- Research article
- Open Access
Cross-sectional study of Schmallenberg virus seroprevalence in wild ruminants in Poland at the end of the vector season of 2013
© Larska et al.; licensee BioMed Central. 2014
- Received: 25 August 2014
- Accepted: 14 December 2014
- Published: 21 December 2014
In view of recurrent Schmallenberg virus (SBV) infections all over Europe between 2011 and 2013, a lively scientific debate over the importance of the sylvatic transmission cycle of the virus has emerged. The study presents results of serosurvey which included wild ruminants representing species of red deer (Cervus elaphus), roe deer (Capreolus capreolus), European bison (Bison bonasus), fallow deer (Dama dama), mouflon (Ovis orientalis musimon) hunted or immobilized at 34 different locations of Poland in the autumn/winter 2013.
Out of 580 sera, 145 (25%) were considered positive for SBV antibodies. The overall SBV seroprevalence calculated using district probability weights was estimated at 27.7% (95% CI: 24.0-31.4). The seroprevalences at the district level varied between 0 and 80.0% (95% CI: 24.5-135.0%) with the mean within-district prevalence of 24.0% (95% CI: 16.5-31.4). Significantly higher seroprevalence was observed in animals from the Eastern provinces (36.6%) compared to the Western provinces (22.8%). SBV infection impact varied significantly between different species (higher SBV seroprevalence in larger species such as European bison), population type (free-ranging; captive), age, body weight, percent of the district forest area, part of Poland, and the densities of wild and domestic ruminants at the district and province level. Using statistical multivariable logistic model, population type, age, part of Poland and domestic ruminant density were identified as the main risk factors for SBV infection in wild ruminants in Poland.
SBV seroprevalence in wild ruminants, similarly to the epizootic situation in domestic ruminants in the country, varied significantly between districts and provinces. Association between SBV seropositivity, species, animal body weight and age group expressed by a higher prevalence in larger ruminants may be explained by more frequent exposure to midge-vector bites of the latter, however it might also be related to the different species susceptibility to SBV infection. The positive effect of higher domestic ruminant density on the risk of SBV infection in wildlife and lower SBV seroprevalences in the latter suggested that the sylvatic cycle of SBV transmission is an effect of the pathogen spillover from the domestic animals.
- Schmallenberg virus
- Wild ruminants
- Risk factors
Schmallenberg virus (SBV) is a novel Orthobunyavirus infecting ruminants which emerged in North Rhine-Westphalia, Germany in August/September 2011 . SBV transmission occurs through midge vectors and infection is mostly sub-clinical in adult animals . The virus infections in pregnant domestic ruminants were observed to provoke abortions, stillbirths and, most frequently, congenital musculoskeletal and neural malformations observed in new-born animals leading to their death shortly after birth . In view of recurrent SBV infections all over Europe, lively scientific debate over the importance of the sylvatic transmission cycle of the virus has emerged . SBV infection has been already reported in the wildlife in Poland . This study presents wider perspective of SBV infections in wild ruminants in Poland.
Serological ID Screen Schmallenberg Virus Competition Multi-Species ELISA (ID.vet, France) for the detection of antibodies against SBV nucleoprotein was performed according to the manufacturer’s instruction. The results were interpreted on the S/P values calculated from the optical density (O.D.) values measured at 450 nm using the following equation: O.D. sample tested – O.D. negative control / O.D. positive control – O.D. negative control × 100. The S/P values below and equal to 40% were considered positive, S/P value above 50% - negative, and the S/P value between 40% and 50% doubtful.
