Co-infection of porcine dendritic cells with porcine circovirus type 2a (PCV2a) and genotype II porcine reproductive and respiratory syndrome virus (PRRSV) induces CD4(+)CD25(+)FoxP3(+) T cells in vitro

Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases in the global swine industry. Porcine circovirus type 2 (PCV2) is the primary causative agent, however co-infection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV) is often required to induce the full spectrum of clinical PCVAD. While the specific mechanisms of viral co-infection that lead to clinical disease are not fully understood, immune modulation by the co-infecting viruses likely plays a critical role. We evaluated the ability of dendritic cells (DC) infected with PRRSV, PCV2, or both to induce regulatory T cells (T(regs)) in vitro. DCs infected with PCV2 significantly increased CD4(+)CD25(+)FoxP3(+) T(regs) (p<0.05) and DCs co-infected with PRRSV and PCV2 induced significantly higher numbers of T(regs) than with PCV2 alone (p<0.05). Cytokine analysis indicated that the induction of T(regs) by co-infected DCs may be dependent on TGF-β and not IL-10. Our data support the immunomodulatory role of PCV2/PRRSV co-infection in the pathogenesis of PCVAD, specifically via T(reg)-mediated immunosuppression.     

Evaluation of a porcine circovirus type 2-specific antigen-capture enzyme-linked immunosorbent assay for the diagnosis of postweaning multisystemic wasting syndrome in pigs: comparison with virus isolation, immunohistochemistry, and the polymerase chain reaction.

Quantitative virus isolation, immunohistochemistry, polymerase chain reaction (PCR) assay, and a porcine circovirus 2 (PCV2)-specific antigen-capture enzyme-linked immunosorbent assay (ELISA) were used for differentiation between clinical and subclinical PCV2 infections of swine. Tissue samples from pigs experimentally infected with PCV2 and field cases of postweaning multisystemic wasting syndrome and PCV2-associated reproductive disorders were used in this evaluation. In initial studies on 6 PCV2 pools using 3 previously published PCR protocols for PCV2 detection, quantitative virus isolation, and antigen-capture ELISA, substantial differences in sensitivity were identified among these procedures. Examination of tissue samples from diseased and clinically normal pigs indicated that immunohistochemistry, quantitative virus isolation, and antigen-capture ELISA could be used to differentiate between clinical and subclinical PCV2 infections, but the PCR assay could not. Because subclinical infections of pigs with PCV2 are common, the use of nonquantitative PCR as a diagnostic tool for PCV2-related diseases should be discouraged and the PCV2-specific antigen-capture ELISA evaluated further.     

Fatal bronchopneumonia in a Metastrongylus elongatus and Porcine circovirus type 2 co-infected pig

Porcine circovirus type 2 (PCV2) infection is distributed worldwide and PCV2-associated disease (PCVAD) is considered among the most economically relevant ones to the global swine industry. PCV2 is known to play a causal role in the porcine respiratory disease complex, usually in close association with a large plethora of other biologic agents. We describe herein a case of fatal parasitic bronchopneumonia by Metastrongylus elongatus in a PCV2-infected pig. Metastrongylosis may still represent a major concern for outdoor herds. Our recent experience suggests that a concurrent PCVAD condition may trigger metastrongylosis, which may subsequently result, at its turn, in severe, sometimes fatal, pulmonary disease.     

Porcine Circovirus Type 2 and Porcine Circovirus-Associated Disease

Porcine circovirus type 2 (PCV2) belongs to the viral family Circoviridae and to the genus Circovirus . Circoviruses are small, single-stranded nonenveloped DNA viruses that have an unsegmented circular genome. PCV2 is the primary causative agent of several syndromes collectively known as porcine circovirus-associated disease (PCVAD). Many of the syndromes associated with PCVAD are a result of coinfection with PCV2 virus and other agents such as Mycoplasma and porcine reproductive and respiratory syndrome virus. PCV2 infection is present in every major swine-producing country in the world, and the number of identified cases of PCVAD is rapidly increasing. In the United States, the disease has cost producers an average of 3–4 dollars per pig with peak losses ranging up to 20 dollars per pig. The importance of this disease has stimulated investigations aimed at identifying risk factors associated with infection and minimizing these risks through modified management practices and development of vaccination strategies. This paper provides an overview of current knowledge relating to PCV2 and PCVAD with an emphasis on information relevant to the swine veterinarian.     

