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Hepatitis E virus (HEV) is an emerging zoonotic pathogen with pigs and wild boar serving as reservoirs for human infection through direct contact with infected animals or the consumption of raw or undercooked pork products. The liver is considered the main target site of HEV replication in swine and an important organ in the pathogenesis of the disease. The aim of this study was to characterize the target liver cells for HEV entry in naturally infected wild boar and to evaluate the type and severity of the pathological changes in order to reach a better understanding of the hepatic pathogenic mechanisms involved in hepatitis E. In total, 58 livers from hunted wild boar were histopathologically evaluated. The presence of specific HEV antibodies in serum was determined by indirect ELISA. Immunohistochemistry was used for the detection of HEV antigen and Real time RT-PCR to detect HEV RNA in liver and serum. HEV seroprevalence in these animals was of 5.197% (CI95%: 1.77-14.14). By Real time RT-PCR, HEV was detected in the liver tissue of four wild boar (6.8%; CI95%: 2.7-16.4) and only one animal was also positive in serum (1.7%; CI95%: 0.3-9.1). The non-viremic animals naturally infected with HEV presented evidence of liver infection, mainly in Kupffer cells and liver sinusoidal endothelial cells, without apparent associated hepatitis lesions. This study supports the hypothesis that low viral titers may persist in the liver of non-viremic individuals, giving thus the possibility of consumption of contaminated liver of animals diagnosed as HEV-negative in serum. Further immunopathogenic studies are necessary to elucidate the mechanisms responsible for this process and to evaluate the protocols of HEV diagnosis in animals destined for human consumption.

Schmallenberg disease is an emerging disease that affects domestic and wild ruminants in Europe. An epidemiological survey was carried out to assess exposure to Schmallenberg virus (SBV) in wild artiodactyls in Spain between 2006 and 2015. A total of 1751 sera from wild artiodactyls, including 1066 red deer, 304 fallow deer, 192 mouflon, 109 wild boar, 49 roe deer and 31 Spanish ibex were tested for antibodies against SBV by ELISA and confirmed by virus neutralization test. SBV was not detected between the 2006/2007 and the 2010/2011 hunting seasons. Overall seroprevalence (including samples collected between the 2011/2012 and 2014/2015 hunting seasons) was 14.6% (160/1099; 95%CI: 12.7-16.6). Mean SBV seroprevalence was 13.3±2.6% in red deer, 23.9±4.2% in fallow deer, 16.4±6.1% in mouflon and 2.8±3.1% in wild boar. No antibodies against SBV were found in roe deer or Spanish ibex. The presence of SBV RNA was confirmed in three of 255 (1.2%) spleen samples from wild ruminants analysed by rRT-PCR. In a multivariate mixed-effects logistic regression model, the main risk factors associated with SBV seroprevalence were: species (fallow deer, red deer and mouflon), age (adults) and interactions between hunting areas of more than 1000 hectares and hunting season (2012/2013, 2013/2014 and 2014/2015). The hypothesis of endemic circulation of SBV in the last few years is supported by the detection of SBV RNA in animals sampled in 2011 and 2015, as well as antibodies detected at low level in juveniles in 2012, 2013 and 2014. The results indicate that SBV circulated in wild ruminant populations in Spain during the same period when the virus was first reported in northern Europe, and at least five months before the first case was officially reported in livestock in Spain.

Understanding the spread of Toxoplasma gondii (T. gondii) in wild birds, particularly in those with opportunistic feeding behavior, is of interest for elucidating the epidemiological involvement of these birds in the maintenance and dissemination of the parasite. Overall, from 2009 to 2011, we collected sera from 525 seagull chicks (Yellow-legged gull (Larus michahellis) and Audouin's gull (L. audouinii)) from 6 breeding colonies in Spain and tested them using the modified agglutination test (MAT) for the presence of antibodies against T. gondii. Chick age was estimated from bill length. Main food source of seagull chicks was evaluated using stable isotope analyses from growing scapular feathers. Overall T. gondii seroprevalence was 21.0% (IC95% 17.5-24.4). A generalized linear mixed-effects model indicated that year (2009) and food source (freshwater) were risk factors associated to the individual risk of infection by T. gondii, while age (days) was close to significance. Freshwater food origin was related to the highest seroprevalence levels, followed by marine origin, supporting freshwater and sewages as important routes of dispersion of T. gondii. Year differences could indicate fluctuating rates of exposure of seagull chicks to T. gondii. Age ranged from 4 to 30 days and seropositivity tended to increase with age (P = 0.07), supporting that seropositivity is related to T. gondii infection rather than to maternal transfer of antibodies, which in gulls is known to sharply decrease with chick age. This study is the first to report T. gondii antibodies in Yellow-legged and Audouin's gulls, thereby extending the range of intermediate hosts for this parasite and underscoring the complexity of its epidemiology.

