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Associations between Torque teno sus viruses (TTSuVs) and the occurrence of postweaning multisystemic wasting syndrome (PMWS) have been reported with controversial results. Currently, no studies have been performed comparing simultaneously viral loads of TTSuVs and PCV2. To examine the role for TTSuVs in PMWS-affected animals, a SYBR Green-based quantitative PCR (qPCR) was designed to detect and quantify TTSuV1, TTSuV2 and PCV2 genomes in swine sera. TTSuV1 genome loads were significantly higher in healthy adults than in young and SPF animals (p < 0.05) suggesting that the prevalence of TTSuV1 infection increases with age and bears no association with PMWS. Regarding TTSuV2, no significant variation was detected in viral loads within any of the groups. As expected, PCV2 genome loads were higher in PMWS-affected swine than in healthy or SPF animals (p < 0.001). These findings provide clear evidence to indicate that neither TTSuV1 nor TTSuV2 viral loads have any correlation with the occurrence of PMWS. [ABSTRACT FROM AUTHOR]

Highlights: [•] The existence of Torque teno sus virus 2 nonstructural proteins was validated. [•] Circular genomic DNA can mimic Torque teno sus virus 2 infection. [•] Torque teno sus virus 2 ORF2/2/3 showed peri-nuclear localization. [Copyright &y& Elsevier]

Porcine circovirus type 2 (PCV2) infection is the cause of postweaning multisystemic wasting syndrome (PMWS). It has been speculated whether cell types permissive of replication are found in the primary lymphoid organs and whether infection of these tissues has an important role in the pathogenesis of PMWS. The aim of this study was to determine if primary lymphoid organ cells support viral replication during PCV2 infection. This was done by histopathological examination of thymus and bone marrow from pigs experimentally inoculated with PCV2 (n = 24), mock-infected pigs (n = 12), pigs naturally affected by PMWS (n = 33), and age-matched healthy control animals (n = 29). In situ hybridization (ISH) techniques were used to detect PCV2 nucleic acid irrespective of replicative status (complementary probe, CP) or to detect only the replicative form of the virus (replicative form probe, RFP). PCV2 was not detected in the experimentally PCV2-inoculated pigs or the control animals. Among the PMWS-affected pigs, 19 of 20 (95%) thymuses were positive for PCV2 by CP ISH, and 7 of 19 (37%) of these also supported viral replication. By CP ISH, PCV2 was detected in 16 of 33 (48%) bone marrow samples, and 5 of 16 (31%) of these also supported replication. The 2 ISH probes labeled the same cell types, which were histiocytes in both organs and lymphocytes in thymus. The RFP labeled fewer cells than the CP. Thus, PCV2 nucleic acids and replication were found in bone marrow and thymus of PMWS-affected pigs, but there was no evidence that primary lymphoid organ cells are major supporters of PCV2 replication. [ABSTRACT FROM PUBLISHER]

Abstract: Porcine respiratory disease complex (PRDC) is the name used to describe a clinical presentation characterized by respiratory signs and poor growth in growing-finishing pigs. Porcine circovirus type 2 (PCV2) is one of the pathogens potentially involved in the presentation of this complex, although, it has been mainly linked to PCV2-systemic disease (PCV2-SD). However, some reports have suggested that PCV2 can cause respiratory lesions in the absence of systemic lymphoid damage; this apparently different condition has been referred as PCV2-lung disease (PCV2-LD). The present study analysed retrospectively the relation between PRDC and PCV2 infection from a pathological point of view. Of the 317 selected pigs suffering from PRDC and submitted for necropsy between 1998 and 2011, the most prevalent lesions were subacute interstitial pneumonia (27.7%) and the combination of subacute interstitial pneumonia plus catarrhal-purulent bronchopneumonia (28.4%). Two hundred and twenty-six pigs were positive for PCV2 by in situ hybridization (ISH); using immunohistochemical techniques, 77 were positive for porcine reproductive and respiratory syndrome virus and 3 for swine influenza virus. Among positive pigs for PCV2 ISH, 184 animals suffered from PCV2-SD. The rest of 42 PCV2 ISH positive pigs had no lymphoid lesions and low amount of viral nucleic acid in these tissues and, therefore, did not qualify for PCVD-SD; only 9 had concomitant PCV2 ISH positivity in the lung, also to a low amount. In conclusion, all PRDC-PCV2 ISH positive cases had evidence of viral systemic infection. These results suggest that PCV2-LD is probably a negligible condition and PCV2 mainly contributes to PRDC in relation to PCV2-SD occurrence. [Copyright &y& Elsevier]

Abstract: Porcine circovirus type 2 (PCV2) infection has been suggested as an acquired immunodeficiency disorder. However, the immunopathogenesis of PCV2 infection is still not fully clarified. In the present study, 35 inguinal lymph nodes (LNs) with different levels of PCV2 load obtained from postwaening multisystemic wasting syndrome (PMWS)-affected pigs and 7 from healthy subclinically PCV2-infected pigs were selected. The LNs were subsequently ranked by their PCV2 loads to mimic the progression of PCV2 infection-associated lesion development. The expressions of 96 selected immune genes in these LNs were assessed by the integration of several reverse transcription quantitative real-time polymerase chain reaction experiments. Hierarchical cluster analysis of the gene expression profiles resulted in 5 major clusters (A, B, C, D, and E). Different clusters of immune gene expression profiles were compatible with the divergent functions of various immune cell subpopulations. 61 out of 96 selected genes belonged to cluster C and were mainly involved in the activation of dendritic cells and B and T lymphocytes. The expression levels of these genes were generally up-regulated in the LNs obtained from PMWS-affected pigs with relatively lower PCV2 loads. However, the up-regulated level tended to reduce or turned into down-regulation as the PCV2 load increased. Genes belonging to cluster B, involved in T cell receptor signaling, became silenced as the PCV2 load increased. The expression profiles of macrophage-associated genes were either independent from or positively correlated with the PCV2 load, such as those in clusters A and E and in cluster D, respectively. In addition, the principle component analysis of the expression of the 96 selected genes in the 42 inguinal LNs revealed that 53.10% and 72.29% of the total data variants could be explained by the top-3 and top-7 principle components, respectively, suggesting that the disease development of PCV2 infection may be associated with a few major and some minor factors. In conclusion, assessment of immune gene expression profiles in LNs supports a close interaction between immune activation and suppression during the progression of PMWS development. [Copyright &y& Elsevier]