Genomic comparison of foot-and-mouth disease virus R strain and its chick-passaged attenuated strain

Porcine circovirus type 2 (PCV2) is a single-stranded circular DNA virus that is the causative agent of porcine circovirus associated disease (PCVAD), a disease complex affecting swine around the world. Although this virus is believed to negatively affect the host's immune system, the mechanism by which PCV2 induces disease is not completely understood. This report describes a series of PCV2 experiments using the gnotobiotic pig model in which a relationship was demonstrated between abnormal leukograms and development of clinical disease in PCV2-infected pigs. When compared to control pigs the leukogram was characterized by a decrease in lymphocytes within 14 days post inoculation (dpi) followed by an increase in neutrophils 7-14 days later. No significant changes in the circulating monocyte, basophil, and eosinophil cell populations were detected. The combination of an absolute neutrophilia and lymphopenia produced a neutrophil/lymphocyte ratio that was predictive of clinical disease and was inversely correlated with the presence of neutralizing antibodies. Based on previous reports, the lymphopenia may be attributed to a direct cytolytic effect of the virus and could negatively affect the pig's immune response. The role of the neutrophilia in the pathogenesis of PCVAD in gnotobiotic pigs is unknown.     

猪圆环病毒2型IgM抗体间接ELISA的建立

猪圆环病毒2型(PCV2)是断奶仔猪多系统衰竭综合症的重要原发性病原,其感染的早期检测有助于及时采取防控措施。以重组衣壳蛋白(Cap蛋白)作为包被抗原,通过对反应条件的优化,建立了PCV2 IgM抗体间接ELISA,其最适抗原包被浓度为1.25 μg／mL,最适血清稀释度为1∶100,临界值为0.35。该检测方法与其他5种常见猪疫病阳性血清无交叉反应,特异性良好。批内、批间重复试验的变异系数均在2%~6%,重复性好。对100份临床样本的检测结果表明,该检测方法与国外同类试剂盒相比,敏感性为90.3%,特异性为92.8%,总符合率为92%。试验结果表明,所建立的PCV2 IgM间接ELISA特异性好和敏感性高,适用于PCV2感染早期的流行病学调查。     

Detection and molecular characterization of porcine circovirus type 2 (PCV2) from piglets with exudative epidermitis in Uruguay

Porcine circovirus type 2 (PCV2) is an economically important emerging pathogen associated with distinct syndromes and diseases in swine, collectively known as porcine circovirus associated diseases (PCVAD). The main purpose of this study was to investigate the presence of PCV2 in piglets affected with exudative epidermitis (EE) in Uruguay. In addition we aimed to analyze the phylogenetic relationships of the isolated strains. In June 2011 an outbreak of EE detected in a small herd was reported. Piglets presented skin lesions compatible with EE and symptoms associated with postweaning multisystemic wasting syndrome (PMWS) were also observed. Sera from affected and healthy animals were tested for the presence of viral DNA. Exclusively, diseased piglets were infected with PCV2. Phylogenetic analysis showed that PCV2 isolates belonged to PCV2b genotype. We report the detection and molecular characterization of PCV2 strains for the first time in Uruguay.     

Preliminary study of Porcine circovirus type 2 and Torque teno sus virus coinfection frequencies in Brazilian pig herds

Torque teno sus virus (TTSuV) is emergent in swine herds. Recent studies have shown an increased frequency of TTSuV2 in Porcine circovirus type 2 (PCV2)-associated diseases (PCVAD), which are endemic in many swine-producing countries, including Brazil. Coinfection with several other viral and bacterial agents results in an increased incidence of more severe PCVAD. Given the limited information on TTSuV and PCV2 coinfection, especially in Brazilian swine herds, this study made a preliminary estimation of the occurrence of coinfection in swine herds by testing samples from different categories. Between 2008 and 2009, 111 samples of feces and 23 serum samples from 5 swine herds were tested for PCV2 and TTSuVs and the results analyzed for associations between these agents. No significant differences in coinfection frequency were observed for PCV2 + TTSuV1 or for PCV2 + TTSuV2 between nursery piglets (P = 0.730), growing pigs (P = 0.331), or sows (P = 0.472). However, a significant difference was observed for PCV2 + TTSuV1 + TTSuV2 between nursery piglets and growing pigs (P = 0.004; Fisher’s exact test). Phylogenetic studies agreed with the grouping of TTSuV1 and TTSuV2 into 2 different clades, with no distinct pattern of clustering of these isolates with the animal categories.     