Andalusia (Southern Spain) is considered one of the main routes of introduction of bluetongue virus (BTV) into Europe, evidenced by a devastating epidemic caused by BTV-1 in 2007. Understanding the pattern and the drivers of BTV-1 spread in Andalusia is critical for effective detection and control of future epidemics. A long-standing metric for quantifying the behaviour of infectious diseases is the case-reproduction ratio (Rt), defined as the average number of secondary cases arising from a single infected case at time t (for t>0). Here we apply a method using epidemic trees to estimate the between-herd case reproduction ratio directly from epidemic data allowing the spatial and temporal variability in transmission to be described. We then relate this variability to predictors describing the hosts, vectors and the environment to better understand why the epidemic spread more quickly in some regions or periods. The Rt value for the BTV-1 epidemic in Andalusia peaked in July at 4.6, at the start of the epidemic, then decreased to 2.2 by August, dropped below 1 by September (0.8), and by October it had decreased to 0.02. BTV spread was the consequence of both local transmission within established disease foci and BTV expansion to distant new areas (i.e. new foci), which resulted in a high variability in BTV transmission, not only among different areas, but particularly through time, which suggests that general control measures applied at broad spatial scales are unlikely to be effective. This high variability through time was probably due to the impact of temperature on BTV transmission, as evidenced by a reduction in the value of Rt by 0.0041 for every unit increase (day) in the extrinsic incubation period (EIP), which is itself directly dependent on temperature. Moreover, within the range of values at which BTV-1 transmission occurred in Andalusia (20.6°C to 29.5°C) there was a positive correlation between temperature and Rt values, although the relationship was not linear, probably as a result of the complex relationship between temperature and the different parameters affecting BTV transmission. Rt values for BTV-1 in Andalusia fell below the threshold of 1 when temperatures dropped below 21°C, a much higher threshold than that reported in other BTV outbreaks, such as the BTV-8 epidemic in Northern Europe. This divergence may be explained by differences in the adaptation to temperature of the main vectors of the BTV-1 epidemic in Andalusia (Culicoides imicola) compared those of the BTV-8 epidemic in Northern Europe (Culicoides obsoletus). Importantly, we found that BTV transmission (Rt value) increased significantly in areas with higher densities of sheep. Our analysis also established that control of BTV-1 in Andalusia was complicated by the simultaneous establishment of several distant foci at the start of the epidemic, which may have been caused by several independent introductions of infected vectors from the North of Africa. We discuss the implications of these findings for BTV surveillance and control in this region of Europe.

Wild and domestic ruminants are susceptible to Bluetongue virus (BTV) infection. Three BTV serotypes (BTV-4, BTV-1 and BTV-8) have been detected in Spain in the last decade. Even though control strategies have been applied to livestock, BTV circulation has been frequently detected in wild ruminant populations in Spain. The aim of the present study is to assess the role for wild ruminants in maintaining BTV after the vaccination programs in livestock in mainland Spain. A total of 931 out 1,914 (48.6%) serum samples, collected from eight different wild ruminant species between 2006 and 2011, were BTV positive by ELISA. In order to detect specific antibodies against BTV-1, BTV-4 and BTV-8, positive sera were also tested by serumneutralisation test (SNT). From the ELISA positive samples that could be tested by SNT (687 out of 931), 292 (42.5%) showed neutralising antibodies against one or two BTV serotypes. For each BTV serotype, the number of outbreaks in livestock (11,857 outbreaks in total) was modelled with pure autoregressive models and the resulting smoothed values, representing the predicted number of BTV outbreaks in livestock at municipality level, were positively correlated with BTV persistence in wild species. The strength of this relationship significantly decreased as red deer (Cervus elaphus) population abundance increased. In addition, BTV RNA was detected by real time RT-PCR in 32 out of 311 (10.3%) spleen samples from seropositive animals. Although BT outbreaks in livestock have decreased substantially after vaccination campaigns, our results indicated that wild ruminants have been exposed to BTV in territories where outbreaks in domestic animals occurred. The detection of BTV RNA and spatial association between BT outbreaks in livestock and BTV rates in red deer are consistent with the hypothesis of virus circulation and BTV maintenance within Iberian wild ruminant populations.

