in wild boar (sus scrofa) - BioOne

Journal of Wildlife Diseases, 48(3), 2012, pp. 612–618 # Wildlife Disease Association 2012

PREVALENCE OF INFECTION AND GENETIC DIVERSITY OF PORCINE CIRCOVIRUS TYPE 2 (PCV2) IN WILD BOAR (SUS SCROFA) IN POLAND Michał Fabisiak,1,4 Anna Szczotka,2 Katarzyna Podgo´rska,2 and Tomasz Stadejek2,3 1 Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences–SGGW, Nowoursynowska 159c, 02-776 Warsaw, Poland 2 Department of Swine Diseases, National Veterinary Research Institute, Aleja Partyzanto´w 57, 24-100 Puławy, Poland 3 Current address: Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences–SGGW, Nowoursynowska 159c, 02-776 Warsaw, Poland 4 Corresponding author (email: [email protected])

ABSTRACT: Porcine circovirus type 2 (PCV2) is a widespread, important pathogen of domestic swine and the causative agent of postweaning multisystemic wasting syndrome and other diseases and conditions referred to as ‘‘porcine circovirus diseases.’’ Specific antibodies and DNA to PCV2 have also been detected in European wild boars and North American feral pigs. We collected 312 tonsil samples from wild boars shot in 13 of 16 districts of Poland, and tested them for PCV2 DNA using a real-time PCR. We detected PCV2 DNA in 75.6% of tested tonsils, and in particular, in 60% of samples from the 2006–07 season, and 91% from 2007–08. The phylogenetic analysis that included 12 PCV2 sequences from wild boars revealed that they belonged to two genetic clusters, PCV2b and PCV2a. We present data on prevalence of PCV2 in Polish wild boars and for the first time report the PCV2a genotype in Poland. Key words: Geographic distribution, PCV2, phylogenetic analysis, Poland, Sus scrofa, wild boar.

circovirus type 2, a member of the Circoviridae, is a small, icosahedral, nonenveloped virus that contains a singlestranded circular DNA genome of 1.76 kb (Hamel et al., 1998). The virus is ubiquitous in domestic swine populations with antibody prevalences reaching almost 100%. Porcine circovirus type 2 causes postweaning multisystemic wasting syndrome (PMWS) in domestic pigs (Allan et al., 1998) and other diseases and conditions referred to as ‘‘porcine circovirus diseases’’ (Allan et al., 2002). Diagnosis of PMWS is based on clinical signs (progressive weight loss, respiratory distress, diarrhea, and pallor of the skin), histopathologic lesions in lymphoid tissues (lymphocyte depletion, granulomatous inflammation, or both), and the presence of abundant PCV2 in these lesions (Sorden, 2000). In wild boar populations prevalence of antibody to PCV2 was 50% in Spain (Vicente et al., 2004; Ruiz-Fons et al., 2006) and 43% in the Czech Republic (Sedlak et al., 2008) and PMWS in wild boars has been described in many countries including the USA (Ellis et al., 2003), Brazil (Correa et al.,

INTRODUCTION

In recent years there has been a growing interest in the role of wild boar (Sus scrofa L.) populations in the epidemiology of important infectious diseases of swine (Sus domestica; Corbet and Hill, 1992). This interest occurs due to an increase in the wild boar population density worldwide, leading to a higher probability of disease transmission. In many European countries wild boar population density is high (Ruiz-Fons et al., 2008). In Poland, in the last few years the size of the wild boar population has doubled, from around 118,000 in 2000 to almost 251,000 in 2009 (Central Statistical Office, 2009). Many pathogens are shared by wild boars and domestic swine, including classical swine fever virus (Paton and GreiserWilke, 2003), African swine fever virus (Jori and Bastos, 2009), Aujeszky’s disease virus (Vicente et al., 2005), porcine reproductive and respiratory syndrome virus (Reiner et al., 2009), and porcine circovirus type 2 (PCV2; Ruiz-Fons et al., 2006). Porcine 612

FABISIAK ET AL.—PREVALENCE AND DIVERSITY OF PCV2 IN WILD BOAR IN POLAND

2006), Germany (Schulze et al., 2004), Croatia (Lipej et al., 2007), Greece (Sofia et al., 2008), and Italy (Petrini et al., 2009). We estimated the prevalence of PCV2 infection in Polish wild boars based on real-time PCR and evaluated phylogenetic relationship of PCV2 from wild boars and domestic pigs in Poland. MATERIALS AND METHODS

