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Summary: During the last decade, West Nile virus (WNV) outbreaks have increased sharply in both horses and human in Europe. The aims of this study were to evaluate characteristics and spatio‐temporal distribution of WNV outbreaks in horses in Spain between 2010 and 2016 in order to identify the environmental variables most associated with WNV occurrence and to generate high‐resolution WNV suitability maps to inform risk‐based surveillance strategies in this country. Between August 2010 and November 2016, a total of 403 WNV suspected cases were investigated, of which, 177 (43.9%) were laboratory confirmed. Mean values of morbidity, mortality and case fatality rates were 7.5%, 1.6% and 21.2%, respectively. The most common clinical symptoms were as follows: tiredness/apathy, recumbency, muscular tremor, ataxia, incoordination and hyperaesthesia. The outbreaks confirmed during the last 7 years, with detection of WNV RNA lineage 1 in 2010, 2012, 2013, 2015 and 2016, suggest an endemic circulation of the virus in Spain. The spatio‐temporal distribution of WNV outbreaks in Spain was not homogeneous, as most of them (92.7%) were concentrated in western part of Andalusia (southern Spain) and significant clusters were detected in this region in two non‐consecutive years. These findings were supported by the results of the space–time scan statistics permutation model. A presence‐only MaxEnt ecological niche model was used to generate a suitability map for WNV occurrence in Andalusia. The most important predictors selected by the Ecological Niche Modeling were as follows: mean annual temperature (49.5% contribution), presence of Culex pipiens (19.5% contribution), mean annual precipitation (16.1% contribution) and distance to Ramsar wetlands (14.9% contribution). Our results constitute an important step for understanding WNV emergence and spread in Spain and will provide valuable information for the development of more cost‐effective surveillance and control programmes and improve the protection of horse and human populations in WNV‐endemic areas. [ABSTRACT FROM AUTHOR]

Myxoma (MYXV) and rabbit hemorrhagic disease (RHDV) viruses are pathogens of economic relevance for cuniculture and conservation concern for wild European rabbits (Oryctolagus cuniculus), recently classified as 'Endangered' in its native range. Large-scale serological surveys, facilitated by sample collection using dried blood spots (DBS), allow monitoring seroprevalence in the wild reservoir but require evaluating the technique for the host and pathogen of interest. This study aimed to evaluate Protein Saver 903 DBS for MYXV and RHDV (genotype GI.2) serological surveys in European rabbits. Paired serum and DBS collected from 172 rabbits harvested or found dead in the Iberian Peninsula were tested for IgG antibodies specific against MYXV and RHDV GI.2 using indirect ELISA. We found an almost perfect agreement between serum and DBS for MYXV (Cohen's κ=0.914, CI 95 0.847 - 0.981) and a strong agreement for RHDV GI.2 (Cohen's κ=0.808, CI 95 =0.722 - 0.893). The diagnostic sensitivity of DBS was 95.4 % (CI 95 90.3 - 97.9 %) for MYXV and 82.1 % (CI 95 73.2 - 88.5 %) for RHDV GI.2. The diagnostic specificity and positive predictive value were 100 % for both pathogens. This study supports DBS as a suitable sampling strategy for serological surveys of antibodies specific to MYXV and RHDV GI.2 in European rabbits, which generally agrees with results from other hosts and pathogens where this technique was evaluated., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Hepatitis E virus (HEV) is an emerging zoonotic virus of public health concern, of which pigs, wild boar and red deer are the main reservoirs. The European Food Safety Authority (EFSA) has recently prioritized the development of monitoring programs of HEV at different stages of the pig food chain, including outdoor pig farming. Pigs managed under these extensive production systems frequently share habitat and natural resources with wild boar and red deer during fattening stages and cross-species transmission of HEV among these species has previously been suggested. In this context, we aimed to (I) to evaluate the risk of HEV circulation within the production phases of extensively raised pigs and at the domestic-wildlife interface, and (II) to identify the genotypes circulating within these hosts. A total of 1452 pigs from seven different pig farms were longitudinally sampled during the breeding, rearing, and fattening production phases. In addition, 138 and 252 sympatric wild boar and red deer, respectively, were analysed. Anti-HEV antibodies were found in 1245 (85.7 %) out of the 1452 Iberian pigs sampled. The seroprevalence was 30.4 % in the breeding phase, 95.4 % in the rearing phase and 97.0 % in the fattening phase. Statistically significant differences (P < 0.05) were found among the three production phases. The seroprevalence was significantly higher (P < 0.001) in fattening pigs compared to those found in sympatric wild boar (31.9 %) and red deer (2.0 %). Three (1.0 %) out of the 293 serum pools analysed were positive for viral RNA. One of them was identified in pigs at the rearing phase (genotype 3 f) and two in wild boar (genotypes 3 f and 3 m). The high seroprevalence detected in extensively raised pigs, together with the detection of the zoonotic HEV-3 f and HEV-3 m subtypes in sympatric domestic and wild swine, highlights the risk of zoonotic transmission and the need to establish surveillance programs and control measures, particularly in breeding and rearing phase, in these epidemiological scenarios., Competing Interests: Declaration of Competing Interest None of the authors of this study has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The European Food Safety Authority has recently listed Q fever as a priority for setting up a coordinated surveillance system. Although Spain is the country with the highest human incidence of the disease in the European Union, updated data on Coxiella burnetii in ruminants are still limited. A total of 780 serum samples from small ruminants and 605 sera from wild ruminants were collected in the Mediterranean ecosystems of southern Spain during the period 2015-2023. Anti- C. burnetii antibodies were detected using a commercial indirect ELISA. The overall individual seroprevalence in the small ruminants was 49.1% (383/780; 95% CI: 45.6-52.6). Antibodies against C. burnetii were identified in 40.0% (156/390) of sheep and in 58.2% (227/390) of goats. At least one seropositive animal was observed in all sheep (100%) and in 92.3% of goat flocks. The species (goat) and the existence of reproductive disorders in primiparous females were potential risk factors for C. burnetii exposure in small ruminant farms. In the wild ruminants, the overall seroprevalence against C. burnetii was 1.5% (9/605; 95% CI: 0.8-2.8). Anti- C. burnetii antibodies were found in 1.8% (2/110) of mouflon, 1.5% (6/390) of red deer, and 1.0% (1/105) of Iberian ibex. The high exposure of the small ruminants to C. burnetii , particularly in goats, detected in the present study is of animal and public health concern. Our results denote that wild ruminants only play a minor role in the epidemiology of this bacterium in southern Spain and suggest an independent epidemiological cycle of C. burnetii in domestic and wild ruminant species in the study area.