Abstract: The present study describes the effects of sow and/or piglet porcine circovirus type 2 (PCV2) vaccination on viraemia, antibody response and production parameters (average daily weight gain [ADWG] and mortality) of piglets from a PCV2 subclinically infected farm. Four hundred seventy-six piglets born from vaccinated (V) or non-vaccinated (NV) sows were further subdivided in a total of four groups: NV sows–NV pigs (NV–NV, n =134), NV sows–V pigs (NV–V, n =135);V sows–NV pigs (V–NV, n =104) and V sows–V pigs (V–V, n =103). A single vaccination of sows before mating was able to confer significantly higher antibody titres to their piglets at 4 weeks of age and a different PCV2 dynamics infection compared to piglets coming from NV sows. Piglet vaccination (independently of sow treatment) caused an earlier seroconversion and lower percentages of PCV2 infected pigs compared to the NV ones throughout their life. The double PCV2 vaccination strategy was able to reduce PCV2 infection but apparently caused some interference in piglet humoral response development. PCV2 vaccination was able to overcome this interference since the ADWG was improved in both groups of vaccinated piglets, independently of the sow treatment, being the highest ones obtained in the double vaccination group. [Copyright &y& Elsevier]

Abstract: Two prospective longitudinal studies in 13 postweaning multisystemic wasting syndrome (PMWS)-affected farms from Spain (n =3) and Denmark (n =10) were performed. Blood samples from pigs were longitudinally collected from 1st week until the occurrence of the PMWS outbreak. Wasted and healthy age-matched pigs were euthanized, necropsied and histopathologically characterised. PMWS diagnosis was confirmed by means of lymphoid lesions and detection of porcine circovirus type 2 (PCV2) in these tissues by in situ hybridization or immunohistochemistry. Serological analyses were performed in longitudinally collected serum samples to detect antibodies against, PCV2, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), swine influenza virus (SIV) and Lawsonia intracellularis (law), Mycoplasma hyopneumoniae, Aujeszky’s disease virus (ADV) and Salmonella spp. A Cox proportional hazards model was used to investigate the simultaneous effects of seroconversion and maternal immunity against the studied pathogens. Results showed that high levels of maternal immunity against PCV2 had a protecting effect in farms from both countries. Moreover, for the Danish dataset, seroconversion against law had an overall protecting effect, but for animals with very low levels of maternal antibody levels against this pathogen, the effect appeared neutral or aggravating. Otherwise, for the Spanish dataset, maternal immunity against PPV and PRRSV gave protective and aggravating effects, respectively. In conclusion, the present study reflects the complex interaction among different pathogens and their effects in order to trigger PMWS in PCV2 infected pigs. [Copyright &y& Elsevier]

Abstract: Porcine circovirus type 2 (PCV2) has been recognized as an important vial pathogen for global swine industry. The virus is associated with postweaning multisystemic wasting syndrome (PMWS) and other syndrome diseases collectively known as porcine circovirus-associated disease (PCVAD). PCV2 infection and PMWS have caused great impact on the pig production in China during the past 10 years. This review will involve in the history of PCVAD, serological epidemiology of PCV2 infection, and clinical aspect of PCVAD, and the genomic characteristics, variation and genotyping of PCV2, as well as control situation of PCVAD in China. [Copyright &y& Elsevier]

Abstract: The present study describes the effects of a commercially available vaccine against Porcine circovirus type 2 (PCV2) on clinical, pathological and virological outcomes of 3-week-old piglets from two farms with a clinical history of postweaning multisystemic wasting syndrome (PMWS). The study was a controlled, double-blinded, parallel group (1:1) and randomized trial (with a negative control) involving a total of 1239 animals. The study period comprised from weaning age (time of vaccination or PBS inoculation) until the first shipment of pigs to the slaughterhouse. The vaccine product was able to reduce clinical signs, PCV2 viral load in sera and faeces, and overall mortality in nurseries and fattening units. Moreover, average daily gain was significantly higher in vaccinated versus non-vaccinated piglets during the trial period. On the other hand, it was shown that maternally derived antibodies interfered with the development of an active humoral immune response after PCV2 vaccination. [Copyright &y& Elsevier]

Abstract: Torque teno viruses (TTVs) are vertebrate infecting, small viruses with circular single stranded DNA, classified in the Anelloviridae family. In pigs, two different TTV species have been described so far, Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2). TTSuVs have lately been linked to postweaning multisystemic wasting syndrome (PMWS). In the present study, TTSuV1 and TTSuV2 prevalence and DNA loads in longitudinally collected serum samples of healthy and PMWS affected pigs from Spanish conventional, multi-site farms were analyzed. Serum samples were taken at 1, 3, 7, 11 and around 15 weeks of age (age of PMWS outbreak) and viral DNA loads determined by quantitative PCR. For both TTSuV species, percentage of viremic pigs increased progressively over time, with the highest prevalence in animals of about 15 weeks of age. TTSuV1 and TTSuV2 viral DNA loads in healthy and TTSuV1 loads in PMWS affected animals increased until 11 weeks of age declining afterwards. On the contrary, TTSuV2 DNA loads in PMWS affected pigs increased throughout the sampling period. It seems that TTSuV species differ in the in vivo infection dynamics in PMWS affected animals. [Copyright &y& Elsevier]

