Detection of porcine circovirus type 2 and its association with PMWS in wild boars and domestic pigs in Germany: a histopathological, immunohistochemical and molecular biological study.

Beside domestic pigs wild boars can also be affected by postweaning multisystemic wasting syndrome (PMWS). For the first time a nationwide survey of wild boars (n = 356) and domestic pigs (n = 340) was carried out in Germany by histopathology, immunohistochemistry (IHC) and quantitative PCR (qPCR). Whereas 102/340 domestic pigs were immunoreactive for PCV2 antigen in at least one examined tissue, only 8/356 wild boars reacted positively. Similar findings could be found in qPCR: all domestic pigs showed viral DNA in at least one tissue, while in the examined tissues of 170 wild boars PCV2-DNA was not detectable. The specimens were examined histologically for histiocytosis and depletion of lymphocytes, both typical for PMWS. Based on these findings, six wild boars and 69 domestic pigs were assumed to be affected by PMWS.

Development and evaluation of single-tube nested PCR (STNPCR) for the detection of porcine circovirus type 2 (PCV2).

Porcine circovirus 2 (PCV2) is a common virus in pig population and is associated with the postweaning multisystemic wasting disease (PMWS). In this study, it was developed and evaluated the single-tube nested PCR (STNPCR) method for the detection of PCV2 DNA. PCV2 reference controls and swine tissue samples were used, and primers were selected for targeting specific regions of the viral genome. In comparison of the methods, STNPCR was 10 times more sensitive than conventional PCR and showed the same sensitivity to nested PCR (NPCR), but with reduction in the risk of cross-contamination. In clinical application, 55 tissue samples were analysed by conventional PCR and resulted in 67% (37/55) of positive reactions, while the NPCR and STNPCR were able to identify the presence of viral DNA in 100% (55/55) of the samples. The high sensitivity combined with the elimination of cross-contamination makes the STNPCR method suitable for the epidemiological studies of PCV2 and can aid in the diagnosis of PMWS.

Automatic detection and recognition of pig wasting diseases using sound data in audio surveillance systems.

Automatic detection of pig wasting diseases is an important issue in the management of group-housed pigs. Further, respiratory diseases are one of the main causes of mortality among pigs and loss of productivity in intensive pig farming. In this study, we propose an efficient data mining solution for the detection and recognition of pig wasting diseases using sound data in audio surveillance systems. In this method, we extract the Mel Frequency Cepstrum Coefficients (MFCC) from sound data with an automatic pig sound acquisition process, and use a hierarchical two-level structure: the Support Vector Data Description (SVDD) and the Sparse Representation Classifier (SRC) as an early anomaly detector and a respiratory disease classifier, respectively. Our experimental results show that this new method can be used to detect pig wasting diseases both economically (even a cheap microphone can be used) and accurately (94% detection and 91% classification accuracy), either as a standalone solution or to complement known methods to obtain a more accurate solution.

Vero cells expressing porcine circovirus type 2-capsid protein and their diagnostic application.

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS) in swine. Although the incidences of PCV2-related diseases are ubiquitous throughout the world, the serological tools are rather limited, mainly because the virus does not induce any cytopathic effects in cells. The purpose of this study was to develop a rapid, sensitive and easy quantitative immunofluorescence assay (QIFA) using the recombinant PCV2 nucleocapsid protein (NCP) for the detection of PCV2-specific antibodies in pig sera. The recombinant PCV2 NCP was expressed in Vero cells by a lentivirus system. The performance of QIFA using these Vero cells as a diagnostic antigen was compared with currently available C-ELISA and I-ELISA; the relative sensitivity turned out to range from 92.5% up to 99.3%. The relative specificity was 93.3% when compared to C-ELISA as the gold standard. The serological experiment also indicated the inverse relationship between QIFA and the viral load in serum, semen, feces samples from 7 PCV2-positive boars. In addition, the PCV2 sequence detected from bone marrow cells shows 99% of sequence identity with PCV2 genome, confirming the infectivity of PCV2.

