(PCV2) infection in the European wild boar

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Epidemiological study on porcine circovirus type 2(PCV2) infection in the European wild boar (Sus scrofa)

Joaquin Vicente, Joaquim Segalés, Ursula Höfle, Mònica Balasch, JoanPlana-Durán, Mariano Domingo, Christian Gortázar

To cite this version:Joaquin Vicente, Joaquim Segalés, Ursula Höfle, Mònica Balasch, Joan Plana-Durán, et al.. Epi-demiological study on porcine circovirus type 2 (PCV2) infection in the European wild boar (Susscrofa). Veterinary Research, BioMed Central, 2004, 35 (2), pp.243-253. <10.1051/vetres:2004008>.<hal-00902829>

https://hal.archives-ouvertes.fr/hal-00902829

https://hal.archives-ouvertes.fr

243Vet. Res. 35 (2004) 243–253© INRA, EDP Sciences, 2004DOI: 10.1051/vetres:2004008

Original article

Epidemiological study on porcine circovirus type 2 (PCV2) infection in the European wild boar

(Sus scrofa)

Joaquin VICENTEa, Joaquim SEGALÉSb*, Ursula HÖFLEa, Mònica BALASCHc, Joan PLANA-DURÁNc, Mariano DOMINGOb,

Christian GORTÁZARa

a Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13005 Ciudad Real, Spain

b Centre de Recerca en Sanitat Animal (CReSA) – Departament de Sanitat i d’Anatomia Animals, Facultat de Veterinària, Universitat Autónoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

c Fort Dodge Veterinaria, S.A., Carretera Camprodon s/n, La Riba, 17813 Vall de Bianya, Girona, Spain

(Received 7 August 2003; accepted 18 November 2003)

Abstract – Porcine circovirus type 2 (PCV2) is considered as the causative agent of postweaningmultisystemic wasting syndrome (PMWS) in domestic pigs, where the virus is ubiquitous asevidenced by serological surveys. We present the results of the first nationwide sero-survey on thepresence of PCV2 antibodies in European wild boars, and report the first PMWS case in a wild boarfrom Spain. Sera from 656 hunter harvested wild boars from 45 different geographical sites and22 additional imported animals were analysed by means of an immunoperoxidase monolayer assay(IPMA). We also examined the tissues from 55 healthy and one diseased wild boars for the presenceof PCV2 nucleic acid and PMWS lesions by in situ hybridisation and histopathology, respectively.Additionally, abundance estimates of wild boars and field interviews were carried out on 30 samplingsites. The prevalence of medium to high PCV2 serological titres among the examined wild boarswas 47.89 ± 1.9%. Seropositive wild boars appeared in all but one of the geographical regionsanalysed. Seroprevalence and titre of PCV2 antibodies were closely related to the management ofthe wild boar populations. Wild boars from intensively managed, farm-like populations had higherprevalence than wild boars living in more natural situations. The effect of wild boar abundance andmanagement on PCV2 antibody prevalence was further evidenced by the high correlation existingbetween the relative abundance estimates of animals and the percentage of wild boars with mediumto high levels of PCV2 antibodies. PCV2 nucleic acid was detected in the tissues of three wild boars.One of these was diagnosed as PMWS. The results, in addition to information on piglet mortalities,suggest a potential role of PMWS in piglet mortality in intensively managed wild boar populations.

European wild boar / porcine circovirus type 2 / epidemiology / postweaning multisystemicwasting syndrome

* Corresponding author: [email protected]

244 J. Vicente et al.

1. INTRODUCTION

Porcine circovirus type 2 (PCV2) isnowadays considered as the etiologicalagent of postweaning multisystemic wastingsyndrome (PMWS), a recently describeddisease of nursery and growing domesticpigs [15]. The disease was initially describedin Canada [8] but, during the last sevenyears, PMWS cases have been describedworldwide, except in Oceania [21]. Thiscondition is clinically characterised bywasting, respiratory distress, pallor of theskin and, very occasionally, jaundice. Dueto the relatively unspecific clinical signs,the establishment of a final diagnosis ofPMWS is based on three different criteria[20, 24]: clinical signs (those cited above),the presence of very specific lesions in lym-phoid tissues (consisting of lymphocytedepletion and granulomatous inflamma-tion) and the presence of PCV2 in these tis-sues. The latter two criteria are very specificfor PMWS and closely associated, since amarked correlation between the severity ofthe lesions and the amount of viral antigenor genome is observed systematically [3, 10].