We collected tonsil samples from wild boars shot in different regions of Poland; 155 tonsils were collected during the 2006–07 hunting season and 157 tonsils during the 2007–08 hunting season. Collections took place at a game purchasing facility where animals hunted in different regions of the country are submitted for further processing. We recorded the geographic origin for each sample. Sections of tonsils of about 0.5–2.0 g were homogenized in a high-speed homogenizer (3620, CAT, Rose Scientific Inc., Edmonton, Canada), at 13,500 rpm, room temperature for 2 min in sterile physiologic saline to obtain 10% homogenates. The samples were then centrifuged at 2,127 3 G for 10 min at 8 C, in a Hettich Rotixa 120R centrifuge (GMI, Inc., Ramsey, Minnesota, USA). Supernatants were collected and stored at 270 C until further analysis. Total DNA was extracted from 250 ml of tonsil homogenates using a genomic kit (A&A Biotechnology, Gdynia, Poland), following the manufacturer’s instructions. Extracted DNA was amplified with SYBR Green I–based realtime PCR using the Stratagene Mx3005PH system and MxPro–Mx3005P v3.20 software (Agilent Technologies, Santa Clara, California, USA). All reactions were run in MicroAmp optical tubes closed with optical caps (Applied Biosystems, Carlsbad, California, USA). Primers ORF2.PCV2.S4 and ORF2.PCV2.AS4 (Ouardani et al. 1999) were used to amplify a 494-nucleotide fragment of a cap (ORF2) gene encoding capsid protein. Reactions were carried out in 25 ml of reaction mixture containing 1 ml of DNA, 13 Quantitect SYBR Green PCR master mix (Qiagen, Hilden, Germany), 10 pmol of each primer and nuclease-free water. Thermal protocol consisted of polymerase activation at 95 C for 15 min and 40 cycles including denaturation, primer hybridization, and elongation (30 sec/95 C, 30 sec/50 C, 30 sec/72 C) and final elongation for 5 min at 72 C. The results were expressed as threshold cycle (Ct) values. Twelve PCR products with Ct values below 36 collected

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during the 2006–07 hunting season were purified with the QIAquick PCR purification kit (Qiagen) and sequenced with the same primers as used for PCR. The sequences were analyzed with the programs from Lasergene 7 program package (DNAStar Inc, Madison, Wisconsin, USA) and CLC Free Workbench software version 3.2.3 (CLC Bio, Aarhus, Denmark). RESULTS

Tonsil samples were collected from 312 wild boars shot in 13 of 16 districts of Poland. Information on the origin of 58 samples (31 from 2006–07 season, and 27 from 2007–08) was not available. We detected PCV2 DNA in 236 of 312 tonsils tested (75.6%), including 93 of 155 (60%) tonsils tested from the 2006–07 season and 143 of 157 (91%) from the 2007–08 season. The Ct values ranged from 15.05 to 39.68. The geographic origin of the samples and the number of positive samples in the 2006–07 and 2007–08 seasons are presented in Table 1. From three districts (Zachodniopomorskie, Warmin´sko-Mazurskie and Dolnos´la˛skie) in both hunting seasons, 13 or more samples were collected allowing analysis of changes in PCV2 prevalence. In all three districts the proportion of PCV2-positive wild boars was significantly higher in 2007–08 than 2006–07. Phylogenetic analysis of the 12 sequenced amplicons (Fig. 1) revealed that they belonged to two genetic clusters, PCV2b and PCV2a (Segales et al., 2008). The identity between the sequences from wild boars clustering with different genotypes (PCV2a and PCV2b) was 88.9– 89.9%, whereas the sequences from wild boars within PCV2b and PCV2a clusters showed 99.1–100% and 99.1–99.6% identity, respectively. The majority of sequences belonged to PCV2b genotype previously detected in domestic pigs in Poland (Podgo´rska and Stadejek, unpubl. data). The sequences of isolates D4, D7, and D12 were clearly different and clustered together with the reference sequences of PCV2a. No such strains were ever detected in

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JOURNAL OF WILDLIFE DISEASES, VOL. 48, NO. 3, JULY 2012

TABLE 1. Geographic origin (district) and a number of wild boars (Sus scrofa) in Poland sampled and tested for DNA to porcine circovirus type 2 during two hunting seasons. The right column contains estimated population of wild boars in respective districts (Central Statistical Office, 2009). Season 2006–07a

a

Season 2007–08a

Total

Positive

Total

Positive

District

Wild boar population (2009)

