Several studies have highlighted the role of certain wildlife species as major reservoirs, depending on the ecosystem and the region of the world [6,7]. Among the wild mammals in Spain, the Eurasian wild boar (Sus scrofa) and the red deer (Cervus elaphus) constitute the most important reservoirs, especially in Mediterranean areas, because they are able to maintain and efficiently transmit the agent in the absence of other domestic or wild hosts [8-10]. The implication of wildlife species in this multi-host cycle, in addition to many environmental and ecological factors, probably hampers the success in the control of bTB and makes the complete eradication of the disease currently not feasible [11-13].

Despite the national and long-lasting coverage of the bTB eradication plans in Spain, the prevalence of bTB varies considerably between regions [14]. In 2009, while disease presence was negligible in the islands and low in the North-East part of the country (prevalence between 0.21 and 0.91%), the herd prevalence in some regions of central, southern and western Spain was considerably higher, reaching values up to 10.27% in Castile-La Mancha [14-16]. There are several epidemiological circumstances that might be favouring this situation. First, cattle herds are farmed especially for beef (and to a lesser extent for bullfighting), comprising breeds of animals difficult to deal with due to their strong character. This kind of cattle is managed in extensive Mediterranean conditions in areas with difficult access, unfavourable topography and lack of resources, which makes it quite difficult to properly test all the animals of the herd. This also induces common grazing and a shared use of scarce water points (such as water holes), and thus, the contact of cattle with other domestic livestock such as sheep, goats, pigs [15] and wildlife [17]. Secondly, some areas, especially in the South Central regions, have experienced a great development in the recreational hunting industry, which leads to overabundance of wild ungulates in which sanitary programs cannot be implemented, leading to high prevalence of diseases [9,18]. All these aspects promote the existence of a wildlife-domestic livestock interface in Mediterranean habitats that could maintain a multihost environmental reservoir for M. bovis[11,13]. However, there is no research on the association between these factors and the status of bTB at either fine or large spatial scales.

The posterior probability of TB occurrence in Ciudad Real estimated by the best fitting model resembled the spatial structure of the disease observed in the data, with areas at the highest risk concentrated in the Northwest corner, the Southwest part and several municipalities in the southern stripe of the region (Figure ​(Figure3).3). Both S_i and U_i were very low (median value of −2.33·10^-3 and −2.96·10^-4, respectively), which may indicate no significant presence in the model of spatial autocorrelation and overdispersion over-and-above the risk factors identified, as well as a good fit of the model (Figure ​(Figure4).4). In addition, convergence of the model was obtained after the first 200 iterations and no problems with autocorrelation were identified for the posterior inferences.

The probability of TB occurrence in Ciudad Real resembled the biogeography of the province as well as game and cattle farming distribution, with areas at high risk concentrated in the municipalities with a higher proportion of forest or natural habitats (Toledo Mountains in the Northwest corner, the Guadiana valley in the East, and Sierra Morena in the South), and the highest big game activity. In fact, the mean number of hunting seasons in which the hunting estates of the municipality have been inspected, which is an indicator of the hunting activity in the municipality, remained statistically significant in the model (see below). Usually, woodlands and savannah-like areas (locally called dehesas) are dedicated to big game, whereas marginal dehesas are located in the valleys in close proximity, where land is not appropriate for agriculture, are mainly dedicated to extensive livestock farming. This distribution, together with particular Mediterranean features that favour the wildlife-livestock interface (i.e. water areas or pasture sharing) may explain how TB could be transmitted from wildlife to livestock and vice versa (Figures ​(Figures11 to ​to3).3). However, from this study we cannot infer the directionality of M. bovis transmission, i.e. whether wildlife reservoirs are infecting cattle, or whether cattle is the responsible of the circulation of M. bovis in both domestic and wild hosts. In fact, it could be the case that some municipalities with a high prevalence of TB in just one species (for example, wild boar) in 2007 may be purely correlated with a high prevalence of TB in only that species (in the example, with no red deer nor cattle infected in that municipality) the previous year. This would indicate no inter-species transmission in some regions. However, Ciudad Real municipalities with this scenario are an exception (9 out of 113) and many TB-infected regions presented TB cases in more than one species (44.19% in 2006 and 28.86% in 2007) (Table ​(Table2).2). This suggests a potential multi-species interaction scenario. Unfortunately, we cannot prove this because the final model did not include any interaction between variables related to hosts. Nevertheless, future studies should reveal whether the presence of the disease in wildlife is associated with increased presence of the disease in livestock and establish the directionality and extent of the TB inter-species transmission.