It is known from serological surveys thatPCV2 is ubiquitous since it is present inboth PMWS affected and non-affectedfarms [14], with a serological prevalenceclose to 100% in finishing pigs worldwide[21]. Furthermore, it is known that the wildboar (Sus scrofa) is also susceptible toPCV2 infection [19]. This latter data alsoapplies to Spain, where a 34.6% PCV2 sero-prevalence was obtained using a limitednumber of sera of wild boars from the Cata-lonian region [22]. Moreover, the first caseof PMWS in a free living European wildboar was recently described in Germany[23], which clearly indicates that both thedomestic pig and the wild boar are suscep-tible not only to PCV2 infection but also tothe development of PMWS. The Europeanwild boar is the most widely distributedungulate in the Spanish mainland, and itsrange and densities have largely increasedin the last three decades [6, 18]. Due to itsabundance, it is also one of the most popular

game species. In order to increase huntingharvest, wild boar populations are increas-ingly managed by high-wire fencing, arti-ficial feeding, and restocking with farmbred individuals. As a result, some wildboar hunting estates resemble extensive pigbreeding facilities, with high densities butalmost no sanitary care. These changes inwildlife management have already risenconcerns regarding the control of infectiousand parasitic diseases [4, 7], and may alsoaffect PCV2 epidemiology.

Thus, considering the ubiquitous distri-bution of PCV2 among domestic pig pop-ulations, and the known risk factors forPMWS in domestic pigs such as poorhygiene and crowding [21], we hypothe-sised (1) that the seropositivity to PCV2 ishigher in intensively managed wild boarpopulations than in more natural ones and(2) that cases of PMWS may eventuallyoccur in farm-like situations, affecting thehunting harvest. Hence, we undertook anation-wide sero-survey for PCV2 antibod-ies in wild boars, tested a sub-sample ofthem for PMWS by in situ hybridisationcombined with histopathology, estimatedwild boar abundance using a faecal pellet-group survey, and examined game manage-ment techniques through direct interviews.

2. MATERIALS AND METHODS

2.1. Sampling sites

Between 2000 and 2003, samples werecollected from 656 hunter-harvested wildboars in 45 Spanish localities (samplingsites) (Fig. 1). Sampling took place duringthe regular hunting season, from Septemberto March. Sampled hunting areas range insize from 408 to 52 000 hectares (ha),means 9 212 ± 18 008 ha for open areas,4 036 ± 3 157 ha for fenced areas, and1 366 ± 1 069 ha for intensively managedones. The number of boars shot on onehunting day ranged from 1 to 120 (means

Epidemiology of PCV2 in the wild boar 245

Figure 1. Map of peninsular Spain showing the sampling sites, the number of sera analysed per siteand the prevalence of wild boars with medium to high titres of antibodies against PCV2 (percentageof grey colour in relation to the whole circle). Dots represent sampling sites where less than10 samples were obtained.


3.43 ± 2.04, 18.89 ± 28.69, 40.33 ± 37.16for open, fenced and intensively managedestates, respectively). If more than 20 wildboars were available for sampling, we arbi-trarily selected a random age and sex strat-ified subset of between 15 and 30 animals,depending on time availability and logisticconstraints.

Sampling sites are representative of thebiogeographically most relevant landscapeunits in peninsular Spain, with a biastowards the Mediterranean shrub-lands ofthe central and southern regions, wherehunting activities are most important [5].Thus, sampling sites were divided into sixgeographic regions, named Asturias, EbroBasin, Guadiana, Jaén, Montes de Toledo,and Sierra Morena (Tab. I). Mediterraneanshrub-lands are dominant in both moun-tainous central areas, Montes de Toledo andSierra Morena. The Guadiana area consistsof a fragmented Mediterranean habitat andis a transition between Montes de Toledoand Sierra Morena. These three areas arecharacterised by large densities of wildboars due to intensive big game manage-ment. Jaén is representative of the Beticmountain chains, which are located insouth-western Spain and are characterisedby extensive olive groves and a lack ofintensive big game management. On thecontrary, the Atlantic climate is representedby Asturias, a mountainous coastal regionfrom the north of Spain, where no feed issupplemented due to the existing conflicts

caused by the damage of wild boars to cro-plands. The last area is the Ebro Basin, withpredominance of Mediterranean agro-eco-systems and diversity of management.

