Modified indirect porcine circovirus (PCV) type 2-based and recombinant capsid protein (ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV.
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Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus (PCV) is a recently reported and economically important disease. Simple and reliable diagnostic methods are needed for detecting antibodies to PCV type 2 (PCV2) for monitoring of PCV infection. Here, we report the development of two modified indirect enzyme-linked immunosorbent assays (ELISAs): a PCV2 ELISA based on cell-culture-propagated PCV2 and an ORF2 ELISA based on recombinant major capsid protein. PCV2 and ORF2 ELISA detected antibodies to PCV2 and the capsid protein, respectively, in sera from pigs experimentally infected with PCV2 as early as 14 and 21 days postinoculation (dpi). The kinetics of the antibody response to PCV2 and the major capsid protein were similar. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs for both assays were less than 30%. To validate the assays, PCV2 and ORF2 ELISAs were performed with 783 serum samples of young and adult pigs collected from different herds in the Midwestern United States and compared with an indirect immunofluorescent assay (IIF). Six out of 60 samples collected from nursery and growing pigs in 1987 were positive by both ELISA and IIF. Compared with IIF, the diagnostic sensitivity, specificity, and accuracy of PCV2 and ORF2 ELISAs were similar (>90%). The tests showed no cross-reactivity with antibodies to porcine parvovirus and porcine reproductive and respiratory syndrome virus. There was good agreement between the two ELISAs and between the ELISAs and IIF. The availability of the two ELISAs should accelerate our understanding of the host immune response to PCV2 and facilitate the development of prevention and control strategies by elucidating the ecology of PCV2 within swine populations. (+info)
Postweaning multisystemic wasting syndrome of pigs in Korea: prevalence, microscopic lesions and coexisting microorganisms.
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A retrospective study was performed on natural cases of postweaning multisystemic wasting syndrome (PMWS), recorded from January 1999 to December 2000, to determine the prevalence, microscopic lesions, and other coexisting pathogens associated with PMWS. PMWS is diagnosed based on three criteria: the presence of clinical signs (retardation of growth), characteristic microscopic lesions (granulomatous inflammation and inclusion body), and the presence of porcine circovirus (PCV)-2 within these lesions. One hundred and thirty three (8.1%) of the 1634 pigs submitted from 1243 pig farms were diagnosed for PMWS. The affected pigs were from 25 to 120 days old, the majority (78 cases, 58.6%) being 60 to 80 days old. PMWS occurred each month during the two-year study period, but the incidence peaked in May (38 cases, 28.6%), followed by April (18 cases, 13.5%) and June (13 cases, 9.8%). The most consistent and characteristic lesions were multifocal, granulomatous inflammation in lymph nodes, liver and spleen, characterized by infiltration of epithelioid macrophages and multinucleated giant cells. The majority of cases (113 cases, 85.0%) was dual infection with other pathogens. The combination of PCV-2 and Hemophilus parasuis (43 cases, 32.3%) was shown to be the most prevalent followed by PCV-2 and porcine reproductive and respiratory syndrome virus (39 cases, 29.3%). The consistent presence of PCV-2, but lower prevalence of other viral and bacterial pathogens in all pigs examined with PMWS, has led to the speculation that PCV-2 is the etiological agent causing PMWS. (+info)
Changes in CD4(+), CD8(+), CD4(+) CD8(+), and immunoglobulin M-positive peripheral blood mononuclear cells of postweaning multisystemic wasting syndrome-affected pigs and age-matched uninfected wasted and healthy pigs correlate with lesions and porcine circovirus type 2 load in lymphoid tissues.
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Forty-one 8- to 12-week-old wasted pigs were selected from several conventional farms with histories of postweaning multisystemic wasting syndrome (PMWS) and classified into two groups according to their porcine circovirus type 2 (PCV2) infection status, as determined by in situ hybridization (ISH). Twenty-four pigs tested positive for PCV2 (PCV2-positive group), while 17 pigs tested negative for PCV2 (PCV2-negative group). In addition, eight uninfected healthy pigs from an experimental farm were used as controls. Heparinized blood samples were taken to obtain peripheral blood mononuclear cells. The CD4(+), CD8(+), CD4(+) CD8(+) (double-positive [DP]), and immunoglobulin M-positive (IgM(+)) cell subsets were analyzed by flow cytometry with appropriate monoclonal antibodies. Histopathological studies were done to evaluate the apparent degrees of lymphocyte depletion in different lymphoid organs (superficial inguinal and mesenteric lymph nodes, Peyer's patches, tonsils, and spleen) and to determine the viral load of the PCV2 genome by using an ISH technique. Animals of the PCV2-positive group showed a significant downshift of the CD8(+) and DP cell subsets compared to the other groups (P < 0.05). Moreover, in PCV2-positive pigs, the amount of PCV2 genome in lymphoid tissues was related to the degree of cell depletion in those tissues (P < 0.05) as well as to the relative decrease in IgM(+) and CD8(+) cells in peripheral blood. These data support the notion that PCV2-positive pigs might have an impaired immune response. (+info)
Rep and Rep' protein of porcine circovirus type 1 bind to the origin of replication in vitro.