The degree of apoptosis in the livers of pigs with hepatitis due to naturally-occurring postweaning multisystemic wasting syndrome (PMWS) was evaluated semi-quantitatively by immunohistochemical detection of the apoptotic marker cleaved caspase-3 (CCasp3). The amount and distribution of porcine circovirus type 2 (PCV2) virus in the liver was evaluated using in situ hybridisation. Livers with mild, stage 1 hepatitis exhibited similar degrees of apoptosis to controls; those with stage II lesions had variable apoptotic rates, ranging from mild to high, and in livers with more severe, stage HI hepatitis, high levels of hepatocyte apoptosis was a feature. Statistical analyses indicated a positive association between the rate of apoptosis, the severity of the hepatitis and the amount of PCV2 DNA in the liver. Double immunolabelling for CCasp3 and PCV2 DNA revealed a predominance of cells labelling only for PCV2, followed by fewer cells labelling only for CCasp3, and the least number labelling for both. The findings suggest that apoptosis, possibly triggered by PCV2 infection and/or hepatic inflammation, plays a key role in the pathogenesis of hepatitis in pigs with naturally-occurring PMWS. [ABSTRACT FROM AUTHOR]

Abstract: The immunopathogenesis of porcine circovirus type 2 (PCV2) infection in conventional pigs is complicated by various environmental factors and individual variation and is difficult to be completely reproduced experimentally. In the present field-based study, a tissue microarray (TMA) consisting of a series of lymphoid follicles having different PCV2-loads was constructed using formalin-fixed and paraffin-embedded superficial inguinal lymph nodes (LNs) from 102 pigs. Using the TMA, a wide range of parameters, including co-infected viral pathogens, immune cell subsets, and cell apoptosis/proliferation activity by immunohistochemical (IHC) staining or in situ hybridization (ISH) were measured, characterized, and compared. The signal location and area extent of each parameter were interpreted by pathologists, semi-quantified by automated image analysis software, and analyzed statistically. The results herein demonstrated a significant negative correlation between PCV2 and CD79a (p <0.001) and a significant positive correlation between PCV2 and lysozyme (p <0.001) or TUNEL (p <0.001) using Pearson correlation analysis. The amount of porcine respiratory and reproductive syndrome virus (PRRSV) and porcine parvovirus antigens did not correlate with the tissue loads of PCV2 nucleic acid. Multiple regression analysis further predicted that PCV2 contributed major effects on CD79a, lysozyme, and TUNEL but PRRSV showed relatively less effects on these parameters. In addition, the total signal intensity of Ki67 (index of cell proliferation activity) did not change significantly among cases with different PCV2 loads; however, as the loading of PCV2 nucleic acid increased, the main contribution of Ki67 signal gradually shifted from B cells in the germinal center to T cells and macrophages in the interfollicular regions. In the present study, the use of TMA to establish a mathematical model with a wider range of statistical analysis can bring us a step forward to understand the immunopathogenesis of PCV2 infection-associated follicular changes in LNs. [Copyright &y& Elsevier]

Abstract: Our primary objectives were to determine: the relative virulence of porcine circovirus (PCV) 2a and PCV2b, if heterologous infection induces severe illness, and the relative concentration of PCV2a and PCV2b in tissues of heterologously infected pigs. In experiment 1, 18 germ-free piglets served as controls or were infected with PCV2a or PCV2b. Half were immune stimulated with keyhole limpet hemocyanin (KLH) emulsified in incomplete Freund''s adjuvant (2aKLH, 2bKLH). No piglets demonstrated severe illness. Lesion severity did not differ, but PCV2 capsid staining was more intense in 2a- than 2b-infected pigs (P <.05). In experiment 2, 20 germ-free piglets were dual inoculated 7 days apart with PCV2a and PCV2b (2a2b, 2b2a), PCV2b twice (2b2b), or PCV2a (2a2a) twice. Five of 9 heterologous-infected pigs developed severe illness. All heterologously infected pigs demonstrated ascites or edema, and 8/9 developed thymic atrophy. By contrast, 1 of 5 2b2b-infected pigs developed bronchopneumonia and pleural effusion. No 2a2a-infected pig developed illness. Gross lesions were more severe in heterologously infected pigs than in 2b2b pigs (P <.05), and were more severe in 2b2b than 2a2a pigs (P <.05). PCV2 capsid staining intensity did not differ by group. In heterologously infected pigs, higher levels of PCV2 DNA reflective of the first inoculum compared to the second were found in mesenteric lymph node (P =.04), spleen (P =.004) and liver (P =.04). These results indicate that dual heterologous PCV2a/2b inoculation 7 days apart may induce severe clinical illness, but PCV2a and PCV2b when administered singularly or in combination with KLH appear to be of equivalent virulence. [Copyright &y& Elsevier]

Abstract: Porcine circovirus type 2 has been linked to many diseases, such as postweaning multisystemic wasting syndrome and can be found in most commercial swine confinement buildings around the world. Although the exact role of the virus in the appearance of disease in animals is not fully understood, the mechanisms responsible for the transmission of the virus are currently believed to happen mostly by contact. Nevertheless, the possibility of airborne transmission cannot be rejected. This study investigated the presence of the virus, total bacteria and total dusts in aerosols. Air samples were taken with gelatin filters in swine confinement buildings and were analyzed by quantitative polymerase chain reaction. Interestingly, concentrations of airborne PCV2 of up to 107 genomes per cubic meter of air were detected. Airborne dust concentrations were correlated to airborne concentrations of PCV2 and total bacteria. Although the infectivity potential of the airborne viral loads were not evaluated, it is clear that the virus can become airborne in detectable concentrations in commercial swine confinement building environments. The significance of this finding in an epidemiological point of view will need further investigation. [Copyright &y& Elsevier]

Abstract: The present study describes the effects of a commercially available genetically engineered chimeric vaccine against porcine circovirus type 2 (PCV2) on clinical, pathological and virological features in three multi-site farms suffering from postweaning multisystemic wasting syndrome (PMWS). The vaccine product was able to reduce clinical signs, PCV2 viral load in lymphoid organs and/or sera, and overall mortality in nurseries and fattening units. This is the first time in which is shown that a PCV2 vaccine is able to decrease specifically PMWS-associated mortality. Another novelty of this study is the assessment of PMWS-like histological lesions in a large number of vaccinated and non-vaccinated pigs under field conditions. [Copyright &y& Elsevier]