Animal tuberculosis (TB) is often maintained by multi-host communities, including livestock and wildlife. Quantitative studies of such communities require estimating the true prevalence of TB, correcting the apparent prevalence by the diagnostic sensitivity (Se) and specificity (Sp) of the test. The goal of this study was to lay the foundations for estimating the true prevalence of TB in wild ungulate populations (wild boar and two cervids: red deer and fallow deer). We used Bayesian latent class models to assess the Se and Sp of gross pathology, IS6110 real-time PCR in tissues, bacteriological culture, and P22 indirect ELISA. We analyzed 308 harvested wild ungulates (211 wild boar and 97 cervids: 92 red deer and 5 fallow deer). The Se of bacteriological culture (80.4%, CI 95 61.0-96.3%) and gross pathology (87.9%, CI 95 69.5-99.9%) was reasonably good in wild boar. These tests showed lower Se in cervids: 60.2% (CI 95 38.3-82.3%) for bacteriological culture and 81.5% (CI 95 63.6-96.2%) for gross pathology. The Se of the real-time PCR was low (50.7% in wild boar and 53.0% in cervids). These tests showed Sp between 95.2 and 99.1% in both taxa. The P22 ELISA performed reasonably well in wild boar (Se = 71.9%, CI 95 59.2-83.4%; Sp = 98.8%, CI 95 96.9-99.9%) but lacked Sp in cervids (Se = 77.1%, CI 95 62.9-89.7%; Sp = 74.5%, CI 95 65.7-83.3%). The real-time PCR in wild boar and cervids and bacteriological culture in cervids tended to show higher Se in low-prevalence populations, possibly due to a higher proportion of early-stage TB lesions. In cervids, the parallel interpretation of gross pathology and bacteriological culture significantly improved the diagnostic performance (Se = 93.1%, CI 95 84.7-98.9%; Sp = 92.9%, CI 95 86.0-98.3%). Our results allow the estimation of true prevalence from the results of a single diagnostic test applied to harvested wild boar, red deer, and fallow deer, paving the way for more precise quantitative ecological studies of the multi-host TB maintenance community., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cardoso, Jiménez-Ruiz, Perelló Jiménez, Nóvoa, Santos, Correia-Neves, Gortázar and Santos.)

Interspecific interactions are highly relevant in the potential transmission of shared pathogens in multi-host systems. In recent decades, several technologies have been developed to study pathogen transmission, such as proximity loggers, GPS tracking devices and/or camera traps. Despite the diversity of methods aimed at detecting contacts, the analysis of transmission risk is often reduced to contact rates and the probability of transmission given the contact. However, the latter process is continuous over time and unique for each contact, and is influenced by the characteristics of the contact and the pathogen's relationship with both the host and the environment. Our objective was to assess whether a more comprehensive approach, using a movement-based model which assigns a unique transmission risk to each contact by decomposing transmission into contact formation, contact duration and host characteristics, could reveal disease transmission dynamics that are not detected with more traditional approaches. The model was built from GPS-collar data from two management systems in Spain where animal tuberculosis (TB) circulates: a national park with extensively reared endemic cattle, and an area with extensive free-range pigs and cattle farms. In addition, we evaluated the effect of the GPS device fix rate on the performance of the model. Different transmission dynamics were identified between both management systems. Considering the specific conditions under which each contact occurs (i.e. whether the contact is direct or indirect, its duration, the hosts characteristics, the environmental conditions, etc.) resulted in the identification of different transmission dynamics compared to using only contact rates. We found that fix intervals greater than 30 min in the GPS tracking data resulted in missed interactions, and intervals greater than 2 h may be insufficient for epidemiological purposes. Our study shows that neglecting the conditions under which each contact occurs may result in a misidentification of the real role of each species in disease transmission. This study describes a clear and repeatable framework to study pathogen transmission from GPS data and provides further insights to understand how TB is maintained in multi-host systems in Mediterranean environments., (© 2024 The Author(s). Journal of Animal Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society.)

Background: Coxiella burnetii, the causative agent of Q fever, is a zoonotic multi-host vector-borne pathogen of major public health importance. Although the European Food Safety Authority has recently made the monitoring of this bacterium in wildlife a priority, the role of wild lagomorphs in the transmission and maintenance of C. burnetii is poorly understood., Aims: The aims of this study were to determine the prevalence and risk factors associated with C. burnetii circulation in European wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis) and to assess the presence of this pathogen in ticks that feed on them in Mediterranean ecosystems in Spain, the country with the highest number of reported cases of Q fever in Europe., Methods: A total of 574 spleen samples were collected from 453 wild rabbits and 121 Iberian hares, and 513 ticks (processed in 120 pools) between the 2017/2018 and 2021/2022 hunting seasons., Results: C. burnetii DNA was detected in 103 (17.9%; 95% CI: 14.8-21.1) of the 574 wild lagomorphs tested. By species, prevalence was 16.3% (74/453; 95% CI: 12.9-19.7) in the European wild rabbit and 24.0% (29/121; 95% CI: 16.4-31.6) in the Iberian hare. At least one positive lagomorph was found on 47.9% of the 96 hunting estates sampled and in every hunting season since 2018/2019. Two risk factors associated with C. burnetii infection were as follows: outbreak of myxomatosis on the hunting estate in the month prior to sampling and high tick abundance observed by gamekeepers on the hunting estate. C. burnetii DNA was also found in 33 of the 120 (27.5%; 95% CI: 19.5-35.5) tick pools tested. The pathogen was detected in 66.7% (4/6), 29.2% (26/89) and 21.4% (3/14) of Haemaphysalis hispanica, Rhipicephalus pusillus and Hyalomma lusitanicum pools respectively., Conclusions: This study provides new epidemiological data on C. burnetii in European wild rabbits and is the first survey on this zoonotic pathogen performed in Iberian hares. Our results indicate widespread endemic circulation of C. burnetii and highlight the importance of both wild lagomorph species as natural reservoirs of this zoonotic bacterium in Mediterranean ecosystems in southern Spain, which may be of public and animal health concern. The high prevalence and wide diversity of positive tick species suggest the possible role of ticks in the epidemiological cycle of C. burnetii, with the potential risk of transmission to sympatric species, including humans., (© 2024 The Author(s). Zoonoses and Public Health published by Wiley‐VCH GmbH.)

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne zoonotic orthonairovirus of public health concern and widespread geographic distribution. Several animal species are known to seroconvert after infection with CCHFV without showing clinical symptoms. The commercial availability of a multi-species ELISA has led to an increase in recent serosurveillance studies as well as in the range of species reported to be exposed to CCHFV in the field, including wild boar (Sus scrofa). However, development and validation of confirmatory serological tests for swine based on different CCHFV antigens or test principles are hampered by the lack of defined control sera from infected and non-infected animals. For the detection of anti-CCHFV antibodies in swine, we established a swine-specific in-house ELISA using a panel of swine sera from CCHFV-free regions and regions with reported CCHFV circulation. We initially screened more than 700 serum samples from wild boar and domestic pigs and observed a correlation of ≃67% between the commercial and the in-house test. From these sera, we selected a panel of 60 samples that were further analyzed in a newly established indirect immunofluorescence assay (iIFA) and virus neutralization test. ELISA-non-reactive samples tested negative. Interestingly, only a subset of samples reactive in both ELISA and iIFA displayed CCHFV-neutralizing antibodies. The observed partial discrepancy between the tests may be explained by different test sensitivities, antibody cross-reactivities or suggests that the immune response to CCHFV in swine is not necessarily associated with eliciting neutralizing antibodies. Overall, this study highlights that meaningful CCHFV serology in swine, and possibly other species, should involve the performance of multiple tests and careful interpretation of the results., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

We conducted a cross-sectional study in wild boar and extensively managed Iberian pig populations in a hotspot area of Crimean-Congo hemorrhagic fever virus (CCHFV) in Spain. We tested for antibodies against CCHFV by using 2 ELISAs in parallel. We assessed the presence of CCHFV RNA by means of reverse transcription quantitative PCR protocol, which detects all genotypes. A total of 113 (21.8%) of 518 suids sampled showed antibodies against CCHFV by ELISA. By species, 106 (39.7%) of 267 wild boars and 7 (2.8%) of 251 Iberian pigs analyzed were seropositive. Of the 231 Iberian pigs and 231 wild boars analyzed, none tested positive for CCHFV RNA. These findings indicate high CCHFV exposure in wild boar populations in endemic areas and confirm the susceptibility of extensively reared pigs to CCHFV, even though they may only play a limited role in the enzootic cycle.