Virus neutralization test (VNT)
Randomly selected non hemolyzed serum samples giving negative (n = 20), positive (n = 50) and doubtful (n = 12) results by c-ELISA were verified by virus neutralization test. The sera were heat-inactivated at 56°C for 30 min beforehand. Serial two-fold dilutions of the sera (starting from 1:4 up to 1:4096) in Glasgow Minimum Essential Medium (GMEM) were incubated for 1–1.5 h at 37°C in a 96-well microplate with approx. 500 TCID50 of the Polish SBV strain 130/6 isolated from the cerebrum of ovine fetus. The sera were tested in duplicate. The strain was passaged 3 times in baby hamster kidney (BHK-21) cells with the final titer of 8 × 104 TCID50. Back titration of virus was included in each assay by performing four 10-fold dilutions of the virus working solution. Two wells with 1:4 dilution of each serum were left uninfected as a serum cytotoxity control. Similarly diluted negative (commercial foetal bovine serum negative in ELISA) and positive (bovine sample from a confirmed SBV outbreak) serum samples were tested simultaneously. After the incubation, each well was overlaid with approx. 104 BHK-21 cells and the plates were incubated for five days at 37°C in 5% CO2. After fixation in 80% chilled acetone, drying, staining with 0.1% crystal violet and washing, the plates were examined under the microscope. Serum was considered as a VNT positive if the viral CPE inhibition was observed in at least one of two wells at a dilution ≥1:4. The titer of tested serum sample was assumed as the reciprocal of the highest dilution of the sample for which none or one of the wells of a dilution showed any cytopathic effect.
Schmallenberg virus seroprevalence in relation to different characteristics of the wild ruminants and their origin in the univariable analysis
OR 95% CI
95% CI 3
Fallow deer (Dama dama)
Red deer (Cervus elaphus)
Mouflon (Ovis aries musimon)
Roe deer (Capreolus capreolus)
European bison (Bison bonasus)
Gender (n = 440)
Below and equal to one year of age
Over 1 year of age
Body weight group (n = 345)
1 (mean 15.9; range 8–25)
2 (mean 40.2; range 30–50)
3 (mean 76.7; range 55–100)
4 (mean 188.6; range 110–650)
Percent of the forest area in district
Low (mean 25.8; range 20.1-30.9)
Medium (mean 40.8; range 35.2-66.0)
High (mean 85.9; range 67.5-97.3)
District-level density of wild ruminants (animals per km2)
Low (mean 2.8; range 2.1-3.4)
Medium (mean 5.5; range 4.1-6.3)
High (mean 8.3; range 6.7-8.7)
Part of country
Province-level density of domestic ruminants
Low (mean 5.5; range 4.2-14.0)
Medium (mean 21.1; range 17.7-28.5)
High (mean 44.4; range 44.4-44.4)
Using the forest district population weights, the overall SBV seroprevalence was estimated at 27.7% (95% CI: 24.0-31.4). The percentage of SBV seropositive wild ruminants was slightly lower than the apparent seroprevalences for wild ungulates observed at the beginning of the European epizootic in Belgium (43.1%)  and in UK (approx. 56% in fallow deer and 71% in red deer) in 2012 . In Poland, after the first detection of SBV infection in domestic ruminants during summer of 2012 ,, SBV transmission to free-ranging red deer in Western and European bison in Eastern Poland were detected in November 2012 . The seroprevalence in wild ruminants was comparable to the value of 35.6% observed in domestic ruminants in Poland in 2013  which suggests similar exposure to SBV infection. It is consistent with the observation of the ecosystem of Spanish hunting estate where SBV seroprevalence in roe deer (80%) was almost identical to the cattle herds (86.8%) reared in the area .
