrit technique as an immunological monitoring tool in a vaccination and challenge scenario, using porcine circovirus 2 (PCV-2) as pathogen model. The immunocrit ratio was monitored in PCV-2 vaccinated (V) and non-vaccinated (NV) 3-week-old piglets (study day 0, SD0) that were subsequently challenged with this virus at SD21 and followed up to SD42. Additional techniques (PCV-2 IgG ELISA, optical refractometry, and proteinogram) were performed to further characterize the results of the immunocrit analysis.ResultsImmunocrit, γ-globulin concentration and PCV-2 S/P values followed similar dynamics: descending after PCV-2 vaccination but ascending after an experimental PCV-2 inoculation. However, statistically significant differences between V and NV animals were only found with the PCV-2 ELISA. In this case, V animals had significantly higher (p < 0.05) S/P values (S/P ratio = 0.74) than NV (S/P ratio = 0.39) pigs only after challenge at SD42. On the other hand, serum total protein obtained by refractometer (STPr) were maintained from SD0 to SD21 and increased in both groups from SD21 to SD42. Correlations between techniques were low to moderate, being the most robust ones found between immunocrit and optical refractometry (ρ = 0.41) and immunocrit with γ-globulins (ρ = 0.39). In a subset of sera, the proteinogram technique was applied to the whole serum and the supernatant of the immunocrit, with the objective to characterize indirectly the immunocrit fraction. The latter one included all protein types detectable through the proteinogram, with percentages varying between 64.3% (γ-globulins) and 82% (β-globulins).ConclusionThe immunocrit technique represented a fraction of the total serum proteins, with low to moderate correlation with all the complementary techniques measured in this study. Its determination at different time points did not allow monitoring the effect of vaccination and/or infection using PCV-2 as a pathogen model.

Quantitative proteomic analysis of PK-15 cells infected with porcine circovirus type 3 using 4D-DIA approach.

Porcine circovirus type 3 (PCV3) infection is clinically related to various diseases, including porcine dermatitis and nephrotic syndrome (PDNS)-like disease, respiratory disease, reproductive disorders, and gastrointestinal and neurological diseases. Since PCV3 infection was discovered in 2016, it has developed rapidly and has attracted much attention worldwide. However, specific preventive and therapeutic interventions are currently lacking. In this study, four-dimensional (4D) data-independent acquisition (DIA)-based quantitative proteomics detection combined with bioinformatics analysis were employed to quantitatively identify the differentially expressed proteins in PK-15 cells from the PCV3-infected group compared with those from the uninfected control group. A total of 194 cellular proteins were significantly altered in response to PCV3 infection, including 58 upregulated proteins and 136 downregulated proteins. In our Gene Ontology (GO) enrichment analysis, these differentially expressed proteins were mostly associated with cellular anatomical entities, binding, cellular processes, biological regulation, catalytic activity, metabolic processes, developmental processes, protein-containing complexes and responses to stimuli. Our Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEPs were predominantly involved in metabolic pathways, the cAMP signaling pathway, protein processing in the endoplasmic reticulum, the PI3K-Akt signaling pathway, and the calcium signaling pathway. For the experiments, Western blotting (WB) was used to confirm the changes in important molecules. The differentially expressed proteins identified should contribute to a greater understanding of the mechanism of PCV3 replication and pathogenesis, as well as the host response.

Efficacy comparison in cap VLPs of PCV2 and PCV3 as swine vaccine vehicle

As one genotype of porcine circovirus (PCV) identified in 2016, PCV3 has brought huge hidden dangers to the global swine industry together with PCV2. Virus-like particles (VLPs) of capsid protein (Cap) of PCV2 serve as an alternative nano-antigen delivery strategy to efficiently induce antiviral immune response against PCV2 and/or other covalently displayed swine pathogens. However, the current understanding is limited on the capability of PCV3 as a nano-vaccine vehicle. Here we systematically compared the characteristics and the immunogenic efficacy of PCV3 Cap (Cap3) and PCV2 Cap (Cap2) in a VLP form. Cap3 VLPs presented higher internalization efficiency into cells and cytokines production compared to those of Cap2. Meanwhile, cross-reactive immunity between Cap3 VLPs and Cap2 VLPs was detected. Furthermore, to evaluate the function of Cap3 VLPs and Cap2 VLPs as vaccine vehicles carrying foreign proteins, the non-structural protein 6 of porcine reproductive and respiratory syndrome virus (PRRSV) was fused to C-terminus of Cap. Cap3-based chimeric particles induced a higher level of nsp6-specific immune response and PRRSV inhibition. Collectively, these self-assembling, Cap-based VLPs offer a compelling platform for enhancing the effectiveness of subunit vaccinations against newly emerging diseases and hold great promise for the development of Cap3-based chimeric subunit vaccines.

