Porcine parvovirus- and porcine circovirus 2 ... - Springer Link

Trop Anim Health Prod (2010) 42:515–522 DOI 10.1007/s11250-009-9454-0

Porcine parvovirus- and porcine circovirus 2-associated reproductive failure and neonatal mortality in crossbred Indian pigs Rinku Sharma & G Saikumar

Accepted: 21 August 2009 / Published online: 10 September 2009 # Springer Science + Business Media B.V. 2009

Abstract The objective of this study was to detect the presence of porcine parvovirus (PPV) and porcine circovirus 2 (PCV2) in a farm showing reproductive failure and increased mortality in neonatal piglets by histopathological examination, polymerase chain reaction, and demonstration of viral antigen and nucleic acid. Out of 594 piglets farrowed by 70 first-parity gilts, nine (1.51%) mummified fetuses, 13 (2.19%) stillborn, and 572 (96.3%) live-born piglets were recorded. The average litter size at birth was 8.48 piglets per litter. One hundred ninety-four (33.91%) piglets died within 7 days of age. PPV was detected in five litters (7.14%) and two of them revealed coinfection with PCV2. The pathological lesions in the coinfected litters were more severe, indicating a synergistic action between the two viruses. Results of this study suggest for the first time occurrence of PPV and coinfection with PCV2 in crossbred Indian pigs affected with reproductive problem and neonatal mortality. Keywords Pigs . Porcine parvovirus . Porcine circovirus 2 . Coinfection . India R. Sharma Referral Veterinary Polyclinic, Indian Veterinary Research Institute, Bareilly 243 122, India G. Saikumar (*) Division of Pathology, Indian Veterinary Research Institute, Bareilly 243 122, India e-mail: [email protected]

Abbreviations APES DAB DIG HE HRPO ISH NBT–BCIP PALS PCV2 PPV PRRSV RT RE SSC TBE

3-Aminopropyl triethoxysilane Diaminobenzidine Digoxigenin Hematoxylin and eosin Horseradish peroxidase In situ hybridization Nitro blue tetrazolium and 5′bromocresyl-3-indoylphosphate Periarteriolar lymphoid sheath Porcine circovirus 2 Porcine parvovirus Porcine reproductive and respiratory syndrome virus Room temperature Reticuloendothelial Sodium chloride sodium citrate Tris–borate–EDTA

Introduction Porcine parvovirus (PPV) disease is the most important infectious cause of reproductive failure in swine throughout the world. PPV was first associated with reproductive losses in swine by Cartwright and Huck (1967). Besides being the major cause of stillbirth mummification embryonic death infertility syndrome (Thomson and Proszesky 1994), it also causes neonatal mortality (van Leengoed et al. 1983) and occasionally

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abortion (Mengeling 1986). Porcine circovirus 2 (PCV2), a newly recognized virus, has also been associated with late-term abortions, stillbirths, and nonviable neonatal piglets (West et al. 1999; O’Connor et al. 2001). Recent studies suggested that coinfections of PPV with PCV2 or porcine reproductive and respiratory syndrome virus (PRRSV) could occur resulting in more severe reproductive failure and neonatal mortality (Altherr et al. 2003; Garbarino et al. 2003; Ritzmann et al. 2005). Though PPV is thought to be ubiquitous in conventionally raised pigs’ world over, it has not been reported so far from India. However, a recent study provided evidence for the first time that PCV2 was associated with reproductive problems among Indian pigs (Sharma and Saikumar 2008). Therefore, this investigation was planned to ascertain reproductive failure and neonatal mortality caused by PPV infection either singly or as a concurrent infection with PCV2 by detection of viral antigen and nucleic acids in tissues of stillborn and dead neonatal pigs.

Materials and methods A total of 70 litters from a farm housing first-parity crossbred gilts (Landrace × local Indian breed) were closely monitored, and observations on the numbers of piglets born alive, piglets born dead, and piglets that died in the first week of life were recorded and analyzed. A detailed necropsy examination was conducted on dead piglets, and tissue samples (lungs, heart, liver, kidney, lymph nodes, spleen, tonsil, brain) from piglets were collected in 10% buffered neutral formalin fixative and on ice and stored at −70°C after proper labeling till further processing. PCR assay for detection of PPV and PCV2 in tissue specimens Approximately 25 mg of tissue was used for isolating genomic DNA using Tris base–ethylenediaminetetraacetic acid–sodium chloride–sodium dodecyl sulfate solution and slight modification of the method described by Sambrook and Russel (2001). Polymerase chain reaction (PCR) for detection of VP 2 gene of PPV was done as per Arnauld et al. (1998) with slight modification. The expected size of the PCR amplicon was 226 bp. The PCR reaction consisted of 50 µL