For each sampling site, we recorded themanagement conditions to which wildboars were subjected: open (164 samplesfrom 16 sites), fenced (388 samples from22 sites), or intensively managed areas withfencing, translocations and artificial feed-ing (104 samples from 7 sites).

2.2. Interviews and abundance estimates

We visited 30 of the sampling sites inSeptember 2002, immediately before thehunting season, in order to carry out inter-views regarding game management, andobtain field estimates of wild boar relativeabundance based on dropping counts. Noabundance estimates were carried out onsites sampled after September 2002. Througha personal interview, we obtained data fromgamekeepers regarding the presence/absenceof fencing, artificial feeding, and transloca-tions of wild boars in the respective estate.We asked if any unusual disease outbreaks,especially in piglets, were recorded.

Artificial feeding was used in four out ofseven open sites, in three of these sites itwas only used previously to hunting days toattract boars. The other estate supple-mented wild boars throughout the year.Seven out of 18 fenced estates fed boarspreviously to hunting and four out of 18 did

Table I. Number of sampling sites, number of analysed sera (N), number of sera with medium to highantibody titres against PCV2 (No. positive), prevalence and 95% confidence intervals for 678 wildboar sera.

Area No. sites N No. positive Prevalence (%) 1.96 S. E. (95% C.I.)

Asturias 3 30 7 23.33 13.72Ebro Bassin 4 24 12 50 20.384Guadiana 9 124 43 36.67 8.46Jaén 1 13 0 0 0M. Toledo 15 277 156 58.42 5.905S. Morena 13 188 96 51.06 7.056Total 45 656 314 47.87 1.91


it across the year. Finally, all but onefarm-like managed estates indicated artifi-cial feeding over the year, and the remain-der one only supplemented in the summer(the limiting season from the point of viewof food resources in Mediterranean habi-tats). Sanitary management was restrictedto the oral administration of parasiticides(ivermectin) in food in one out of 18 fencedestates and three out of five intensive man-aged estates. No use of vaccines nor antibi-otics was recorded through the interviews.In one occasion we were able to collectsamples from 22 wild boars that had beenillegally imported to a fenced hunting estatefrom two French farms. No other restockingof wild boars was reported in any studyestate. Only in one fenced estate the possi-bility of hybridisation between wild boarsand domestic pigs was reported in the inter-views. The co-existence of wild boars withfree roaming domestic pigs was not com-mon in our study areas.

Since ungulate droppings tend to beaggregated [16], we developed a simplemethod using dropping frequency (insteadof their number) to estimate the relativeabundance of European wild boars. Briefly,each count consisted of n = 40 transects of100 m, divided into 10 sectors of 10 m inlength. Dropping frequency was defined asthe average of the number of 10 m sectorswith wild boar droppings in each transect of100 m (DF = ΣDi/n; where “D” is the numberof dropping-positive sectors and rangesfrom zero to ten, and “n” is the number of100 m transects, usually 40).

There were statistical differences in wildboar abundance indexes between samplingsites depending on the management type.Open hunting areas (n = 7) had a DF of0.05 ± 0.05, fenced hunting areas (n = 18)had 0.44 ± 0.2, and intensively managedones (n = 5) had 1.48 ± 0.8 (F1, 29 = 16.76,P < 0.001, n = 30).

2.3. Field necropsies

Each hunter-harvested wild boar (n =656) was measured, weighed, and its sex

determined. Thus, 348 females and 277 maleswere identified, but no sex was recorded for31 of the sampled animals. Based on tootheruption patterns, boars less than 7 monthsold were classified as piglets, boars between7 and 12 months were classified as juve-niles, those between 12 and 24 months assub-adults, and those over 2 years as adults[17]. We estimated the fat condition usingthe kidney fat index (KFI) [12]. Samples ofselected organs were collected and fixed byimmersion in 10% neutral-buffered forma-lin. One wild boar piglet was found dead inan intensively managed hunting estate andprocessed as the hunter-harvested ones.Blood samples were collected from theheart during necropsy. Serum was obtainedafter centrifugation and stored at –20 °Cuntil use.

