The study area included the southern half of peninsular Spain. This area was selected on the basis of the distribution of C. imicola and the geographic distribution of BTV-4 outbreaks in livestock and the associated animal movement restriction area (www.mapa.es).

Figure 1

Figure 1. Map of Spain showing wild ruminant sampling sites (black dots) and areas (gray) sampled along a south-to-north gradient during 2003–2007. SC, Sistema Central; TO, Toledo Province; MT, Montes de Toledo; GU,...

Blood samples were obtained from 2,233 red deer, 106 fallow deer, 44 roe deer, 72 mouflon, and 10 aoudad during 2003–2007 in 62 locations (Figure 1). Most (n = 1,575) of the red deer samples and all samples from other ruminant species were collected from hunted animals; some samples (n = 658) from red deer were collected on 5 farms. These farms were located in the Alcornocales (ALC), Sierra Morena (SM), Guadiana Valley (GU), Montes de Toledo (MT), and Sistema Central (SC) areas. Most (69%) samples were collected during the hunting season (October–February). Samples were not obtained during certain periods because of logistic surveillance constraints (online Appendix Table, available from www.cdc.gov/EID/content/14/6/951-appT.htm). Sex and age of red deer were determined, the latter according to tooth eruption patterns (14). Animals <1 year of age were classified as juveniles, those 1–2 years of age as subadults, and those >2 years of age as adults.

Blood was collected into sterile tubes from the heart or thoracic cavity from hunted animals and by cervical puncture from farm animals. Samples were centrifuged, and serum was stored at –20°C. Antibodies to BTV in sera were detected by using a competitive ELISA (Pourquier ELISA Bluetongue Serum; Institut Pourquier, Montpellier, France) according to manufacturer’s instructions. The inhibition value of the ELISA for domestic ruminants has been found to be applicable for testing serum from wild ruminants (15).

We expected a south-to-north gradient in the expansion of BTV across Spain as observed in livestock. Thus, sampling sites were grouped into 6 areas according to a south-to-north gradient (Figure 1). These areas included southern (ALC), south-central (SM, GU, and MT), and central (Toledo Province and SC) Spain. On the basis of annual distribution of BT outbreaks in Mediterranean countries (2), a year was defined as the period July–June.

None of the samples collected in 2003–2004 and 2004–2005 had antibodies to BTV; antibody-positive samples were detected in 2005–2006 and 2006–2007. Mean seroprevalence values for ruminant species analyzed are shown in the Table. A total of 17% ±2% of red deer sampled in 2005–2006 and 29% ±4% of those sampled in 2006–2007 had antibodies to BTV. Northernmost red deer (SC area) were antibody positive only in 2006–2007. However, the number of samples (n = 5) tested in this area the previous year was low.

Figure 2

Figure 2. Study areas in Spain showing wild ruminant sampling sites by study year. A) 2003–2004; B) 2004–2005; C) 2005–2006; D) 2006–2007. A year is defined as the period July–June. Animal movement restriction...

BTV seroprevalence in red deer decreased along a south-to-north gradient and increased throughout the years sampled (online Appendix Table, available from www.cdc.gov/EID/content/14/6/951-appT.htm and Figure 2). Mean seroprevalence was slightly higher in males and adults but dependent on the sampling area (online Appendix Table, available from www.cdc.gov/EID/content/14/6/951-appT.htm). Because the first evidence of contact with BTV occurred in 2005, observed seroprevalences can be considered as incidence rates. Thus, in 2005–2006, the incidence rate was higher in subadults than in adults and juveniles.

The presence of antibodies to BTV in wild ruminants has recently been studied in Belgium (A. Linden, pers. comm.) and Germany (www.efsa.europa.eu/EFSA/DocumentSet/appx6_bluetongue_S8_en.pdf). In 2006, BTV seroprevalence was 0.58% in Belgian cervids. The survey in Germany showed BTV seroprevalence rates of 0.09% in red deer, 5.7% in roe deer, and 4.9% in mouflon. Our study provides evidence that BTV is present in wild ruminants in Europe over a large area.

All ruminant species studied were positive for BTV. This result is not surprising because other wild ruminant species have been reported to be susceptible to BTV infection (8–10). We observed some differences in BTV seroprevalence between ruminant species (Table). However, if one considers the epidemic nature of BTV, differences in the number of seropositive ruminant species, and the unequal distribution of C. imicola in the study area (2; www.mapa.es), the observed results may be caused by differences in susceptibility to the vector/pathogen or differences in vector/pathogen distribution across the study area.

We observed similar spatial and temporal BTV patterns in red deer (Figure 2) and livestock. Nevertheless, we found the first evidence of contact with BTV 1 year later in red deer than in livestock. This delay may have been caused by larger numbers of samples from livestock than from wild ruminants. However, our findings suggest that wild ruminants, particularly cervids because of their wider distribution in Europe, could be used as sentinels for surveillance of BTV. Moreover, the high BTV seroprevalence in cervids from the southernmost sampling area suggests that cervids may not interfere with vaccinations given in this region.

This study shows an increased distribution of BTV across Spain and that wild ruminants in Europe can be infected with BTV. Our findings, combined with those of earlier studies, suggest a complex epidemiologic scenario of BTV in Europe with many susceptible hosts, an increase in its main vector because of climate changes, and the appearance of new competent vectors. Nevertheless, more information on the role of susceptible wild ruminant species is needed to clarify the complexity of BTV epidemiology in Europe.