Aims: A cross-sectional study was carried out to assess the seroprevalence and risk factors associated with Trichinella spp. exposure in wild boar and Iberian domestic pigs from Mediterranean ecosystems of southwestern Spain., Methods and Results: Serum samples from 1360 wild boar and 439 Iberian domestic pigs were obtained during 2015-2020, from regions where Iberian pigs are raised under extensive conditions, hence sharing habitat with wild boar. Seropositivity was found in 7.4% (100/1360; 95% CI: 6.1-8.9) of the wild boar analysed. In this species, the individual seroprevalence ranged from 3.6% (8/223) (hunting season 2016-2017) to 11.4% (37/326) (2018-2019). A significant higher seropositivity was observed during the hunting season 2018-2019 (p < 0.009: OR = 3.07; 95% CI = 1.32-7.18) and one statistically significant cluster was detected within the studied area, in south central Andalusia [Relative Risk (RR) = 2.9; p = 0.037]. Females showed a significantly higher seroprevalence than males (8.7% vs. 5.8%) (p < 0.001: OR = 1.58; 95% CI = 1.08-2.32). No seropositivity to Trichinella spp. was detected in Iberian domestic pigs (0.0%; 95% CI: 0.0-0.9)., Conclusions: Although wild boar play an important role as a reservoir of Trichinella sp. in the Mediterranean ecosystems of southwestern Spain, our results suggest that the wild boar production system does not seem to pose a risk of Trichinella exposure to domestic pigs, despite sharing habitats in these ecosystems., (© 2023 The Authors. Zoonoses and Public Health published by Wiley-VCH GmbH.)

Vulture feeding stations (VFS) are strategic sites for the management of ungulate game by-products, with major implications for the conservation of obligate avian scavengers. These feeding points are designed to prevent access by facultative mammalian scavengers and to reduce the risk of trophically transmitted pathogen infection through scavenging behaviour. The aim of this study, using camera trap video recordings, was to evaluate the feeding dynamics around 32 lots of ungulate game by-products deposited in VFS in southern Spain. During the study period (2017-2022), 11 different avian and mammal scavenger species was detected. Also, many feeding events involving all scavenger species (51.1%; 362/709) and mammal scavenger species (23.4%; 166/709) was recorded. A significantly earlier presence of major obligate scavengers (Gyps fulvus and Aegypius monachus) (P = 0.023) and a lower persistence of by-products deposited (P < 0.001) were detected during October-December compared to January-March. Our results confirm that VFS play an important role in vulture conservation in Iberian Mediterranean ecosystems. However, we also point out the potential risk of VFS as hotspots of foodborne pathogens for mammal scavengers, particularly when the hunting offal persists longer. Consequently, we urge the competent authorities to update the regulations affecting VFS management, and hunting estate managers to make every effort to correctly apply those preventing scavenging mammals from gaining access to VFS. Further studies tackling access of non-target species to VFS are also warranted to properly assess the sanitary implications and geographical extent of these problems for mammal species inhabiting areas where VFS are installed., Competing Interests: Declaration of Competing Interest None of the authors of this study has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Wildlife is an important reservoir of zoonotic pathogens. The objective of the present study was to assess the importance of wild ungulates in the epidemiology of Rickettsia spp. Ticks and spleen samples were collected from 262 red deer (Cervus elaphus) and 83 wild boar (Sus scrofa) hunted in southwestern Spain over a 5-year period. DNA was extracted from tick pools (n = 191) and spleens (n = 345), and two nested PCR assays targeting the rOmpA and rOmpB genes were used to detect Rickettsia DNA. Five tick species were identified (Hyalomma lusitanicum, Dermacentor marginatus, Ixodes ricinus, Rhipicephalus bursa and Haemaphysalis sulcata). Rickettsia DNA was detected in 31 (16.2%) tick pools and two red deer spleen samples (0.8%). Four validated Rickettsia species (R. slovaca, R. monacensis, R. helvetica and R. raoultii), one uncultivated species (Candidatus R. rioja) and two uncharacterized Rickettsia spp. were detected in ticks. R. helvetica and R. slovaca were also detected in spleen samples from red deer. The overall prevalence in ungulate spleen samples was lower than in tick pools suggesting that these ungulates do not play a major role in the transmission of Rickettsia spp. However, their importance as spreaders of positive ticks cannot be ruled out. The results present a challenge for the veterinary and public health communities since most of the Rickettsia spp. detected are pathogenic. Furthermore, the new Rickettsia species could be potential pathogens. For these reasons, identifying Rickettsia species present in ticks and wildlife is of particular interest to clarify their sylvatic cycle and establish appropriate control measures., (© 2023 The Authors. Zoonoses and Public Health published by Wiley-VCH GmbH.)

Myxoma virus (MYXV) and rabbit hemorrhagic disease virus (RHDV) are important drivers of the population decline of the European rabbit, an endangered keystone species. Both viruses elicit strong immune responses, but the long-term dynamics of humoral immunity are imperfectly known. This study aimed to assess the determinants of the long-term dynamics of antibodies to each virus based on a longitudinal capture-mark-recapture of wild European rabbits and semiquantitative serological data of MYXV and RHDV GI.2-specific IgG. The study included 611 indirect enzyme-linked immunosorbent assay (iELISA) normalized absorbance ratios for each MYXV and RHDV GI.2 from 505 rabbits from 2018 to 2022. Normalized absorbance ratios were analyzed using log-linear mixed models, showing a significant positive relationship with the time since the first capture of individual rabbits, with monthly increases of 4.1% for antibodies against MYXV and 2.0% against RHDV GI.2. Individual serological histories showed fluctuations over time, suggesting that reinfections boosted the immune response and likely resulted in lifelong immunity. Normalized absorbance ratios significantly increased with the seroprevalence in the population, probably because of recent outbreaks, and with body weight, highlighting the role of MYXV and RHDV GI.2 in determining survival to adulthood. Juvenile rabbits seropositive for both viruses were found, and the dynamics of RHDV GI.2 normalized absorbance ratios suggest the presence of maternal immunity up to 2 months of age. Semiquantitative longitudinal serological data provide epidemiological information, otherwise lost when considering only qualitative data, and support a lifelong acquired humoral immunity to RHDV GI.2 and MYXV upon natural infection. IMPORTANCE This study addresses the long-term dynamics of humoral immunity to two major viral pathogens of the European rabbit, an endangered keystone species of major ecological relevance. Such studies are particularly challenging in free-ranging species, and a combination of longitudinal capture-mark-recapture and semiquantitative serology was used to address this question. Over 600 normalized absorbance ratios of iELISA, obtained from 505 individual rabbits in 7 populations over 5 years, were analyzed using linear mixed models. The results support a lifelong acquired humoral immunity to myxoma virus and rabbit hemorrhagic disease virus upon natural infection and suggest the presence of maternal immunity to the latter in wild juvenile rabbits. These results contribute to understanding the epidemiology of two viral diseases threatening this keystone species and assist in developing conservation programs., Competing Interests: The authors declare no conflict of interest.

