Post‐epidemic investigation of Schmallenberg virus in wild ruminants in Slovenia

Abstract Schmallenberg virus (SBV) is a vector‐borne virus belonging to the genus Orthobunyavirus within the Bunyaviridae family. SBV emerged in Europe in 2011 and was characterized by epidemics of abortions, stillbirths and congenital malformations in domestic ruminants. The first evidence of SBV infection in Slovenia was from an ELISA‐positive sample from a cow collected in August 2012; clinical manifestations of SBV disease in sheep and cattle were observed in 2013, with SBV RNA detected in samples collected from a total of 28 herds. A potential re‐emergence of SBV in Europe is predicted to occur when population‐level immunity declines. SBV is also capable of infecting several wild ruminant species, although clinical disease has not yet been described in these species. Data on SBV‐positive wild ruminants suggest that these species might be possible sources for the re‐emergence of SBV. The aim of this study was to investigate whether SBV was circulating among wild ruminants in Slovenia and whether these species can act as a virus reservoir. A total of 281 blood and spleen samples from wild ruminants, including roe deer, red deer, chamois and European mouflon, were collected during the 2017–2018 hunting season. Serum samples were tested for antibodies against SBV by ELISA; the overall seroprevalence was 18.1%. Seropositive samples were reported from all over the country in examined animal species from 1 to 15 years of age. Spleen samples from the seropositive animals and serum samples from the seronegative animals were tested for the presence of SBV RNA using real‐time RT‐PCR; all the samples tested negative. Based on the results of the seropositive animals, it was demonstrated that SBV was circulating in wild ruminant populations in Slovenia even after the epidemic, as almost half (23/51) of the seropositive animals were 1 or 2 years old.


| INTRODUC TI ON
In late 2011, unspecific clinical symptoms were observed in dairy cattle in the town of Schmallenberg in the state of North Rhine-Westphalia, near the Dutch/German border region (Hoffmann et al., 2012). Using specific RT-qPCR, an orthobunyavirus of the Simbu serogroup was identified as the causative agent and named 'Schmallenberg virus' (SBV). SBV's RNA genome is phylogenetically close to those of Shamonda virus, Aino virus and Akabane virus Hoffmann et al., 2012). Culicoides biting midges play an essential role in the transmission of SBV, and they most likely spread the infection in many European countries (De Regge et al., 2014). SBV infections in adult ruminants are generally asymptomatic or may produce only mild unspecific signs, such as fever, diarrhoea and reduced milk production. When SBV-naive dams are infected during a critical period of pregnancy, the infection can cause premature birth or stillbirth with severe foetal malformation (Bayrou et al., 2014;Hoffmann et al., 2012;. Since its emergence, SBV spread rapidly among European livestock from the initial area of detection (Afonso et al., 2014). Schmallenberg virus is also capable of infecting several wild ruminant species, and early and quick spread of SBV has been observed, although clinical disease has not yet been described in these species (Laloy et al., 2014;Rossi et al., 2017). Thus, most of the published data regarding SBV infections in wildlife are based on the detection of antibodies in serum samples collected from animals without clinical signs characteristic of SBV infection. Regarding wild ruminants, SBV-specific antibodies have been detected in deer, European mouflon, European bison, elk, chamois, Alpine ibex and moose (Chiari et al., 2014;Garcia-Bocanegra et al., 2017;Laloy et al., 2014;Larska, Krzysiak, Kesik-Maliszewska, & Rola, 2014;Larska, Krzysiak, Smreczak, Polak, & Zmudzinski, 2013;Linden et al., 2012;Malmsten et al., 2017;Mouchantat et al., 2015;Rossi et al., 2017), and SBV RNA was detected in two red and one fallow deer in Spain (Garcia-Bocanegra et al., 2017). According to the detection of SBV antibodies in wild ruminants, these species might play a role in the epidemiology of SBV (Garcia-Bocanegra et al., 2017;Larska et al., 2014). Wild ruminants may increase the risk of spillover transmission to livestock, especially in regions where they frequently share the same habitats (Rossi et al., 2017). In areas where conditions are favourable for the vectors and where wild ruminants can act as a reservoir, the virus may also become endemic (Garcia-Bocanegra et al., 2017).
The aim of this study was to investigate whether SBV was or is circulating among wild ruminants in Slovenia and whether these species can act as a potential virus source in the re-emergence of SBV.

| MATERIAL S AND ME THODS
Samples from a total of 281 apparently healthy adult free-range wild ruminants were collected throughout the country during the 2017/2018 hunting season (May 2017 to May 2018). Game wardens and hunters were encouraged to submit samples from animals shot during the regular annual cull. No ethical/welfare authority approval was required as all samples were collected post-mortem. Prior to sampling, the hunters were instructed regarding the procedures and equipped with field sampling kits. Spleen and blood samples were collected from each carcass. Immediately after death, the blood samples were collected from the jugular vein or the heart. Samples were collected from 129 roe deer, 113 red deer, 29 chamois and 10 European mouflons of both sexes and various ages. Age was estimated subsequently by authorized committee of hunters during obligatory annual verification of hunted ungulates (Flajsman, Jerina, & Pokorny, 2017). Eruption patterns and tooth wear were used for age estimation of deer, whereas in chamois and mouflon, a horn growth rings method was used.
The animals were classified into two age groups: yearlings (<1 year old) and adults (>1 year old). Most samples (n = 223) were obtained from animals older than one year, namely, from 1 to 18 years of age, with a median age of 2 years. After sampling, the blood and spleen samples were transported to the Veterinary Faculty, University of Ljubljana. The blood samples were centrifuged for 10 min at 1,200 × g and stored in a −80°C freezer until use. The spleen samples were stored in a −80°C freezer until analysis.