2.4. Immunoperoxidase monolayer assay (IPMA)

A previously published protocol to detectPCV2 antibodies by means of an IPMAtechnique [13] was applied on 656 serafrom the necropsied Spanish wild boars andthe 22 imported ones. Serum dilutions werefrom 1:20 to 1:20 480, making serial four-fold dilutions. For statistical analyses, sero-logic results were grouped as negative orlow (titre below 1:320) or medium to high(titre equal to or higher than 1:320) titres.

2.5. Histopathology and in situ hybridisation

Formalin-fixed samples of lung, lymphnodes, liver, spleen and/or kidney of a sub-sample of eight piglets, 13 juvenile, 23 sub-adult and 12 adult wild boars were dehy-drated, embedded in paraffin wax, sectionedat 4 µm, and stained with haematoxylin andeosin (HE). Correlative tissue sections con-taining the same tissues were placed onProbe On Plus glass microscope slides(Fisher Scientific, Pittsburgh, USA) inorder to perform an in situ hybridisation


technique to detect PCV2 nucleic acidfollowing previously published protocols[15].

2.6. Statistical analyses

Confidence intervals for standard errorsof prevalence were estimated with theexpression S.E.95%C.I. = 1.96[p(1 – p)]/n1/2

[11]. To test for significant differencesin PCV2 seroprevalence between sex, age-classes, and degree of management andinteractions, log-linear analysis of contin-gency tables were employed, reporting thepartial χ2 values from a saturated model [2].We treated both age and management degreesas categorical variables with three classesas described above. An additional numberof analysed piglets were not included due tothe low sample size (n = 20). We also usedSpearman non-parametric correlations tostudy the relationship between density andthe probability of having medium to highantibody titres (as continuous variables)and between the PCV2 antibody titres andKFI. ANOVA was employed to study theeffect of management type on wild boardensity. The level of significance wasestablished at the 5% level.

3. RESULTS

Medium to high titres of antibodies toPCV2 were found in 332 of the 678 ana-lysed sera (49 ± 1.88% of the total sample).Regarding only Spanish wild boars, mediumto high titres of antibodies to PCV2 werefound in 314 of the 656 sera (47.9 ± 1.91%of the total sample). Prevalence differedamong geographical regions as shown inTable I. Figure 1 shows the percentage ofsampled wild boars with medium to highPCV2 antibody titres. As shown in Figure 1,only three out of 35 sites with at least10 sampled animals had no wild boars withmedium or high antibody titres. A highprevalence of medium to high titres wasfound in the imported farmed wild boarsfrom France (81.8 ± 16%).

The proportion of animals with mediumto high titres of antibodies differed signifi-cantly between management condition types(prevalence: partial χ2 = 19.87, P < 0.001).No significant differences were foundbetween the sex or age classes (Fig. 2).Thus, independently of the sex and agestructure, intensively managed wild boarshad the highest values, while open popula-tions had the lowest ones. Juveniles fromintensively managed hunting estates showedan extremely high seropositivity as com-pared to juveniles from fenced or open pop-ulations (Fig. 3). No relationship was foundbetween the kidney fat index and PCV2antibody titres.

The effect of wild boar abundance andmanagement on PCV2 antibody prevalencewas further evidenced by the high correla-tion existing between the relative abun-dance estimates based on dropping counts,and the percentage of wild boars withmedium to high levels of PCV2 antibodies(Spearman correlation, rs = 0.55, P < 0.001,n = 30; Fig. 4).

Only 20 piglet sera were analysed: 12from open populations (all with no or lowPCV2 antibody titres), 5 from fenced estates(one of them with a medium to high titre),and only one from an intensively managedestate, also with a medium to high titre.Even though the probability of samplingpiglets was similar in the three managementtypes, the actual number of sampled pigletswas lower than the expected number in theintensively managed estates (Chi squaretest, χ2 = 19.33, 2 d.f., P < 0.001). In theinterviews, three out of five intensivelymanaged estates reported increased pigletmortality. Dead or sick piglets in these estateswere found mainly at feeding or wateringplaces. In two of these estates the rangersreported that almost no piglets were seen atthe feeding sites or during spotlighting sur-veys. In contrast, no observations regardingincreased piglet mortality (nor lack of pigletsightings) were recorded in the 25 inter-viewed open or fenced hunting estates. Fora subset of piglets from which KFI was


available (n = 17), no correlation was evi-denced between the PCV2 titre and KFI.