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Genome replication of Porcine circovirus type 1 (PCV1) relies upon expression of the full-length protein Rep and a spliced isoform (Rep'), and the presence of a 111-bp genomic fragment comprising the origin of replication. Using an electrophoretic mobility shift assay (EMSA), the capability of both Rep proteins to bind to partial fragments of the origin of replication of PCV1 was investigated in vitro. Both proteins formed complexes with double-stranded DNA origin fragments containing a stem-loop structure with a conserved nonamer and four hexamer repeats (5'-CGGCAG; H1 to H4). Use of truncated EMSA substrates identified minimal binding sites (MBS) for Rep and Rep' protein: The Rep binding site was mapped to the right leg of the stem-loop and the two inner hexamer repeats H1/H2, while binding of Rep' required only the presence of two hexamer repeats. Two differentially retarded complexes were observed with Rep protein, which presumably result from alternative binding to the MBS or to H3/4. (+info)
Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2.
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Postweaning multisystemic wasting syndrome (PMWS) is a disease of nursery and fattening pigs characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates. Porcine circovirus 2 (PCV2) has been demonstrated to be the cause of PMWS. However, other factors are needed for full development of the syndrome, and porcine reproductive and respiratory syndrome virus (PRRSV) infection has been suggested to be one of them. Twenty-four conventional 5-week-old pigs were distributed in four groups: control (n = 5), PRRSV inoculated (n = 5), PCV2 inoculated (n = 7), and PRRSV and PCV2 inoculated (n = 7). The two groups inoculated with PRRSV showed growth retardation. Pigs inoculated with both PRRSV and PCV2 had increased rectal temperature. One of these pigs developed wasting, had severe respiratory distress, and died. The most important microscopic lesion in pigs inoculated with PCV2 was lymphocyte depletion with histiocytic infiltration of the lymphoid organs, more severe and in a wider range of tissues in doubly inoculated pigs. Interstitial pneumonia was observed in the three inoculated groups. PCV2 nucleic acid was found by in situ hybridization in larger amounts and in a wider range of lymphoid tissues in PRRSV- and PCV2-inoculated than in PCV2-inoculated pigs. TaqMan PCR was performed to quantify the PCV2 loads in serum during the experiment. PCV2 loads were higher in doubly inoculated pigs than in pigs inoculated with PCV2 alone. These findings indicate that severe disease can be reproduced in conventional 5-week-old pigs by inoculation of PRRSV and PCV2. Moreover, these results support the hypothesis that PRRSV infection enhances PCV2 replication. (+info)
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.
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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. (+info)
Detection of porcine circovirus types 1 and 2 in serum and tissue samples of pigs with and without postweaning multisystemic wasting syndrome.
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Presence of porcine circovirus type 1 (PCV1) and PCV2 was studied in sera and superficial inguinal lymph nodes from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs by using in situ hybridization and PCR. PCV1 and PCV2 were found in less than 3% and more than 50% of the samples, respectively. The most sensitive technique and site was PCR in superficial inguinal lymph nodes, but in situ hybridization correlated better with presence of characteristic lesions. (+info)
Simultaneous detection of porcine circovirus 2 and porcine parvovirus in naturally and experimentally coinfected pigs by double in situ hybridization.
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A technique for double in situ hybridization to simultaneously detect porcine circovirus 2 (PCV2) and porcine parvovirus (PPV) in the same tissue section was developed and applied to lymph node and spleen from 8 pigs experimentally coinfected with PCV2 and PPV and 20 pigs with naturally occurring postweaning multisystemic wasting syndrome. For double labeling studies, the tissue samples were processed sequentially, first for PPV in situ hybridization using a digoxigenin-labeled probe and then for PCV2 in situ hybridization using a biotinylated probe. Positive cells contained reaction products for PCV2 and PPV, respectively. Both PCV2 DNA and PPV DNA were observed mainly in the cytoplasm but occasionally in the nucleus. With double in situ hybridization, both PCV2 DNA and PPV DNA were simultaneously detected in lymph node and spleen. This double labeling technique for the detection of PCV2 and PPV is suitable both for pathogenesis studies and for diagnostic applications. (+info)