Development and evaluation of an indirect in situ polymerase chain reaction for the detection of porcine circovirus type 2 in formalin-fixed and paraffin-embedded tissue specimens

Taking advantage of the high sensitivity of polymerase chain reaction (PCR) and the cell-localizing ability of in situ hybridization (ISH), an indirect in situ PCR (ISPCR) method was developed for detecting the distribution of porcine circovirus type 2 (PCV2) in formalin-fixed and paraffin-embedded inguinal lymph nodes obtained from clinically healthy PCV2-carrier pigs and postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Comparisons of the relative sensitivity of indirect ISPCR with other routinely used diagnostic methods for PCV2 indicated that nested PCR was the most sensitive method followed by indirect ISPCR, conventional PCR, ISH, and immunohistochemical (IHC) staining. Although indirect ISPCR, ISH, and IHC staining all revealed a similar signal distribution pattern of PCV2, using indirect ISPCR allowed specific amplification and detection of previously uneasily detected PCV2 signal than by routine ISH or IHC staining, particularly in those cells within the germinal center in clinically healthy PCV2-carrier pigs. Furthermore, six different PCV2 signal expression patterns in conjunction with the correlated lymphoid lesion stages were classified to describe the tissue morphological changes and viral infection. The result indicates that indirect ISPCR is a more effective, cell-based diagnostic tool with good specificity to detect limited PCV2 infection in formalin-fixed and paraffin-embedded tissue specimens and it would be a useful tool for further exploring the pathogenesis of PCV2 infection.     

Evaluation of commercial polyclonal- and monoclonal-antibody-based immunohistochemical tests for 2 genotypes of Porcine circovirus type 2 and comparison with in-situ hybridization assays

The objective of the present study was to evaluate polyclonal- and monoclonal-antibody-based immunohistochemical (IHC) tests for the detection of 2 genotypes of Porcine circovirus type 2 (PCV2), a and b, in formalin-fixed, paraffin-embedded lymph-node tissue from pigs with experimental or natural postweaning multisystemic wasting syndrome and to compare the IHC results with those of in-situ hybridization (ISH) assays. The ISH assays proved more sensitive than the IHC tests for the detection of PCV2a and PCV2b. According to these findings, polyclonal-antibody-based IHC testing is the most practical routine diagnostic method for the detection of PCV2 regardless of genotype because IHC testing is less technically complex than ISH testing. However, ISH assays are useful to differentiate between PCV2a and PCV2b in surveillance programs for the monitoring of PCV2 in swine herds.     

A novel method for the detection of porcine circovirus type 2 replicative double stranded viral DNA and nonreplicative single stranded viral DNA in tissue sections.

Porcine circovirus type 2 (PCV2), an economically important pathogen of swine, is the necessary cause of post weaning multisystemic wasting disease (PMWS); PCV2 infection is associated with porcine dermatitis and nephritis syndrome (PDNS). Current immunohistochemical (IHC) methodologies identify PCV2 antigens but are not capable of differentiating replicating virus from nonreplicating virion particles in tissue sections. In this paper, a combination of IHC using commercial monoclonal antibodies specific for single stranded (ss) and double stranded (ds) DNA and PCV2 specific in situ hybridization (ISH) was used to show the specificity of the former for PCV2 DNA in tissue sections from PCV2-infected gnotobiotic pigs. Cold-ethanol-fixed tissue sections were superior to formalin-fixed tissues for detection of PCV2 DNA, presumably due to the lack of protein cross-linking in the latter. These data demonstrate that conventional IHC detects PCV2 DNA forms in experimentally infected PCV2-positive gnotobiotic porcine tissue sections that are minimally compromised by either formalin fixation or the hybridization conditions needed for ISH.     

猪圆环病毒2型原位杂交检测方法的建立

参照GenBank发表的PCV2ORFl基因序列设计了1对引物,利用PCR地高辛探针合成的方法制备了长度为494bp的特异性探针,经检验具有良好的特异性和敏感性,可检测最低质粒DNA质量浓度为0．9728ug／L。用该探针建立了原位杂交组织切片检测方法,并用来检测PCV2感染猪的扁桃体和淋巴结组织,结果表明阳性信号主要存在于巨噬细胞胞浆中,信号强、背景良好,阴性对照无显色,说明该方法可作为PCV2实验室诊断和机理研究的一种有效检测方法。     

Immunopathological characterization of porcine circovirus type 2 infection-associated follicular changes in inguinal lymph nodes using high-throughput tissue microarray

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.     