一例猪圆环病毒2型和大肠杆菌、溶血葡萄球菌混合感染的诊断

山东德州某猪场发生猪高热、呼吸系统疾病甚至死亡的疫情。采集病料提取病变组织总DNA或RNA进行猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒、猪瘟病毒的PCR或RT-PCR检测。PCR扩增出353 bp的猪圆环病毒2型特异性条带。同时进行细菌分离培养、生化鉴定等试验,诊断为猪圆环病毒2型和大肠杆菌、溶血葡萄球菌混合感染。     

Establishment and Preliminary Application of Nested PCR for the Detection of Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) caused significant economic losses of swine industry,it's very important to establish a economy and rapid detection method.To strengthen the prevention and control of porcine circovirus disease,rapid and high sesitive detection method of PCV2 was established using nested PCR.According to PCV2 ORF2 gene conservative region,we designed two pairs of nested PCR primers,constructed ORF2 gene recombinant plasmid for standard template and optimizated the PCR reaction conditions to establish nested PCR detection method of PCV2 and detect the clinical samples of pig farms.Our results showed that the sensitivity and repeatability of this method were very high,the minimum detection level was 10 copies/μL and detection rate of clinical samples was 97.3%.In brief,the established nested PCR detection method of PCV2 in this study provided powerful means for quick and efficient detection of PCV2,and prevention and control of porcine circovirus disease in the swine industry.     

猪圆环病毒2型巢式PCR检测方法的建立与初步应用

猪圆环病毒2型(porcine circovirus type 2,PCV2)给养猪业造成了重大的经济损失,建立经济、快速的检测方法尤为重要。本研究旨在利用巢式PCR技术,建立高灵敏度PCV2检测平台,以加强对猪圆环病毒病的防控。根据PCV2 ORF2基因保守区,设计内、外2对巢式PCR引物,通过优化PCR反应条件,建立巢式PCR检测方法,并对猪场临床样本进行检测。结果显示,本研究建立的巢式PCR检测方法灵敏度高、重复性强,最低检出量为10拷贝/μL,检出率达97.3%。本研究建立的巢式PCR检测方法为快速、高效地检测PCV2以及为猪圆环病毒病的防控提供有力手段。     

Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies.

Porcine circovirus type 2 (PCV2)-associated disease (PCVAD) continues to be an important differential diagnosis on pig farms in the United States and worldwide. Case trend analyses indicate that the incidence of PCVAD is on the rise in the United States. Accurate diagnosis is important in order to implement appropriate intervention strategies. PCVAD can manifest as a systemic disease, as part of the respiratory disease complex, as an enteric disease, as porcine dermatitis and nephropathy syndrome, or as reproductive problems. PCVAD may be only a sporadic individual animal diagnosis; however, PCVAD may also manifest as a severe herd problem accelerated and enhanced by concurrent virus or bacterial infections. This article is intended to discuss the most common disease manifestations, pathogenesis, diagnostic approaches, and intervention strategies associated with PCVAD in North America.     

Porcine circovirus 2-associated disease in Eurasian wild boar.

Porcine circovirus 2 (PCV2) was first identified in high-health herds of domestic swine and was associated with a debilitating disease called postweaning multisystemic wasting syndrome (PMWS). Most subsequent studies have indicated that PCV2 infects only swine but there is little information on porcids other than improved breeds of domestic swine. Multisystemic disease was reported in a group of Eurasian wild boars raised under free-range conditions. Affected young pigs had pneumonia and enteritis and were cachectic. Porcine circovirus 2 was identified in affected tissue by immunohistochemistry and in situ hybridization, and a PCV2-like virus was isolated from pooled organs. The open reading frame (ORF2) of the isolated PCV2 had a 98.7% homology with the ORF2 of a reference PCV2 isolate. These diagnostic data indicate that PCV2 can infect and cause disease in Sus scrofa subspecies other than domestic swine.     