Abstract: The aim of the present longitudinal study was to assess the evolution of two acute phase proteins (APPs), pig-major acute phase protein (pig-MAP) and haptoglobin (HPT), in serum from pigs that developed postweaning multisystemic wasting syndrome (PMWS) in comparison to healthy and wasted non-PMWS affected pigs. In addition, evidence of infection with other pathogens and its relation with variations in APPs concentrations was also assessed. Fourteen independent batches of 100–154 pigs were monitored from birth to PMWS outbreak occurrence in 11 PMWS affected farms. Pigs displaying PMWS-like signs and age-matched healthy controls were euthanized during the clinical outbreak. PMWS was diagnosed according to internationally accepted criteria and pigs were classified as: (i) PMWS cases, (ii) wasted non-PMWS cases and (iii) healthy pigs. At the moment of PMWS occurrence, pig-MAP and HPT concentration in PMWS affected pigs were higher than in healthy ones (p <0.0001). No differences in APPs serum concentrations between subclinically PCV2-infected pigs and healthy non-PCV2-infected pigs (based on quantitative PCR on serum results) were detected. Results showed a significant correlation between PCV2 loads and both pig-MAP (R =0.487–0.602, p <0.0001) and HPT (R =0.326–0.550, p <0.05–0.0001) concentrations in serum of PMWS affected pigs, indicating that the acute phase response in PMWS affected pigs occurred concomitantly to PCV2 viremia. No other pathogen, apart from PCV2, was consistently related with variations in APPs concentrations. A ROC analysis, made to determine the capacity of discrimination of both APPs between PMWS affected and non-affected pigs, showed higher sensitivity and specificity values using pig-MAP compared to HPT. These results suggest that pig-MAP might be a better indicator of PMWS status than HPT. Moreover, the fact that APR occurred some weeks before the start of clinical signs suggests that APPs could provide valuable prognostic information for PMWS development. [Copyright &y& Elsevier]

Abstract: The immunogenicity and efficacy generated by one dose of a PCV2 sub-unit vaccine (Porcilis PCV®) were evaluated in 3-week-old conventional piglets. Vaccination induced both humoral and cell-mediated responses against PCV2, which were increased after the challenge with a PCV2 genotype “b” isolate. High levels of maternally derived antibodies (IPMA≥10log2) at the time of vaccination were found to interfere with the active seroconversion, whereas titres below 8log2 allowed the development of a proper antibody response. Nevertheless, the immunity induced by one dose of the product was partly protective against PCV2 infection, since viremia, shedding and viral load in tissues were significantly reduced in vaccinated pigs compared to controls. [Copyright &y& Elsevier]

Abstract: The interaction between porcine circovirus type 2 (PCV2) and the pig immune system has been suggested to be a determinant event for the pathogenesis of postweaning multisystemic wasting syndrome (PMWS). To gain insight into the host immune mechanisms developed upon PCV2 infection, early innate and adaptive immune responses were examined in 1-week-old, caesarean-derived, colostrum-deprived piglets using a subclinical infection model of PCV2 in combination with lipopolysaccharide (LPS) as a potential immunostimulation factor. The use of LPS did not show any significant effect on the course of PCV2 infection, nor did in the evolution of the immunological parameters evaluated. Ex vivo responses were detected as early as 1 day post-infection (PI) and consisted of an elevation of the plasmatic levels of interleukin (IL)-8 in PCV2-inoculated pigs followed by an increase on plasmatic IFN-α at day 5 PI. Regarding IL-10, only one PCV2-inoculated pig was positive (day 7 PI); this pig was the only one in which viremia persisted until the end of the study. In vitro cytokine determination showed that, regardless of the treatment administrated to the pigs, an IL-10 release was observed when peripheral blood mononuclear cells (PBMC) cultures were stimulated with PCV2. Seroconvertion to PCV2 measured by an immunoperoxidase monolayer assay (IPMA) occurred between 7 and 14 days PI, whereas neutralizing antibodies (NA) did not appear until day 29 PI. PCV2 DNA was first detected in serum at day 7 PI, reaching the peak of viremia between days 14 and 21 PI, followed by a drop in viral load that was found coincident with the appearance of PCV2-specific IFN-γ-secreting cells (PCV2-IFN-γ-SC) and NA. Results from the present work suggest that viral clearance might be mediated by the development of PCV2-IFN-γ-SC in contribution to the PCV2-specific NA. [Copyright &y& Elsevier]

Abstract: Data from a cross-sectional study of 113 British pig herds carried out in 2004 were used to investigate the associations between postweaning multisystemic wasting (PMWS) in pigs and herds and porcine circovirus 2 (PCV2) antigen score and antibody titre, and associated histological signs in lymph nodes. The sensitivity and specificity of published herd definitions for PMWS were tested on the study farms to consider the role of PCV2 in PMWS. Herds were defined as PMWS-affected, -unaffected or -recovered based on current and past postweaning mortality (PWM), grower pigs with clinical signs of rapid wasting, hairiness and pallor and no other known cause of death on the farm. PCV2 antigen and antibody were not used in the definition of PMWS. In each PMWS-affected herd, up to three sick pigs with the clinical signs above and one healthy pig of a similar age were taken for postmortem examination (PME). In all other herds at least one healthy pig was taken for PME. Lymph nodes were analysed for PCV2 antigen and histological changes, and serum samples were analysed for PCV2 antibody. PCV2 antibody was present in all the herds sampled. There was a non-linear association between PCV2 antigen and antibody. There was no association between the presence of high scores of PCV2 antigen in pigs and the presence of high PWM in herds. PCV2 antigen score was significantly higher in sick than healthy pigs within farms, and high PCV2 score was associated with giant cells, coalescence and absence of germinal centres in lymph nodes. These results did not vary by PMWS-affected, -unaffected or -recovered farms. PCV2 antigen was present at high scores in approximately 10% of healthy pigs on all farms. All three herd definitions of PMWS were highly sensitive, defining PMWS-affected herds as affected, but had a specificity ranging from 23% to 43%. We conclude that the current diagnostic tests for PCV2 indicated higher scores of virus in sick pigs but were not useful to define pigs or herds with PMWS. The ubiquity of PCV2 and the lack of specificity of the PCV2 tests indicate that PCV2 may be a necessary but not sufficient cause of PMWS disease. Linking this with the knowledge that the herd breakdowns occurred in a space time epidemic indicates that another infectious co-factor may be necessary for disease to occur. [Copyright &y& Elsevier]