Tick abundance is an essential demographic parameter to infer tick-borne pathogen transmission risks. Spatiotemporal patterns of tick abundance are heterogeneous, so its determinants at small spatial scales need to be understood to reduce their negative effects on hosts. Current knowledge of these determinants is scarce, especially in Mediterranean environments, limiting the possibilities for designing efficient tick control strategies. With the goal of unravelling tick abundance determinants and informing new tick management strategies, we estimated tick burdens on 1965 wild ungulates in Doñana National Park, Spain, annually between 2010 and 2020. Under the hypothesis of a predominant host influence on tick abundance, we modelled the burdens of Rhipicephalus annulatus, Hyalomma lusitanicum, and Ixodes ricinus with relevant predictors grouped into four factors: i) environment; ii) host population; iii) host individual; and iv) land-use. Generalized linear mixed models with a zero-inflated negative binomial distribution were built. Additionally, we analysed the differential contribution to abundance of each factor by deviance partitioning. We finally estimated the similarity in the environmental space of tick species by analysing their niche overlap with the environmental principal component analysis method. Our work hypothesis was confirmed for R. annulatus and H. lusitanicum, but we found that tick abundance at a fine spatial scale is jointly driven by multiple drivers, including all four factors considered in this study. This result points out that understanding the demography of ticks is a complex multifactorial issue, even at small spatial scales. We found no niche differences between the three tick species at the study spatial scale, thus showing similar host and environmental dependencies. Overall results identify that host aggregation areas displaying environmentally favourable traits for ticks are relevant tick and vector-borne pathogen transmission hotspots. Our findings will facilitate the design of new strategies to reduce the negative effects of tick parasitism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Crimean-Congo haemorrhagic fever (CCHF) is an emerging tick-borne human disease in Spain. Understanding the spatiotemporal dynamics and exposure risk determinants of CCHF virus (CCHFV) in animal models is essential to predict the time and areas of highest transmission risk. With this goal, we designed a longitudinal survey of two wild ungulate species, the red deer (Cervus elaphus) and the Eurasian wild boar (Sus scrofa), in Doñana National Park, a protected Mediterranean biodiversity hotspot with high ungulate and CCHFV vector abundance, and which is also one of the main stopover sites for migratory birds between Africa and western Europe. Both ungulates are hosts to the principal CCHFV vector in Spain, Hyalomma lusitanicum. We sampled wild ungulates annually from 2005 to 2020 and analysed the frequency of exposure to CCHFV by a double-antigen ELISA. The annual exposure risk was modelled as a function of environmental traits in an approach to understanding exposure risk determinants that allow us to predict the most likely places and years for CCHFV transmission. The main findings show that H. lusitanicum abundance is a fundamental driver of the fine-scale spatial CCHFV transmission risk, while inter-annual risk variation is conditioned by virus/vector hosts, host community structure and weather variations. The most relevant conclusion of the study is that the emergence of CCHF in Spain might be associated with recent wild ungulate population changes promoting higher vector abundance. This work provides relevant insights into the transmission dynamics of CCHFV in enzootic scenarios that would allow deepening the understanding of the ecology of CCHFV and its major determinants., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

The hepatitis E virus (HEV) is the main cause of viral acute hepatitis in the world, affecting more than 20 million people annually. During the acute phase of infection, HEV can be detected in various body fluids, which has a significant impact in terms of transmission, diagnosis or extrahepatic manifestations. Several studies have isolated HEV in the genitourinary tract of humans and animals, which could have important clinical and epidemiological implications. So, our main objective was to evaluate the presence of HEV in testis of naturally infected wild boars (Sus scrofa). For it, blood, liver, hepatic lymph node and testicle samples were collected from 191 male wild boars. The presence of HEV was evaluated in serum by PCR, as well as in tissues by PCR and immunohistochemistry. Four animals (2.09%; 95%CI: 0.82-5.26) showed detectable HEV RNA in serum, being confirmed the presence of HEV-3f genotype in three of them by phylogenetic analysis. HEV was also detected in liver and/or hepatic lymph nodes of the four animals by RT-PCR, as well as by immunohistochemistry analysis. Only one of these wild boars also showed detectable viral load in testis, observing HEV-specific labelling in a small number of fibroblasts and some Sertoli cells. Our results confirm the presence of HEV genotype 3 in naturally infected wild boar testis, although no associated tissue damage was evidenced. This study does not allow us to discard semen as a possible source of HEV transmission in suids. Future experimental studies are necessary to evaluate the impact of HEV genotype 3 on fertility and the possibility of transmission through sexual contact in this specie., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

A cross-sectional study was carried out to assess the circulation of bluetongue virus (BTV) and Schmallenberg virus (SBV) within the wild and domestic ungulate host community in Doñana National Park (southwestern Spain). A total of 440 animals, including 138 cattle, 102 red deer (Cervus elaphus), 101 fallow deer (Dama dama) and 99 wild boar (Sus scrofa), were sampled in 2015 during the seasonal peak of Culicoides spp. (summer-autumn). Serum and spleen samples were analysed to detect exposure (using commercial blocking ELISAs) and infection (by RT-PCR), respectively, to BTV and SBV. Cattle were not tested by BTV-bELISA because all were previously vaccinated against BTV serotypes 1 and 4. High BTV seroprevalences were found in red deer (97.0%) and fallow deer (64.7%). Antibodies against SBV were detected in 37.0% of cattle, 16.8% of red deer, 23.5% of fallow deer and 2.0% of wild boar. Thirty-eight of the 203 deer (18.7%; 17 red deer and 21 fallow deer) were co-exposed to both viral agents. BTV-4 RNA was confirmed in four red deer and two fallow deer. SBV RNA was found in two fallow deer. Co-infections were not detected. Our results indicate high exposure, widespread distribution and active circulation of BTV and SBV in the ruminant community in the study area. We provide additional evidence for the potential role of wild cervids as reservoirs of these Culicoides-borne viruses in two different epidemiological scenarios: with vaccination (BTV) and without vaccination (SBV) of sympatric livestock. This study highlights the importance of wildlife surveillance, particularly of cervid species, for the proper execution of control programmes of Culicoides-borne diseases in extensively reared livestock., (© 2022 Wiley-VCH GmbH.)

