Apoptosis in postweaning multisystemic wasting syndrome (PMWS) hepatitis in pigs naturally infected with porcine circovirus type 2 (PCV2)

The Veterinary Journal 189 (2011) 72–76

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Apoptosis in postweaning multisystemic wasting syndrome (PMWS) hepatitis in pigs naturally infected with porcine circovirus type 2 (PCV2) Ana R. Resendes a,b, Natàlia Majó a,b, Ted S.G.A.M. van den Ingh c, Enric Mateu a,b, Mariano Domingo a,b, Maria Calsamiglia b, Joaquim Segalés a,b,⇑ a b c

Centre de Recerca en Sanitat Animal (CReSA), Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain Departament de Sanitat i d’Anatomia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands

a r t i c l e

i n f o

Article history: Accepted 25 June 2010

Keywords: Porcine circovirus type 2 (PCV2) Postweaning multisystemic wasting syndrome (PMWS) Apoptosis Cleaved caspase-3 Immunohistochemistry

a b s t r a c t The degree of apoptosis in the livers of pigs with hepatitis due to naturally-occurring postweaning multisystemic wasting syndrome (PMWS) was evaluated semi-quantitatively by immunohistochemical detection of the apoptotic marker cleaved caspase-3 (CCasp3). The amount and distribution of porcine circovirus type 2 (PCV2) virus in the liver was evaluated using in situ hybridisation. Livers with mild, stage I hepatitis exhibited similar degrees of apoptosis to controls; those with stage II lesions had variable apoptotic rates, ranging from mild to high, and in livers with more severe, stage III hepatitis, high levels of hepatocyte apoptosis was a feature. Statistical analyses indicated a positive association between the rate of apoptosis, the severity of the hepatitis and the amount of PCV2 DNA in the liver. Double immunolabelling for CCasp3 and PCV2 DNA revealed a predominance of cells labelling only for PCV2, followed by fewer cells labelling only for CCasp3, and the least number labelling for both. The findings suggest that apoptosis, possibly triggered by PCV2 infection and/or hepatic inflammation, plays a key role in the pathogenesis of hepatitis in pigs with naturally-occurring PMWS. Ó 2010 Elsevier Ltd. All rights reserved.

Introduction Porcine circovirus type 2 (PCV2) belongs to the genus Circovirus of the Circoviridae family (small non-enveloped viruses) with a circular single-stranded DNA genome of approximately 1.76 kb (Pringle, 1999). PCV2 is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), a multifactorial disease of swine (Krakowka et al., 2000; Bolin et al., 2001). PMWS affects nursery and fattening pigs of 5–18 weeks of age and clinical signs include weight loss, anaemia, dyspnoea and, occasionally, diarrhoea and jaundice (Clark, 1997; Rosell et al., 1999). Mortality rates vary from 1% to 30%, and the disease has had a significant economic impact on pig production worldwide (Harding, 2004; Segalés et al., 2005). Gross lesions include non-collapsed, tan-mottled lungs, enlarged lymph nodes, and less frequently, multifocal necrosis of lymph nodes, jaundice, hepatic atrophy and multiple white foci in kidney cortices (Rosell et al., 1999; Segalés et al., 2004). Microscopic hallmarks of PMWS include lymphocyte depletion and histiocytic infiltration of lymphoid or⇑ Corresponding author at: Centre de Recerca en Sanitat Animal (CReSA) and Departament de Sanitat i d’Anatomia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain. Tel.: +34 93 581 45 63; fax: +34 93 581 44 90. E-mail address: [email protected] (J. Segalés). 1090-0233/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2010.06.018

gans, occasionally accompanied by multinucleated giant cells and basophilic cytoplasmic inclusion bodies, interstitial pneumonia, hepatitis and nephritis (Ellis et al., 1998; Rosell et al., 2000; Darwich et al., 2003; Segalés et al., 2004). PCV2 is a relatively recently recognised cause of infectious hepatitis in pigs (Rosell et al., 2000), occurring in up to 44% of PMWSaffected animals. Four stages of hepatic damage have been described based on the amount of lymphohistiocytic infiltration and hepatocyte injury (cytoplasmic swelling and vacuolation, necrosis, karyomegaly and apoptotic bodies), as well as on the extent of the disorganisation of the hepatic plates and perilobular fibrosis (Harding and Clark, 1997; Rosell et al., 1999, 2000). Hepatitis is classified as severe (lesion stages III and IV) in 15% of all cases of hepatitis associated with PMWS, and appears to develop late in the course of disease (Rosell et al., 2000; Segalés et al., 2004). Other viral causes of hepatitis in pigs include swine hepatitis E virus (HEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV) and Aujeszky’s disease virus (ADV), although HEV, PRRSV or PPV do not cause severe hepatitis under natural or experimental infections (Krakowka et al., 2000; Halbur et al., 2001; Paul et al., 2003; de Deus et al., 2007; Lee et al., 2009). Four patterns of PCV2 distribution are associated with PMWS hepatitis and infected cells include hepatocytes, Kupffer-cells, sinusoidal endothelial cells, histiocytes and mononuclear cells