Trop Anim Health Prod (2010) 42:515–522

mixture consisting of 10× PCR buffer (5.00 µL), 50 mM MgCl2 (1.50 µL), 2.5 mM deoxyribonucleotide triphosphate (2.00 µL), 10 pmol/µL PPV1F primer (2.50 µL), 10 pmol/µL PPV1R primer (2.50 µL), Taq DNA polymerase (2.5 U), DNA (3.00 µL), and nuclease-free water to make up to 50 µL. The thermal cycling conditions consisted of initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 30 s and final extension at 72°C for 10 min. The PCR amplicon was electrophoresed along with 100 bp molecular weight marker on a 1.5% agarose gel. The duplicate tissue specimens were subjected to PCR for detecting PCV2 in the affected litters as previously described (Larochelle et al. 1999). Histopathological examination Fixed tissues were subjected to histopathological processing and hematoxylin and eosin staining as per standard procedures. Individual sections were microscopically examined, and the histopathological alterations were recorded and digitally microphotographed (Olympus). The tissues from mummified fetuses were not processed for histopathology due to advanced autolysis. Demonstration of PPV antigen by direct fluorescent antibody technique and indirect immunoperoxidase test Thin sections (5–8 µm) were cut from frozen tissue specimens using a cryomicrotome (MICROM, Germany) onto 3-aminopropyl triethoxysilane (APES)treated glass slides. After sectioning, the slides were dried at room temperature (RT) and postfixed in chilled (4°C) acetone for 10 min. After fixation, the slides were stained with fluorescent-labeled PPV conjugate (VMRD Inc.) using the direct fluorescent antibody technique as prescribed by the manufacturer. PPV antigen was demonstrated in the paraffinembedded sections by procedure as previously described (Kennedy et al. 2000). Five–eight-micrometer-thin sections were cut from paraffin-embedded tissue blocks on a rotary microtome (Leica) onto APES-treated glass slides. After sectioning, the slides were dried at RT for 1 h and incubated at 56°C for 30 min. The sections were stained by indirect immunoperoxidase technique


using PPV specific mouse monoclonal antibody (3C9D11H11, VMRD Inc.; 1:250) and chicken antimouse horseradish peroxidase (HRPO) conjugate (Bethyl; 1:500). The antigen–antibody reaction was visualized by application of diaminobenzidine (DAB) substrate solution (Sigma). The slides were counterstained with Mayer’s hematoxylin and examined under a high-resolution microscope (Olympus BX41). Demonstration of PCV2 antigen by indirect immunoperoxidase test The PCV2 antigen was demonstrated in the paraffinembedded sections by procedure as previously described (Kennedy et al. 2000). Five–eight-micrometer-thin sections were cut from paraffin-embedded tissue blocks on a rotary microtome (Leica) onto APES-treated glass slides. After sectioning, the slides were dried at RT for 1 h and incubated at 56°C for 30 min. The sections were stained by indirect immunoperoxidase technique using PCV2 specific polyclonal sera, used undiluted (VMRD Inc.) and goat antipig HRPO conjugate (Bethyl; 1:500). The antigen–antibody reaction was visualized by application of DAB substrate solution (Sigma). The slides were counterstained with Mayer’s hematoxylin and examined under a high-resolution microscope (Olympus BX41). Demonstration of PPV nucleic acid by in situ hybridization using digoxigenin labeled DNA probe The PPV probe preparation and the in situ hybridization (ISH) procedure were as previously described (Choi and Chae 1999) with certain modifications. Digoxigenin (DIG)-labeled DNA probe of 226 bp targeting the VP 2 region was generated by a PCR labeling reaction as described, employing the recombinant plasmid carrying cloned PCR amplicon from VP 2 region of PPV-Ind-Bly-05 isolate as template and DIG-11-dUTP (Roche) as the nonisotopic label. The DNA probe was purified using column-based MinElute PCR product purification kit (Qiagen) as per manufacturer’s recommended procedure to remove unincorporated nucleotides, enzymes, and other reaction components. Purified probe was electrophoresed along with 1 µg of quantitative 200-bp–10-kb DNA molecular weight marker (BioTools) on a 1.5% agarose gel. Following electrophoresis, the quantity of the