PCV2-DNA in formalin-fixed and paraffin embedded lymph nodes of wild boar (Sus scrofa ssp. scrofa): one sampling approach for two laboratory techniques.

Superficial inguinal lymph nodes from 72 wild boars examined in a previous immunohistochemical (IHC) study on porcine circovirus type 2 (PCV2) were selected for a PCV2 polymerase chain reaction (PCR) analysis. Four of these lymph nodes were PCV2-IHC strongly positive with PMWS histological lesions (outcome 1), 6 weak to mild PCV2-IHC positive without PMWS histological lesions (outcome 2) and 62 PCV2-IHC negative. Considering IHC the gold standard for diagnosis, the aims of the study were to evaluate the suitability of the PCV2-DNA extraction from formalin-fixed and paraffin-embedded (FFPE) tissue and the sensitivity and specificity of PCR under two IHC interpretations criteria: (A) the sample was considered positive if the result was outcome 1; (B) the sample was considered positive if the result was outcome 1 or 2. Under (A) criteria, sensitivity and specificity of PCR were 100% and 89.7%, respectively; the Cohen's Kappa coefficient was 0.49. Under (B) criteria, sensitivity and specificity of PCR were 80.0% and 95.2%, respectively; the Cohen's Kappa coefficient was 0.72. The high Cohen's Kappa coefficient under the (B) interpretative criteria indicates good agreement between the two methods. In conclusion, 1) DNA extracted from FFPE specimens of wild boar is suitable for PCR and further represents a screening test for PCV2/PCVD (PCV2 Diseases) investigations in wild boar as well; 2) routine histological sampling can also be useful for PCV2 virological studies in wild boar.

Acute phase proteins as a tool for differential diagnosis of wasting diseases in growing pigs.

The concentrations of haptoglobin (Hp), C-reactive protein (CRP) and serum amyloid A (SAA) were measured in wasted pigs, first to evaluate their usefulness in the diagnosis of infectious, wasting diseases in pigs, and second, to evaluate whether their concentrations can distinguish the lymphoid depletion score in the lymph tissues of wasted affected pigs. Fifty-three wasted pigs and seven specific pathogen free (SPF) pigs were postmortem examined. Gross lesions were evaluated and samples for histopathological, immunohistochemical, molecular biology and microbiological analysis were taken. Thirty-one pigs were diagnosed as postweaning multisystemic wasting syndrome (PMWS) and 22 as porcine respiratory disease complex (PRDC). Lymphoid depletion degree in lymph tissues of PMWS and PRDC affected pigs was determined. Serum Hp was significantly higher in pigs with PRDC in comparison with the PMWS affected pigs. Serum CRP concentration was significantly lower in pigs with PRDC than in PMWS affected pigs (P<0.001). CRP and SAA levels increased with the lymphoid depletion score, presenting statistical differences between pigs with no depletion and pigs with low, moderate or severe lymphoid depletion (P<0.05, P<0.05 and P<0.001 for CRP and P<0.01, P<0.01 and P<0.01 for SAA, respectively). Hp was higher in pigs with no or low depletion compared with the pigs suffering severe lymphoid depletion (P<0.001 and P<0.05, respectively).

Theoretical and experimental approaches to estimate the usefulness of pooled serum samples for the diagnosis of postweaning multisystemic wasting syndrome.

Classical postweaning multisystemic wasting syndrome (PMWS) diagnosis is based on postmortem findings (histopathology plus viral detection in lymphoid tissues). Because one of the major differences between PMWS-affected and nonaffected pigs is Porcine circovirus-2 (PCV-2) load in serum and tissues, real-time quantitative polymerase chain reaction (qPCR) has been suggested as a potential diagnostic technique for the disease. The objective of the present study was to assess the applicability of qPCR to quantify PCV-2 loads in pooled serum samples as an easy-to-use PMWS diagnostic tool at the herd level. The experimental design included two simulation studies with several serum pool sizes from pigs already screened for PMWS (by histopathology and detection of PCV-2 by qPCR). Several qPCR thresholds were defined and validated with experimental pools created in the laboratory. Quantitative PCR on pooled serum samples did not result in a sufficiently reliable alternate method to the classical PMWS diagnosis method based on individual clinical, histopathological, and PCV-2 detection criteria. However, serum pools seemed to be an alternative at a low economic cost for the quantification of PCV-2 loads in suspicious herds. A targeted (including only clinically diseased animals) sampling approach did not give better estimates compared with a random sampling approach.

