Detection of PCV2 Virus of Viral Antigen
- Virus Isolation (VI)
- Immunohistochemistry (IHC)
- Electron Microscopy (EM)
- Indirect and Direct Fluorescent Antibody Assays (IFA/FA) on Tissue Sections
- Antigen-Capture ELISA
PK-15 cells support PCV2-replication in vitro and these cells can be inoculated with body fluids or homogenate from pigs suspected to be infected with PCV2 (Pogranichnyy et al., 2000). Glucosamine treatment of the cells has shown to be effective in increasing PCV2-replication (Tischer et al., 1987). A PCV2-induced cytopathic effect is typically not observed and in order to determine viral replication, immunofluorescent or immunoperoxidase staining has to be performed. VI is not routinely done for PCV2 because it is time consuming and not always efficient since viable virus is required and prolonged transit time and autolysis can have negative effects. Applications may include determining if PCV2 shed in semen is infectious. Another version of virus isolation is the quantitative virus isolation (McNeilly et al., 1999). For this assay, 10-fold dilutions of clinical specimens (serum, tissue homogenates) are inoculated on PK-15 cells. This test has been found useful in discriminating subclinical PCV2-infection from clinical PCV2-infection (McNeilly et al., 1999).
|Click to view a larger image|
IHC uses polyclonal antibody to detect PCV2 antigen in formalin-fixed, paraffin-embedded tissue sections (McNeilly et al., 1999; Sorden et al., 1999). This method allows localization of the antigen within a tissue section. It has been determined that an estimated minimum viral load of 108 PCV2 genomes per 500 ng DNA was required to give visible staining in IHC (Brunborg et al., 2004). A comparison of ISH and IHC on tissues from diseased pigs that were stored for up to 6 month in 10% neutral buffered formalin before being embedded in paraffin found that both techniques were able to detect antigen or nucleic acids in all tissues examined (McNeilly et al., 1999). ISH was found to be more specific than IHC especially when compared to IHC performed with polyclonal antibodies (Kim and Chae, 2004).
This method is used to demonstrate circovirus-like particles directly within a cell and to study the virus structure and size. Intracytoplasmic inclusions in macrophages in affected lymph nodes have been shown to be electron dense, round to ovoid bodies with sharp margins. The matrix was described as being heterogenous, with different areas being granular, crystalline in a herringbone pattern or crystalline in cross-sectional arrays on non-enveloped, small, icosahedral-viral particles (Kiupel et al., 1998). EM is not routinely done in diagnostic labs because it is time consuming and expensive. The overall sensitivity is low and there has to be an abundance of virus present in the tissue in order to be detectable by EM.
|Immunofluorescent antibody assay:
Apple-green staining is PCV2 antigen
Indirect and Direct Fluorescent Antibody Assays (IFA/FA) on Tissue Sections
IFA/FA uses a monoclonal antibody or polyclonal antiserum to detect antigen(s) in frozen tissue sections (McNeilly et al., 2002). The assay is fast but antigen can not be confidently associated with lesions and the assay is relatively subjective. Studies using polyclonal antisera and monoclonal antibodies against PCV1 and PCV2 isolates on cells infected with either PCV1 or PCV2 have shown that there was no cross-reaction (Allan et al., 1999).
Antigen-capture ELISA on tissue homogenates has been described and the results were found to be comparable to quantitative virus isolation and IHC (Allan et al., 2000). An antigen detection capture ELISA optimized for fecal samples was also developed (Synbiotics Europe, Lyon, France) and tested on samples with and without a history of PMWS (Lopez et al., 2005). The main advantage of this kit is that fecal samples are readily available. In contrast, PCV2 is not always detected in serum, bleeding pigs might be inconvenient, and PCR on large numbers of samples is expensive.
Allan GM, McNeilly F, Meehan BM, Ellis JA, Connor TJ, McNair I, Krakowka S, Kennedy S: A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation. J Vet Med B Infect Dis Vet Public Health. 47:81-94, 2000
Allan GM, Mc Neilly F, Meehan BM, Kennedy S, Mackie DP, Ellis JA, Clark EG, Espuna E, Saubi N, Riera P, Bøtner A, Charreyre CE: Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland. Vet Microbiol. 66:115-23, 1999
Brunborg IM, Moldal T, Jonassen CM: Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR. J Virol Methods. 122:171-178, 2004
Kim J, Chae C: 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. J Vet Diagn Invest. 16:45-50, 2004
Kiupel M, Stevenson GW, Mittal SK, Clark EG, Haines DM: Circovirus-like viral associated disease in weaned pigs in Indiana. Vet Pathol. 35:303-307, 1998
Lopez P, Guillossou S, Deshaies E, Brajon N, Bestul K, Leterme S: Porcine circovirus type 2: enzyme linked immunosorbent assay (ELISA) for the detection of antigen and antibodies in feces. In: Proc Intern Conf “Animal Circoviruses and Associated Diseases”, Belfast, UK, p 91, 2005
McNeilly F, McNair I, O'Connor M, Brockbank S, Gilpin D, Lasagna C, Boriosi G, Meehan B, Ellis J, Krakowka S, Allan GM: Evaluation of a porcine circovirus type 2-specific antigen-capture enzyme-linked immunosorbent assay for the diagnosis of postweaning multisystemic wasting syndrome in pigs: comparison with virus isolation, immunohistochemistry, and the polymerase chain reaction. J Vet Diagn Invest. 14:106-112, 2002
McNeilly F, Kennedy S, Moffett D, Meehan BM, Foster JC, Clarke EG, Ellis JA, Haines DM, Adair BM, Allan GM: A comparison of in situ hybridization and immunohistochemistry for the detection of a new porcine circovirus in formalin-fixed tissues from pigs with post-weaning multisystemic wasting syndrome (PMWS). J Virol Methods. 80:123-128, 1999
Pogranichnyy RM, Yoon KJ, Harms PA, Swenson SL, Zimmerman JJ, Sorden SD: Characterization of immune response of young pigs to porcine circovirus type 2 infection. Viral Immunol. 13:143-153, 2000
Sorden SD, Harms PA, Nawagitgul P, Cavanaugh D, Paul PS: Development of a polyclonal-antibody-based immunohistochemical method for the detection of type 2 porcine circovirus in formalin-fixed, paraffin-embedded tissue. J Vet Diagn Invest. 11:528-530, 1999
Tischer I, Peters D, Rasch R, Pociuli S: Replication of porcine circovirus: induction by glucosamine and cell cycle dependence. Arch Virol. 96:39-57, 1987