A longitudinal study was carried out to determine the seroprevalence of avian influenza viruses (AIVs) in waterfowl used as decoys in Andalusia, southern Spain. A total of 2319 aquatic birds from 193 flocks were analyzed before and after the hunting season 2011-2012. In the first sampling, 403 out of 2319 (18.0%, CI95%: 15.8-19.0) decoys showed antibodies against AIVs by ELISA. The AI seroprevalence was significantly higher in geese (21.0%) than in ducks (11.7%) (P<0.001). Besides, the spatial distribution of AIVs was not homogeneous as significant differences among regions were observed. The prevalence of antibodies against AIVs subtypes H5 and H7 were 1.1% and 0.3%, respectively, using hemagglutination inhibition test (HI). The overall and H5 seroprevalences slightly increased after the hunting period (to 19.2% and 1.4%, respectively), while the H7 seroprevalence remained at the same level (0.3%). The proportion of flocks infected by AIVs was 65.3%, while 11.2% and 4.9% of flocks were positive for H5 and H7, respectively. Viral shedding was not detected in any of the 47 samples positive by both ELISA and HI, tested by RRT-PCR. The individual incidence after the hunting season was 3.4%. The fact that 57 animals seroconverted, 15 of which were confirmed by HI (12 H5 and 3 H7), was indication of contact with AIVs during the hunting period. The results indicate that waterfowl used as decoys are frequently exposed to AIVs and may be potentially useful as sentinels for AIVs monitoring. The seroprevalence detected and the seropositivity against AIVs H5 and H7, suggest that decoys can act as reservoirs of AIVs, which may be of animal and public health concern.

In recent years, a number of zoonotic flaviviruses have emerged worldwide, and wild birds serve as their major reservoirs. Epidemiological surveys of bird populations at various geographical scales can clarify key aspects of the eco-epidemiology of these viruses. In this study, we aimed at exploring the presence of flaviviruses in the western Mediterranean by sampling breeding populations of the yellow-legged gull (Larus michahellis), a widely distributed, anthropophilic, and abundant seabird species. For 3 years, we sampled eggs from 19 breeding colonies in Spain, France, Algeria, and Tunisia. First, ELISAs were used to determine if the eggs contained antibodies against flaviviruses. Second, neutralization assays were used to identify the specific flaviviruses present. Finally, for colonies in which ELISA-positive eggs had been found, chick serum samples and potential vectors, culicid mosquitoes and soft ticks (Ornithodoros maritimus), were collected and analyzed using serology and PCR, respectively. The prevalence of flavivirus-specific antibodies in eggs was highly spatially heterogeneous. In northeastern Spain, on the Medes Islands and in the nearby village of L'Escala, 56% of eggs had antibodies against the flavivirus envelope protein, but were negative for neutralizing antibodies against three common flaviviruses: West Nile, Usutu, and tick-borne encephalitis viruses. Furthermore, little evidence of past flavivirus exposure was obtained for the other colonies. A subset of the Ornithodoros ticks from Medes screened for flaviviral RNA tested positive for a virus whose NS5 gene was 95% similar to that of Meaban virus, a flavivirus previously isolated from ticks of Larus argentatus in western France. All ELISA-positive samples subsequently tested positive for Meaban virus neutralizing antibodies. This study shows that gulls in the western Mediterranean Basin are exposed to a tick-borne Meaban-like virus, which underscores the need of exploring the spatial and temporal distribution of this flavivirus as well as its potential pathogenicity for animals and humans.

The control of bovine tuberculosis (bTB) is at a critical point in the last stage of eradication in livestock. Wildlife species recently have emerged infected with TB in Europe, particularly ungulates in the Iberian Peninsula. Epidemiological information regarding TB in wild ungulates including affected species, prevalence, associated risk factors and appropriate diagnostic methods need to be obtained in these countries. A cross-sectional study was carried out on wild artiodactyl species, including Eurasian wild boar (Sus scrofa) red deer (Cervus elaphus), roe deer (Capraelus capraelus), fallow deer (Dama dama), Spanish ibex (Capra pyrenaica hispanica) and mouflon (Ovis musimon), in Spain to assess the seroprevalence against Mycobacterium bovis or cross-reacting members of the Mycobcaterium tuberculosis complex (MTBC), and to provide information on associated risk factors. Previously, two in-house indirect enzyme linked immunosorbent assays (bPPD-ELISA and MPB83-ELISA) were developed using known TB status sera. Positive reference sera were selected from infected animals confirmed by culture. The M. bovis isolates belonged to spoligotypes SB0121, SB0120, SB0295, SB0265 and SB0134. Two hundred and two out of 1367 (7.5%; 95% CI: 6.1-8.9) animals presented antibodies against M. bovis by both bPPD-ELISA and MPB83-ELISA. Significantly higher TB seroprevalence was observed in wild boar compared to the other species analyzed. Interestingly, seropositivity against M. bovis was not found in any out of 460 Spanish ibex analyzed. The logistic regression model for wild boar indicated that the seropositivity to M. bovis was associated with age, location and year of sampling, while the only risk factor associated with M. bovis seroprevalence in red deer and fallow deer was the age. The seroprevalence observed indicates a widespread exposure to MTBC in several wild artiodactyl species in southern Spain, which may have important implications not only for conservation but also for animal and public health.