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probe was visually assessed. Prehybridization was carried out for 60 min in hybridization buffer at 60°C under moist conditions (2× sodium chloride–sodium citrate as moist chamber solution) in hybridization oven. For hybridization, DIG-labeled DNA probe was diluted at 1 ng/µL (1/100 µL) in hybridization buffer and heat denatured for 10 min at 95°C on a heating block and quenched immediately on ice before application to the tissue sections. Hybridization was carried out overnight for 18 h at 45°C in hybridization oven. For detection of probe hybridization to target, the sections were incubated with anti-DIG alkaline phosphatase conjugate (Roche; 1:250). Colorimetric detection comprised of layering of nitro blue tetrazolium and 5-bromocresyl-3-indolylphosphate (NBT/BCIP) substrate solution over the sections till satisfactory color development under low light conditions. The sections were counterstained for 1 min with 0.5% aqueous methyl green and examined microscopically under a high-resolution microscope (Olympus BX41).

Results A total of 70 farrowings pertaining to first-parity gilts were recorded in the farm under study. The reproductive failure in sows characterized by mummified fetuses, small litter size, and weak, nonviable neonatal piglets was observed. Out of the 594 piglets delivered from 70 farrowings, a total of nine (1.51%) mummified fetuses, 13 (2.19%) stillborn, and 572 (96.3%) live-born piglets were recorded. Out of the live born piglets, 194 (33.91%) piglets from 65 litters died within 7 days of age. The average litter size at birth was 8.48 piglets per litter. In 13 litters, the litter size was either five or less than five piglets with the lowest being one piglet in one litter. PCR assay for detection of PPV and PCV2 The PCR assay resulted in amplification of the expected 226-bp fragment of VP 2 gene of PPV in five of the 70 litters (7.14%) tested by PCR. The virus could be detected in the lung and tonsil tissues of the piglets. Two of the five PPV positive litters revealed coinfection with PCV2. The lungs, tonsils, lymph nodes, and brain tissues of the piglets in these two litters revealed positive amplification for PCV2. PCV2 infection alone was detected in piglets of 12

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litters (data not included). The tissue samples were also tested for the presence of PRRSV, classical swine fever virus, and pseudorabies virus by reverse transcriptase-PCR/PCR and were found negative (data not included). Gross and histopathology findings in PVV infected litters In one of the PPV-affected litters which comprised four mummified fetuses and no live-born piglets, the fetuses were of different sizes, indicating in utero death at different stages of gestation (Fig. 1). The visceral organs were in advanced stage of autolysis in all the fetuses, and serosanguineous fluid was present in the body cavities. The litter size of the second PPVaffected litter was six in which all the piglets died within a day of birth. Only two piglets were farrowed in the third affected litter where one piglet died within a day and the other on the third day. The gross findings in piglets from these two PPV-affected litters were almost similar with minor differences. Small amount of straw-colored fluid was observed in the body cavities. Lungs were noncollapsing, edematous, and congested with focal hemorrhages. Heart revealed thickened opaque pericardium and pale streaks in the myocardium (Fig. 2). Liver revealed either intense congestion or pale/yellowish discoloration. Gross changes in kidneys varied from congestion and petechial hemorrhages to pale yellowish discoloration of the surface and renal papillae. Stomach contained traces of curdled milk, and the mucosa showed mild to severe edematous changes. Other minor changes

Fig. 2 Enlarged heart of a day-old piglet showing pale white streaks on the epicardium and vascular congestion

included congestion of tonsils, intestinal mucosa, and meninges. Histopathologically, lungs revealed mild to moderate interstitial pneumonia and patchy areas of alveolar necrosis. Myocarditis characterized by diffuse areas of mononuclear cells infiltration, edema, and degeneration of myocardiocytes, and mild hemorrhages was observed at a few places. Liver revealed disorientation of hepatic cords, severe degeneration of hepatocytes, congested sinusoids, mild to moderate Kupffer cell proliferation, moderate to severe hemorrhages in the parenchyma, and mild fatty changes in centrilobular areas. Kidneys revealed mild focal interstitial nephritis due to infiltration of mononuclear cells, degeneration of epithelial lining cells of proximal convoluted tubules, and hemorrhages in the interstitium. Spleen revealed multiple areas of small to medium diffuse hemorrhages and mild depletion of periarteriolar lymphoid sheath (PALS) and proliferation of reticuloendothelial (RE) cells. Lymph nodes revealed multiple focal hemorrhages mainly in the cortical region, depletion of lymphocytes and hemorrhages in lymphoid follicles, and infiltration of RE cells. Brain showed focal accumulation of glial cells mainly in the vicinity of blood vessels in the cerebral cortex and in the subependymal region. Gross and histopathology findings in PPV and PCV2 coinfected litters

