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.     

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.     

A comparison of immunohistochemistry and in situ hybridization for the detection of porcine circovirus type 2 in pigs

The aim of this study was to develop and to optimize an immunohistochemistry (IHC) method for PCV2 identification and to compare it with an in situ hybridization (ISH) technique. The results demonstrated that both ISH and IHC successfully detected PCV2 viral antigens or nucleic acid in the examined tissues. Most of the slides identified previously in ISH as PCV2-positive were also positive in IHC. In the case of nearly half of the slides the results of IHC examination revealed an increase in the intensity of staining. IHC presented higher sensitivity and specificity than ISH. No negative impact of the time of paraffin block storage on ISH detection results was observed. In addition, IHC results were easier to interpret due to better image quality after staining. Overall results confirmed IHC was a reliable and useful technique for PMWS diagnosis.     

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。     

Retrospective study on porcine circovirus type 2 infection in pigs from 1985 to 1997 in Spain

A retrospective survey was performed to detect lesions of Postweaning multisystemic wasting syndrome (PMWS) and nucleic acid of porcine circovirus type 2 (PCV2) in archived formalin-fixed, paraffin-embedded tissues from 189 pigs, and antibodies to this virus in sera of 388 pigs from the Spanish livestock between the years 1985 and 1997. PCV2 nucleic acid was detected by in situ hybridization (ISH) in tissues from 78 of 189 (41.3%) examined pigs. Variable amount of viral genome was detected in association with slight to severe microscopic lymphoid lesions consisting of lymphocyte depletion and histiocytic infiltration. The first positive case of PMWS with typical lesions and ISH positive corresponded to a pig necropsied in 1986. Two hundred and eighty-two of 388 (72.7%) sera were positive by immunoperoxidase monolayer assay. Serological and pathological data of the present study indicate that PCV2 was a enzootic infection in Spain since 1985, suggesting that the introduction of this virus in the livestock occurred previously.     

Double in situ hybridization for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV).

A double in situ hybridization method for the simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV) genomes in the same tissue section was applied to lung tissues from 9 pigs in which PRRSV and PCV coinfection had been previously demonstrated. Paraffin-embedded tissue sections were simultaneously hybridized with a digoxigenin-labeled antisense RNA probe for PRRSV and a fluorescein-labeled antisense RNA probe for PCV, and hybridization was detected with anti-digoxigenin alkaline phosphatase/fast red and anti-fluorescein peroxidase/diaminobenzidine, respectively. PRRSV and PCV genomes were identified in the same pulmonary cell types as reported previously in all 9 pigs. In all pigs, PCV-positive cells outnumbered PRRSV-positive cells. A small proportion of alveolar macrophages contained both PRRSV and PCV genomes.     

A comparison of virus isolation, polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine circovirus 2 and porcine parvovirus in experimentally and naturally coinfected pigs.

Virus isolation, polymerase chain reaction (PCR), immunohistochemistry, and in situ hybridization were compared for the detection of porcine circovirus 2 (PCV2) and porcine parvovirus (PPV) from experimentally and naturally coinfected pigs. All coinfected pigs developed postweaning multisystemic wasting syndrome (PMWS), characterized by sudden onset of depression and anorexia. Microscopically, granulomatous inflammation with intracytoplasmic inclusion bodies was present in lymph node from all coinfected pigs at 32 days postinoculation. Of the 200 tissues from 20 experimentally coinfected pigs evaluated, 99 and 58 tissues were positive for PCV2 and PPV, respectively, by 4 techniques. Virus isolation, PCR, immunohistochemistry, and in situ hybridization identified PCV2 infection in 137, 148, 103, and 129 tissues and PPV infection in 107, 132, 59, and 94 tissues. Of the 200 tissues from 20 naturally coinfected pigs evaluated, 109 and 45 tissues were positive for PCV2 and PPV, respectively, by 4 techniques. Virus isolation, PCR, immunohistochemistry, and in situ hybridization identified PCV2 infection in 144, 155, 113, and 139 tissues and PPV infection in 93, 109, 45, and 82 tissues. Because the characteristic microscopic lesions are important criteria for the diagnosis of clinical PMWS, immunohistochemistry and in situ hybridization for the detection of PCV2 and PPV in formalin-fixed, paraffin-embedded tissues provide confirmation of a histopathological diagnosis of PMWS.     

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。     

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.     

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.     

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.     

In situ hybridization for the detection of feline interleukin 1 alpha mRNA on the paraffin-embedded section using biotin-labeled probes.

In situ hybridization (ISH) technique with a biotin-labeled probe was established for detecting feline interleukin 1 (IL-1) alpha mRNA in necropsied specimens. Homology between human IL-1 alpha cDNA used as a probe and feline IL-1 alpha mRNA was confirmed by means of dot blot hybridization using the biotin-labeled probe. Hence, we tried by this biotinylated probe to detect mRNA of IL-1 alpha in paraffin-embedded sections. The following results were obtained for the routine procedures: 1) coating slides with poly-L-lysine and/or heating at 60 degrees C at least for 6 hours gave an excellent result for the adhesion of the tissue sections, 2) 10 micrograms/ml solution of proteinase K treatment for 30 minutes or 50 to 100 micrograms/ml solution of proteinase K treatment for 10 to 30 minutes at 37 degrees C gave the good results in the detection of ISH signal, 3) suitable denaturation time of probes at 70 to 90 degrees C was 5 to 15 minutes, and 4) effective hybridization was obtained by incubation for 24 hours at 4 degrees C, for 18 to 24 hours at 25 degrees C or for 5 to 24 hours at 37 degrees C.     

