Our findings confirm that wild ruminants were actively exposed to BTV in the study area. A large number of the samples tested were positive by ELISA but negative by SNT. The differences between the tests, likely reflect that ELISA and SNT measure distinct antibody populations. The ELISA used in the present study detects both IgM and IgG antibodies to BTV, and it has been proven to be highly sensitive and specific in livestock . Even though the quality of sera may also influence the results of both tests, it is particularly important for SNT. In fact, a high number of samples positive by ELISA could not be analyzed by SNT due to cytotoxicity. BTV seroprevalence was determined only from samples positive by both ELISA and SNT and therefore could have been underestimated.
To our knowledge, this is the first study on BTV serotypes in different wild ruminant species. The seroprevalence levels indicate widespread circulation of BTV in red deer, fallow deer and mouflon, which is in agreement with what was previously reported [6–8]. The results support the idea that sero-surveillance on these species would be useful to detect virus circulation, especially in areas where a vaccination program has been implemented in livestock [9, 10]. Furthermore, the low seropositivity detected in roe deer is in agreement with previous studies [7, 12, 22], suggesting that it is not a relevant species in the dissemination of BTV. Differences among species might be related to the natural resistance of the hosts, population densities, geographical distribution, sampling period or management factors.
The higher seroprevalence in adult animals was probably due to a greater exposure of this age group to the virus over time [8, 10, 12]. Interestingly, seropositivity was found in young animals (juveniles and sub-adults) sampled long after any previous outbreak in domestic ruminants. This was particularly clear in the case of BTV-4: even though the last outbreak in domestic ruminants was reported in October 2005, juveniles and sub-adults of the different wild ruminant species analyzed presented antibodies against BTV-4 between the 2008/2009 and the 2010/2011 hunting seasons in all the provinces where outbreaks had been reported in livestock. Similar findings were observed for BTV-1 and BTV-8, and evidence the ability of BTV to circulate despite no cases reported in domestic ruminants. Whether that circulation indicates the maintenance of BTV within the wild ruminant population despite vaccination of domestic ruminants, or is the consequence of repeated introductions of the virus, merits further studies.
No antibodies against BTV-4, BTV-1 or BTV-8 were detected in wild ruminants sampled prior to the detection of the first outbreak of each serotype in livestock. Between the 2008/2009 and the 2010/2011 hunting seasons, seroprevalence against the different serotypes increased in the majority of areas, even when the same species and ages were compared. These findings could indicate the persistence of antibodies against BTV for very long periods or, more likely, a longer BTV maintenance and circulation in wild ruminants compared to livestock. The lower seropositivity against BTV-8 (3.4%) was not unexpected taking into account that only 24 outbreaks had been reported in livestock in the study area .
The risk of being a seropositive animal was 2.3 and 3.9 times higher in the western and central regions, respectively, as compared to the eastern, which is in accordance with the geographical distribution of BTV observed in domestic ruminants (Figure 2). Vector density, host density and environmental factors are possibly implicated in the spatial distribution of BTV [23, 24]. However, BTV-1, BTV-4 and BTV-8 seropositive animals were detected in regions where these serotypes were not reported in livestock. This was particularly evident for BTV-8 (Figure 2) and confirms a different spatial distribution of BTV in wild ruminants as compared to livestock [8, 15].
The presence of BTV-1 and BTV-4 RNA in red deer and mouflon confirms the susceptibility of these species to BTV infection [9, 10]. In addition, the low cycles (Ct) RT-PCR values obtained in 12 animals (Ct lower than 35) support the potential reservoir role of these species. Three of BTV-1 RNA positive wild ruminants were found in November 2009, even though the last BTV-1 outbreak in livestock was reported in December 2008. One of these BTV-1 RNA positive animals showed no antibodies by ELISA or SNT, which could suggest a fresh infection in this animal [12, 13]. Furthermore, BTV-1 RNA positive wild ruminants sampled in the 2010/2011 hunting season were detected in locations different from those where the last BTV-1 outbreak was reported in livestock . The BTV-4 RNA positive animal was detected on November 2010, one month after this serotype was first detected on a sentinel farm located at 60 km distance.
Vaccination has been suggested as one of the most important control measures for BT . In this sense, the limited number of cases reported in livestock despite the circulation observed in wild ruminants suggests that vaccination seems to be effective to prevent the clinical disease. However, to prevent outbreaks in livestock, the vaccination of the domestic population would have to be maintained until virus circulation within the wild ruminant population has ceased.
Our results confirm that wild ruminant populations from southern Spain were exposed to BTV-1, BTV4 and BTV-8. The high seroprevalence to BTV-1 and BTV-4 found in the present study, the detection of both BTV seropositive and RNA positive animals, including juvenile animals, years after the last outbreak was reported in livestock, and the presence of the different BTV serotypes in areas where BTV outbreaks had never been reported in domestic ruminants, indicate that wild ruminants seem to be implicated in the dissemination and persistence of BTV, and probably play a significant role as reservoirs for BTV.