Development of a Polyclonal-Antibody-Based Immunohistochemical Method for the Detection of Type 2 Porcine Circovirus in Formalin-Fixed, Paraffin-Embedded Tissue

Abstract

Abstract. An immunohistochemical method for the detection of type 2 porcine circovirus (PCV2) in paraffin-embedded tissue was developed. Rabbits were inoculated with purified PCV2 to obtain a polyclonal antiserum. Antiserum was applied to sections of porcine tissue that contained lesions consistent with postweaning multisystemic wasting syndrome and in which PCV2 genome had been demonstrated by in situ hybridization. In all cases (18/18), the density and distribution of positive cells detected by in situ hybridization or immunohistochemistry were identical. The immunohistochemical method is more rapid and less expensive than in situ hybridization and is thus more suitable for routine diagnostic use.A newly emerged porcine disease syndrome, postweaning multisystemic wasting syndrome (PMWS), has been reported throughout North America and Europe and is associated with infection by a novel strain of porcine circovirus (PCV). 2,3,5,7-10 PMWS is characterized clinically by progressive dyspnea, emaciation, and occasionally icterus and pathologically by a wide range of inflammatory lesions that most often include lymphohistiocytic to granulomatous lymphadenitis, interstitial pneumonia, hepatitis, and interstitial nephritis. 2,3 The hallmark of PMWS is depletion of lymphoid tissues and the consistent presence in those tissues of a newly identified strain of PCV. 2,3,5,[7][8][9][10] The genotype and serotype of the PCV isolated from PMWS lesions (PCV2) is markedly different from that of PCV1, an apparently ubiquitous, nonpathogenic virus initially identified over 20 years ago as a contaminant of the PK15 cell line. 1,6,11,12 Although pigs with PMWS are consistently infected with PCV2, it remains uncertain whether PCV2 infection alone is sufficient to induce PMWS. 4 Detection of PCV2 in tissues has been achieved by 1) identification of macrophages with large basophilic to amphophilic intracytoplasmic inclusion bodies that ultrastructurally correspond to arrays of PCV particles, 7 2) demonstration of viral genome via in situ hybridization (ISH), 2,9 or 3) demonstration of viral antigen via immunohistochemistry. 3,4,7 Because characteristic inclusion bodies are an inconsistent feature of PCV2 infection, reliable methods for the detection of PCV2 genome or antigen are highly desirable. Compared with immunohistochemistry, ISH is more complex and less suited to a busy diagnostic laboratory. Others have reported the immunohistochemical detection of PCV in tissues using rabbit antisera raised against PCV1 3,4 or monoclonal antibodies to PCV1. 4 Immunohistochemistry using rabbit antisera to a presumptive PCV2 isolate was previously reported, 7 but detailed methodology was not presented. Herein, the production of polyclonal Received for publication April 19, 1999. rabbit antiserum to purified PCV2 and utilization of this antiserum in an immunohistochemical assay suitable for routine diagnostic use is described. A well-characterized PCV2 isolate (ISU-31) was propagated in glucosamine-treated PCV-free PK15 cells. 9 When an indirec...

Absence of Evidence of Porcine Circovirus Infection in Piglets with Congenital Tremors

“…22 Tissue sections from a Spanish case of PMWS were used as a positive control. 21 Immunohistochemical labeling for PrV was done on brain sections from all CT cases.…”

A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection

“…For this purpose, a 96-well microplate (Medisorp, Nunc, Waltham, USA) was coated with each one of the aliquots/well at a concentration of 10ng/μL with PBS, pH 7.4. Rabbit anti-PCV2 serum diluted at 1:5000 (Sorden et al 1999) was used as the detector antibody. The horseradish peroxidase (HRP)-conjugated bovine anti-rabbit immunoglobulins (Santa Cruz Biotechnology, Dallas, USA) was used at a 1:5000 dilution.…”

Gastric ulcers in pigs affected with postweaning multisystemic wasting syndrome

“…A polyclonal antibody against PCV2 4 at 1:1.000 dilution in the streptavidin-biotinimmunoperoxidase 5 technique, and with diaminobenzidine 5 as chromogen was applied (Sorden et al 1999). Anti-Helicobacter pylori IHC staining was performed in the cardiac and antral sections similarly as in anti-PCV2 IHC, but at 1:200 dilution and with a rabbit anti-H. pylori antibody 5 .…”