Crimean-Congo haemorrhagic fever virus (CCHFV) continues to cause new human cases in Iberia while its spatial distribution and ecological determinants remain unknown. The virus remains active in a silent tick-animal cycle to which animals contribute maintaining the tick populations and the virus itself. Wild ungulates, in particular red deer, are essential hosts for Hyalomma ticks in Iberia, which are the principal competent vector of CCHFV. Red deer could be an excellent model to understand the ecological determinants of CCHFV as well as to predict infection risks for humans because it is large, gregarious, abundant and the principal host for Hyalomma lusitanicum. We designed a cross-sectional study, analysed the presence of CCHFV antibodies in 1444 deer from 82 populations, and statistically modelled exposure risk with host and environmental predictors. The best-fitted statistical model was projected for peninsular Spain to map infection risks. Fifty out of 82 deer populations were seropositive, with individual population prevalence as high as 88%. The highest prevalence of exposure to CCHFV occurred in the southwest of the Iberian Peninsula. Climate and ungulate abundance were the most influential predictors of the risk of exposure to the virus. The highest risk regions were those where H. lusitanicum is most abundant. Eight of the nine primary human cases occurred in or bordering these regions, demonstrating that the model predicts human infection risk accurately. A recent human case of CCHF occurred in northwestern Spain, a region that the model predicted as low risk, pointing out that it needs improvement to capture all determinants of the CCHFV infection risk. In this study, we have been able to identify the main ecological determinants of CCHFV, and we have also managed to create an accurate model to assess the risk of CCHFV infection., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Autochthonous cases of hepatitis E (HE) associated with zoonotic genotypes HEV-3 and HEV-4 have significantly increased in industrialized countries over the last decade. Suidae are generally recognized as the main reservoirs of these genotypes. Susceptibility to HE virus (HEV) infection and zoonotic potential have also been confirmed in other species, including sheep and goat. However, the information about their role in the epidemiology of HEV remains very scarce. The objective of this study was to assess the prevalence, spatial distribution and risk factors associated with HEV exposure in sheep and goats in southern Spain, the country with the highest census of small domestic ruminants in the European Union. Blood samples from 240 sheep and 240 goats were collected between 2015 and 2017. Sera were analysed in parallel using a commercial double-antigen ELISA and real-time PCR. A total of 38 (7.9%; 95%CI: 5.5-10.3) out of 480 sampled animals showed anti-HEV antibodies. By species, the seroprevalences found in sheep and goats were 2.1% (5/240; 95%CI: 0.3-3.9) and 13.8% (33/240; 95%CI: 9.4-18.1) respectively. Anti-HEV antibodies were found on 19 (59.4%; 95%CI: 42.4-76.4) of the 32 sampled farms. The GEE model showed that species (goat) and number of small ruminants in the farm (≤348 animals and ≥538 animals) were risk factors potentially associated with HEV exposure in small ruminants in the study area. HEV RNA was not detected in any of the 480 (0.0%; 95%CI: 0.0-0.8) tested animals. Our results confirm that sheep and goats are naturally, but not equally exposed to HEV and indicate the widespread spatial distribution of HEV among small ruminant populations in southern Spain. Further studies are required to elucidate the role of sheep and goat in the epidemiology of HEV and their potential implications for public health., (© 2022 The Authors. Zoonoses and Public Health published by Wiley-VCH GmbH.)

Paratuberculosis is a worldwide, chronic infectious disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) that mainly affects ruminant species. This disease has a significant economic impact on small ruminant production due to the costs of implementing control measures and production losses. A cross-sectional study was carried out to determine the seroprevalence, spatial distribution and risk factors associated with MAP exposure in sheep and goats in Andalusia (southern Spain). Serum samples from 4134 small ruminants (2266 sheep and 1868 goats) in 153 flocks were tested by an in-house ELISA for antibodies against MAP using paratuberculosis protoplasmic antigen 3 (PPA3) as coating antigen. Antibodies against MAP were detected in 8.1% (183/2266; 95% CI: 7.0-9.2%) of sheep and 20.0% (374/1868; 95% CI: 18.2-21.8%) of goats. The true individual seroprevalence was 8.4% (95% CI: 6.9-10.1%) in sheep and 25.2% (95% CI: 22.7-27.8%) in goats. Seropositivity was detected in 66.3% (55/83; 95% CI: 56.1-76.4%) of sheep herds and 90.0% (63/70; 95% CI: 83.0-97.0%) of goat herds. Spatial analysis identified three statistically significant clusters (p < 0.05) associated with areas with higher seroprevalence of MAP. The main risk factors potentially associated with MAP exposure were: species (goat) and absence of perimeter livestock fencing. The results of this study show that MAP is widespread in small ruminant populations in southern Spain and suggest that goats may play a more important role than sheep in the transmission and maintenance of MAP. Because of animal health concerns and the economic consequences of paratuberculosis, appropriate surveillance and control programs are required to reduce the risk of MAP infections in small ruminant flocks in this country., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

A cross-sectional study was carried out to evaluate shared pathogens that can be transmitted by close or non-close contact at the domestic-wild ruminants' interface. During summer-autumn 2015, a total of 138 cattle and 203 wild ruminants (red deer, Cervus elaphus, and fallow deer, Dama dama) were sampled in Doñana National Park (DNP, south-western Spain), a Mediterranean ecosystem well known for the interaction network occurring in the ungulate host community. Pestiviruses, bovine respiratory syncytial virus (BRSV; Bovine orthopneumovirus), bovine herpesvirus 1 (BoHV-1; Bovine alphaherpesvirus 1) and Mycobacterium tuberculosis complex (MTC) were assessed using serological, microbiological and molecular techniques. The overall seroprevalence against viruses in cattle was 2.2% for pestiviruses, 11.6% for BRSV and 27.5% for BoHV-1. No virus-specific antibodies were found in wildlife. MTC incidence in cattle was 15.9%, and MTC seroprevalence in wild ruminants was 14.3%. The same Mycobacterium bovis spoligotypes (SB1232, SB1230 and SB1610) were identified in cattle, red deer and fallow deer. The serological results for the selected respiratory viruses suggest epidemiological cycles only in cattle. Surveillance efforts in multi-host epidemiological scenarios are needed to better drive and prioritize control strategies for shared pathogens., (© 2021 Wiley-VCH GmbH.)