54 20 13 17 7 3 6 1 1 1 1 — — — — — 31

37 11 6 8 3 3 2 1 1 1 0 — — — — — 20

37 16 24 4 17 18 5 4 — — 2 1 2 — — — 27

35 16 20 3 15 17 5 3 — — 2 1 2 — — — 24

Zachodniopomorskie (1) Dolnos´la˛skie (2) Warmin´sko-Mazurskie (3) Wielkopolskie (4) Lubuskie (5) Opolskie (6) Kujawsko-Pomorskie (7) Podlaskie (8) Ło´dzkie (9) Mazowieckie (10) Pomorskie (11) S´la˛skie (12) Podkarpackie (13) Lubelskie (14) Małopolskie (15) S´wie˛tokrzyskie (16) Unknown

35,900 22,900 23,800 24,100 16,400 10,600 10,800 9,200 7,200 13,500 16,700 9,000 7,700 12,800 4,000 3,700 —

Dashes indicate no boars were sampled.

domestic pigs in Poland (Podgo´rska, 2008). The geographic origin of samples that were subjected to sequencing and formed the phylogenetic tree is shown in Figure 2 (as available). DISCUSSION

Most of the published data on PCV2 in wild boars is based on serology but four studies analyzed the presence of PCV2 DNA in samples collected from wild boars. In Hungary, Csagola et al. (2006) analyzed 307 organ samples and found 20.5% positive for PCV2 DNA. In Germany, Knell et al. (2005) tested 238 samples of spleen from wild boars and 43 (18.1%) were positive. Also Reiner et al. (2010) analyzed 349 samples from German wild boars and found 63.1% (by standard PCR) and 45.4% (real-time PCR) PCV2 positive. The most recent study describing PCV2 prevalence in wild boar populations by real-time PCR came from Romania (Cadar et. al., 2010), where 13.5% of 469 samples from wild boars from the Transylvania region were positive. The

proportion of PCV2-positive wild boars identified in our study (76.6%) was distinctly higher. In the study by Reiner et al. (2010), the prevalence of PCV2 ranged from 12.5% (state of Berlin and Schleswig-Holstein) to 100% (Lower Saxony and Brandenburg). Also in the study from Romania (Cadar et al., 2010) the prevalence of PCV2 infection varied widely, from 7% to 30%, among counties. In seven, mostly western and northern, districts of Poland from which the number of samples tested ranged between 11 and 91, the prevalence of PCV2 was 52.4% to 95%. We observed a much higher proportion of PCV2-positive wild boars in the 2007–08 season (91%) than in 2006–07 (60%). This increase was evident when comparing the total number of samples analyzed but also when comparing the results from the three major hunting districts with highest wild boar populations, which were represented by a relatively large number of samples. In Zachodniopomorskie there was an increase from 69% (37 positive of 54 samples tested) to 95% (24 of 27); in Dolnos´la˛skie, from


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FIGURE 2. Geographical origin (district) of Polish wild boars (Sus scrofa) from which porcine circovirus type 2 (PCV2) was amplified and sequenced. Sequence designations as in Figure 1 (without a year of isolation); PCV2b isolates are marked with black dots, and PCV2a isolates are marked with white squares. Isolates D6, D10, and D12 are not shown because of their unknown geographic origin. Numbers in parentheses and italics refer to the districts in Table 1. Shaded districts are those from which more than 10 wild boars were sampled.

FIGURE 1. Phylogenetic tree based on partially sequenced cap gene of 12 porcine circovirus type 2 (PCV2) isolates derived from wild boars (Sus scrofa) D1–D12 (hunting season indicated) and selected PCV2 sequences from domestic pigs (Sus domestica; country and year of isolation indicated). Accession numbers of PCV2 sequences obtained from wild boars: D1 GU731181, D2 GU731181, D3 GU731181, D4 GU731181, D5 GU731181, D6 GU731181, D7 GU731181, D8 GU731181, D9 GU731181, D10 GU731181, D11 GU731181, D12 GU731181. Accession numbers of PCV2 sequences obtained from Polish domestic pigs: PL1 EU444004, PL2 EU444005, PL3 EU444006, PL4 EU444007, PL5 EU444008, PL6 EU444009, PL7 EU444010.

