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Synthetic Peptide-derived Antibody-basedImmunohistochemistry for the Detection of PorcineCircovirus 2 in Pigs with Postweaning Multisystemic

Wasting Syndrome

Y. Ha, K. Jung, C. Choi, K.-K. Hwang* and C. Chae

Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul

National University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 141-742 and *Laboratory of Veterinary Public Health

and Laboratory Animal Science, Cheju National University, Ara-Dong, Jeju-city, Jeju-do, Republic of Korea

Summary

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Porcine circovirus 2 (PCV2) was detected consistently in formalin-fixed paraffin wax-embedded lymphnode and spleen from experimentally and naturally infected pigs by synthetic peptide-derived polyclonalantibody-based immunohistochemistry. Synthetic peptides were generated from open reading frame 2 ofPCV2 by solid-phase peptide synthesis, purified by high performance liquid chromatography, and injectedinto rabbits to produce polyclonal antibody. Positive cells had large nuclei with abundant cytoplasm, andresembled macrophages. In serial sections, a similar distribution of PCV2 antigen and DNA was confirmedin virus-infected cells by immunohistochemistry and in-situ hybridization, respectively. The immunohis-tochemical method described was successfully applied to formalin-fixed, paraffin wax-embedded tissuesand should prove helpful in diagnosing postweaning multisystemic wasting syndrome.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: PCV2; pig; PMWS diagnosis; porcine circovirus; postweaning multisystemic wasting syndrome; synthetic peptide; viral infection

Porcine circovirus 2 (PCV2) is now recognized asthe causal agent of postweaning multisystemicwasting syndrome (PMWS) (Chae, 2004). Clini-cally, PMWS is characterized by progressive weightloss, respiratory signs, and jaundice (Choi et al.,2000; Chae, 2004). In contrast to PCV2, PCV1 is apersistent contaminant of the porcine kidney cellline, PK-15, and is considered nonpathogenic(Tischer et al., 1986).

Since clinical signs of PMWS are nonspecific andvariable, the presence of PCV2 DNA or antigen inlymphoid tissues, demonstrated by in-situ hybridiz-ation (ISH) or immunohistochemistry (IHC),respectively (Choi et al., 2000; Kim and Chae,2001b), together with moderate to severe lymphoid

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pondence to: C. Chae.

depletion or granulomatous lymphadenitis, orboth, are used as diagnostic criteria (Chae, 2004).However, the use of in-situ hybridization is limitedbecause this method is more technically complexand expensive than immunohistochemistry. PCV2antigens have been detected in swine tissues bymonoclonal and polyclonal antibody-based immu-nohistochemical procedures (Ellis et al., 1999; Choiet al., 2000). The objective of this study was todevelop synthetic peptide-derived polyclonal anti-body-based immunohistochemistry for the detec-tion of PCV2 in formalin-fixed, paraffin wax-embedded tissues from pigs with PMWS.

Tissue culture-propagated PCV1 (strainSNUVR000462; 2nd passage) and PCV2 (strainSNUVR000463; 2nd passage) were used as thesources of viral inocula. For inoculation, a suspen-sion of PCV1 or PCV2 containing 1.2!105

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Y. Ha et al.202

TCID50/ml was prepared as previously described(Kim et al., 2003). Thirteen colostrum-deprivedconventional piglets aged 1 day were used. All wereseronegative for PCV, porcine parvovirus, andporcine reproductive and respiratory syndromevirus (PRRSV). Five piglets were inoculated intra-nasally with 2 ml of a 1 in 20 dilution of PCV1suspension, five received 2 ml of 1 in 20 dilution ofPCV2 suspension, and three (negative controls)received PCV-free PK-15 cell lysates. The groupswere held in separate isolators and examined atregular intervals. All 13 piglets were subjected toeuthanasia by electrocution at 25 days post-inocu-lation (dpi), at which time they were known to showconsistent and intense labelling for PCV. Samplesof inguinal lymph node and spleen were collectedand fixed in 10% (v/v) phosphate-buffered for-malin for 1–2 days before processing for histo-pathological examination.