One out of 56 wild boars had markedlymphocyte depletion with moderate histi-ocytic infiltration of lymphoid tissues, whichare the hallmark lesions for PMWS. Thisanimal was a 6-month-old piglet showingwasting that had been found dead in anintensively managed enclosure from Toledo

province, central Spain. No serology wasavailable for this animal. A marked amountof viral genome was detected in the tissuesof this animal; the labelling was mainlylocated in the cytoplasm of macrophage-like cells in the lymph nodes, spleen, lungand liver, while a low amount of the PCV2genome was also detected within the cyto-plasm of epithelial tubular cells of the

Figure 2. Percentage (and 95% C.I.) of wild boars with medium to high antibody titres againstPCV2, depending on their age and sex. Piglets are not included.

Figure 3. Percentage (and 95% C.I.) of wild boars with medium to high antibody titres againstPCV2, depending on their age and on the management type of the sampling site.


kidney. Two more wild boars, juvenile indi-viduals that had been shot, showed PCV2nucleic acid in their tissues. One of themcame from an intensive enclosure in Mon-tes de Toledo and the other came from afenced estate in Sierra Morena. They had avery low amount of viral nucleic acid withinthe cytoplasm of cells in the lymphoid fol-licles (dendritic-like cells) of lymph nodes;no labelling was observed in other tissuesof these animals. It is remarkable that bothindividuals had high titres to PCV2(1:20 480). The rest of the 53 wild boars hadnegative results by in situ hybridisation.

Other microscopic lesions observed inthe studied group of animals were a variabledegree of hyperplasia of the broncho-alve-olar lymphoid tissue (n = 18; the presenceof nematodes with morphological charac-teristics typical of Metastrongylus spp. wereobserved associated to these lesions), tuber-culosis-like granulomatous lesions, consist-ing of a necrotic centre (mineralised or not)surrounded by epithelioid and scatteredmultinucleated giant cells in the lymphnodes, lung and/or liver (n = 14), catarrhal-purulent bronchopneumonia (n = 8) andfibrino-necrotising pleuropneumonia (n = 1).

4. DISCUSSION

This paper presents the first large-scalestudy of PCV2 infection in European wildboars. The high seroprevalence to PCV2,along with its widespread geographical dis-tribution, suggest an endemic status ofPCV2 in the wild boar in Spain. This is inagreement with a previous report that alreadyshowed the PCV2 antibody presence innorth-eastern Spanish wild boars [22].Moreover, we report the first confirmedPMWS case in a Spanish wild boar, furtherconfirming that European wild boars candevelop PMWS [23].

The three localities with no animals withmedium to high titres were placed inthe northern, southern, and south-westernextremes of the study area, and corre-sponded to open, low density wild boarpopulations. In contrast, high titres werefrequently observed in the Mediterraneanwoodlands of Montes de Toledo and SierraMorena, where wild boars are abundant,populations are not fragmented, and man-agement is often intensive. Indeed, the per-centage of wild boars with medium to highantibody titres was maximal in intensively

Figure 4. Mean seroprevalence of medium to high PCV2 antibody titres, plotted against abundanceindexes (mean number of droppings every 100 m in 4 km transects) in 30 wild boar populations.

open, fenced, intensively managed.


managed populations. This fact shows thatmanagement conditions have a clear effecton PCV2 prevalence in wild boars. Thisdensity dependence was expected since theseroprevalence in domestic swine rearedunder intensive conditions is also extremelyhigh [14]. Moreover, since wild boar man-agement conditions are becoming increas-ingly intensive in Spain, the risk of increasingPCV2 prevalence in wild boars is alsogrowing; this situation also applies to manyEuropean countries [18], since natural wildboar populations are increasing.

In our opinion, while the wild boar isconsidered a potential reservoir of virusesfor the domestic swine, such as classicalswine fever virus, Aujeszky disease virusand bovine viral diarrhoea virus [1, 9, 25,27], it is likely that this situation is the con-trary for PCV2.