Singular PCV2a or PCV2b infection results in apoptosis of hepatocytes in clinically affected gnotobiotic pigs

Porcine circovirus type 2 (PCV2) is clinically associated with respiratory disease, failure-to-thrive, hepatitis, and diarrhea; however, the precise pathogenesis of PCV2-associated disease and in particular its involvement in apoptosis is still controversial. The objectives of this study were (1) to determine whether PCV2 is associated with apoptosis by examining and comparing hepatic tissues from clinically affected or unaffected gnotobiotic pigs that were experimentally infected with PCV2, (2) to determine if there are differences between PCV2a and PCV2b in inducing hepatocyte apoptosis, and (3) to determine if there are differences between apoptosis detection systems. Forty-eight gnotobiotic pigs were separated into five groups based on inoculation status and development of clinical disease: (1) sham-inoculated, clinically-unaffected (n=4), (2) inoculated with PCV2a, clinically-unaffected (n=10), (3) inoculated with PCV2a, clinically-affected (n=6), (4) inoculated with PCV2b, clinically-unaffected, (n=13) and (5) inoculated with PCV2b, clinically-affected (n=15). Formalin-fixed, paraffin-embedded sections of liver from all pigs were analyzed for signs of apoptosis [presence of single strand DNA breaks in the nucleus by the terminal transferase dUTP nick end labeling (TUNEL) assay or presence of intra-nuclear cleaved caspase 3 (CCasp3) demonstrated by CCasp3 immunohistochemistry (IHC)]. In addition, the liver tissues were also tested for presence of cytoplasmic and intra-nuclear PCV2 antigen by an IHC assay. Specific CCasp3 and TUNEL labeling was detected in the nucleus of hepatocytes in PCV2a and PCV2b infected pigs with significantly (P<0.05) higher levels of apoptotic cells in clinically-affected pigs. Regardless of PCV2 subtype (PCV2a; PCV2b), there were higher levels of PCV2 antigen in clinically-affected pigs compared to clinically-unaffected pigs. There was no significant difference in detection rate of apoptotic cells between the TUNEL assay and CCasp3 IHC. When high amounts of PCV2 antigen were present, the incidence of CCasp3 and TUNEL staining also increased regardless of the PCV2 genotype. This suggests that PCV2-induced apoptosis of hepatocytes is important in the pathogenesis of PCV2-associated lesions and disease.     

Utilization of a rate enhancement hybridization buffer system for rapid in situ hybridization for the detection of porcine circovirus in cell culture and in tissues of pigs with postweaning multisystemic wasting syndrome.

A rapid in situ hybridization (ISH) technique for the detection of porcine circovirus (PCV) nucleic acid in cell culture and formalin-fixed paraffin-embedded tissues was developed. A fluorescein-labeled RNA probe was transcribed from a plasmid containing 530 bp of the ORF1 of a PCV isolated from a pig with postweaning multisystemic wasting syndrome (PMWS). Hybridization using standard hybridization buffer was performed at 42 C for 16 hours and was compared to hybridization using rate enhancement hybridization (REH) buffer at 67 C for 2 hours. Hybridization was detected with an alkaline phosphatase-conjugated antifluorescein antibody. In both cultured cells and tissues from pigs with PMWS, the signal intensity and number of labeled cells in sections hybridized with REH buffer were equal to those of sections hybridized with standard hybridization buffer. The total time required for ISH using the REH buffer is 7-8 hours, thus making this protocol suitable for application in routine PCV diagnosis.     