The SBV seroprevalences of the 34 forest districts included in the study ranged between 0 and 80.0% (95% CI: 24.5-135.0%) with the mean within-district prevalence of 24.0% (95% CI: 16.5-31.4) (Figure 1). No seropositive wild ruminants were found in nine (26.5%) districts of which seven were located in Western Poland. Significant associations between SBV seroprevalence and environment conditions such as part of the country where the animals originated from, the percent of forest area in the district and wild and domestic ruminant densities were also found (Table 1). Higher proportion of SBV seropositive wild ruminants was found in Eastern Poland, in the districts with higher percent of forest area and in the provinces characterized by the highest densities of domestic ruminants. However, strong dependence between wild and domestic ruminant densities (ρS = −0.6; p < 0.0001), or their relation with the part of Poland (ρS = −0.5; p < 0.0001 and ρS = 0.6; p < 0.0001, respectively), or association between the part of country and the percent of forest area (ρS = 0.3; p < 0.0001) should be considered when interpreting those results. High geographic variation in SBV seroprevalence in wildlife was described also in France and Belgium ,. The differences may be related to the vector exposure of wild host species in relation to their habitat, vector abundance or population structure. In the Spanish study, SBV infection rate of roe deer inhabiting the lowlands was significantly higher than the low prevalence in Pyrenean chamois and mouflon residing in the high mountains of the same national park . Another factor determining the differences in SBV exposure may be connected to the species and size of the animal (age and body weight). In our study, SBV seroprevalence differed significantly between the species and body weight of wild ruminants, with European bison (81.9%) and the heaviest animals (42.4%) showing the highest seroprevalences (Table 1). It is consistent with the previous study which showed lower seroprevalences in smaller ruminants (sheep and goats) in respect to cattle . At this point, the limitation of the study should also be considered. Table 1 presents the results of the univariable analysis, however some variables such as wild and domestic ruminant densities, part of Poland and forest area were cross-correlated and therefore might influence the interpretation of the results. Despite the assumption of homogenous distribution of the different species in the country, one should be aware that sampling of large wildlife species is restricted by local regulations or practices which might affect the interpretation of statistical analysis. Whether the differences in SBV seropositivity are connected to the species susceptibility, the animal size or age is difficult to determine with certainty, especially as those characteristics are closely related. The differences of species susceptibility to SBV infection have been studied experimentally. Poskin et al.  and Wernike et al.  have shown that a lower infectious dose of SBV is required to infect cattle in comparison to sheep which would need ten times higher dose of the virus to get seroconversion in all inoculated animals. Therefore the higher seroprevalence in bison may reflect their higher susceptibility as in cattle. Another explanation might be the higher production of carbon dioxide in the larger ruminant species which is one of the strongest midge attractants and may promote higher exposure to their bites and hence to SBV infection. Studies on the feeding behavior confirm the observation. According to Lassen et al.  and Ayllón et al.  among others, most Culicoides species feed preferentially on cattle. In relation to high SBV infection rate in European bison, the specific environment of the tested population should also be considered important. The European bison included in the study originated from a reserve at the Białowieża Forest which is known for their vast marshes and therefore abundant in the midge vectors. The area is neighboring with Polish Eastern borders. Interestingly, the SBV prevalence in wild ruminants was increased in the Eastern part of the country which is known for higher density of cattle reared extensively in smaller size herds, often pastured enabling SBV transmission at the interface between domestic and wild ruminant populations . In contrast to Eastern Poland, the density of domestic ruminant population on the Western side of Vistula river is lower, while the population size of wild ungulates is significantly higher. Those observations suggest that SBV infection in wild ruminants is more likely to be the effect of spillover from domestic livestock and not the opposite. Probably, the environment characteristics such as humidity, temperature, variation in temperature during night and day affecting the presence and density of vector influence the possible transmission and consequently the prevalence in both domestic and wild animals. Perhaps the role of SBV wildlife reservoirs threatening domestic ruminants should not be overemphasized . In case of endangered and protected species such as European bison, the threat to the reproduction and health of the animals should not be underestimated. A single clinical case connected to SBV infection have been already described in an elk calf in Poland, however the association could not be proven . Species susceptibility should be investigated further.