The PMWS affected animal was a 6-month-old wild boar within the age cate-gory of piglets. However, the presentationof PMWS in the domestic swine is around2 to 4 months of age [21], while it is rare orvery rare at slaughter age, which would bethe equivalent age for this diseased wildboar piglet. The same situation applied tothe already described case of PMWS ina German wild boar [23] which was a10-month-old juvenile; histopathologicallesions observed in both cases were identi-cal to those described in domestic pigs suf-fering from PMWS. At present it is notpossible to assess the reasons for the differ-ence in age presentation. Living conditions,age of infection and infection load withPCV2 are potential factors that influencethe progress of the disease resulting fromthis viral infection. Furthermore, it isknown that a non-specific activation of theimmune system and concurrent viral/bacte-rial infections can trigger or worsen PMWSpresentation [21]. Taking into account thegeneral histopathological results of thestudied wild boars, where evidence ofbacterial and parasitic infections occurred,it would not be surprising that these con-comitant processes may influence the final

outcome of PCV2 infection towards thedevelopment of PMWS in certain animals.Also, an inverse relationship may occursince PCV2 infection may trigger the devel-opment of other processes, such as tubercu-losis. However, further studies on youngwild boars, and especially in those with agecomparable to the natural occurring PMWSin domestic swine, are needed.

The present study was based on hunter-harvested animals and not on clinical cases,except for the piglet with PMWS. There-fore, it was not surprising to detect only fewindividuals with PCV2 nucleic acid in theirtissues, although in low amounts. This latterfinding probably represents a subclinicalinfection and/or a recovery phase of the dis-ease, as suggested for domestic swine witha similar amount of viral nucleic acid inlymphoid tissues [20]. A third possibilitysuggested for the domestic pigs, an initialphase of infection, is probably non-applica-ble since most of the cases were older thanseven months of age.

Piglets are less interesting to hunters dueto their small size and trophy, and becauseSpanish hunting regulations forbid shoot-ing “reproducing animals”. Thus, wild boarpiglets are less frequently shot than elderage classes. Although the number of wildboars with definitive diagnosis of PMWS isminimal, three facts suggest that PMWSmight play a role in the population dynam-ics of intensively managed populations:first, fewer piglets than expected were sam-pled from intensively managed areas; sec-ond, juveniles from these conditions had anextremely high seroprevalence (suggestingrelatively recent past PCV2 infection),which contrasts with data from other man-agement types; and third, a number of thesefarm-like areas reported abnormal pigletmortalities. However, if PMWS is of impor-tance in wild boar piglets, the control of thedisease would be very difficult in this pop-ulation since the control of the disease indomestic pig farms is carried out throughall in-all out management, strict hygieneand other zootechnical measures [21]. These


options are very difficult to apply in inten-sively managed wild boar populations, andalmost impossible in non-intensively man-aged ones.

The main weaknesses of this study arethe lack of information from part of conti-nental Spain, including the north-west andthe whole Mediterranean coast, and thelimited information on piglets less thanseven months of age, which are most likelyto develop PMWS. Moreover, retrospec-tive studies on old wild boar sampleswould be needed to elucidate the temporalevolution of PCV2 presence in Spanishwild boar populations. The abundance indexused in this study needs a validation regard-ing factors such as habitat, time of the yearand age and sex related differences in def-ecation behaviours, that exceeds the frameof this survey.

Our study highlights the need to includeall host classes and management situationsin the sampling of wildlife species for epi-demiological surveys [26]. In order toimprove our knowledge on PCV2 epidemi-ology, and particularly in order to deter-mine the importance of PMWS as a mortalityfactor in wild boar piglets, future studiesshould pay special attention to the pigletage-class and should consider radio-tag-ging in order to permit true mortality esti-mates and a prompt retrieval of the carcasses.

ACKNOWLEDGEMENTS

The authors thank Isabel G. Fernández-de-Mera, Fran Ruiz and Diego Villanúa for theirassistance in the field, Mónica Pérez, fromCReSA, for technical assistance, Pelayo Acevedofor producing the map, and Daryl Williams forreviewing the written English language. Part ofthis work has been a contribution derived fromthe agreements between Yolanda Fierro andUCLM, and between CSIC and Principado deAsturias. The study was supported by projectAGL2001-3947, Ministerio de Ciencia y Tec-nología and FEDER and partly funded by theProject QLRT-PL-199900307 from the FifthFramework Programme 1998–2002 of the Euro-pean Commission.

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(PCV2) infection in the European wild boar