Forty archived formalin-fixed, paraffin wax-embedded tissues from pigs in which naturalPMWS had been diagnosed on the basis of clinicalsigns and histopathology were used. Five cases fromeach of the years 1997 to 2004 were selected.Formalin-fixed, paraffin wax-embedded tissuesfrom pigs infected experimentally with PRRSV,porcine epidemic diarrhoea virus (PEDV), ortransmissible gastroenteritis virus (TGEV) infec-tion, or naturally with classical swine fever or swineinfluenza, were used as negative controls.

The synthetic peptide antigen from the openreading frame (ORF) 2 of PCV2 (GenBank, genepeptide accession number AAC35299) was used inthis study. The synthetic peptide antigen, NH2-(C)KATALTYDPYVNYSS-COOH (peptide number132–146), was prepared by solid-phase peptidesynthesis and purified by high performance liquidchromatography (Takara Korea Life Science andBiotechnology, Seoul, Republic of Korea) aspreviously described (Sabirov et al., 1998).

The PCV2 synthetic peptides were dissolved inphosphate-buffered saline (PBS; pH 7.4) andcoupled to keyhole limpet haemocyanin,suspended in PBS at a molar ratio of 20:1, 0.2%glutaraldehyde being used as a coupling reagent asdescribed by Harlow and Lane (1998). Aliquotsequivalent to 100 mg of coupled peptide wereemulsified in Freund’s complete adjuvant andinjected intradermally into 10 female New Zealandrabbits. After 14 days, the immunization wasrepeated with conjugate emulsified in Freund’sincomplete adjuvant. Thereafter, three boosterinjections were performed at monthly intervalsand blood samples were taken from the ear vein7–10 days after the final injection.

Two serial sections (4 mm) of each sample wereplaced on positively charged slides (Superfrost/Plus slides; Erie Scientific Company, Portsmouth,NH, USA), and stored at room temperature. Onesection was processed for IHC and othe other forISH of PCV2. For IHC, sections were dewaxed inxylene and rehydrated in PBS (pH 7.4, 0.01 M) for5 min. De-proteinization was carried out in 0.2 NHCl for 20 min at room temperature. Tissues werethen digested at 37 8C for 20 min in proteinase K(100 mg/ml) (Gibco BRL, Grand Island, NY, USA)in PBS. After rinsing twice with PBS, endogenousalkaline phosphatase was quenched with 20%glacial acetic acid solution for 2 min at 4 8C. Allslides were then incubated with normal goat serumin PBS (0.1 M, pH 7.4) for 30 min at roomtemperature to saturate nonspecific protein-bind-ing sites. Polyclonal rabbit antibody to PCV2 ORF2synthetic peptide was used at a dilution of 1 in 50 inPBS containing Tween 20 0.1%, the antibody beingcoated on the slides and incubated for 1 h at roomtemperature.

After three washes with Tween 20, sections wereflooded and incubated for 1 h at 36 8C withbiotinylated goat anti-rabbit IgG (Dako, Glostrup,Denmark) diluted 1 in 200 in Tween 20. The slideswere then washed with Tween 20 before beingflooded and incubated for 1 h at 36 8C withstreptavidin-alkaline phosphatase conjugate(Roche Molecular Biochemicals, Mannheim,Germany). They were then equilibrated with Tris-buffer (pH 8.2) for 5 min at room temperature.The final reaction was produced by immersing thesections in a solution of red substrate (BoehringerMannheim, Indianapolis, IN, USA) for 10 min atroom temperature. The sections were lightlycounterstained with Mayer’s haematoxylin.