猪圆环病毒检测技术研究进展

猪圆环病毒病是由猪圆环病毒(PCV)感染引起的,以免疫抑制为特征的一类病毒性传染病,临床表现主要是由PCV-2引起的仔猪断奶后多系统衰竭综合征。文章就近几年国内外对该病毒检测技术,包括病毒分离、电镜观察、聚合酶链式反应、间接免疫荧光试验、免疫组织化学技术、酶联免疫吸附试验、原位核酸杂交试验等的研究进展做了阐述。     

Detection of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, swine influenza virus and Aujeszky's disease virus in cases of porcine proliferative and necrotizing pneumonia (PNP) in Spain

Proliferative and necrotizing pneumonia (PNP) is a severe form of interstitial pneumonia characterised by hypertrophy and proliferation of pneumocytes type 2 and presence of necrotic cells within alveoli lumen. Many viral agents have been linked to PNP aetiology, with especial emphasis on porcine reproductive and respiratory syndrome virus (PRRSV). To gain knowledge on PNP causality, a retrospective study on 74 PNP cases from postweaning pigs from Spain was carried out. Coupled with histopathological examinations, the presence of porcine circovirus type 2 (PCV2) by in situ hybridization (ISH), and PRRSV, swine influenza virus (SIV) and Aujeszky's disease virus (ADV) by immunohistochemical (IHC) methods, were investigated. PCV2 was the most prevalent viral agent in PNP cases (85.1%) followed by PRRSV (44.6%); 39.1% of PNP cases showed PCV2 as the solely detected agent, while only 4.1% had PRRSV as the unique pathogen. SIV and ADV were very sporadically detected in PNP cases, and always in co-infection with PCV2. Therefore, present data indicate that PCV2 is the most important aetiological agent in PNP cases from Spain and that PRRSV is not essential for the development of PNP. Taking into account the presented results and available literature, we suggest that PCV2 is possibly the main contributor to PNP cases in Europe while PRRSV could play a similar role in North America.     

猪圆环病毒2型原位杂交检测技术的建立与应用

参照GenBank发表的猪圆环病毒2型（PCV2）ORF2基因序列设计引物，利用PCR扩增得到PCV2BF株341bp的核酸片段，用随机引物法制备出地高辛标记的核酸探针。制备的探针与PCV1、PRRSV、PPV、PRV等不发生反应，可检测的最低PCV2DNA含量为1．78Pg。对30份临床组织样本进行了检测，并与PCR比较，结果表明，阴性符合率为100％，阳性符合率为88．9％。应用原位杂交技术分析了PCV2在人工感染仔猪主要组织中的分布，结果表明，感染后3d，从仔猪的淋巴结、胸腺、肺脏、脾脏、鼻黏膜可检测到阳性信号，感染后21d，肝脏、肾脏、胰腺和回肠可检出阳性信号，至感染后42d，可从心脏、胃、脑检出阳性信号。在整个试验过程中会厌软骨、膀胱、皮肤、肌肉等组织均为阴性。本研究结果表明，建立的PCV2原位杂交技术具有良好的敏感性和特异性，可用于PCV2的实验室诊断和感染靶细胞的定位分析。     

猪圆环病毒检测技术研究进展

猪圆环病毒是近年来新发现的畜禽病毒之一,疑为一种潜在人兽共患病病原,具有重要的经济意义。文章就其检测技术,如病毒分离、电镜观察、聚合酶链式反应(PCR)、多重PCR、多重套式PCR、定量竞争性PCR、PCR 限制性片段长度多态性分析(PCR RFLP)、免疫组织化学技术(IHC)、原位杂交技术(ISH)等的研究进展做一阐述。     

Development of probes to differentiate porcine circovirus types 1 and 2 in vitro by in situ hybridization

Porcine circovirus type 1 (PCV1), a PK-15 cell line contaminant, and porcine circovirus type 2 (PCV2), associated with post-weaning multisystemic wasting syndrome (PMWS), are genetically and antigenically related. Several techniques have been developed to detect PCV, including in situ hybridization (ISH). Previously reported probes used for ISH may hybridize with both PCV1 and PCV2 nucleic acids. We attempted to produce probes for ISH that can detect and differentiate PCV2 from PCV1 in PCV-infected cells. Riboprobes were synthesized from the sense and antisense strands of both open reading frames 1 and 2 (ORF1 and ORF2) of PCV2. At 42 and 58 degrees C, the ORF1 antisense probe hybridized with nucleic acid from both PCV1- and PCV2-infected cells. At 58 degrees C, the ORF2 antisense probe hybridized with PCV2 nucleic acid but not with PCV1 nucleic acid. The ORF1 and ORF2 sense probes bound only with PCV2 nucleic acid. Both antisense strand probes produced stronger signals than the sense strand probes. The results showed that the PCV2 ORF1 antisense probe is the most likely probe to detect both PCV types while the ORF2 antisense probe is capable of discriminating between PCV1 and 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.     