Fig. 1 Four variable-sized mummified fetuses infected with PPV

In the first coinfected litter, eight of the 12 delivered piglets died within 7 days of birth. The gross findings in these piglets were multiple reddish blue areas of congestion on the skin of thoracic, abdominal, pelvic


region, and limbs. The carcass lymph nodes were enlarged and tan colored in two piglets. Thoracic cavity and pericardial sac contained increased amount of dirty straw-colored fluid. The lesions in visceral organs were more severe in nature as compared to the PPV-alone-affected litters. In addition, fibrinous adhesions were observed on the heart, liver, and intestines. Histopathologically, the piglets revealed either mild interstitial pneumonia characterized by macrophage proliferation and presence of a few syncytial cells in the interstitium and alveolar spaces or fibrinous bronchointerstitial pneumonia. Variable-sized, multiple basophilic inclusion bodies were observed in the cytoplasm and nucleus of alveolar macrophages. Heart revealed congestion, focal hemorrhages, moderate intermyofibrillar edema, loss of striations, and indistinct nuclei of myocardiocytes and multifocal infiltration of mononuclear cells in the epicardium and myocardium. Severe multifocal hepatitis characterized by mononuclear cell infiltration along with presence of basophilic, intranuclear inclusion bodies in degenerating hepatocytes was observed. Lesions in kidneys were those of interstitial nephritis. Spleen revealed moderate to severe lymphoid depletion, proliferation of macrophages, and formation of a few syncytial cells in the PALS region. Lymph nodes revealed pronounced follicle depletion, proliferation of macrophages, occasional syncytial cell, and mild eosinophilic infiltration together with presence of intracytoplasmic basophilic inclusion bodies in macrophages and large mononuclear cells. In some lymph nodes, especially the superficial inguinal lymph nodes, syncytial cells, and intracytoplasmic and intranuclear basophilic inclusion bodies in macrophages in the follicles, a few eosinophils and hemorrhages were noticed. Focal mononuclear cell meningitis and multifocal areas of malacia were observed in the cerebrum. The malacic foci showed complete loss of neurons and infiltration of glial cells. In the second litter, along with two mummified fetuses, one live-born piglet that died soon after birth was farrowed. The gross and histopathological findings in these piglets were similar to those observed in the other coinfected litter.

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signals in the lungs were observed mainly in the alveolar septal cells. PPV antigen in the heart was demonstrated in the myocardiocytes (Fig. 3). Indirect immunoperoxidase test (IPT) on formalin-fixed, paraffin-embedded tissues revealed antigen-positive cells in lungs, heart, and kidneys. The positive reaction was noticed mainly in the nucleus and occasionally in the cytoplasm of cells. The antigenpositive cells were diffusely scattered as individual cells or small foci. PPV antigen was demonstrated in the alveolar septal macrophages, myocardiocytes, and degenerating and necrotic epithelial cells of proximal convoluted tubules of kidneys. PCV2 antigen demonstration in tissues The PCV2 antigen was successfully demonstrated in the lymph nodes, lungs, and spleen of PPV coinfected day-old and 7-day-old piglets. In lymph nodes, the antigen was demonstrated in the follicular and interfollicular regions of the cortex and paracortex and located in the cytoplasm of macrophages and other reticuloendothelial cells. In lungs, the PCV2 antigen was located in the alveolar macrophage-like cells and cellular debris in the alveolar lumen (Fig. 4). PCV2 antigen in the spleen was noticed in the mononuclear and syncytial cells in the PALS region. PPV nucleic acid demonstration In situ hybridization demonstrated the PPV nucleic acid in lungs, heart, liver, kidney, and spleen of dayold piglets. Hybridization signals were noticed both in the nucleus and the cytoplasm of cells. The PPV

PPV antigen demonstration in tissues Positive fluorescent signals were observed in lungs and heart tissues of day-old piglets. The fluorescent

Fig. 3 Heart of a day-old piglet showing PPV antigen in the myocardiocytes. IPT–DAB substrate–MH ×250