Specific, simple and rapid detection of porcine circovirus type 2 using the loop-mediated isothermal amplification method.

BACKGROUND: Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS), and porcine dermatitis and nephropathy syndrome (PDNS). It has caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS. RESULTS: A loop-mediated isothermal amplification (LAMP) assay was used to detect PCV2 in this study. Three pairs of primers were specially designed for recognizing eight distinct sequences of the ORF2 gene. This gene lies in the PCV2 virus genome sequence, and encodes the Rep protein that is involved in virus replication. Time and temperature conditions for amplification of PCV2 genes were optimized to be 55 min at 59°C. The analysis of clinical samples indicated that the LAMP method was highly sensitive. The detection limit for PCV2 by the LAMP assay was 10 copies, whereas the limit by conventional PCR was 1000 copies. The assay did not cross-react with PCV1, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, transmissible gastroenteritis of pigs virus or rotavirus. When 110 samples were tested using the established LAMP system, 95 were detected as positive. CONCLUSION: The newly developed LAMP detection method for PCV2 was more specific, sensitive, rapid and simple than before. It complements and extends previous methods for PCV2 detection and provides an alternative approach for detection of PCV2.

Recent advances in the epidemiology, diagnosis and control of diseases caused by porcine circovirus type 2.

Post-weaning multisystemic wasting syndrome (PMWS) emerged as a significant disease affecting pig production in the 1990s although the causal agent, porcine circovirus type 2 (PCV2), and the disease itself, had existed in swine for many years prior to this. The important multifactorial 'triggers' of PMWS include the immune and infection status of the sow, the timing of PCV2 infection, variations in the virulence of PCV2, co-infections, immune modulation as well as host genetics and management factors. In terms of diagnosis, histopathological examination and the detection of PCV2 within lymphoid tissue remains the 'gold standard' as quantitative PCR techniques are currently not specific or sensitive enough. The recent commercial availability of PCV2 vaccines provides an excellent tool for reducing the impact of PMWS and other porcine circovirus-related diseases. This review assesses recent advances in the epidemiology, diagnosis and control of PMWS.

Comparative evaluation of a competitive polymerase chain reaction (PCR) and a SYBR Green-based real-time PCR to quantify Porcine circovirus-2 DNA in swine tissue samples.

Porcine circovirus-2 (PCV-2) is considered the major etiological agent of post-weaning multisystemic wasting syndrome (PMWS) in pigs. The clinical manifestations of the disease are correlated with moderate to high amounts of PCV-2 DNA in biological samples of affected pigs. A threshold of 10(7) DNA copies/ml is suggested as the trigger factor for symptoms. A comparative study was conducted to determine which quantitative method would be more suitable to estimate the PCV-2 DNA load. Two polymerase chain reaction (PCR) assays were developed: a competitive PCR (cPCR) and a SYBR Green-based real-time PCR. The assays were compared for their capacity to detect PCV-2 in DNA samples extracted from liver, lung, spleen, mesenteric lymph nodes, and kidney of PMWS-affected (n = 23) or non-PMWS-affected pigs (n = 9). Both assays could successfully quantify PCV-2 DNA in all tissue samples and were able to detect significant differences between the numbers of PCV-2 DNA copies found in tissues of PMWS-affected and non-PMWS-affected pigs (≥ 10(2.5)). The highest mean viral loads were detected by the SYBR Green real-time PCR, up to 10(7.0 ± 1.5) copies/100 ng of total DNA sample, while the cPCR detected up to 10(4.8 ± 1.5). A mean difference of 10(1.8) was found between the amounts of PCV-2 DNA detected, using the SYBR Green real-time PCR and the cPCR, suggesting that the viral load threshold for PMWS should be determined for each particular assay.