| ELISA
For SBV antibody detection in the serum samples, a commercial

ELISA kit (ID Screen Schmallenberg Virus Indirect Screening Test
Multi-Species, ID. vet Innovative Diagnostics, France) was used according to the manufacturer's instructions. The results were interpreted according to the manufacturer's instructions.
The real-time RT-PCR reaction included a reverse transcription step at 55°C for 10 min, followed by a denaturation step at 95°C

| Statistical analysis
The probability (and confidence intervals (CI); p = .05) of prevalence of antibodies against SBV (seroprevalence) was estimated taking into account the binominal distribution of data (antibodies detected, non-detected) for each of the species × tests separately.
The predicted number of seropositive animals for the population in the entire country was recalculated from sample to the entire population. Estimates of the population size for the study species were extracted from a professional report ) that applied age-at-harvest and pellet group counting method to estimate abundancy of all species of ungulates in the entire country.

| RE SULTS
Based on the ELISA results, of the 281 serum samples, 51 (18.1%) were considered positive, and 230 (81.9%) were considered negative for the presence of SBV antibodies (Table 1). Seropositivity was detected in all the examined animal species, with the seroprevalence ranging from 10% in mouflon to 19.5% in roe deer. Detailed information regarding seroprevalence is presented in Table 3. Of the 51 positive samples, 50 samples were from adult animals from 1 to 15 years of age ( Table 2). The geographical locations where positive animals were detected are shown in Figure 1.
The estimated population size of red deer, roe deer, chamois and mouflon in Slovenia is 20,000, 110,000, 11,000 and 1,500, respectively. The predicted number of seropositive animals in the entire country, which was calculated from sample to entire population, was 3,900, 21,000, 1,100 and 150 for red deer, roe deer, chamois and mouflon, respectively, with more detailed information presented in Table 3.
In all the spleen and serum samples, the presence of SBV RNA using real-time RT-PCR was not detected (Table 1). There is one survey from Spain reporting the detection of SBV RNA in spleen samples from deer, but the prevalence (1.2%) of SBV was very low (Garcia-Bocanegra et al., 2017). The most reliable explanation for this finding is that ruminants experience short-term viremia during the first week after SBV infection Wernike, Eschbaumer, et al., 2013;; thus, it is almost impossible to detect SBV in wild ruminants in a post-epidemic survey.

| D ISCUSS I ON
Red and roe deer in Slovenia usually inhabit the lowland; mouflon habitats include steep, sunny, mountainous slopes near tree lines; and chamois inhabit extensive and continuous areas in the Alps.
The seropositivity in red and roe deer from this study was almost 20%, while seropositivity in chamois and mouflon was half of that of deer. As in Italy and Spain (Chiari et al., 2014;Fernandez-Aguilar et al., 2014), the seroprevalence in the present study was, on average, higher in lowland species than in highland wild ruminants. In contrast, Rossi et al. (2017) showed that the seroprevalence above 800 m occurred, on average, 1 year after domestic outbreaks, and sampling mountain wild ruminants in the same year may produce contradictory interpretations, possibly explaining the results from Italy and Spain. According to Toplak et al. (2015), the highest seroprevalence in domestic ruminants in Slovenia was observed in 2013; this is probably the result of exposure of the naive population to SBV infection during 2012, which was 5 years before the start of sampling among wild ruminants.
The majority of SBV-positive samples (n = 50) in this study were collected from adult animals; only one roe deer was under one year of age. According to Elbers et al. (2014), maternal antibodies against SBV in calves can persist for 5-6 months, which may also be the case in the present study. There have been several reports of low-level SBV circulation among ruminants following the Schmallenberg epidemic in Europe (Collins, Barrett, Doherty, McDonnell, & Mee, 2017;Larska, 2018;Stavrou, Daly, Maddison, Gough, & Tarlinton, 2017), resulting in a reduced seroprevalence and probably immunity (Meroc et al., 2015;Wernike, Holsteg, Sasserath, & Beer, 2015). The mechanism by which SBV infection persists from season to season (overwinters) remains unclear (European Food Safety Authority, 2014). Some data suggest that Culicoides biting midges play an essential role in the transmission of SBV, and SBV may overwinter in these vectors (Larska, Lechowski, Grochowska, & Zmudzinski, 2013). Similarly, data on SBV-positive wild ruminants suggest that these species might play a role in the epidemiology of SBV (Garcia-Bocanegra et al., 2017;Larska et al., 2014;Stavrou et al., 2017) and thus are a possible source for the re-emergence of SBV. When the population-level immunity declines and the population of immunologically naive animals increases, the re-emergence of SBV is predicted (Collins, Barrett, Doherty, Larska, & Mee, 2016;Stavrou et al., 2017).
In conclusion, the present study revealed that SBV was circulating among wild ruminants in Slovenia after the epidemic and might still be circulating.

ACK N OWLED G EM ENTS
The authors are grateful to all the hunters for their participation in the study by collecting the samples. The authors acknowledge financial support from the Slovenian Research Agency (research core funding No. P4-0092) and Hunting Association of Slovenia.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.

E TH I C A L A PPROVA L
No ethical/welfare authority approval was required, as all samples were collected post-mortem.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared. TA B L E 3 SBV seroprevalence in wild ruminants and predicted number of SBV seropositive animals in the entire country