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Fig. 4 Lungs of a day-old piglet showing presence of PCV2 antigen in the cytoplasm of alveolar septal macrophage-like cells and cellular debris in lumen. IPT–DAB substrate–MH ×400

nucleic acid in the lungs was found scattered in macrophage-like cells in the alveolar septae and interstitium (Fig. 5). In the heart, the positive signals were most often found in myocardiocytes and epicardium. Nucleic acid labeling in liver was widespread and positive cells were observed mainly as individual cells or occasionally concentrated as small foci in the degenerating and necrotic hepatocytes mainly around the central veins. In the kidneys, the PPV nucleic acid was observed in the degenerating tubular epithelial cells and occasionally in the glomerular tufts. The hybridization signals in spleen were detected mainly in macrophage-like cells in the PALS region.


technique in piglets of five litters (7.14%). After initial screening of suspected cases by PCR targeting the VP 2 gene of PPV (Molitor et al. 1991; Arnauld et al. 1998) and PCV2 (Larochelle et al. 1999), additional tests for demonstration of viral antigen or nucleic acids in association with histopathological lesions were applied for confirmation of the diagnosis. The clinical, gross, and histopathological findings in the PPV-infected piglets in the present study included weak live-born piglets, presence of straw-colored or serosanguineous fluid in the body cavities, mild necrotizing interstitial pneumonia, myocarditis, pale friable liver, mild to moderate Kupffer cell proliferation, renal degeneration, interstitial nephritis, and nonsuppurative meningoencephalitis. These findings were similar to those reported by earlier workers (Narita et al. 1975; van Leengoed et al. 1983; Bolt et al. 1997). Detection of PPV antigen and nucleic acids in lungs, heart, liver, spleen, and kidneys of affected piglets especially in the cells of monocyte–macrophage series corroborated previous observations on target organs and sites of replication of the virus (Cutlip and Mengeling 1975; Oraveerakul et al. 1993; Choi and Chae 2000; Kennedy et al. 2000; Kim et al. 2003). PPV-infected litters were found coinfected with PCV2 as demonstrated by PCR and indirect immunoperoxidase test. PCV2-associated reproductive failure has been recorded in Canada (West et al. 1999; O’Connor et al. 2001), Italy (Garbarino et al. 2003), Germany (Altherr et al. 2003), Denmark (LadekjaerMikkelsen et al. 2001), Korea (Kim et al. 2004), and

Discussion PPV and PCV2 induced reproductive failure characterized by decrease in litter size, increase in stillbirths, mummified fetuses, and neonatal deaths as observed in the present study have been well documented (Donaldson-Wood et al. 1977; van Leengoed et al. 1983; Kim et al. 2004). In the present study, PPV infection could be demonstrated by PCR, direct fluorescent antibody technique, indirect immunoperoxidase test, and DIG-labeled in situ hybridization

Fig. 5 Serial section from lungs shown in Fig. 4, showing presence of PPV nucleic acids labeling in macrophage-like cells in the alveolar septae and interstitium. ISH–NBT/BCIP–MH ×400


India (Sharma and Saikumar 2008). The pathological lesions in the coinfected litters were more severe than in litters infected with PPV alone. In addition to the changes observed in litters infected with PPV alone, the coinfected litters showed presence of PCV2 intracytoplasmic basophilic inclusion bodies in the macrophages of lymph nodes and malacic foci in brains. These results indicated synergistic effect of PPV and PCV2 concurrent infection. Previous study to elucidate the role of these two viruses in the development of PMWS showed that during the immunoproliferative phase of PPV convalescence, activated macrophages and other immune cells support heightened PCV2 replication (Krakowka et al. 2000). Coinfections of PPV and PCV2 in pigs showing reproductive failure have been recorded occasionally (Garbarino et al. 2003; Kennedy et al. 2000; Altherr et al. 2003). In the farm investigated in the present study, all breeding females were first-parity gilts and no preventive vaccination against PPV was practiced, as the disease had not been reported so far in the country. It is an established fact that PPV induces reproductive failure mainly in gilts and could be transmitted through the semen of infected boars (Joo and Johnson 1976). Similarly, PCV2 infection can also be transmitted vertically (Harms et al. 1999; Jolie et al. 2000) by infected boars (Schmoll et al. 2008). The reproductive failure in the farm under study could possibly be due to use of boars infected with PPV and PCV2, as the farm did not practice artificial insemination. The results of this study suggests that PPV, a major cause of reproductive failure in swine world over but not recognized so far in India, is causing disease along with PCV2 (an emerging viral pathogen), resulting in reproductive failure in gilts and high mortality among neonatal piglets. The situation warrants further studies to ascertain the contributory role of these viruses to reproductive failure among Indian pigs. Acknowledgments The authors acknowledge Director, IVRI, Izatnagar for providing necessary facilities during the course of this investigation.

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