A picture of trends in Aujeszky’s disease virus exposure in wild boar in the Swiss and European contexts

Study area

We selected five different study units (A-E, Fig. 1) in Switzerland (41,284 km
²
) with the aims of: (1) covering the main wild boar habitat; (2) including northern
and southern wild boar populations; (3) covering all representative bioregions of
Switzerland, i.e. i) the Jura mountains (approx. 4,307 km
²
), shaped by forests and pastures, ii) the densely populated Swiss Plateau (approx.
11,168 km
²
), iii) the Alps (approx. 23,000 km
²
), of which a large part reaches altitudes above the timber line, and iv) the part
of Ticino located south from the Alps (approx. 2,812 km
²
); (4) covering most of the Swiss border to France, Germany and Italy; and (5) complementing
former studies on wild boar pathogens in Switzerland 77], 78]. Contacts are possible among wild boar in the study units A-D (i.e., northern population)
whereas wild boar in study unit E (Ticino, i.e., southern population) are separated
from the northern population by the Alps and can only interact with Italian wild boar
populations.

Sample collection and laboratory analysis

Blood samples collected from 1,228 wild boar over six hunting seasons (2008–2013)
were available for this study. In accordance with the national hunting law 54] a hunting season was defined as lasting from July 1
^st
to June 30
^th
of the following year, with most of the hunting bag being harvested from December
to February. Samples from wild boar shot before 2012 had been collected in the frame
of former projects 30], 79] and stored in the archive of the Centre for Fish and Wildlife Health (FIWI Bern,
Switzerland), while samples from 2012–2013 were collected for the purpose of the present
study. Calculation of the target sample size per hunting season and study unit was
derived from the regional hunting bags and performed with the WinEpiscope 2.0 software
package. Since 2011 samples sizes have been calculated with the aim of estimating
prevalence and assuming a prevalence of 50 %, with a confidence level of 95 % and
an accepted absolute error of 5 % 78]. Efforts were made towards an even age and sex distribution among units. Blood samples
were collected either by local hunters and game wardens with provided sampling kits
and sent to the FIWI or were obtained by FIWI collaborators at game check points.
Blood was collected from the thoracic cavity or the cavernous sinusoid 80].

This study did not involve purposeful killing or capture of animals and was exempt
from ethical approval according to Swiss legislation. Samples originated from dead
wild boar either shot for population regulation purposes (regular hunt, culling by
professional game-wardens; 922.0 hunting law) or killed in traffic accidents. Nine
samples originated from wild boar found dead submitted to the FIWI for pathological
examination.

Information on weight, sex and body condition of the animals as well as the location,
circumstances (found dead, hunted or culled) and date of sampling were systematically
collected with a standardized datasheet. According to Hebeisen 81], wild boar were classified into four age classes: Piglets: 20 kg, striped coat,
n?=?64; Juveniles: 20-40 kg, reddish coat, n?=?342; Subadults: 40-60 kg, black coat, n?=?370; Adults: 60 kg, black or silver coat, n?=?385; and no age data were delivered for 67 animals. Sex ratio of the sample was
balanced, with 597 males and 611 females. Sex was undetermined for 20 animals.

Blood samples were centrifuged immediately after arrival at the FIWI. Serum aliquots
were stored at -20 °C until analysis. Sera were tested for antibodies against ADV
with a commercial competitive ELISA kit (IDEXX PRV/ADV gI, IDEXX, Inc., USA) successfully
applied in former studies in Spain and Germany 1], 19], 34], 37]. According to the manufacturer’s instructions, samples with a sample/negative (S/N)-value
greater than 0.6 and less or equal to 0.7 were classified as doubtful, and samples
with S/N-values greater than 0.7 as positive. All doubtful and positive samples were
retested with the same ELISA.

Literature review

We performed a review of internationally available scientific articles about serosurveys
of ADV. In a first step, three online databases (PubMed, EBSCOhost and Google Scholar)
were searched using the key words “wild boar”, “Sus scrofa”, “Aujeszky’s disease” and “pseudorabies”. In a second step, we screened references
mentioned in the obtained publications selecting studies conducted between 1995 and
2014 on free-ranging wild boar in Europe and providing seroprevalences obtained by
ELISA.

Data management

Data handling and coding was carried out with Microsoft Office Excel 2010 (Microsoft
Corporation, Redmond, Washington, USA). Two time periods were defined, both for the
Swiss data and the literature review, starting arbitrarily 20 years ago and using
the first year of the wild boar sampling campaign carried out by the FIWI as a threshold:
1995–2007 (historical data) and 2008–2014 (samples available for the current study).
Prevalence calculations and statistical tests were performed with the NCSS 2007 software
(J. L. Hintze, Kaysville, Utah, USA). Prevalences were calculated assuming test sensitivity
and specificity of 100 % and excluding doubtful ELISA results. The Fisher’s exact
test (FET) was used to test for differences in seroprevalence among sexes, age classes,
hunting seasons, study units and populations (north and south). Level of significance
was set at P??0.05.

Maps were designed with the free QGIS- Software (QGIS Development Team, 2012. Versions
1.8.0, 2.0.1 and 2.2.0; QGIS Geographic Information System. Open Source Geospatial
Foundation Project, http://qgis.osgeo.org) and Microsoft PowerPoint 2010 (Microsoft Corporation, Redmond, Washington, USA).