Isolation of porcine circovirus 3 using primary porcine bone marrow-derived cells

Porcine circovirus 3 (PCV3) was first reported in the United States in 2016; this virus is considered to be involved in diverse pathologies, such as multisystem inflammation, porcine dermatitis and nephropathy syndrome, and reproductive disorders. However, successful isolation of PCV3 using cultured cells has been rare. In this study, we aimed to isolate PCV3 using primary porcine bone marrow-derived cells. Mononuclear cells were isolated from the femur bones of clinically healthy pigs. These primary cells were cultured for 6–10 days post-seeding and infected with PCV3-containing tissue homogenates. The cells were cultured for up to 37 days, and the culture medium was changed every 3–4 days. The growth curve of PCV3 in porcine bone marrow cells revealed a decline in growth during the first 10 days post-infection, followed by an increase leading to > 1010 genomic copies/mL of the cell culture supernatant; moreover, the virus was capable of passaging. The indirect fluorescent antibody assay for PCV3 infection revealed the presence of PCV3 capsid protein in the cytoplasm and nuclei of infected cells. Bone marrow cells were passaged for more than 20 generations (over 5 months), and PCV3 persistently infected the cells. PCV3-infected bone marrow cells expressed mesenchymal markers. These results reflect that primary porcine bone marrow-derived mesenchymal cells are permissive to PCV3 and continuously replicate a high copy number of the PCV3 genome. These findings regarding the high replication rate of PCV3 in bone marrow-derived mesenchymal cells could enhance our understanding of PCV3 pathogenicity.

Molecular detection of emerging porcine circovirus in Taiwan

Porcine Circovirus type (PCV) 2 is an important pathogen that has been circulating worldwide and has cuased serious economic loss in pig industry. However, both PCV3 and PCV4 are newly emerging viruses. In Taiwan, PCV2 has been one of the critical pathogens in pig frams and PCV3 has been detected since 2016; however, the epidemiolog of PCV3 in Taiwan remains unclear and PCV4 has yet to be identified. Therefore, in order to detect the positive rate of PCV2, to investigate the epidemiolog of PCV3 in the pig farms, and to examine whether pigs were infected with PCV4 in Taiwan, a total of 128 samples from 46 clinical cases of pigs were collected from September 2020 to December 2021. The case detection rates were 54.3 % for PCV2, 43.5 % for PCV3, and 2.2 % for PCV4. The results suggested that the positivity rates for both PCV2 and PCV3 were still high in Taiwan. In addition, PCV3 was detected among cases from all 7 sampled counties and in 11 of the 16 sampling months, suggesting that PCV3 may lead to endemic pig disease in Taiwan. Surprisingly, the PCV4 was also detected, suggesting the first PCV4 case in Taiwan. The complete genomes derived from the identified PCV3 and PCV4 strains were subsequently sequenced followed by phylogenetic analysis. The results suggested that the 17 identified PCV3 strains could be divided into Taiwanese-like and Japanese-like strains. In addition, the amino acid residues at positions 27, 80, and 212 in the identified PCV4 cap protein were asparagine, isoleucine, and methionine, respectively, and thus the identified PCV4 was catalorized into clade PCV4b. Consequently, it is concluded that (i) the prevalence of PCV2 and PCV3 is still high in Taiwanese pigs, (ii) PCV3 has may be an endemic infection in Taiwan and can be classified into Japanese-like and Taiwanese-like strains, (iii) PCV4 was detected for the first time in Taiwanese pigs and can be classified into PCV4b. It remains unclear how PCV2, PCV3, and PCV4 were introduced to Taiwan, and thus continuous investigation of emerging pathogens in pigs is needed.

Characterization of capsid protein of porcine circovirus 4 by in-silico approach

Porcine Circovirus 4 (PCV4) has been detected in pigs from China only in 2019. PCV4 has been found to be associated with several forms of clinical syndromes in pigs. Like other Porcine Circoviruses, the only structural protein of PCV4 is the capsid protein. Therefore, it could be a key vaccine candidate or diagnostic marker. The present study aims to characterize the capsid protein of PCV4 by using several bioinformatics tools. The in-silico analysis revealed that the molecular weight of PCV4 capsid protein is about 27.3 kDa which is made up of 228 amino acids. The capsid protein of PCV4 contains a nuclear localization signal at N-terminal and several post-translational modifications could be predicted at different residues of the protein. The secondary structure of capsid protein contains coil, beta strand, and alpha helix. The 3D structure of the protein was predicted with a high level of confidence. The capsid protein of PCV4 also contains several potential B-cell epitopes. Thus, the in-silico characterization of the capsid protein will be beneficial in designing diagnostics and vaccine candidates in future to combat PCV4 infection in pigs.