Porcine circovirus-associated disease in weaner pigs in Western Australia

Objective To report the occurrence and pathology of porcine circovirus type 2 (PCV2)‐associated disease (PCVAD) of postweaning pigs in two Australian pig herds. Methods Mortality data from two commercial piggeries that experienced higher than normal postweaning illthrift and mortalities were examined. Gross and histopathological examinations were performed on the index cases, and at weekly intervals thereafter for a period of 10 weeks. Specimens were submitted to the laboratory for routine diagnostic testing and for exclusion of porcine reproductive and respiratory syndrome virus (PRRSV). The genomes of two strains of PCV2 isolated during testing were sequenced. Results Mortality rates in weaned, 5–12‐week‐old pigs spiked significantly during mid to late 2007. This increase in the mortalities was mainly attributed to salmonella‐associated diarrhoea and illthrift. Salmonellosis was diagnosed in 73/110 cases inclusive of both piggeries. Many pigs also had chronic granulomatous lymphadenitis and diffuse histiocytic interstitial pneumonia consistent with PCVAD and associated with varying amounts of PCV2 antigen and inclusion bodies. All samples tested for PRRSV were negative. Sequence analysis of the PCV2 isolates showed strain differences between piggeries. Conclusion This report describes the first outbreaks of PCVAD in growing pigs in Western Australia (WA) and describes lesions not previously seen in this laboratory. It also describes the first isolation of a PCV2 group 1 virus in WA associated with PCVAD. Although the outbreaks of PCVAD occurred with concurrent salmonellosis, the two diseases were unrelated. Neither of the outbreaks met the Australian case definition for the diagnosis of postweaning multisystemic wasting syndrome.     

Assessment of performance of selected serological tests for diagnosing brucellosis in pigs

Swine brucellosis due to Brucella suis is considered an emerging zoonotic disease whose control is based on serological testing and the subsequent culling of seropositive animals or the full depopulation of affected flocks. Here we assessed the performance of several serological tests (Rose Bengal Test [RBT], indirect ELISA [i-ELISA], blocking ELISA [b-ELISA], and two competitive ELISAs [c-ELISA]) for diagnosing swine brucellosis caused by B. suis biovar 2. Both frequentistic and Bayesian statistical inference were used. A frequentistic analysis, using sera from known gold standard (GS) populations (i.e., from truly infected or brucellosis free animals), resulted in maximum (100%) diagnostic sensitivity (Se) and specificity (Sp) in the RBT, i-ELISA and b-ELISA tests. However, c-ELISAs resulted in lower diagnostic Se (ranging from 68.5% to 92.6%, according to the different cut-offs selected). A Bayesian analysis of tests yielding the best diagnostic performance with GS sera (RBT, i-ELISA and b-ELISA), but using a large collection of field sera, resulted in similar Se among tests but markedly lower (≈ 80%) than that resulting from the frequentistic analysis using the GS serum populations. By contrast, the estimated Sp in the Bayesian analysis was only slightly lower than 100%, thus similar to that obtained frequentistically. Our results show that adequate diagnostic tests for brucellosis in swine are available, but also emphasize the need for more extensive validation studies before applying these tests under field conditions.     

猪圆环病毒2型与猪TTV混合感染的流行病学调查

根据GenBank_h发表的猪圆环病毒2型（Porcinecircovirustype2,PCV2）基因组序列和TTV（Torquetenovirus）1、2型的UTR序列设计合成引物,建立了分别用于检测PCV2和TTV1、TTV2的PCR及巢式PCR方法。应用建立的PCR方法对送检的广东、福建和江西等7个省份258份血液和组织样品进行了PCV2、TTV1和TTV2的检测,确定猪群中PCV2与TTV1和／或TTV2混合感染情况。结果表明,94份样品表现为PCV2和TTV1的混合感染,占样品总数的36．4％;193份样品表现为PCV2和TTV2的混合感染,占74．8％;另外,还有一些样品为三重感染,占34．5％。由此可以看出,猪群中PCV2和／或TTV1和／或TTV2的混合感染很普遍。     