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(Rosell et al., 2000). Given that the severity of the hepatitis has been associated with the amount and distribution of PCV2 present, and that PCV2 can replicate in hepatocytes and macrophages (Pérez-Martín et al., 2007), it has been suggested that the extent of hepatocyte infection is directly attributable to lesion severity (Rosell et al., 2000; Hirai et al., 2003; Krakowka et al., 2005). A number of viruses encode pro-apoptotic genes, activating their transcription to facilitate the release of viral progeny during acute infection (O’Brien, 1998; Benedict et al., 2003; Irusta et al., 2003). The potential role of virus-induced apoptosis in the pathogenesis of the hepatitis associated with PMWS remains unclear. Hirai et al. (2003) demonstrated apoptosis in cases of severe hepatitis in pigs experimentally infected with PCV2 by both electron microscopy and using the TUNEL-assay, whereas Krakowka et al. (2004) did not associate hepatocyte loss with apoptosis in gnotobiotic pigs with PMWS. Furthermore, Kiupel et al. (2001) found evidence of apoptosis in lymphoid tissues of mice infected with PCV2. The objective of the present study was to investigate the possible role of apoptosis in the development of hepatitis in pigs that had naturally developed PMWS using an in situ detection system that identifies early apoptotic events (Hengartner, 2000; Eckle et al., 2004). Materials and methods Case selection and sampling Liver samples from 16 pigs, 2–3.5 months of age and affected by PMWS with different degrees of hepatitis, were selected from archived formalin-fixed, paraffin-embedded material at the Veterinary Pathology Service of the Veterinary School of Barcelona. PMWS had been diagnosed in these animals using established criteria (Segalés and Domingo, 2002) and all were negative for PRRSV, ADV and swine influenza virus (SIV) by immunohistochemistry, although tissues had not been evaluated for infection with PPV or HEV. Five livers from clinically normal 3.5 month-old pigs used in a previous study were used as controls (Resendes et al., 2004). PCV2 was not detected in these animals using PCR on serum and in situ hybridisation (ISH) on tissues, respectively (Quintana et al., 2002). The control pigs came from a high health status farm free of PMWS and seronegative to PRRSV, ADV, PPV, SIV and Mycoplasma hyopneumoniae, and were euthanased using IV pentobarbital in line with European guidelines on the welfare of experimental animals (Directive 86/609 EEC). A complete necropsy was performed on each pig, a blood sample was collected and the tissues were fixed by immersion in 10% neutral-buffered formalin for approximately 24 h. Tissues were then processed routinely and stained using haematoxylin and eosin for histopathological examination.