Identification of a conserved B-cell epitope on the capsid protein of porcine circovirus type 4.

Porcine circovirus type 4 (PCV4) is a novel circovirus. Although PCV4 has been identified in several countries, including China, Korea, Thailand, and Spain, no vaccine is available. Given the potential pathogenic effects of PCV4 on pigs, PCV4 could threaten the global pig farming industry, highlighting the urgency for further investigation. Thus, epitopes of PCV4 remain to be determined. Our finding of a conserved epitope significantly advances vaccine development and pathogen detection.

An in vitro self-ligation-based method for constructing infectious DNA clone of porcine circovirus type 2

The aim of this study was to establish a rapid method for constructing infectious clones of porcine circovirus type 2 (PCV2). In this study, we constructed circular infectious clones of PCV2 by seamless cloning technology, using the clinically isolated strain PCV2-LX as a template. Meanwhile, this method was compared with the conventional restriction-ligation approach, focusing on the in vitro circularization (self-ligation) process of the genome and the growth characteristics of rescued viruses. The results showed that this method eliminates the need to analyze and introduce restriction endonuclease sites, thus avoiding the complexities associated with traditional restriction enzyme-based cloning steps. It offers a simple and rapid operation, enabling more efficient editing of the PCV2 genome. The infectious clones constructed using this method could be successfully rescued through liposome transfection, resulting in the production of recombinant viruses that could be stably passaged. Moreover, the recombinant viruses rescued by this method exhibited enhanced proliferative capacity in PK-15 cells and 3D4/31 cells (immortalized porcine alveolar macrophages). In conclusion, this study has established a novel reverse genetics system for PCV2, providing a new strategy for the development of PCV2 genetic engineering vaccines. Additionally, it serves as a reference for the construction of infectious clones for other emerging circoviruses such as PCV3 and PCV4.

E3 ligase RNF2 inhibits porcine circovirus type 3 replication by targeting its capsid protein for ubiquitination-dependent degradation.

Porcine circovirus type 3 (PCV3) is closely associated with various diseases, such as the porcine dermatitis, nephropathy syndrome, and multisystemic clinicopathological diseases. PCV3-associated diseases are increasingly recognized as severe diseases in the global swine industry. Ring finger protein 2 (RNF2), an E3 ubiquitin ligase exclusively located in the nucleus, contributes to various biological processes. This ligase interacts with the PCV3 Cap. However, its role in PCV3 replication remains unclear. This study confirmed that the nuclear localization signal domain of the Cap and the RNF2 N-terminal RING domain facilitate the interaction between the Cap and RNF2. Furthermore, RNF2 promoted the binding of K48-linked polyubiquitination chains to lysine at positions 139 and 140 (K139 and K140) of the PCV3 Cap, thereby degrading the Cap. RNF2 knockdown and overexpression increased or decreased PCV3 replication, respectively. Moreover, the RING domain-deleted RNF2 mutant eliminated the RNF2-induced degradation of the PCV3 Cap and RNF2-mediated inhibition of viral replication. This indicates that both processes were associated with its E3 ligase activity. Our findings demonstrate that RNF2 can interact with and degrade the PCV3 Cap via its N-terminal RING domain in a ubiquitination-dependent manner, thereby inhibiting PCV3 replication.IMPORTANCEPorcine circovirus type 3 is a recently described pathogen that is prevalent worldwide, causing substantial economic losses to the swine industry. However, the mechanisms through which host proteins regulate its replication remain unclear. Here, we demonstrate that ring finger protein 2 inhibits porcine circovirus type 3 replication by interacting with and degrading the Cap of this pathogen in a ubiquitination-dependent manner, requiring its N-terminal RING domain. Ring finger protein 2-mediated degradation of the Cap relies on its E3 ligase activity and the simultaneous existence of K139 and K140 within the Cap. These findings reveal the mechanism by which this protein interacts with and degrades the Cap to inhibit porcine circovirus type 3 replication. This consequently provides novel insights into porcine circovirus type 3 pathogenesis and facilitates the development of preventative measures against this pathogen.