Abstract: Several real-time PCR assays for quantification of PCV2 DNA (qPCR) have been described in the literature, and different in-house assays are being used by laboratories around the world. A general threshold of 107 copies of PCV2 per millilitre serum for postweaning multisystemic wasting syndrome (PMWS) diagnosis has been suggested. However, neither inter-laboratory nor inter-assay comparisons have been published so far. In the present study, two different qPCR probe assays used routinely in two laboratories were compared on DNA extracted from serum, nasal and rectal swabs. Results showed a significant linear association between the assays (p <0.0001), and a systematic difference of 1.4log10 copies of PCV2 per millilitre of sample (p <0.0001). This difference indicated that the assay from laboratory 1 yielded a higher output than the one from laboratory 2. Results also showed that there was no linear association between the amount of PCV2 DNA and the amount of total DNA, neither in nasal (p =0.86) nor in rectal (p =0.78) swabs, suggesting that normalizing of PCV2 DNA load in swab samples to total DNA concentration is not suitable. The present exploratory study highlights the need for the performance of ring trials on qPCV2 protocols between laboratories. Meanwhile, the proposed thresholds for PMWS diagnosis should only be considered reliable for each particular laboratory and each particular assay. [Copyright &y& Elsevier]

Abstract: The main aim of the present study was to describe new methods for the identification of antibodies against the PCV2 capsid (Cap) and replicase (Rep) proteins in pig sera. Specifically, two new indirect enzyme-linked immunosorbent assays (ELISA) were developed based on recombinant PCV2 Cap (rCap) and Rep/Rep′ (rRep) proteins expressed in baculovirus and produced in Trichoplusia ni insect larvae. Both assays were validated by testing serum samples in a longitudinal study of 107 animals with different clinico-pathological features of PCV2 infection: pigs with postweaning multisystemic wasting syndrome (PMWS), wasted pigs without a diagnosis of PMWS and healthy animals. Longitudinal antibody profiles indicated that healthy animals had significantly higher anti-Cap and anti-Rep antibody levels than the rest of the animal groups at 11 weeks of age. Moreover, PMWS affected pigs could be distinguished from the rest of the pig groups by their lower anti-Rep antibody levels at 11 weeks of age and at necropsy. The results demonstrate the potential of these two ELISAs for large-scale serological studies. This study represents the first longitudinal study of the induction of anti-Cap and anti-Rep antibodies in farms affected by PMWS, from 1 week of age until the occurrence of disease. [Copyright &y& Elsevier]

Abstract: Porcine circovirus type 2 (PCV2) infections in pigs have diverse clinical presentations and are considered economically important diseases worldwide. However, despite intensive research, the early pathogenesis of PCV2 and the primary target cells for PCV2 infections and replication are still unknown. Rolling-circle amplification (RCA) is an amplification technique for small, circular DNA templates that essentially mimics rolling-circle replication in vitro. In this study, the amplification of PCV2-specific DNAs using randomly primed RCA has been demonstrated. This novel approach has circumvented the normal requirement for conventional virus isolation procedures for the characterization of PCV2 DNAs from clinical samples. In addition, the potential utility of a strand-specific derivative of RCA was further investigated. Specifically, strand-specific RCA for the detection of active virus replication following the amplification of complementary sense PCV2 DNAs, which occur as double-stranded replicative intermediates that are present only during de novo viral DNA replication both in vitro and in vivo has been demonstrated. [Copyright &y& Elsevier]

Abstract: Porcine circovirus type 2 (PCV2) is a single-stranded circular DNA virus infecting domestic pigs worldwide. Interaction of this virus with the immune system apparently modulates the immune response of the host. In the present study, the implication of different components of PCV2 in the modulation of the immune response of the host were investigated by using PCV2 viral-like particles (VLPs) and 16 novel oligodeoxyribonucleotides containing CpG motifs (CpG-ODNs) based on the PCV2 genomic sequence. The role of these viral components was studied by evaluating the cytokine profiles (IFN-α, IFN-γ, IL-10, IL-2 and IL-12) on porcine peripheral mononuclear cell (PBMC) and bone marrow-derived dendritic cell (BMDC) cultures. Also, the effect of PCV2 and its elements were examined in recall antigen (pseudorabies virus, PRV) responses. While PCV2 was a potent inducer of IL-10 by PBMCs, such effect was not observed using CpG-ODNs or VLPs. However, IFN-γ and IL-2 production by recall antigen was repressed in presence of PCV2 and most of the studied CpG-ODNs. VLPs did not have such repressive effect. In BMDC cultures, PCV2 and most of CpG-ODNs were able to inhibit IFN-α secretion induced by PRV. Interestingly, CpG-ODNs with inhibitory effect were located within the PCV2 Rep gene. Additionally, PCV2 virus was a very strong IL-12 inducer in BMDC cultures. Whereas, IFN-α modulation on BMDC after PCV2 VLP treatment was neglectable, PCV2 VLPs were potent IL-12 inducers. Our data shows that PCV2 viral elements can distinctly regulate cytokine production depending on the cell population studied. Thus, the final immune response upon PCV2 infection seems to depend on the fine balance between the regulatory elements present in viral DNA and structural protein within the host immune system. [Copyright &y& Elsevier]