Risk factors associated with SBV infection in wild ruminants in Poland in 2013 estimated by multivariable mixed-effects logistic regression model with species as fixed effect ( p < 0.0001)
P > | z |
OR 95% CI
Part of country
Province-level density of domestic ruminants
Wild ruminants might play a role in SBV transmission in Poland, however lower seroprevalence in relation to the domestic ruminants suggests the spill-over effect from the latter, rather than inverse. The higher SBV seroprevalence in the Eastern part of the country suggests eastbound transmission of SBV in Europe, while low SBV prevalence in the wild ruminants below one year old may indicate possible decline of SBV epidemic in Poland. The species and size of the ruminant seem to be significant risk factors for SBV infection, probably due to the different species susceptibility and/or increased attraction of midges to larger animals.
This work was supported by NCBiR projects: 208020 and NR12-0126-10. The authors thank Agnieszka Nowakowska, Maria Sudowska, and Karol Stasiak for their technical assistance, Artur Jabłoński for the coordination of animal sampling. We are grateful to Magdalena Konsencjusz-Białowąs and Jan Błaszczyk from General Directorate of National Forest Holding for providing current data on population of free-living animals in Poland.
- Hoffmann B, Scheuch M, Höper D, Jungblut R, Holsteg M, Schirrmeier H, Eschbaumer M, Goller KV, Wernike K, Fischer M, Breithaupt A, Mettenleiter TC, Beer M: Novel orthobunyavirus in Cattle, Europe, 2011. Emerg Infect Dis. 2012, 18: 469-472. 10.3201/eid1803.111905. doi:10.3201/eid1803.111905PubMed CentralView ArticlePubMedGoogle Scholar
- van den Brom R, Luttikholt SJ, Lievaart-Peterson K, Peperkamp NH, Mars MH, van der Poel WH, Vellema P: Epizootic of ovine congenital malformations associated with Schmallenberg virus infection. Tijdschr Diergeneeskd. 2012, 137: 106-111.PubMedGoogle Scholar
- Tarlinton R, Daly J, Dunham S, Kydd JH: Schmallenberg virus: could wildlife reservoirs threaten domestic livestock?. Vet J. 2013, 198: 309-10. doi:10.1016/j.tvjl.2013.10.003View ArticlePubMedGoogle Scholar
- Larska M, Krzysiak M, Smreczak M, Polak MP, Żmudziński JF: First detection of Schmallenberg virus in elk (Alces alces) indicating infection of wildlife in Białowieża National Park in Poland. Vet J. 2013, 198: 279-81. doi:10.1016/j.tvjl.2013.08.013View ArticlePubMedGoogle Scholar
- General Directorate of National Forest Holding: The summary of the hunting plans for 2013/2014. National Forestry Database (SILP), 2014.Google Scholar
- Central Statistical Office: Forestry. Warsaw 2013.Google Scholar
- Ministry of Environment: Regulation on the detailed conditions for the performing hunting and marking carcasses (Dz.U. z 2005 Nr 61 poz. 548). Warsaw, 2005.Google Scholar
- Krzysiak M, Larska M: Pharmacological immobilization of European bison (Bison bonasus). Medycyna Wet. 2014, 70: 172-175.Google Scholar
- Polish General Directorate for Environmental Protection: Regulations DOP-OZGIZ.6401.06.7.2012.1 s and DOPOZ. 6401.06.7.2012.1 s1.Warsaw, 2012.Google Scholar
- Central Statistical Office [http://www.stat.gov.pl/gus]
- Casaubon J, Chaignat V, Vogt HR, Michel AO, Thür B, Ryser-Degiorgis MP: Survey of bluetongue virus infection in free-ranging wild ruminants in Switzerland. BMC Vet Res. 2013, 9: 166. doi:10.1186/1746-6148-9-166PubMed CentralView ArticlePubMedGoogle Scholar