Quantification of porcine circovirus type 2 (PCV2) DNA in serum and tonsillar, nasal, tracheo-bronchial, urinary and faecal swabs of pigs with and without postweaning multisystemic wasting syndrome (PMWS)

The present study focused on PCV2 quantification by TaqMan PCR in nasal (n=99), tonsillar (n=108), tracheo-bronchial (n=72), urinary (n=91) and faecal (n=42) swabs, as well as in serum (n=57), from a total of 146 pigs received at the Pathological Diagnostic Service at the Veterinary School of Barcelona (Spain). Animals were classified into three categories based on histopathological and in situ hybridisation (ISH) results: PMWS affected pigs (Group A, n=42), PCV2 subclinically infected pigs (Group B, n=29), and non-PMWS with PCV2 ISH negative pigs (Group C, n=75). Overall, tracheo-bronchial swabs had the higher PCV2 load followed by serum, tonsillar, nasal, faecal and, finally, urinary swabs. PCV2 genome was also detected in different proportions in all three categories of pigs; in all tested sites, viral load means were significantly higher (P0.05) were observed among tested specimens when age-groups (pigs younger than 1.5 months, and equal or older than 1.5 months of age) were compared. In summary, PCV2 is presumably excreted through respiratory (nasal and tracheo-bronchial) and oral (tonsillar) secretions, urine and faeces of both PMWS and non-PMWS affected pigs, with higher viral loads being associated with the presence of PMWS lesions.     

Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome.

Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Recently, the disease has been reproduced with inocula containing a newly described porcine circovirus (PCV), designated PCV 2, and porcine parvovirus (PPV). In order to determine if these viruses interact in naturally acquired PMWS, affected tissues from field cases were examined by immunohistochemistry (IHC) and polymerase chain reaction (PCR) for PCV 2 and PPV, as well as by PCR for the other recognized porcine circovirus, PCV 1. Porcine circovirus 2 was detected by PCR or IHC in affected fixed or frozen tissues from 69 of 69 cases of PMWS collected over 3 years from 25 farms. Porcine parvovirus was detected in 12 of the same cases, and PCV 1 was detected in 9 of 69; however, an apparent decrease was found in the sensitivity of the PCRs used to detect the latter 2 viruses when fixed tissue from the same cases were compared with the use of frozen tissues. Porcine circovirus 2 was not detected by PCR in affected tissues from 16 age-matched pigs that had Streptococcus suis-associated disease. Electron microscopic examination of plasma pooled from 15 pigs with PMWS revealed the presence of PCV and PPV, whereas these viruses were not observed in pooled plasma from 5 age-matched clinically normal pigs. These results confirm and extend previous findings documenting a consistent association of PCV 2 with PMWS. As well, infection by PPV or PCV 1 or both may be an important cofactor in the pathogenesis of some, but apparently not all, cases of PMWS.     

Characterization of interstitial nephritis in pigs with naturally occurring postweaning multisystemic wasting syndrome

Kidney samples with interstitial nephritis from 26 pigs affected by postweaning multisystemic wasting syndrome (PMWS) were selected. A histologic evaluation was carried out to describe the type of inflammation and its relationship with viral load, as assessed by in situ hybridization (ISH). Of 26 cases, 10 revealed a tubulointerstitial, lymphoplasmacytic nephritis, 11 an interstitial granulomatous nephritis, and 5 both types of inflammation (mixed type). In 4 cases of granulomatous inflammation, the pattern was not classically nodular, and a population of macrophages and lymphocytes was present (interstitial lymphohistiocytic nephritis). ISH confirmed the presence of porcine circovirus type 2 (PCV2) nucleic acid in all cases. The epithelium of the renal tubules was the most constantly ISH-positive structure. In tubulointerstitial nephritis, the higher the number of positive inflammatory cells, the more severe the inflammation. The ISH reaction was more heterogeneous and unpredictable in granulomatous nephritis, with some epithelioid and giant cells positive by ISH. To quantify macrophages distributed in the three patterns of nephritis, immunohistochemical methods using anti-major histocompatibility complex II (anti-MHC-II) and anti-lysozyme antibodies were undertaken, and semiquantitative evaluation was carried out. MHC-II was mainly expressed by lymphocytes in tubulointerstitial nephritis, but did not always stain macrophages in cases of granulomatous (including lymphohistiocytic) nephritis; the anti-lysozyme antibody revealed macrophages when present in tissues. The amount of PCV2 nucleic acid was not apparently associated with the pattern of inflammation (tubulointerstitial or granulomatous). PCV2 load seems to reflect the severity of the lymphoplasmacytic inflammation but not that of granulomatous and lympho histiocytic types.     