Abstract: Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS). Despite first sequencing studies did not find any association between PCV2 sequences and PMWS occurrence, recent works have suggested the opposite. In the present study, 87 open reading frame 2 (ORF2) sequences obtained from pigs with different clinical conditions and coming from farms with different PMWS status were analyzed. Results further confirmed the existence of two genogroups and the definition of two PCV2 genotypes (1 and 2) is proposed. All sequences included in genotype 1 came from pigs from PMWS affected farms, while all sequences obtained from non-PMWS affected farms corresponded to genotype 2. Moreover, infection of single pigs from PMWS affected farms harbouring both genotypes is described. Present results suggest that PCV2 genotype 1 may potentially be more pathogenic than PCV2 genotype 2. [Copyright &y& Elsevier]

Abstract: Previous studies have described a “litter effect” associated with mortality in postweaning multisystemic wasting syndrome (PMWS) affected farms. The main objective of this study was to evaluate litter mortality in different PMWS affected farms and to characterize it in relation to three variables of the sow: parity, porcine circovirus type 2 (PCV2) infectious status and PCV2 antibody titres. The study was performed in seven farms that experienced PMWS in nurseries and/or fattening areas. Fifteen sows from each farm were randomly selected from the same farrowing batch. Serum samples were analyzed for antibodies to PCV2 and for genomic detection of PCV2. Four piglets from each sow (60 piglets per farm) were selected and ear-tagged at birth. Out of 420 initial piglets, 104 (25%) died. Sixty three of them (60%) were necropsied, and 40 (63%) diagnosed as PMWS based on case definition criteria. Our results show that sow PCV2 viremia was significantly related to piglet mortality since more piglets per litter died from viremic than from non-viremic sows. Additionally, a significantly greater proportion of animals died from sows that had low antibody titres against PCV2 (39% vs. 18% from sows with medium to high antibody titres). The present study, of exploratory nature, confirms previous results and further characterizes the so called “litter effect” by establishing that the sow PCV2 status had a significant effect on litter mortality in PMWS affected farms. [Copyright &y& Elsevier]

Abstract: Lymphoid depletion of various lymphoid organs is one of the major lesions in pigs suffering from postweaning multisystemic wasting syndrome (PMWS). The co-existence of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) in PMWS-affected pigs along with the more severe and wider range of lymphocyte depletion of lymphoid organs in PCV2 and PRRSV dually-inoculated pigs imply that PCV2 and PRRSV may interact in the pathogenesis of PMWS. The mechanism for the development of lymphoid depletion in PMWS-affected pigs remains controversial. The objective of the present study was to evaluate and compare the effects of inoculation of both viruses, singularly or in combination, on swine splenic macrophages (SMs) and co-cultured splenic (SLs) and peripheral blood (PBLs) lymphocytes in vitro. A significant reduction in the survival rate and increase in the apoptotic rate of the co-cultured SLs and PBLs and concurrent increase in the expression levels of Fas ligand (FasL) in SMs and Fas in co-cultured SLs and PBLs were demonstrated in PRRSV alone- and PCV2 and PRRSV dually-inoculated groups with the latter more prominent. The increased FasL was proven capable of inducing Fas/FasL-mediated apoptosis in co-cultured FasL-sensitive Jurkat T cells. The de novo expression and production of FasL in PCV2 and PRRSV dually-inoculated SMs and concurrently increased surface expression of Fas in co-cultured lymphocytes may contribute, at least partially, to lymphoid depletion in PMWS-affected pigs with PCV2 and PRRSV dual infection. [Copyright &y& Elsevier]

Abstract: Monocyte/macrophage lineage cells are major target cells of porcine circovirus type 2 (PCV2). From tissues of field pigs suffering from postweaning multisystemic wasting syndrome (PMWS), both intracytoplasmic and intranuclear PCV2 signals, including antigens and nucleic acid, were easily detected in the monocyte/macrophage lineage cells. However, there was a high incidence of intracytoplasmic PCV2-positive signals, but lack of intranuclear signals and PCV2 replication in these cells in vitro. Concurrent infection with bacteria and activation of immune system are suggested to promote viral replication. In this study, lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate (PMA) were used to stimulate PCV2-inoculated alveolar macrophages (AMs). A decrease in intracytoplasmic but increase in intranuclear PCV2-positive signals, including antigens and nucleic acid, were detected in LPS-treated PCV2-inoculated AMs, but not in PMA-treated cells. Additionally, the replication product corresponding to PCV2 spliced major capsid protein (Cap) mRNA and a significant elevation in PCV2 titer were demonstrated in the LPS-treated PCV2-inoculated AMs. The results imply that Gram-negative bacterial co-infection in PCV2-infected pigs may be an important factor in promoting PCV2 replication and contributing, at least partially, to the full development of PMWS. [Copyright &y& Elsevier]

Abstract: Porcine dermatitis and nephropathy syndrome (PDNS) is broadly discussed as a porcine circovirus type 2 (PCV2)-associated disease, although PCV2, in contrast to postweaning multisystemic wasting syndrome (PMWS), has to date not been proven to be the aetiologic agent. In order to better understand the complex immunopathology of PDNS, the systemic cytokine expression profiles of (i) five pigs suffering from PDNS, (ii) five animals suffering from naturally acquired PMWS and (iii) five controls were investigated at mRNA and protein levels by means of multiplex real-time RT-PCR and flow cytometric intracellular cytokine detection, respectively. IL-1α, IL-6 and IFN-γ mRNA expressions were found to be elevated in PDNS pigs. At the protein level, an increased capacity of peripheral blood mononuclear cells to produce IL-2, IL-4, IL-6, IL-12, TNF-α and IFN-γ was evident. Hematological investigations revealed a hypochromic anemia while basophils and monocytes were relatively and neutrophils absolutely increased in PDNS pigs. PCV2 antibody levels did not differ significantly between PDNS and PMWS affected animals. Taken results together, the cytokine profile of the PDNS affected animals together with hematological data pointed towards a proinflammatory condition supporting a Th1 bias. Cytokine data of PMWS affected animals exhibited only minor non-significant differences when compared to controls, only IL-10 was significantly decreased at the mRNA level. [Copyright &y& Elsevier]