Background: To control the transmission of relevant shared diseases, such as animal tuberculosis (TB) and African swine fever (ASF), it is essential to reduce the risk of interaction between livestock and wild ungulates. In Eastern and Central Europe, the current spread of ASF virus affecting wild boar and domestic pigs (especially those raised outdoors and/or in backyards) has devastated the pig sector in affected regions and is seriously threatening other exporting countries. Here, we evaluated the risk of wildlife-livestock interactions on 45 outdoor pig farms in Spain, the second largest pork producer in the EU and then proposed biosecurity-related actions. An integrated, systematic wildlife risk mitigation protocol based on interviews, questionnaires and field audits was developed and applied on each farm., Results: Most of the interaction risk points were associated with water sources (84.2%; 701/832), mainly springs and ponds, which accounted for almost all the specific points with high or very high risk scores. The risk of interaction at feeding points (6.9%; 57/832) and those associated with facilities for livestock and/or game management (8.9%; 74/832) were rated as low and very low risk, respectively. Wild boar were present and hunted on 69% of the farms. Supplementary feeding for wild ungulate species (mainly wild boar) was provided on almost half (48.9%; 22/45) the surveyed farms. Risk mitigation actions were categorised to target water access, waterers, food, other livestock species, grazing, wildlife, and offal disposal. Of the total number of actions (n = 2016), 82.7% were identified as priority actions while 17.3% represented alternative options which were identified less cost-effective. On average, 37.1 (median: 32; range 14-113) action proposals per study farm were made and 2.0 (median: 1; range 0-4) per risk point. The mean estimated cost of implementing the proposed priority actions was 14,780 €/farm (25.7 €/hectare and 799.4 €/risk point)., Conclusions: This study expands the knowledge of interaction risks between domestic pigs and wild ungulates in outdoor pig farming systems and highlights the importance of considering local risks and management practices when designing and prioritising adapted wildlife risk mitigation and biosecurity actions. This practical and feasible protocol developed for Mediterranean ecosystems is easily transferable to professionals and can be adapted to extensive (outdoor) production or epidemiological systems in other European regions., (© 2022. The Author(s).)

Toxoplasmosis is a parasitic zoonosis caused by Toxoplasma gondii which infects warm-blooded species worldwide. Humans can be infected through ingestion of tissue cysts from raw or undercooked meat, including game meat. A nationwide large-scale cross-sectional study was conducted to assess exposure to T. gondii in seven wild ruminant species in Spain. A total of 2,040 serum samples from 77 sampling sites randomly distributed in the five bioregions (BRs) covering mainland Spain were tested for antibodies against T. gondii using the modified agglutination test. The overall seroprevalence was 22.0% (449/2,040). Seroprevalence by species in decreasing order was as follows: 39.6% (141/356) in roe deer (Capreolus capreolus), 37.1% (138/372) in fallow deer (Dama dama), 16.6% (92/553) in red deer (Cervus elaphus), 14.0% (26/186) in Southern chamois (Rupicapra pyrenaica), 11.5% (24/209) in mouflon (Ovis aries musimon), 7.8% (27/346) in Iberian wild goat (Capra pyrenaica) and 5.6% (1/18) in Barbary sheep (Ammotragus lervia). Seropositivity was detected in 74.0% (57/77) of the sampling sites. Results indicate widespread but not homogeneous exposure to T. gondii in wild ruminant populations in Spain during the last two decades and highlight differences related to animal species and spatial distribution of these species in this country; this implies potential consequences of T. gondii for animal health, conservation and public health., (© 2021 The Authors. Zoonoses and Public Health published by Wiley-VCH GmbH.)

The importance of biosecurity as a strategy to prevent and control infectious diseases has increased substantially over the last few decades. Several studies have reported a low implementation level of biosecurity measures (BSM), particularly in cattle farms. In addition, a recent study demonstrated that cattle farmers are well aware of the recommended BSM and recognize them as more effective (in terms of time and costs) than treatment for disease. Therefore, other factors must be considered when it comes to understanding the decision-making process followed by a farmer regarding the adoption of BSM. This study analysed the possible influence of five mental constructs described in the health belief model (HBM) on the adoption of BSM and assessed the possible association of these constructs with different demographic and socio-psychological factors. Through an online survey, 988 questionnaires were completed by cattle farmers originating from Belgium, France, Germany, Spain and the Netherlands. The study revealed that the actual implementation of the BSM seems to be significantly influenced by the farmers' perception of the measures' benefits and the perception of health responsibility. Both constructs are influenced by the farmers' personality in terms of risk aversion and biosecurity knowledge. It was also found that organic farmers had a significantly lower perception of the BSM benefits and of their responsibility towards animal, public and environmental health when compared with other types of farmer. Organic farmers in this study seemed less likely to implement biosecurity measures. To increase the adoption of BSM by cattle farmers, it is therefore important to emphasise the actual evidence-based benefits of the measures and to investigate further how to strengthen cattle farmers' sense of responsibility towards animal, public and environmental health., (© 2020 Wiley-VCH GmbH.)

Bluetongue is a vector-borne disease affecting domestic and wild ruminants, with a major socioeconomic impact. Endemic circulation of the bluetongue virus serotype 4 (BTV-4) and BTV-1 have occurred in Spain since 2004 and 2007, respectively. However, epidemiological studies have seldom been approached from a long-term perspective in wild ruminants. A total of 881 deer (red deer and fallow deer) were necropsied from 2005 to 2018 as part of the DNP health-monitoring program. Serum samples were tested for antibodies against BTV with the aims of assessing the temporal trend and to evaluate the modulating factors: individual, populational, environmental, and stochastic. Red deer displayed statistically significant higher seroprevalences of BTV (SBT; 78.6 ± 3.8%) than fallow deer (53.1 ± 4.7%). The detection of BTV-1 and BTV-4 by the serum neutralization test in calves suggested the circulation of both serotypes over the study period. For red deer, wet years together with high densities could provide suitable conditions for vector borne BTV transmission. Moreover, proximity to high suitability habitat for Culicoides, permanent pasturelands, was associated with higher SBT. The differences in the ecology and behaviour of deer species influencing the exposure to the vectors could determine the differences found in the SBT patterns. This study evidences the role that deer species may play in the maintenance of BTV, however, elucidating the epidemiological role of host in different contexts as well as the consequences of climate change on the competent vector populations and its potential effect on the dynamics of BTV infection in hosts communities deserve further research., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The knowledge regarding the spatial ecology of red deer (Cervus elaphus) in different environments is crucial if effective management actions are to be designed. However, this knowledge continues to be scarce in the complex contexts of mixed land use and management circumstances. This study describes the spatial ecology of red deer monitored using GPS collars in Mediterranean ecosystems of South-Central Spain, considering the effect of individual and seasonal (food shortage period, rut, hunting season and food abundance period) factors on different land use and management scenarios, namely protected areas, mixed farms and fenced hunting estates. Our results showed less activity (ACT), a shorter daily range (DR) and a smaller home range (HR) during the food shortage period: ACT: 0.38 ± (SD) 0.12; DR: 3010.9 ± 727.3 m; and weekly HR: 122.2 ± 59.6 ha. With regard to land use, individuals were less ACT and had a smaller DR on fenced hunting estates (ACT: 0.24 ± 0.12; DR: 1946.3 ± 706.7 m) than in protected areas (ACT: 0.59 ± 0.12; DR: 4071.4 ± 1068.2 m) or on mixed farms (ACT: 0.57 ± 0.29; DR: 5431.1 ± 1939.5 m) in all the periods studied. Red deer selected land cover with forage and shelter when foraging and resting, respectively. When drive hunt events occurred (mixed farms and fenced hunting estates), the deer were more prone to select safer habitats (scrublands) and avoid open areas (crops or grasslands) than were their counterparts in protected areas. The patterns observed can be explained by sexual and seasonal differences as regards requirements, the response to disturbances and, interestingly, population management. Our results provide useful information with which to design scientifically-based species adaptive management in response to relevant and timely situations in Europe, such as the potential transmission of shared infections, vehicle collisions, and damage to crops and ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The effective management of shared pathogens between wild ungulates and livestock requires the understanding of the processes of interaction between them. In this work, we studied the interspecific frequency of interaction (ifreq) and its spatiotemporal pattern between wild and domestic ungulates that coexist in free-ranging farms. For this purpose, 6 red deer, 6 wild boar, 8 Iberian pigs and 3 cattle were monitored using GPS devices during the "montanera" period (the period in which Iberian pigs are maintained in extensive conditions to feed on acorn). The ifreq was quantified for two spatiotemporal windows: 30 m - 10 min, for inferring potential direct interactions (short window), and 30 m - 12 days for indirect interactions (large window). Secondly, the variation in the ifreq was modelled with regard to 2 temporal (time of the day and week of the year) and 4 environmental factors (distance to water, distance to vegetation cover, Quercus density and distance to feeding points). The interactions at the short window were scarce (N = 13); however, they were very frequent at the large one (N = 37,429), with the red deer as the species with the greatest involvement in the interactions. Models showed that the time of the day and distance to water were the variables that best predicted the ifreq and they were conditioned by differences in the activity pattern of the targeted species. Food resource availability also predicted the ifreq, especially at the short window and between wild species. The results presented here highlight the role that wild ungulates may play in the transmission of pathogens to extensive livestock in general and pigs in particular and show the epidemiological risk of certain areas, periods of time and management practices (for wildlife and livestock) as well as providing useful information in the prevention of the transmission of shared pathogens., (© 2020 Wiley-VCH GmbH.)