55% (11 of 20) to 100% (16 of 16); and in Warmin´sko-Mazurskie, from 46% (6 of 13) to 83% (20 of 24) in 2006–07 and 2007–08, respectively. These regions have the largest wild boar populations in Poland (Table 1). Increasing prevalence of PCV2 in wild boars also can be suspected in at least some states of Germany. Knell et al. (2005) detected 18.1% (43 of 238) PCV2-positive wild boars in the states of RheinelandPalatinate and Hesse in samples from the 2002–03 season. In samples collected in the same regions of Germany in the seasons 2004–05 to 2006–07 by Reiner et al. (2010) 39% of 45 wild boars from RheinelandPalatinate were PCV2-positive, as were 50% of 40 samples from Hesse. The differences between the various countries in prevalence of PCV2-positive wild boars may indicate true variation in the virus’ distribution in wild boar populations or may be because different tissues were used for the analyses. In the studies

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of Csagola et al. (2006), Knell et al. (2005), and Cadar et al. (2010) the tissues analyzed were only spleen in the study by Knell, and kidneys, liver, spleen, and lymph nodes in two other studies. In the study by Reiner et al. (2010), tonsils were included in the analysis, as well as lungs, spleen, and lymph nodes. In our study, the tonsils were the only tissue analyzed. High prevalence of PCV2 infection identified in tonsil samples also raises the question of how long PCV2 can persist in this tissue, and what the impact could be on sustaining infection and spreading the virus in populations of wild boars or domestic pigs. In domestic pigs the phylogenetic analysis of isolated PCV2 sequences is of great importance for molecular epidemiology, allowing following of genetic change in circulating strains, and possibly the emergence of new strains. Two main phylogenetic groups of PCV2 have been recognized, PCV2a and PCV2b. The PCV2a strain predominated on pig farms with and without PMWS in some countries from 1997 until 2003. After 2003 PCV2b strains became more common in outbreaks of PMWS in North America and some European countries (Segales et al., 2008). The third genotype, PCV2c, is only represented by three sequences obtained from Denmark from samples from the 1980s (Dupont et al., 2008). Recently the new PCV2d genotype was reported from China (Guo et al., 2010). In previous papers concerning the sequence analysis of PCV2 from wild boar the isolates were found to be identical or very similar to those previously identified in domestic swine in Slovenia (Toplak et al., 2004), Germany (Schulze et al., 2004; Knell et al., 2005), and Hungary (Csagola et al., 2006). However, one of the isolates from Germany (Knell et al., 2005) was closely related to a Chinese isolate from domestic pig, and the second to an isolate from French domestic pigs. In Hungary, some isolates had the closest relationship with a Canadian isolate, and the others were similar to a German isolate. In Greece, Sofia et al. (2008) showed that

the two Greek strains from wild boars were closely related to each other and were clustered with two isolates from wild boars from Hungary. Csagola et al. (2006) and Sofia et al. (2008) postulated that observed genetic similarities suggest an epidemiologic link between wild boar and domestic pig populations. However, in recent studies, no direct evidence was found for PCV2 transmission between domestic pig and wild boar (Reiner et al. 2010). We provided the first report of the PCV2a genotype in Poland. Previously we showed that all analyzed PCV2 strains from diseased and healthy domestic pigs in Poland belonged to the PCV2b genotype (Podgo´rska, 2008). Nine of the wild boar sequences from PCV2b showed very high identity to those from Polish domestic pigs. The three wild boar sequences that belonged to PCV2a showed highest similarity to the isolate from Hungary. Taken together these findings suggested that the PCV2 strains in wild boars in Poland are more diverse than those from domestic swine and future discovery of new PCV2 variants can not be excluded. Taking into account that not all of the districts of Poland were included in this study, potentially even higher diversity in PCV2 in wild boar could be expected. At present the role of wild boars as PCV2 vectors to domestic pigs is unknown but the occurrence of novel variants described elsewhere in domestic pigs, sometimes causing acute disease, makes further monitoring of the PCV2 evolution in both species highly important. According to Firth et al. (2009) the rate of nucleotide substitution for PCV2 is the highest yet recorded for a single-stranded DNA virus, and this high rate of evolution may allow PCV2 to maintain evolutionary dynamics closer to those of single-stranded RNA viruses than to those of doublestranded DNA viruses. Despite the high prevalence of PCV2 in wild boars no clinical cases of PMWS or other PCV2-associated diseases in Polish wild boars have been described. Cases of PMWS have been identified in several


countries and included farmed wild boars in North America (Ellis et al., 2003) and Brazil (Correa et al., 2006) and free-living animals in Croatia and Greece (Lipej et al., 2007; Sofia et al., 2008). Based on analysis of the sequences obtained from wild boars and domestic pigs, the diversity of PCV2 in Polish wild boars could be higher than in domestic pigs. High similarity of several wild boar– derived sequences to others obtained from Polish pig farms suggests that epidemiologic relationships could exist between these populations. More intensive and systematic investigations are needed. ACKNOWLEDGMENTS

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