Abstract: An exploratory case–control study was carried out in Spain in 2002/2003, involving 62 pig farms of different production systems to assess risk factors that, in association with PCV2 infection, induce postweaning multisystemic wasting syndrome (PMWS) expression. To achieve this objective two groups of farms selected according to their PMWS status were compared: “cases” (farms with clinical PMWS, n =32) and “controls” (farms without clinical PMWS, n =30). A filled-in questionnaire and 45 blood samples (15 sows, and two groups of 15 pigs of 12 and 20 weeks of age, respectively) were obtained from each farm. Additionally, two to three diseased pigs were necropsied and relevant tissues to diagnose PMWS collected when PMWS was clinically suspected (“case” farms). A statistical analysis to compare “case” versus “control” farms was performed with the variables obtained from the questionnaire (191 variables) and the serologic test results (20 variables). Data were analysed using conditional logistic regression with a nested n:m matched design taking into account the farm size. Three variables were found significant in the final model: two related to vaccination scheme and one to PCV2 seroprevalence in growing pigs. Vaccination of gilts against PRRSV increased the odds of PMWS expression and vaccination of sows against atrophic rhinitis was related to decreased odds of the disease; however, the possibility that those two factors could be spurious effects (due to the small sample size) or confounding variables cannot be ruled out. On the other hand, a higher prevalence of antibodies to PCV2 at 12 weeks of age was observed in pigs from “case” farms than in pigs from “control” farms. This result suggests that an earlier infection with PCV2 might be a risk factor for PMWS expression. [Copyright &y& Elsevier]

Postweaning multisystemic wasting syndrome (PMWS) is an economically important disease in pigs caused by porcine circovirus type 2 (PCV2). Development of this disease is presumably associated with an impairment of the immune system. We, therefore, investigated the systemic expression of relevant cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, TNF-α, IFN-γ) and IL-2Rα at mRNA (semiquantitative RT–PCR) and at protein level (flow cytometric intracellular cytokine detection after short-time stimulation of peripheral blood mononuclear cells) in 10 feeder pigs aged 14 weeks suffering from natural PMWS and in 10 clinically healthy pen-mates. Hematological examination revealed a significant (p<0.001) relative lymphopenia in the diseased animals when compared to reference pigs. IL-1α and IL-10 mRNA levels were notably increased in the affected pigs, whereas IL-2 and IL-2Rα (CD25) mRNA levels tended to be down-regulated. IL-8, TNF-α and IFN-γ mRNA expressions appeared to be slightly increased. Intracellular cytokine levels as measured by flow cytometry revealed an increase of IL-1β, IL-2, and IL-6, whereas IL-12 and TNF-α expressions were not affected. IFN-γ was slightly decreased in the diseased animals. In conclusion, despite the assumption, that the cellular immune response to PMWS as a virus-induced disease should be characterized by either a Th1 driven cytokine profile or a cytokine profile indicative of T cell immunosuppression, our results did not support that hypothesis. Nevertheless, data from intracellular cytokine detection suggest an even increased percentage of the remaining lymphocytes capable to produce IL-2 upon in vitro stimulation, which is in contrast to the slightly diminished IL-2 mRNA levels reflecting the in vivo situation at least at the mRNA level. [Copyright &y& Elsevier]

Postweaning multisystemic wasting syndrome (PMWS) affects nursery and growing pigs, and is characterized by wasting, failure to thrive, pale skin, respiratory distress, diarrhoea and sometimes jaundice. Macroscopic findings are aspecific, but lymphocyte depletion in lymphoid tissues is one of the histological hallmarks [Vet. Q. 24 (2002) 109]. Spontaneous cases of PMWS were studied to evaluate proliferative activity and apoptosis as mechanisms involved in the pathogenesis of cell depletion in lymph nodes. The presence of Porcine Circovirus type 2 (PCV2) genome in the processed material was confirmed by in situ hybridization (ISH). The lymph node pattern of depletion was graded as initial, intermediate or final stage according to histological criteria in 10 superficial inguinal nodes from piglets with PMWS which died spontaneously or were slaughtered by euthanasia. The apoptotic and proliferative fraction were investigated by monoclonal antibody MIB1 immunohistochemistry and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end labeling) methods, respectively, and compared to three normal cases. One-way analysis of variance (ANOVA) comparison between the MIB1 index (number of positive cells per 100 nuclei) in controls and PMWS cases revealed a decrease of proliferation in both lymphoid and medulla-like tissues in the initial group (respectively, P=0.0017 and 0.024) but not in the intermediate (respectively, P=0.25 and 0.88) or final (respectively, P=0.47 and 0.81) cohorts. The apoptotic index (number of apoptosis/apoptotic bodies in 100 cells) revealed a statistically significant decrease only in the initial group (one-way ANOVA P=0.05). The proliferation/apoptosis ratio (MIB1/APO ratio) assessed to determine cell turnover disclosed a significant decrease of cell turnover from initial to final PMWS cases (Spearman’s rank test: P=0.027). Decreased cell proliferation and not increased apoptosis seems to be the most important variable leading to cell depletion in PMWS lymphoid tissues. [Copyright &y& Elsevier]