Comparison of bacterial culture, polymerase chain reaction, and a mix-enzyme-linked immunosorbent assay for the detection of Salmonella status in grow-to-finish pigs in western Canada with a Bayesian approach

Among grow-to-finish pigs from 10 herds in Alberta and Saskatchewan, 23 (16%) of 144 fecal samples were culture-positive and 40 (28%) of 144 pigs were seropositive for Salmonella. With a Bayesian model specifying dependence between the 2 tests, the sensitivity (Se) of culture and real-time polymerase chain reaction (RT-PCR) was 79% to 86%, depending on the cut-off value for the enzyme-linked immunosorbent assay (ELISA). Culture specificity (Sp) was assumed to be 100%; RT-PCR Sp was found to be 94%. The ELISA Se was 76% and 51% at optical density cut-off values ≥ 20% and ≥ 40%, respectively; the Sp was 94% at each cut-off value. The model showed some sensitivity to ELISA prior information, the ELISA Se being approximately 8% lower when informative prior information was specified in the model. When there was no adjustment for dependence between culture and RT-PCR, the posterior estimates for both culture and RT-PCR Se were 11% higher than with the conditional-dependence model and had considerably narrower probability intervals, which suggests that correlation between culture and PCR is important and should be adjusted for in future studies.     

Evaluation of the diagnostic accuracy of the modified agglutination test (MAT) and an indirect ELISA for the detection of serum antibodies against Toxoplasma gondii in sheep through Bayesian approaches

The diagnostic accuracies of the modified agglutination test (MAT) and indirect ELISA test for the detection of serum antibodies against Toxoplasma gondii in sheep were evaluated through Bayesian approaches on two populations of sheep created from three different groups of animals (T. gondii-aborted ewes, colostrums-deprived newborn lambs, and ewe-lambs and adult ewes with unknown T. gondii infection status). Tests showed a high degree of agreement (kappa statistic = 0.93; 95% confidence interval = 0.87, 0.98) and a significant specificity (Sp) correlation (gamma(Sp) = 0.26; 95% credibility interval = 0.017, 0.61). When prior information was used for all unknown parameters the posterior medians for the sensitivity (Se) and Sp of the MAT and ELISA were, respectively, 92.6% (95% credibility interval = 85.2, 96.9), 95.5% (89.9, 98.7), 90.5% (83.4, 95.6), and 97.8% (94.2, 99.5). These estimates remained similar when uninformative priors were included. The Se estimates of the MAT and ELISA were higher than those obtained on pigs in other study using the same approach (Se = 80.6% and Sp = 89.5% for the MAT, and Se = 71.5% and Sp = 85.5% for the ELISA [Georgiadis, M.P., Wesley, O.J., Gardner, I.A., Singh, R., 2003. Correlation-adjusted estimation of sensitivity and specificity of two diagnostic tests. Appl. Stat. 52, 63-78]. This finding supported the believe that test performances may vary when applied on different animal species. Thus, if these tests are planned to be used on animal species other than sheep or pigs, their diagnostic accuracy should be re-assessed to prevent biased inferences from their results.     

Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica

Porcine circovirus (PCV) was recently divided into 2 antigenically distinct types that differ (65% amino acid identity) in the protein encoded by open reading frame 2 (ORF2). Porcine circovirus 1 is apparently non-pathogenic and, in contrast, PCV2 is associated with porcine multisystemic wasting syndrome (PMWS). Our objective was to determine the extent of exposure of normal pigs in Canada and Costa Rica to PCV2. Recombinant DNA techniques were used to produce an antigen from ORF2 of PCV2 that was suitable for the detection of antibody in swine sera. The presence of PCV2 nucleotide sequences was detected using polymerase chain reaction (PCR) techniques. Using these tests, specific antibody and nucleotide sequences were demonstrated in sera from a cohort of pigs during a PMWS outbreak. Antibody was detected in normal, healthy hogs slaughtered in Canada (82.4% of 386) and in Costa Rica (14.6% of 322). This is the first report indicating the presence of PCV2 in Latin America. More than 50% of these sera also contained PCV2 nucleotide sequence. Although these hogs were healthy when slaughtered, they were infected with PCV2 and may have previously been ill. The widespread occurrence of PCV2 in swine suggests that this virus is adapted to replication in porcine tissue.     

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.