Simultaneous Detection of Porcine Circovirus 2 and Porcine Parvovirus in Naturally and Experimentally Coinfected Pigs by Double in Situ Hybridization

Abstract

Abstract.A technique for double in situ hybridization to simultaneously detect porcine circovirus 2 (PCV2) and porcine parvovirus (PPV) in the same tissue section was developed and applied to lymph node and spleen from 8 pigs experimentally coinfected with PCV2 and PPV and 20 pigs with naturally occurring postweaning multisystemic wasting syndrome. For double labeling studies, the tissue samples were processed sequentially, first for PPV in situ hybridization using a digoxigenin-labeled probe and then for PCV2 in situ hybridization using a biotinylated probe. Positive cells contained reaction products for PCV2 and PPV, respectively. Both PCV2 DNA and PPV DNA were observed mainly in the cytoplasm but occasionally in the nucleus. With double in situ hybridization, both PCV2 DNA and PPV DNA were simultaneously detected in lymph node and spleen. This double labeling technique for the detection of PCV2 and PPV is suitable both for pathogenesis studies and for diagnostic applications.Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in pigs that was first described in 1991. 1 Since then, PMWS has been recognized in pigs in Asia, North America, and Europe. 17 The disease occurs in swine herds that are usually in otherwise good health. The disease has low morbidity but a relatively high case fatality rate among 5-12-week-old pigs. 1,11 Clinically, PMWS is characterized by progressive weight loss, respiratory signs, and jaundice. 3,7,9 The microscopic lesions of PMWS include granulomatous interstitial pneumonia, lymphadenopathy, hepatitis, nephritis, and pancreatitis. 1,11 Porcine circovirus 2 (PCV2) antigen and nucleic acid have been detected consistently in tissues of pigs with PMWS. 3,5,7,9,13 The demonstration of PCV2 antigen and nucleic acid closely associated with lesions in a wide range of tissues from diseased pigs has led to speculation that PCV2 may be an etiologic agent of PMWS. However, coinfection of PCV2 and porcine parvovirus (PPV) has been reported in some experimental and natural cases of PMWS. 2,8,10,15,16 Multiplex polymerase chain reaction (PCR) techniques have been used for the simultaneous detection of both PCV2 and PPV. 8,14 However, PCR does not allow localization of the 2 viruses in infected tissues or cells. In contrast, in situ hybridization provides cellular detail and histologic architecture so that the presence of the 2 viruses in lesions may be studied in the same section. Although single in situ hybridization can be used to detect either PCV2 or PPV in formalin-fixed, paraffin-embedded tissues, 5,13,15,17 simultaneous detection of PCV2 and PPV by double in situ hybridization has not been reported. The double in situ hybridization technique for the simultaneous detection of both PCV2 and PPV combines the sensitivity and specificity of in situ hybridization and eliminates the need to test clinical specimens separately for each virus. The objective of the present study was to develop a method of double in situ hybridization for si- multaneous detection of both PCV2 DNA ...

Canine Circovirus 1 (CaCV-1) and Canine Parvovirus 2 (CPV-2)

“…A synergistic relationship between circovirus and parvovirus was first reported in pigs, where dual infections with PCV-2 and porcine parvovirus (PPV) were shown to reproduce clinical disease consistent with PMWS. 2,8,11,14 While dual infection with circovirus and parvovirus may represent a common coinfection that causes clinical disease in different species, nonspecific immunostimulation is sufficient to cause severe circoviral disease in pigs. 17 It has been speculated that induction of host cell proliferation is the main requirement for S-phase-dependent circovirus replication, 7,27 and the trigger mechanism for such proliferation can be highly variable.…”

Perspectives on the Evolution of Porcine Parvovirus

“…PPV was prevalent in South Korea and was continuously surveyed due to significant losses in the swine industry. Although the vaccine reduced infection rates dramatically [ 11 ], there have been no recent investigations of PPV. The nucleotide substitution rate of PPV was approximately 10 −5 substitutions per site per year for the NS1 gene and 10 −4 substitutions per site per year for the VP1 and VP2 genes.…”

Simultaneous Detection and Differentiation between Porcine Circovirus and Porcine Parvovirus in Boar Semen by Multiplex Seminested Polymerase Chain Reaction

“…Although PCV2 and PPV have previously been differentiated from fresh and formalin-fixed tissues using the multiplex polymerase chain reaction (PCR) and in situ hybridization [14,16], no one has, as yet, reported detection and differentiation among PCV1, PCV2, and PPV in semen using multiplex PCR. However, virus isolation from semen on continuous cell lines is difficult [15].…”

“…In contrast to the generally accepted dogma of PPV pathogenesis in reproductive failure, PPV/PCV2 co-infections have been demonstrated in a significant portion of field cases of PMWS in Korea [5,17] and Canada [8]. Co-infection with PCV2 and PPV induces more severe lesions and clinical disease [2,11,18].Although PCV2 and PPV have previously been differentiated from fresh and formalin-fixed tissues using the multiplex polymerase chain reaction (PCR) and in situ hybridization [14,16], no one has, as yet, reported detection and differentiation among PCV1, PCV2, and PPV in semen using multiplex PCR. However, virus isolation from semen on continuous cell lines is difficult [15].…”