The finding that municipalities with a high proportion of cattle farms becoming bTB positive in 2006 were at high risk for TB occurrence in 2007 (OR=1.86; 95% CI=1.06-3.39) is an indicator that TB until 2007 repeatedly tends to appear in the same municipalities. These findings are in agreement with the trend of incidence and persistence of bTB in South Central Spain during 2006–2009, particularly in Ciudad Real, observed in previous studies [14]. This indicates the persistence of the pathogen over consecutive sanitary campaigns despite the eradication efforts and the culling of positive animals. Herd-level research is needed to elucidate whether barriers imposed to prevent the transmission of M. bovis into bTB-free farms are not as efficient as expected, or diagnostics were not reliable enough in presumed free farms. Both of these risk factors have not been explored in this study. Most of the extensive farms and cattle census in Ciudad Real province are located in municipalities where some of the risk factors identified in this model may operate, such as wildlife TB reservoir abundance (see below). Although the TB inspection in Spain is compulsory in all cattle farms, these aspects also show that the efforts in controlling M. bovis infection in the whole cattle stock should not be relaxed, especially in extensive farms where enclosing and handling the whole stock is more difficult. Our final model suggests that the higher the number of cattle submitted to the sanitary campaigns, the higher the likelihood of the municipality to be TB positive the following year (OR=1.94; CI=1.11-3.40). As Brooks-Pollock and Keeling [20] reported, many elements associated with herd size could contribute to disease persistence within a herd, such as an increased number of movements or larger land coverage with increased risk of environmental contamination, or more densely stocked cattle. Nonetheless, this should be evaluated at the herd level.

Although the information regarding TB prevalence in wildlife is usually not available and more difficult to obtain than in cattle, we believe that it is also crucial to monitor the TB status of wild reservoirs in a region [19]. In fact, one of the significant covariates in the final model is the mean number of hunting seasons in which the hunting estates of the municipality have been inspected (OR=1.69; 90% CI=1.19-2.42), which is related to the intensity and sustainability of the hunting activity performed in each estate over time, and therefore the abundance of big game. In addition, the analysis revealed two additional major risk factors related to wild hosts: the apparent prevalence of TB in wild boar (OR=1.94; 95% CI=1.20-3.59) and the number of TB-positive red deer (OR=1.69; 90% CI=1.05-2.90) both in the previous year. The implication of wild boar and red deer in the TB presence and maintenance in South Central Spain has been previously suggested [10]; the wild boar showing higher TB prevalence than red deer [9,21]. In addition, wild boar has been proposed as the most relevant wild host of M. bovis in Spain [10] becoming a true TB reservoir in the Iberian Peninsula [22]. This importance is enhanced by its abundance in Europe, the high levels of infection observed in this species, its ability to disseminatethe pathogen, its scavenging behaviour and, mostly, its potential ability for crossing fences and contacting with livestock [22]. Given this situation, physical biosafety practices may serve as a means to cost-effectively prevent contact between wild and domestic mammals and thus TB inter-species transmission. This is particularly useful in those areas where the hunting industry plays an important role in the local economy, such as Ciudad Real. Also, any action conducted to reduce TB prevalence and mycobacteria spreading in wild ungulates should contribute to reduce the M. bovis transmission risk at the wildlife-livestock interface. Options include the management of host density, spatial aggregations at supplementary feeding sites or waterholes, the safe disposal of carrion and viscera by hunters, and oral vaccination [5,21].

There is evidence supporting the inter-species circulation of M. bovis[13] and several studies have recently confirmed the presence of the same M. bovis spoligotypes in cattle and wild fauna sharing the same area [8,11,22-26]. M. bovis transmission between wild and domestic animals may occur directly via close contact, or indirectly via contamination of food or the environment with M. bovis. Most of the bTB control programmes in developed countries are mainly directed towards livestock not including wildlife, even where it is obviously involved in disease maintenance. The lack of integral control strategies is more evident in extensive cattle, which is the sector mostly exposed to wildlife contacts [15]. This was supported previously [16] and in our results, since the proportion of cattle farms classified as extensive beef breeding farms (OR=1.47; 90% CI=1.02-2.14) and the number of farms devoted to bullfighting cattle were revealed as risk factors in the final model (although this latter covariate is not highly significant, 90% CI=0.97-1.87, probably because there are scarce and very disperse observations). Both types of farms are integrated in natural pasture agrosystems in close proximity with wildlife. However, the inclusion of these two covariates related to extensive cattle farming reinforces the idea that sanitary programs should incorporate the wildlife interface, especially in those cattle farms most exposed to M. bovis infection, as has been done in Spain since 2011 ( http://rasve.mapa.es/Publica/Programas/Normativa.asp).