The hepatitis E virus (HEV) is an emerging zoonotic pathogen whose main reservoir is suids. Most of the ecological and epidemiological aspects of its sylvatic cycle remain unknown. Thus, in this work, we study the drivers of HEV exposure in the wild boar population of Doñana National Park (DNP, southwest Spain) operating in the medium and long-term (2005-2018). Anti-HEV antibodies are widely distributed throughout the wild boar (46.7 ± 3.8%, 327 out of 700 sampled), showing a statistically significant age-increasing pattern. The temporal pattern displayed important interannual fluctuations. This could be mediated by marked variations in the population control of the wild boar, and subsequent changes in abundance rates, and its interplay with climatic conditions; as wet years together with a low abundance of wild boar led to the lowest seroprevalence. The fact that seroprevalence is high during conditions of high abundance, and not affected by rainfall level, is probably due to the increased interactions among the animals, and possibly, the subsequent higher environmental contamination with HEV particles. The proximity to the marshland (the main water body of the study area) is associated with a higher risk of testing positive, which is probably mediated by the preferential use of this area during the dry season and the favourable environmental conditions for the survival of HEV particles. A deeper understanding of the epidemiology of HEV in host communities deserves future research concerning other susceptible species. Most importantly, wild boar population control remains a challenge at the international level, and an increase of shared pathogen-related conflicts associated with this species is expected, as exemplified by HEV. Therefore, surveillance of wild boar diseases, including integrated population monitoring and sustainable population control programmes, will be essential to control the associated risks.

A large-scale study was carried out to determine the prevalence of antibodies against Pestivirus species in wild ruminants and describe their spatial variation in mainland Spain. Serum samples of 1,874 wild ruminants from different regions of this country were collected between the years 2000 and 2017. A total of 6.6% (123/1,874) animals showed antibodies against Pestivirus by both blocking ELISA (bELISA) and virus neutralization tests (VNT). The prevalence of antibodies against pestiviruses was different both among species and regions. Seroprevalence by species was 30.0% (75/250) in Southern chamois (Rupicapra pyrenaica), 7.0% (25/357) in fallow deer (Dama dama), 2.5% (10/401) in red deer (Cervus elaphus), 2.4% (8/330) in Iberian wild goat (Capra pyrenaica), 1.1% (4/369) in roe deer (Capreolus capreolus) and 0.8% (1/130) in mouflon (Ovis aries musimon), not detecting seropositivity (0/37) in Barbary sheep (Ammotragus lervia). The results confirm that exposure to pestiviruses was detected throughout mainland Spain, with significantly higher seroprevalence in Northern regions associated with the presence of Southern chamois. This indicates an endemic circulation of pestiviruses in Southern chamois and a limited circulation of these viruses in the remaining wild ruminant species during the last two decades, thus suggesting that non-chamois species are not true Pestivirus reservoirs in Spain. Nonetheless, the high spatial spread of these viruses points out that new epidemic outbreaks in naïve wild ruminant populations or transmission to livestock may occur, evidencing the usefulness of monitoring pestiviruses in wild ruminants, especially at the wildlife-livestock interface., (© 2020 Wiley-VCH GmbH.)

Animal tuberculosis (TB), which is caused by the Mycobacterium tuberculosis complex (MTC), is a zoonotic disease of global concern, and has a wide variety of wild and domestic reservoirs that can establish complex epidemiological systems. Of all the strategies employed to control TB, reducing the risks of interaction at the wildlife-livestock interface is a cornerstone. However, detailed protocols with which to assess and implement farm-specific preventive actions that can be employed against interactions with wildlife are lacking for extensive production systems. We describe an On-farm Wildlife Risk Mitigation Protocol that is applicable to beef cattle farming in Mediterranean environments in order to control the wildlife-livestock interaction and MTC transmission through the use of Farm-specific Action Plans (FsAP). We assessed the implementation and verification of FsAP in terms of its practical feasibility and acceptability by farmers (n=55 farms). Of the potential risk points, waterers (41.3 %) and waterholes (24.4 %) were the most common. Waterholes and springs were identified as the points with the greatest risks. Actions related to water management were essential on most farms (99 % of the high-risk points), as were those regarding wildlife management (36.4 % of the farms provided wild boar or cervids with supplementary food for hunting purposes). Overall, 75 % of the farmers adopted the plans to some extent, with an average of 31.8 % of actions implemented, but with high variability depending on the type of actions proposed. Farmers prioritised low-cost measures. Our results, in their entirety, indicate that the adoption of this On-farm Wildlife Risk Mitigation Protocol is practical and feasible in Mediterranean ecosystems, and can be easily transferred to professionals and adapted to other bioregions or epidemiological systems. The subsequent evaluation of FsAPs in terms of efficacy and cost-effectiveness, along with increasing their acceptance by farmers, are necessary steps for the further development of TB Risk Mitigation Programmes at a nationwide level., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Schmallenberg disease (SBD) is an emerging vector-borne disease that affects domestic and wild ruminants. A long-term serosurvey was conducted to assess exposure to Schmallenberg virus (SBV) in all the wild ruminant species present in mainland Spain. Between 2010 and 2016, sera from 1,216 animals were tested for antibodies against SBV using a commercial blocking ELISA. The overall prevalence of antibodies was 27.1% (95%CI: 24.7-29.7). Statistically significant differences among species were observed, with significantly higher seropositivity found in fallow deer (Dama dama) (45.6%; 99/217), red deer (Cervus elaphus) (31.6%; 97/307) and mouflon (Ovis aries musimon) (28.0%; 33/118) compared to Barbary sheep (Ammotragus lervia) (22.2%; 8/36), Iberian wild goat (Capra pyrenaica) (19.9%; 49/246), roe deer (Capreolus capreolus) (17.5%; 34/194) and Southern chamois (Rupicapra pyrenaica) (10.2%; 10/98). Seropositive animals were detected in 81.4% (57/70; 95%CI: 70.8-88.8) of the sampled populations. SBV seroprevalence ranged from 18.8% (48/256) in bioregion (BR)2 (north-central, Mediterranean) to 32.3% (31/96) in BR1 (northeastern or Atlantic, Eurosiberian). Anti-SBV antibodies were not found before 2012, when the first outbreak of SBD was reported in Spain. In contrast, seropositivity was detected uninterruptedly during the period 2012-2016 and anti-SBV antibodies were found in yearling animals in each of these years. Our results provide evidence of widespread endemic circulation of SBV among wild ruminant populations in mainland Spain in recent years. Surveillance in these species could be a useful tool for monitoring SBV in Europe, particularly in areas where wild ruminants share habitats with livestock., (© 2020 Blackwell Verlag GmbH.)