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of mononuclear cells were observed in the parenchyma along with a variable degree of hepatic plate disorganisation and frequent hepatocyte necrosis and apoptosis in all lobules. Immunohistochemical examination and semi-quantitative scoring Cleaved caspase-3 (CCasp3) was detected using a polyclonal rabbit anti-cleaved caspase-3 antibody (anti-Asp175) diluted 1/50 (Cell Signaling Inc.) (Resendes et al., 2004). Briefly, 4 lm thick sections of liver on silane-coated slides (3-[trietoxysilyl]propylamine), were dewaxed in xylene, rehydrated through graded alcohols, placed in distilled water and processed using the routine immunohistochemical avidin– biotin peroxidase (ABC) method. Lymphoid tissue and murine liver where apoptosis had been induced by ischaemia were used as positive controls. An isotype-matched antibody was used in the negative control. Positive labelling was counted in 10, randomly-selected microscopic fields at 200 magnification. Positive hepatocytes, apoptotic bodies, endothelial cells, leucocytes and cells of undetermined type were counted. Positive labelling within inflammatory aggregates in portal areas was not counted. This semi-quantitative analysis resulted in four categories: (1) no or minimal apoptosis (1–2 positive figures in some lobules); (2) low level of apoptosis (2–6 positive figures in some lobules); (3) moderate level of apoptosis (6–15 positive figures in most lobules), and (4) high level of apoptosis (>15 positive figures in most/all lobules). Double-immunostaining for CCasp3 and PCV2 Double-immunostaining to detect PCV2 DNA and CCasp3 simultaneously was carried out by adjusting the described ISH (Rosell et al., 1999, 2000) and CCasp3 immunolabelling (Resendes et al., 2004) protocols. Two cases with severe hepatitis and large amounts of both PCV2 infected and CCasp3-positive hepatocytes were assessed. Tissue sections were first tested using the ISH protocol and, after staining with nitroblue tetrazolium, were intensively washed once with 10 mM Tris (pH 8) buffer (Sigma, Trizma base T-1503), twice with 1 mM EDTA and twice with Tris-buffered saline (TBS) (pH 7.4). Immunohistochemical examination for CCasp3 was then carried out on the sections as described above, using a routine avidin–biotin peroxidase protocol and detection method (0.05% diaminobenzidine, 3% hydrogen peroxide in TBS for 10 min). Tissue sections were counterstained with fast green prior to dehydration and mounting for examination. Positive and negative controls for PCV2 and CCasp3 were included in the protocols, respectively. Semi-quantitative assessment of positive labelling for CCasp3, PCV2, and for both components was expressed as low, moderate and high. Statistical analysis Associations between rates of apoptosis (minimal, mild, moderate and high), the severity of the hepatitis (stages I–III), amounts of PCV2 in the liver (negative, mild, moderate and high), were assessed using the v2 test. Differences were considered significant when P < 0.05. Statistical analyses were performed using the Stats Direct v.2.5.4 program.

Histopathological examination and in situ hybridisation for PCV2

Results PCV2 DNA was detected using ISH (Rosell et al., 1999, 2000). In brief, the hybridisation procedure used a 40 nucleotide-long probe (50 -CCTTCCTCATTACCCTCCTCGCCAACAATAAAATAATCAAA-30 ) that was complementary to the Rep gene sequence (ORF1). The positive control tissue was a lymph node exhibiting histopathological evidence of PMWS and positive for PCV2 on PCR. Lymph node tissue from a pig negative for PCV2 on PCR was used as a negative control. The number and distribution of PCV2-positive cells in the liver was categorised as previously described (Rosell et al., 2000), as follows: (1) small amounts of virus (labelled hepatocytes rarely observed, small numbers of labelled Kupffer-cells with a diffuse distribution and small numbers of positive mononuclear cells in periportal areas); (2) moderate amounts of virus (diffuse cell labelling with positive Kupffercells, intermediate numbers of labelled hepatocytes throughout all lobules and positive mononuclear cell infiltrates in perilobular areas); (3) large amounts of virus (diffuse cell labelling with large numbers of positive hepatocytes and Kupffer-cells in all lobules, and periportal labelling of mononuclear cells). Hepatic injury was categorised according to the severity and distribution of: periportal and parenchymal mononuclear inflammatory infiltration; hepatocyte swelling, vacuolation, necrosis, karyomegaly, shrinkage with chromatin condensation and apoptosis; and disorganisation of hepatic plates and periportal fibrosis (Rosell et al., 2000). These evaluations were carried out by two pathologists without prior knowledge of the sample group. Three stages of lesion severity were identified: stage I (mild hepatitis), characterised by mild, multifocal lymphohistiocytic infiltrates in the portal tracts and irregularly distributed in the parenchyma; stage II (moderate hepatitis), characterised by intermediate to intense lymphohistiocytic inflammation of the portal tracts often accompanied by foci of mononuclear inflammatory cells in the parenchyma and scattered hepatocyte necrosis in some lobules, and stage III (severe hepatitis), where intense, multifocal to coalescing infiltrations