Many wild ruminants such as Spanish ibex (Capra pyrenaica) are susceptible to Bluetongue virus (BTV) infection, which causes disease mainly in domestic sheep and cattle. Outbreaks involving either BTV serotypes 1 (BTV-1) and 8 (BTV-8) are currently challenging Europe. Inclusion of wildlife vaccination among BTV control measures should be considered in certain species. In the present study, four out of fifteen seronegative Spanish ibexes were immunized with a single dose of inactivated vaccine against BTV-1, four against BTV-8 and seven ibexes were non vaccinated controls. Seven ibexes (four vaccinated and three controls) were inoculated with each BTV serotype. Antibody and IFN-gamma responses were evaluated until 28 days after inoculation (dpi). The vaccinated ibexes showed significant (P<0.05) neutralizing antibody levels after vaccination compared to non vaccinated ibexes. The non vaccinated ibexes remained seronegative until challenge and showed neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of non vaccinated ibexes from 2 to the end of the study (28 dpi) and in target tissue samples obtained at necropsy (8 and 28 dpi). BTV-1 was successfully isolated on cell culture from blood and target tissues of non vaccinated ibexes. Clinical signs were unapparent and no gross lesions were found at necropsy. Our results show for the first time that Spanish ibex is susceptible and asymptomatic to BTV infection and also that a single dose of vaccine prevents viraemia against BTV-1 and BTV-8 replication.

Design, sampling and data interpretation constitute an important challenge for wildlife surveillance of avian influenza viruses (AIV). The aim of this study was to construct a model to improve and enhance identification in both different periods and locations of avian species likely at high risk of contact with AIV in a specific wetland. This study presents an individual-based stochastic model for the Ebre Delta as an example of this appliance. Based on the Monte-Carlo method, the model simulates the dynamics of the spread of AIV among wild birds in a natural park following introduction of an infected bird. Data on wild bird species population, apparent AIV prevalence recorded in wild birds during the period of study, and ecological information on factors such as behaviour, contact rates or patterns of movements of waterfowl were incorporated as inputs of the model. From these inputs, the model predicted those species that would introduce most of AIV in different periods and those species and areas that would be at high risk as a consequence of the spread of these AIV incursions. This method can serve as a complementary tool to previous studies to optimize the allocation of the limited AI surveillance resources in a local complex ecosystem. However, this study indicates that in order to predict the evolution of the spread of AIV at the local scale, there is a need for further research on the identification of host factors involved in the interspecies transmission of AIV.

Toxoplasma gondii is a zoonotic intracellular protozoan parasite of worldwide distribution that infects many species of warm-blooded animals, including birds. To date, there is scant information about the seropositivity of T. gondii and the risk factors associated with T. gondii infection in wild bird populations. In the present study, T. gondii infection was evaluated on sera obtained from 1079 wild birds belonging to 56 species (including Falconiformes (n=610), Strigiformes (n=260), Ciconiiformes (n=156), Gruiformes (n=21), and other orders (n=32), from different areas of Spain. Antibodies to T. gondii (modified agglutination test, MAT titer ≥1:25) were found in 282 (26.1%, IC(95%:)23.5-28.7) of the 1079 birds. This study constitute the first extensive survey in wild birds species in Spain and reports for the first time T. gondii antibodies in the griffon vulture (Gyps fulvus), short-toed snake-eagle (Circaetus gallicus), Bonelli's eagle (Aquila fasciata), golden eagle (Aquila chrysaetos), bearded vulture (Gypaetus barbatus), osprey (Pandion haliaetus), Montagu's harrier (Circus pygargus), Western marsh-harrier (Circus aeruginosus), peregrine falcon (Falco peregrinus), long-eared owl (Asio otus), common scops owl (Otus scops), Eurasian spoonbill (Platalea leucorodia), white stork (Ciconia ciconia), grey heron (Ardea cinerea), common moorhen (Gallinula chloropus); in the International Union for Conservation of Nature (IUCN) "vulnerable" Spanish imperial eagle (Aquila adalberti), lesser kestrel (Falco naumanni) and great bustard (Otis tarda); and in the IUCN "near threatened" red kite (Milvus milvus). The highest seropositivity by species was observed in the Eurasian eagle owl (Bubo bubo) (68.1%, 98 of 144). The main risk factors associated with T. gondii seropositivity in wild birds were age and diet, with the highest exposure in older animals and in carnivorous wild birds. The results showed that T. gondii infection is widespread and can be at a high level in many wild birds in Spain, most likely related to their feeding behaviour., (© 2011 Cabezón et al.)