An ELISA based on a truncated soluble ORF2 protein for the detection of PCV2 antibodies in domestic pigs.

Postweaning multisystemic wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA) based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.

Fast diagnosis and quantification for porcine circovirus type 2 (PCV-2) using real-time polymerase chain reaction.

BACKGROUND/PURPOSE: The postweaning multisystemic wasting syndrome, caused by the porcine circovirus type 2 (PCV-2), is a major disease that poses a significant threat to the global swine industry. The purpose of this study was to establish a real-time polymerase chain reaction (PCR) method for the quantification of PCV-2 and to enable the rapid differentiation of porcine circoviruses type 1 and 2 (PCV-1 and PCV-2). Such a method would significantly speed up the process of clinical diagnosis, and could also be used to study the pathogenic mechanisms of diseases associated with PCV-2. METHODS: Multiplex real-time PCR, together with LightCycler PCR data analysis software, was used for the quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2. A 263-bp DNA fragment was amplified from the 3' end of the open reading frame-2 of PCV-2 by nested PCR, and its DNA sequence was verified as having 100% identity with a PCV-2 standard (NCBI accession number: AF055394). The 263-bp DNA fragment was cloned into the pGEM-T easy vector, and the recombinant plasmid was serially diluted and quantified using real-time PCR. A standard curve was then constructed for quantification of the PCV-2 levels in field samples. The differentiation of PCV-1 and PCV-2 was carried out by analyzing the melting temperatures of the genotype-specific PCR products. RESULTS: To quantify the PCV-2 levels in field samples, a standard curve (1 x 10(2) -1 x 10(9) copies/microL) was constructed. PCV-2 concentrations as low as 1 x 10(2) copies/microL could be detected in specimens taken from the lymph nodes or infected tissues in samples of PCV-2-infected pigs. The diagnosis of PCV-1 and PCV-2 infections and the quantification of the viral load in the field samples could be completed within 45 minutes after extracting the viral DNA using a commercial extraction kit. CONCLUSION: This study demonstrate that real-time PCR is a clinically feasible method for the accurate quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2.

Antimicrobial use in Danish pig herds with and without postweaning multisystemic wasting syndrome.

A retrospective cohort study was performed on 130 pig herds in Denmark, to assess the effect of PMWS on the use of antimicrobial drug. The study comprised 65 herds diagnosed with PMWS during 2003-2004, and matched by the veterinary practitioner with 65 herds free from PMWS. Information on antimicrobial use 1 year before and 1 year after the diagnosis was achieved from the National Prescription Medicine Monitoring Database, VetStat, and summarized on quarter within age group and herd. The multiple linear regression analysis comprised antimicrobial use as the outcome variable with (1) quarter relative to diagnosis of PMWS in the positive herd (same date for the negative match), (2) diagnosis of PMWS (same date used for matched PMWS(-) herd), (3) season and (4) temporal trend as fixed effects. Relative to the unaffected herds, the antimicrobial use in the sow units in the PMWS(+) herds was elevated significantly by 35% in the last quarter and 43% in the fourth quarter before positive diagnosis in the herds (p<0.05). In weaner pigs, the antimicrobial use increased significantly two quarters before, and one quarter after the positive diagnosis, by 68%, 91% and 124% respectively. In weaner pigs, effects were seen of herd size and season. The study support that increased morbidity occur for an extended period prior to the diagnosis of PMWS, both in the sow units and the weaner pig units and further indicate that the syndrome cease after the diagnosis, with a decrease in need for antimicrobial treatment.

Possible pathogenic interplay between Chlamydia suis, Chlamydophila abortus and PCV-2 on a pig production farm.