The results of virus detection and its association with hepatitis severity are summarised in Table 1. Nine pigs had stage I hepatitis, and all had small amounts of PCV2 DNA. Three animals had stage II hepatitis and moderate amounts of viral DNA, and four pigs had stage III hepatitis with moderate to large amounts of PCV2. All five controls were negative for PCV2 DNA and none had liver lesions. Apoptosis was minimal in the control livers (Table 1). Although very occasional labelling was scattered throughout the parenchyma, most lobules had no positive staining (Fig. 1A). CCasp3-labelled figures were leucocytes within sinusoids, hepatocytes (some with marginated chromatin), apoptotic bodies outside or within hepatocytes (both chromatin-containing and chromatin-free) and endothelial cells. Similar proportions for each cell type were detected. All labelled cells generally displayed intense, diffuse cytoplasmic and/or nuclear immunostaining. These apoptotic rates and the distribution of CCasp3-labelling were taken as the normal ‘background’ expression of this protein in normal pig liver. Apoptotic labelling associated with each stage of hepatitis is also detailed in Table 1. In cases of stage I hepatitis (n = 9) there were minimal to low levels of apoptosis. Labelled figures and their distribution were similar to those detected in controls. Variable

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Table 1 Details of hepatitis stage, viral load (as determined by in situ hybridisation) and apoptosis score (as determined by immunolabelling for the apoptotic marker cleaved caspase-3) in the livers of pigs with postweaning multisystemic wasting syndrome. PCV2, porcine circovirus type 2. Stage of hepatitis (number of pigs)

Number of pigs Amount of PCV2 in liver

Control (5) I (9) II (3) III (4)

Apoptosis score

None

Low

Moderate

High

Minimal

Low

Moderate

High

5 – – –

– 9 – –

– – 3 1

– – – 3

5 5 –

– 4 1 –

– – 1 –

– – 1 4

Fig. 1. Photomicrographs illustrating apoptosis in: (A) control pig liver, (B and C) pig liver with severe porcine circovirus type 2-associated hepatitis, labelled for the apoptotic marker cleaved caspase-3 (CCasp3) using the immunoperoxidase method with haematoxylin counterstaining. (B) Numerous scattered light brown-red CCasp3-labelled apoptotic bodies are visible. (C) Arrowheads indicate chromatin-containing CCasp3-labelled apoptotic bodies and apoptotic nuclei with chromatin margination from a hepatocyte-like cell. Arrow in inset highlights an endothelial cell with both cytoplasmatic and nuclear CCasp3-labelling. Scale bars: A, 100 lm; B, 100 lm; C, 25 lm.

amounts of CCasp3 immunostaining (ranging from mild to high) were detected in cases of stage II hepatitis (n = 3). Animals with stage III hepatitis (n = 4) exhibited large amounts of CCasp3 immunostaining. Positive figures in sections with stages II and III hepatitis were mainly hepatocytes, with positive staining in both the cytoplasm and nucleus (Fig. 1B and C), and frequently were apoptotic bodies both outside and inside hepatocytes and Kupffer-cells (Fig. 1B and C). Some labelled hepatocytes were shrunken with margined nuclear chromatin (Fig. 1B and C) and rarely, cytoplasmic labelling for CCasp3 was observed within endothelial cells (Fig. 1C). Double-labelling for PCV2 and CCasp3 was only observed in hepatocytes with CCasp3 detected in the cytoplasm and virus detected in the nucleus, respectively (Figs. 2A and B). Our semi-quantitative evaluation indicated that double-labelled cells were present in low numbers compared to cells labelling for either CCasp3 or PCV2 DNA (Fig. 2A). Hepatocytes and mononuclear cells labelling solely for viral DNA predominated within tissue sections (Fig. 2A). Statistical analysis indicated a positive association between the degree of apoptosis, the severity of the hepatitis, and the amount of PCV2 DNA (P < 0.05). Larger amounts of virus were associated with more severe hepatitis and higher rates of apoptosis (P < 0.05). Discussion The results of this study indicated that apoptosis plays a role in the pathogenesis of the hepatitis associated with PMWS, with increased hepatocyte apoptosis associated both with more severe tissue injury and with increased viral loads. Moreover, the find-