Porcine circovirus type 2 in muscle and bone marrow is infectious and transmissible to naïve pigs by oral consumption

Pork products are a possible source of introduction of PCV2 isolates into a pig population. However, limited work has been done on the transmission through meat of porcine circovirus type 2 (PCV2), a virus associated with several disease syndromes in pigs. The objectives of this study were to determine if pork products from PCV2-infected pigs contain PCV2 DNA/antigen and to determine if the PCV2 present in the tissues is infectious by performing in vitro and in vivo studies. Skeletal muscle, bone marrow, and lymphoid tissues from pigs experimentally inoculated with PCV2 were collected 14 days post-inoculation (DPI). The tissues were tested for presence of PCV2 DNA by quantitative real-time PCR, for PCV2 antigen by immunohistochemistry (IHC), and for presence of infectious PCV2 by virus isolation and inoculation of PCV2 naïve pigs. Lymphoid tissues contained the highest amount of PCV2 (positive by PCR, IHC, and virus isolation), bone marrow contained a lower amount of PCV2 (positive by PCR and IHC but negative by virus isolation), and skeletal muscle contained the lowest amount of PCV2 (positive by PCR but negative by IHC and virus isolation). Naïve pigs fed for three consecutive days with either skeletal muscle, bone marrow, or lymphoid tissues all became PCV2 viremic as determined by quantitative real-time PCR on serum starting at 7 DPI. The pigs also seroconverted to PCV2 as determined by PCV2 IgM and IgG ELISA. In addition, PCV2 antigen was detected by IHC stains in lymphoid tissues and intestines collected from the majority of these pigs. Results from this study indicate that uncooked PCV2 DNA positive lymphoid tissues, bone marrow, and skeletal muscle from PCV2 viremic pigs contain sufficient amount of infectious PCV2 to infect naïve pigs by the oral route.     

Experimental infection of 3-week-old conventional colostrum-fed pigs with porcine circovirus type 2 and porcine parvovirus

This report describes an experimental infection with porcine circovirus type 2 (PCV2) in combination with porcine parvovirus (PPV) in 3-week-old conventional colostrum-fed pigs with maternal antibodies to both viruses. Two groups of four pigs each were inoculated with PCV2 and PPV. One of the groups received also a commercial inactivated vaccine against porcine pleuropneumonia to evaluate possible effects of the stimulation of the immune system of pigs on the infection. Another group of four pigs was kept as uninfected control. Clinical signs, rectal temperatures and body weights were recorded. Serum antibody titers to PCV2 and PPV were determined at weekly intervals. Pigs were killed 42 days after inoculation and tissue samples were examined for the presence of gross and microscopic lesions. Tissues were also analyzed for the presence of PCV2 and PPV DNA by PCR, and for the presence of PCV2 antigen by immunohistochemistry (IHC). All the pigs had serum antibodies to PCV2 and PPV at the beginning of the trial. None of them developed clinical symptoms or pathological lesions typical of post-weaning multisystemic wasting syndrome (PMWS), a disease associated to PCV2 infection. However, IHC and/or PCR analyses showed that clinically silent PCV2 infection developed in five of the eight inoculated pigs, regardless of the administration of the vaccine. In particular, PCV2 DNA and/or antigen were detected in most of the tissues examined in the two pigs with the lowest titer of maternal PCV2 antibodies at the beginning of the trial. PPV DNA was not detected in any of the samples examined. The five pigs with PCR and/or IHC evidence of PCV2 infection had a mean weight gain during the experiment lower than that of the inoculated PCR-negative pigs considered together and that of the control pigs. In conclusion, it would appear that passive immunity against PCV2 can play a role in preventing the development of PMWS, but is not able to prevent the establishing of clinically silent PCV2 infections. The dissemination and persistence of the virus in the tissues may depend on the level of PCV2 antibodies at the time of inoculation.