- Barlow A, Green P, Banham T, Healy N: Serological confirmation of SBV infection in wild British deer. Vet Rec. 2013, 172: 429. doi:10.1136/vr.f2438View ArticlePubMedGoogle Scholar
- Chiari M, Sozzi E, Zanoni M, Alborali LG, Lavazza A, Cordioli P: Serosurvey for schmallenberg virus in alpine wild ungulates. Transbound Emerg Dis. 2014, 61: 1-3. doi:10.1111/tbed.12158View ArticlePubMedGoogle Scholar
- Fernández-Aguilar X, Pujols J, Velarde R, Rosell R, López-Olvera JR, Marco I, Pumarola M, Segalés J, Lavín S, Cabezón O: Schmallenberg virus circulation in high mountain ecosystem, Spain. Emerg Infect Dis. 2014, 20: 1062-4. doi:10.3201/eid2006.130961PubMed CentralView ArticlePubMedGoogle Scholar
- Laloy E, Bréard E, Sailleau C, Viarouge C, Desprat A, Zientara S, Klein F, Hars J, Rossi S: Schmallenberg virus infection among red deer, France, 2010–2012. Emerg Infect Dis. 2014, 20: 131-4. doi:10.3201/eid2001.130411PubMed CentralView ArticlePubMedGoogle Scholar
- Linden A, Desmecht D, Volpe R, Wirtgen M, Gregoire F, Pirson J, Paternostre J, Kleijnen D, Schirrmeier H, Beer M, Garigliany MM: Epizootic spread of Schmallenberg virus among wild cervids, Belgium, Fall 2011. Emerg Infect Dis. 2012, 18: 2006-8. doi:10.3201/eid1812.121067PubMed CentralView ArticlePubMedGoogle Scholar
- Kaba J, Czopowicz M, Witkowski L: Schmallenberg virus antibodies detected in Poland. Transbound Emerg Dis. 2013, 60: 1-3. doi:10.1111/tbed.12039View ArticlePubMedGoogle Scholar
- Larska M, Polak MP, Grochowska M, Lechowski L, Związek JS, Żmudziński JF: First report of Schmallenberg virus infection in cattle and midges in Poland. Transbound Emerg Dis. 2013, 60: 97-101. doi:10.1111/tbed.12057View ArticlePubMedGoogle Scholar
- Larska M, Kęsik-Maliszewska J, Kuta A: Spread of Schmallenberg virus infections in the ruminants in Poland between 2012 and 2013. Bull Vet Inst Pulawy. 2014, 58: 169-176. doi:10.2478/bvip-2014-0026View ArticleGoogle Scholar
- Poskin A, Martinelle L, Mostin L, Van Campe W, Dal Pozzo F, Saegerman C, Cay AB, De Regge N: Dose-dependent effect of experimental Schmallenberg virus infection in sheep. Vet J. 2014, 201: 419-22. doi:10.1016/j.tvjl.2014.05.031View ArticlePubMedGoogle Scholar
- Wernike K, Eschbaumer M, Breithaupt A, Hoffmann B, Beer M: Schmallenberg virus challenge models in cattle: infectious serum or culture-grown virus?. Vet Res. 2012, 43: 84. doi:10.1186/1297-9716-43-84PubMed CentralView ArticlePubMedGoogle Scholar
- Lassen SB, Nielsen SA, Kristensen M: Identity and diversity of blood meal hosts of biting midges (Diptera: Ceratopogonidae: Culicoides Latreille) in Denmark. Parasit Vectors. 2012, 5: 1-9. doi:10.1186/1756-3305-5-143View ArticleGoogle Scholar
- Ayllón T, Nijhof AM, Weiher W, Bauer B, Allène X, Clausen PH: Feeding behaviour of Culicoides spp. (Diptera: Ceratopogonidae) on cattle and sheep in northeast Germany. Parasit Vectors. 2014, 7: 34. doi:10.1186/1756-3305-7-34PubMed CentralView ArticlePubMedGoogle Scholar
- Elbers AR, Stockhofe-Zurwieden N, van der Poel WH: Schmallenberg virus antibody persistence in adult cattle after natural infection and decay of maternal antibodies in calves. BMC Vet Res. 2014, 10: 103. doi:10.1186/1746-6148-10-103PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.