ings suggested that apoptotic events are related to PCV2 infection of hepatocytes and/or to inflammatory cell infiltration. The immunohistochemical identification of an execution caspase has previously been used to detect apoptosis in human tissue and in the rat (Labat-Moleur et al., 1998; Bantel et al., 2001; Dukers et al., 2002; Eckle et al., 2004), and our findings indicate such a methodology is appropriate in detecting apoptosis in the porcine liver. Apoptosis has been demonstrated in cases of hepatitis in pigs associated with both natural and experimental PCV2 infection (Rosell et al., 2000; Hirai et al., 2003). In the present study, increased apoptosis was associated with more extensive viral infection of hepatocytes, increased hepatocyte injury and with more severe tissue inflammation. Stage I lesions, where PCV2 infection was largely restricted to Kupffer-cells and periportal mononuclear cell infiltrates, exhibited similar degrees of apoptosis to those of controls, whereas severe, stage III hepatitis, was associated with widely distributed hepatocyte apoptosis and PCV2 infection. The reason for the variability in the extent of apoptosis in stage II lesions is not clear but may reflect the variable inflammation observed in such lesions. The essential differences between stage II and III hepatitis were the severity of the inflammation, the viral tissue load and both parenchymal disruption and hepatocyte injury. Overall, our findings indicated that the distribution and extent of apoptosis closely maps to hepatic injury and viral infection and suggests a link between these events, in line with the findings of previous studies (Rosell et al., 2000; Hirai et al., 2003). However, given that hepatocyte necrosis was also detected in these livers, it is possible that this process may also play a part in the pathogenesis of the hepatitis (Jaeschke et al., 2004).

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Fig. 2. Photomicrographs illustrating double immunolabelling of a liver with severe porcine circovirus type 2-associated hepatitis for viral nucleic acid and for the apoptotic marker cleaved caspase-3 (CCasp3): (A) large amounts of virus (blue) and a small number of brown-staining CCasp3-positive apoptotic bodies (arrows) visible; (B) arrow in inset highlights an apoptotic hepatocyte surrounded by PCV2-positive inflammatory cells. Arrowhead points to a PCV2-positive hepatocyte that does not label for CCasp3. Scale bars: A, 50 lm; B, 25 lm.

Although double-immunostaining found that a proportion of hepatocytes exhibited both PCV2 infection and evidence of apoptosis, the majority of hepatocytes demonstrated either one or other of these processes suggesting a potentially more complex association between these processes, perhaps dependent on the stage of the cell cycle and/or on the stage of viral replication. PCV2 can replicate in and activate the transcription of pro-apoptotic proteins such as the ORF3 in both murine and porcine hepatocytes (Liu et al., 2005; Karuppannan et al., 2009). It is also possible that immune-mediated mechanisms are involved as exemplified by the expression of pro-apoptotic Fas-ligand by cytotoxic T-lymphocytes in hepatitis B in humans (Mochizuki et al., 1996; Hayashi and Mita, 1999). It must be considered that co-infections with swine HEV, PPV and/or bacteria could have contributed to the development of the hepatitis in these cases, as both HEV and PPV infection have been linked to mild hepatic lesions in pigs (Krakowka et al., 2000; Halbur et al., 2001; Paul et al., 2003; Lee et al., 2009), and dual experimental infections with PPV and PCV2 result in severe hepatic lesions (Krakowka et al., 2000). However, evidence against such a role for co-infections include (1) the fact that severe hepatitis has been induced in PCV2-inoculated gnotobiotic and conventional pigs that did not receive any other pathogen (Krakowka et al., 2001; Fenaux et al., 2002; Ladekjaer-Mikkelsen et al., 2002); (2) pigs with PMWS infected with HEV usually have mild stage I and II hepatitis and this lesion exists in 48.2% of HEV-negative animals with PMWS (Martin et al., 2007), and (3) PMWS has only sporadically been linked to infection with PPV infection under field conditions (Larochelle et al., 2003). It remains possible that other, noninfectious factors are involved in the pathogenesis of this hepatitis.

Conclusions This study suggests that apoptosis, possibly triggered by PCV2 infection and/or hepatic inflammation, plays a key role in the pathogenesis of hepatitis in pigs with naturally-occurring PMWS.

Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

Acknowledgements The authors would like to thank Merche Mora, Mónica Perez, Blanca Perez, Marina Sibila and Alex Olvera from CReSA and Unitat d’ Histologia i Anatomia Patològica, Universitat Autònoma de Barcelona for their excellent technical assistance. The first author was granted a scholarship for mobility in research from Agencia de Gestió d’Ajuts Universitaris i de Recerca (AGAUR), Generalitat de Catalunya (Spain) and the work was funded by QLRT-PL199900307 from European Commission’s Fifth Framework Programme (1998–2002) and 2-FEDER-1997-1341 of the Plan Nacional I+D (Spain).

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