A concurrent outbreak of chlamydial disease in boars, sows and gilts and postweaning multisystemic wasting syndrome (PMWS) in weaned piglets was investigated on a large pig production farm in Estonia. Chlamydia suis DNA was detected in conjunctival swabs from boars, sows and gilts, but also in the faeces of boars and sows. Chlamydophila abortus DNA was found in semen, and in conjunctival swabs from sows; DNA was demonstrated by microarrays. Serum samples from boars were examined using a Chlamydiaceae-specific recombinant ELISA. All 10 serum samples examined were positive (1:960 to 1:3840). Chlamydiosis was characterised by reproductive failure and conjunctivitis. Piglets were not examined for Chlamydiaceae, as eye problems were not observed. Piglets showed wasting, respiratory signs, diarrhoea, enlargement of lymph nodes and increased mortality (10 per cent). Porcine circovirus type 2 (PCV-2) was detected in the lymph nodes of piglets by immunohistochemistry, and PCV-2 antibodies were demonstrated in all 10 serum samples from sows examined using an immunoperoxidase monolayer assay.

Rapid detection of porcine circovirus type 2 using a TaqMan-based real-time PCR.

Porcine circovirus type 2 (PCV2) and the associated disease postweaning multisystemic wasting syndrome (PMWS) have caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS. To establish a sensitive, specific assay for the detection and quantitation of PCV2, we designed and synthesized specific primers and a probe in the open reading frame 2. The assay had a wide dynamic range with excellent linearity and reliable reproducibility, and detected between 102 and 1010 copies of the genomic DNA per reaction. The coefficient of variation for Ct values varied from 0.59% to 1.05% in the same assay and from 1.9% to 4.2% in 10 different assays. The assay did not cross-react with porcine circovirus type 1, porcine reproductive and respiratory, porcine epidemic diarrhea, transmissible gastroenteritis of pigs and rotavirus. The limits of detection and quantitation were 10 and 100 copies, respectively. Using the established real-time PCR system, 39 of the 40 samples we tested were detected as positive.

Heterologous expression of full-length capsid protein of porcine circovirus 2 in Escherichia coli and its potential use for detection of antibodies.

A capsid protein of porcine circovirus 2 (PCV 2) serves as a diagnostic antigen for the detection of PCV 2-associated disease known as a postweaning multisystemic wasting syndrome (PMWS). In this report, a bacterial expression system was developed for the expression and purification of the full-length PCV 2 capsid (Cap) protein from a codon-optimized cap gene. Replacement of rare arginine codons located at the 5' end of the cap reading frame with codons optimal for E. coli was found to overcome the poor expression of the viral protein in the prokaryotic system. The Cap protein was purified to greater than 95% homogeneity by using a single cation-exchange chromatography at a yield of 10 mg per litre of bacterial culture. Despite the failure of the E. coli-expressed Cap protein to self-assemble into virus-like particles (VLPs), the immunization of mice with recombinant Cap yielded antibodies with the same specificity as those raised against native PCV 2 virions. In addition, the antigenic properties of the purified Cap protein were employed in a subunit-based indirect ELISA to monitor the levels of PCV 2 specific antibodies in piglets originating from a herd which was experiencing PCV 2 infection. These results pave the way for a straightforward large-scale production of the recombinant PCV 2 capsid protein and its use as a diagnostic antigen or a PCV 2 subunit vaccine.

Ring tests to evaluate the performance of Porcine circovirus-2 (PCV-2) polymerase chain reaction (PCR) assays used in North American diagnostic laboratories.

Two laboratory studies involving 11 laboratories were undertaken to assess the performance of North American Porcine circovirus-2 (PCV-2) polymerase chain reaction (PCR) assays. Laboratories received identical submissions containing randomly coded positive and negative control samples, and serially diluted PCV-2-spiked samples. In study 1 and 2, respectively, spiked samples contained measured amounts of PCV-2 virus or DNA. All but 1 assay detected DNA in the most concentrated spiked sample. There were no statistical differences in the proportion of positive or negative samples reported by quantitative (n = 7) versus non-quantitative (n = 6) assays. Across both studies, the false positive rate was 17% (4 out of 23), and 17% (2 out of 12) of assays cross-reacted with PCV-1. The most sensitive assay detected PCV-2 DNA levels about 100 000 times lower the least sensitive assay. This study demonstrated that the PCR assays available in North American diagnostic labs vary considerably in their detection limits and quantification.