Postweaning multisystemic wasting syndrome (PMWS) in pigs in Croatia: detection and characterisation of porcine circovirus type 2 (PCV2)

The objective of this study was to characterise porcine circovirus type 2 (PCV2) from pigs with naturally occurring postweaning multisystemic wasting syndrome (PMWS) in Croatia, and to determine the epizootiological, clinical and pathomorphological features of the disease. During a systematic health monitoring programme conducted in the period from January 2002 to June 2003, PMWS was suspected on eight different pig-producing farms in Croatia. The diagnosis of PMWS met all three key criteria: the presence of compatible clinical signs, the presence of the characteristic microscopic lymphoid lesions, and the detection of PCV2 within the lesions by polymerase chain reaction (PCR) and by in situ hybridisation (ISH). Moreover, PCV2 DNA from swine tissues was extracted and sequenced. The phylogenetic analysis of 4 Croatian PCV2 strains showed close relationship to PCV2 strains isolated in Slovenia, France, the Netherlands, the United Kingdom, China and Hungary. PCV2 was also demonstrated by electron microscopy in the lymph node of an affected animal. This is the first demonstration of PMWS in Croatia based on all scientifically accepted diagnostic criteria.     

Genetic characterisation of Australian strains of porcine circovirus types 1 and 2

OBJECTIVE: As post-weaning multi-systemic wasting syndrome (PMWS) has not been identified within Australia, to determine if the absence of disease was associated with genetic differences between the strains of porcine circovirus (PCV) present in Australia and those from countries in association with PMWS. DESIGN: Pig tissues were obtained from weaned pigs found dead or presenting with clinical signs of illthrift and also from neonatal pigs with congenital tremors and used as a source of virus DNA for sequence analysis. PROCEDURE: DNA was extracted from the tissues and PCV detected by polymerase chain reaction (PCR). PCR with PCV type-specific primers was used to amplify the entire genome from selected tissues. The genomes of three strains of PCV1 and seven strains of PCV2 from three Australian states were sequenced and subjected to phylogenetic analysis using standard procedures. RESULTS: The three Australian PCV1 strains had 98 to 99% nucleotide identity to strains in other countries and the seven Australian PCV2 strains had 94 to 99% identity to PCV2 strains in other countries where PMWS has occurred. Six of the seven Australian PCV2 strains were genetically similar to each other, while the seventh was more distantly related. There were no consistent differences in the predicted amino acid sequence of the Australian strains of PCV2 and strains associated with PMWS in other countries. CONCLUSION: There were no consistent differences between Australian strains of PCV and those that have been associated with PMWS in other countries and it appears likely that other factors are responsible for the absence of PMWS in Australia.     

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.     

Apoptosis and proliferative activity in lymph node reaction in postweaning multisystemic wasting syndrome (PMWS)

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.     

Optimal enhancement of in situ hybridization for the detection of porcine circovirus 2 in formalin-fixed,paraffin-wax-embedded tissues using a combined pretreatment of thermocycler and proteinase K

Optimal enhancement of the hybridization signal was developed for the detection of porcine circovirus (PCV) 2 in formalin-fixed, paraffin-wax-embedded tissues from pigs with postweaning multisystemic wasting syndrome. The hybridization signal obtained after thermocycler pretreatment was very uniform across the section, whereas the signal obtained after either proteinase K or microwave pretreatment not only was weaker but was of variable intensity across sections. Thermocycler pretreatment combined with brief proteinase K digestion can enhance signal detection for target viral nucleic acid in formalin-fixed, paraffin-wax-embedded tissues. A strong hybridization signal was detected in the cytoplasm of macrophages and multinucleated giant cells in lymph node and spleen without background staining and morphological damage. The technical improvement results, therefore, in an identical background at the same time as an increased signal and, thus, may help detect lower levels of PCV2 DNA in formalin-fixed, paraffin-wax-embedded tissues.     

Double in situ hybridization for simultaneous detection and differentiation of porcine circovirus 1 and 2 in pigs with postweaning multisystemic wasting syndrome

Double in situ hybridization using a digoxigenin-labelled porcine circovirus 1 (PCV1) and biotinylated PCV2 probe, was developed for the simultaneous detection and differentiation of PCV1 and PCV2 in formalin-fixed, paraffin-embedded tissues from pigs with postweaning multisystemic wasting syndrome. The combination of an alkaline phosphatase conjugated antidigoxigenin system with alkaline phosphatase conjugated streptavidin-biotin system allowed identification of PCV1 and/or PCV2. No evidence of cross-reaction was observed. Positive cells exhibited a red or dark brown reaction product for PCV1 and PCV2, respectively. Both PCV DNAs were observed mainly in the cytoplasm but occasionally in the nucleus. Co-localization of hybridization signal for both PCV1 and PCV2 was present in macrophages and multinucleated giant cells of the lymph node and spleen. This double-labelling technique for the differentiation between PCV1 and PCV2 is suitable for pathogenesis studies and diagnostic applications.     

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.