Immunometabolic features of the formation of post-vaccination immunity against porcine circovirus type 2 in sows

Immunometabolic features of the formation of post-vaccination immunity against porcine circovirus type 2 in sows

Soluble expression and immunogenicity analysis of capsid proteins of porcine circoviruses types 2, 3, and 4

Porcine circoviruses (PCVs) contain four types: PCV1, PCV2, PCV3, and PCV4, all of which can infect pigs. Among them, PCV1 is non-pathogenic, and PCV2 can cause porcine circovirus diseases (PCVD) or porcine circovirus-associated diseases (PCVAD). Although the pathogenicity of PCV3 and PCV4 is still controversial, increasing evidence shows that PCV3 and PCV4 can cause PCV-related disease. However, mixed infection of PCV2, PCV3, and PCV4 with other pathogens often occurs in large-scale pig breeding, bringing severe economic losses to the global pig industry. In this study, the soluble recombinant proteins of PCV2, PCV3, and PCV4 Cap were expressed by the prokaryotic expression system and biotinylated to combine with the Streptavidin magnetic beads, followed by immunogenicity evaluation of the recombinant proteins. Furthermore, we also assessed the efficacy and immunogenicity of trivalent recombinant proteins conjugated with different adjuvants in mice. The results showed that the highly effective anti-PCV serum was successfully prepared, and the recombinant proteins conjugated with different adjuvants produced various degrees of humoral and cellular immunity in mice. Three recombinant proteins are effective immunogens, and the trivalent proteins coupled with the aluminum adjuvant or GM-CSF-CpG for two-dose immunization can stimulate prominent humoral and cellular immunity against PCVs in vivo. The soluble recombinant proteins are the most promising candidate for developing a trivalent vaccine against PCVs (PCV2, PCV3, and PCV4) infection simultaneously.

Immunological characteristics of a recombinant alphaherpesvirus with an envelope-embedded Cap protein of circovirus.

Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion. We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus. An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells. Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.

Application of Methods Detecting Xenotransplantation-Relevant Viruses for Screening German Slaughterhouse Pigs.

Detection methods have been developed to prevent transmission of zoonotic or xenozoonotic porcine viruses after transplantation of pig organs or cells to the recipient (xenotransplantation). Eleven xenotransplantation-relevant viruses, including porcine cytomegalovirus, porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpesviruses -1, -2, -3 (PLHV-1, 2, 3), porcine parvovirus (PPV), porcine circovirus 2, 3, 4 (PCV2, 3, 4), hepatitis E virus genotype 3 (HEV3), porcine endogenous retrovirus-C (PERV-C), and recombinant PERV-A/C have been selected. In the past, several pig breeds, minipigs, and genetically modified pigs generated for xenotransplantation had been analyzed using these methods. Here, spleen, liver, and blood samples from 10 German slaughterhouse pigs were screened using both PCR-based and immunological assays. Five viruses: PCMV/PRV, PLHV-1, PLHV-3, and PERV-C, were found in all animals, and PCV3 in one animal. Some animals were latently infected with PCMV/PRV, as only virus-specific antibodies were detected. Others were also PCR positive in the spleen and/or liver, indicative of an ongoing infection. These results provide important information on the viruses that infect German slaughterhouse pigs, and together with the results of previous studies, they reveal that the methods and test strategies efficiently work under field conditions.

Detection of Lawsonia intracellularis by oral fluids and fecal samples in Canadian swine

Objectives: The study objectives were to 1) describe the proportion of Lawsonia intracellularis-positive samples in unvaccinated and vaccinated Canadian swine herds during the mid- and late-finishing phases; 2) compare the probability of detecting L intracellularis by quantitative polymerase chain reaction using fecal samples (FS) and oral fluids (OF); and 3) investigate risk factors of L intracellularis detection using FS and OF. Material and methods: Site demographics and vaccination protocols were obtained from 40 Canadian swine sites via questionnaire. Three OF and 3 FS were collected per site once during the mid-finisher (15-17 wk of age) and once during the late-finisher (20-22 wk of age) production stages. Results: Half of all investigated production sites were positive for L intracellularis. A 2-fold increase in L intracellularis detection rate was observed for OF compared to FS (odds ratio = 2.36; 95% CI, 1.24-4.49; P = .009). The presence of porcine circovirus type 2 (PCV2) had a 5-fold increased risk of L intracellularis positivity compared to sites without PCV2 (incidence rate ratio [IRR] = 4.99; 95% CI, 1.29-20.23; P = .02). A higher positive rate was found for sites with L intracellularis outbreaks within the last 2 years (IRR = 3.08; 95% CI, 1.51-6.37; P = .002). Implications: This study presents evidence that OF may have a higher detection rate compared to FS for L intracellularis. Herds with PCV2 or exposure to recent L intracellularis outbreaks may be at increased risk of harboring L intracellularis and warrant additional investigation.

Molecular characterization and phylogenetic analysis of Porcine Circovirus-2 isolated from infected pigs in the Southern region of India

Molecular characterization and phylogenetic analysis of Porcine Circovirus-2 isolated from infected pigs in the Southern region of India