Porcine circovirus type 2 (PCV2) has been associated with a newly identified and described disease in swine called the postweaning multisystemic wasting syndrome (PMWS). An association of PCV2 with various other clinical conditions in pigs are also increasingly being reported. To date molecular studies to determine the extent of genetic variations of this virus have been limited. To more fully understand the extent of genetic diversity we report the sequencing of 34 PCV2 strains from Eastern Canada originating from a variety of clinical conditions spanning years 1990–2001 along with the phylogenetic analysis of these strains and that of 36 PCV2 sequences published in GenBank. Sequence analysis of the complete genome indicated that the Canadian PCV2 strains analyzed in the present study are closely related to each other but also to other PCV2 strains originating from Western Canada, US, Europe and Asia. Sequence analysis of ORF1 and ORF2 genes of the 34 strains revealed that the extent of nucleotide variation was greater for the ORF2 than for the ORF1. The amino acid sequences alignment of the PCV2 capsid protein identified three major regions of amino acid heterogeneity, two of which correspond with dominant immunoreactive areas. No repeatable amino acid motifs for these two regions could be associated with PCV2 strains identified from cases of PMWS or other clinical conditions. Phylogenetic analysis of all 70 strains revealed one large cluster composed of strains from Europe, Taiwan, China and Canada. This large cluster could be divided in several sub clusters, in two of which Canadian strains were found closely related to strains from Germany. The remaining strains from Canada and the US were spread in small groupings along the phylogenetic tree and an association with geographic origin could not be established. The genomic characterizations performed in this study indicate that PCV2 strains associated with PMWS are scattered throughout the phylogenetic tree often in groupings including PCV2 strains identified from cases other that PMWS such as, porcine reproductive and respiratory syndrome (PRRS), generalized tremors, porcine dermatitis and nephropathy syndrome (PDNS), arthritis, nervous signs, erysipelas and even healthy pigs. [Copyright &y& Elsevier]

Postweaning multisystemic wasting syndrome (PMWS) in swine is causally associated with the newly recognised pathogen, porcine circovirus type 2 (PCV2). In this study, 3-week-old SPF PCV2-seronegative piglets were inoculated intranasally with PCV2. The effect of immunostimulation on the induction of PMWS was investigated by immunisation with keyhole limpet hemocyanin (KLH) emulsified in incomplete Freunds adjuvant. The study was terminated 5 weeks after inoculation. While disease was not observed in the age-matched controls, two out of five non-immunised PCV2-infected piglets died on postinoculation day (PID) 21, and one was euthanized on PID 25 in moribund condition. These animals had appeared lethargic with persistent fever from PID 12 onwards. The euthanized pig appeared smaller than littermates and suffered from jaundice. At postmortem examination, gastric ulceration, icterus, and liver and thymus atrophy were observed. Furthermore, histological lesions of degenerating hepatocytes and hepatitis in combination with lymphoid depletion and syncytial cells in lymph nodes were consistent with the diagnosis of PMWS. One out of five immunostimulated PCV2-infected piglets was euthanized on PID 22 with convulsions after a period with wasting. This pig was lethargic from PID 14 onwards with persistent fever from PID 8 and transient dyspnoea. No differences in clinical signs, gross pathologic or histological findings were observed for the remaining non-immunostimulated and immunostimulated PCV2-infected piglets. All 10 PCV2-inoculated piglets seroconverted to PCV2 within 14 days after inoculation. By virus isolation, quantitative polymerase chain reaction (Q-PCR), and immunostaining of cryostat sections, it was demonstrated that lymphoid tissue contained abundant PCV2 antigen. Viral DNA load in serum samples was assessed by Q-PCR. All four PMWS-affected piglets had high levels of PCV2 DNA in serum, suggesting that there was a correlation between high levels of viral DNA in serum and the development of PMWS. In conclusion, infection with PCV2 caused PMWS in SPF piglets, however, the immunostimulation did not seem to play a critical role. [Copyright &y& Elsevier]

Abstract: The discovery of porcine circovirus type 2 (PCV2) in 1998 initiated intensive research on arguably the most economically important pathogen facing the global swine industry today. PCV2 infection is now widespread worldwide, and increasing numbers of disease conditions have been linked to PCV2 infection in pigs. In this special issue of Virus Research, leading experts in the field review the history, epidemiology, transmission, clinical and pathological features, immunology, pathogenesis, molecular biology and vaccine development of PCV2 and porcine circovirus associated disease (PCVAD). In addition, circovirus-like DNA sequences recently identified from humans and other animal species and their biological significances are also reviewed. The articles in this special issue identify gaps in current PCV2 research and offer insights for the direction of future research. [Copyright &y& Elsevier]

Objective: The objective of the present study was to investigate whether a combined or concurrent application of two vaccines against Mycoplasma hyopneumoniae (M. hyo.) and porcine circovirus type 2 (PCV2) in suckling piglets can be as effective as the single use of both products., Material and Methods: A total of 598 piglets were allocated to five groups. In the 1st and 3rd weeks of life the placebo Porcilis® Diluvac forte and the two vaccines Porcilis® M HYO ("M HYO") and Porcilis® PCV ("PCV") were administered according to the following scheme: group A: placebo/PCV; group B: M HYO/M HYO; group C: placebo/placebo; group D: M HYO/M HYO + PCV (combined single dose); group E: placebo/M HYO + PCV (different injection sites). Lung lesions due to M. hyo. infection were recorded at slaughter, and average daily weight gain, morbidity, mortality, serum PCV2 load and specific humoral immune responses were compared between the groups. Local and systemic side effects were recorded., Results: Sporadic impairment of the herd health status due to piglet diarrhoea (n = 111) from the 1st to 3rd weeks of life were not associated with M. hyo. or PCV2. A tendency towards a higher average daily weight gain was found in vaccinated pigs compared to the control group. Slight differences between groups in terms of lung lesions, morbidity and mortality were not significant. M. hyo. and PCV2 antibody-titers were significantly higher in vaccinated than in non-vaccinated pigs. One pig from both group A (PCV2) and group C (placebo) displayed local reactions at the vaccination site., Conclusion and Clinical Relevance: A positive effect on animal health can be achieved by vaccination against M. hyo. and PCV2 in herds with suboptimal health status. A simultaneous vaccination either by a combined or concurrent application has no negative effect on health status. Simultaneous vaccination yielded the same positive effect on average daily weight gain as single vaccinations. Therefore, a simultaneous vaccination against M. hyo. and PCV2, which reduces workload and is beneficial for animal welfare, can be recommended.