A 570 base pair (bp) DNA fragment from openreading frame (ORF) 1 (GenBank Accession No.Y09921) was used as PCV1 probe. The forwardand reverse primers were 5 0-CTCGGCAGCGTCAGTGAAAA-3 0 (nucleotide positions 800–819), and5 0-AAATTACGGGCCCACTGGCT-3 0 (nucleotidepositions 1350–1369), respectively (Kim et al.,2003). A 547 bp DNA fragment from ORF 2(GenBank Accession No. AF027217) was used as aPCV2 probe. The forward and reverse primers were5 0-CAGTTCGTCACCCTTTCCC-3 0 (nucleotides939 to 957) and 5 0-GGGGGACCAACAAAATCTCT-3 0 (nucleotides 1466 to 1485), respectively (Kimand Chae, 2001).

Polymerase chain reaction (PCR) products werepurified with a 30-kD cut-off membrane filter. Thenucleotide sequences of the purified PCR productswere determined by means of BigDye chemistry

Fig. 1. Lymph node of pig inoculated with the porcine circovirus 2 (PCV2); 25 days post-inoculation. PCV2 antigen (red reaction)detected in the cytoplasm of macrophages. IHC, fast red, haematoxylin counterstain. !400.

Immunohistochemical Diagnosis of PMWS 203

with the ABI Prism Sequencer (Applied Biosystems,Foster City, CA, USA). Sequencing was performedon the purified PCR products before they werelabelled by random priming with digoxigenin-dUTP (Boehringer Mannheim), according to themanufacturer’s instructions. ISH was performed aspreviously described (Kim and Chae, 2001a).

PCV2 antigen was detected by IHC in tissuesfrom all pigs infected experimentally or naturallywith PCV2. Positive cells typically exhibited a redreaction product, mainly in the cytoplasm butoccasionally in the nucleus, without backgroundstaining. Positive cells, which had large nuclei withabundant cytoplasm, resembled macrophages.PCV2 was detected consistently in lymph node(Fig. 1) and spleen.

A hybridization signal for PCV2 was also detectedin tissues from all pigs infected experimentally ornaturally with PCV2. Positive cells typically exhib-ited a black reaction product, mainly in thecytoplasm but occasionally in the nucleus, withoutbackground staining. A strong hybridization signalfor PCV2 was detected in the cytoplasm ofmacrophages and multinucleated giant cells inlymph node and spleen. Serial sections examinedby IHC and ISH confirmed a similar distribution ofPCV2 antigen and DNA, respectively, in virus-infected cells.

Sections from pigs experimentally or naturallyinfected with viruses other than PCV2 gave negativeresults by IHC and ISH. No hybridization signalsfor PCV2 were seen in sections subjected to priortreatment with a solution of DNase A.

This study demonstrated that PCV2 antigencould consistently be detected by synthetic

peptide-derived polyclonal antibodies in cells ofhistiocyte or macrophage lineage in lymphoidtissues from pigs with PMWS. ISH and IHC ofconsecutive sections of lymphoid tissues indicatedthat most areas containing numerous PCV2 DNA-positive cells also contained numerous PCV2antigen-positive cells, suggesting that the immuno-histochemical signals were specific for PCV2. Inaddition, synthetic peptide-derived polyclonal anti-bodies against PCV2 did not react with lymphoidtissues from pigs experimentally infected withPCV1 or other swine viruses.

In the present study, PCV2 antigen was detectedin formalin-fixed, paraffin wax-embedded lym-phoid tissues by the IHC procedure. However, notall monoclonal and polyclonal antibodies aresuitable for use in IHC, due to the cross-linkingeffect of the formalin fixation, whichrenders certain epitopes undetectable (Hainesand Chelack, 1991). To establish the diagnosis ofPMWS, techniques are required that link virus andtissue lesions, such as IHC and ISH, but not thePCR or virus isolation because the frequentoccurrence of subclinical PCV2 infection maylead to false positive diagnoses (Chae, 2004). TheIHC method described was successfully applied toformalin-fixed, paraffin wax-embedded tissues andshould prove helpful in diagnosing PMWS.

Acknowledgments

This research was supported by the Ministry ofAgriculture, Forestry and Fisheries—Special Grants

Y. Ha et al.204

Research Program (MAFF-SGRP) and Brain Korea21 Project, Republic of Korea.

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Received; June 14th; 2004

Accepted; January 3rd; 2005

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