Toxoplasma gondii is an obligate intracellular protozoan which infects warm-blooded vertebrates, including humans, worldwide. In the present study, the epidemiology of T. gondii was studied in the wild ungulate host community (wild boar, red deer, and fallow deer) of Doñana National Park (DNP, south-western Spain) for 13 years (2005-2018). We assessed several variables which potentially operate in the medium and long-term (environmental features, population, and stochastic factors). Overall, the wild ungulate host community of DNP had high seroprevalence values of T. gondii (STG; % ± confidence interval (CI) 95%; wild boar ( Sus scrofa ) 39 ± 3.3, n = 698; red deer ( Cervus elaphus ) 30.7 ± 4.4, n = 423; fallow deer ( Dama dama ) 29.7 ± 4.2, n = 452). The complex interplay of hosts and ecological/epidemiological niches, together with the optimal climatic conditions for the survival of oocysts that converge in this area may favor the spread of the parasite in its host community. The temporal evolution of STG oscillated considerably, mostly in deer species. The relationships shown by statistical models indicated that several factors determined species patterns. Concomitance of effects among species, indicated that relevant drivers of risk operated at the community level. Our focus, addressing factors operating at broad temporal scale, allows showing their impacts on the epidemiology of T. gondii and its trends. This approach is key to understanding the epidemiology and ecology to T. gondii infection in wild host communities in a context where the decline in seroprevalence leads to loss of immunity in humans.

Microsporidia are obligate intracellular protist-like fungal pathogens that infect a broad range of animal species, including humans. This study aimed to assess the presence of zoonotic microsporidia ( Enterocytozoon bieneusi , Encephalitozoon intestinalis , Encephalitozoon hellem , and Encephalitozoon cuniculi ) in organ meats of European wild rabbit ( Oryctolagus cuniculus ) and Iberian hare ( Lepus granatensis ) consumed by humans in Spain. Between July 2015 and December 2018, kidney samples from 383 wild rabbits and kidney and brain tissues from 79 Iberian hares in southern Spain were tested by species-specific PCR for the detection of microsporidia DNA. Enterocytozoon bieneusi infection was confirmed in three wild rabbits (0.8%; 95% CI: 0.0-1.7%) but not in hares (0.0%; 95% CI: 0.0-4.6%), whereas E. intestinalis DNA was found in one wild rabbit (0.3%; 95% CI: 0.0-0.8%) and three Iberian hares (3.8%; 95% CI: 0.0-8.0%). Neither E. hellem nor E. cuniculi infection were detected in the 462 (0.0%; 95% CI: 0.0-0.8%) lagomorphs analyzed. The absence of E. hellem and E. cuniculi infection suggests a low risk of zoonotic foodborne transmission from these wild lagomorph species in southern Spain. To the authors' knowledge, this is the first report of E. intestinalis infection in wild rabbits and Iberian hares. The presence of E. bieneusi and E. intestinalis in organ meats from wild lagomorphs can be of public health concern. Additional studies are required to determine the real prevalence of these parasites in European wild rabbit and Iberian hare.

Peste des Petits Ruminants (PPR) is a highly infectious disease caused by a virus member of the genus Morbillivirus, which mainly affects goats, sheep and wild ruminants. It is considered one of the most significant transboundary infectious diseases and represents an animal health concern in developing countries. Spain is considered a PPR-free country. Nevertheless, given its geographical proximity to countries in North Africa where PPR virus (PPRV) has been circulating in recent years, the south of Spain can be considered a risk area for the introduction of PPRV. The aim of the present study was to assess circulation of PPRV in domestic and wild ruminant species in this country. During the period 2015-2017, a total of 910 sera from domestic, wild and captive ruminants were analysed using a commercial blocking ELISA to detect antibodies against PPRV. None of the 910 (0.0%; 95% CI: 0.0-0.3) animals tested were positive for anti-PPRV antibodies. To the best of our knowledge, this is the first serosurvey study of PPR in Spain. The results indicate absence of circulating PPRV in the south of Spain during the study period. Due to the risk of PPRV introduction into European countries of the Mediterranean basin, epidemiological surveillance should be maintained and extended in this region., (© 2020 Blackwell Verlag GmbH.)

In recent decades, cases of autochthonous hepatitis E (HE) have sharply increased in European countries where foodborne transmission is considered the main route of HE virus (HEV) transmission. Although rabbits are considered the main reservoir of the zoonotic HEV-3ra subtype, information on the role of wild lagomorphs in the epidemiology of HEV remains scarce. The aim of this study therefore was to assess the circulation of HEV in European wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis), the most important lagomorph species in Spanish Mediterranean ecosystems. Liver samples from 372 wild rabbits and 78 Iberian hares were analysed using a broad-spectrum RT-PCR that detects HEV genotypes 1-8. None of the 450 lagomorphs tested were positive for HEV infection. To the best of our knowledge, this is the first study to assess HEV circulation in wild rabbits in Spain and the first to evaluate HEV infection in Iberian hares. Our results indicate absence of HEV circulation in wild rabbits and Iberian hares in southern Spain during the study period, which suggests that the risk of transmission of HEV from wild lagomorphs to other species, including humans, is low., (© 2020 Blackwell Verlag GmbH.)

Bluetongue (BT) is a reportable re-emerging vector-borne disease of animal health concern. Enzyme-linked immunosorbent assays (ELISA) are frequently used in BT surveillance programs in domestic ruminants, but their diagnostic accuracy has not been evaluated for wild ruminants, which can play an important role as natural reservoirs of bluetongue virus (BTV). The aim of this study was to assess two commercial ELISAs for BT diagnosis in wild ruminants using control sera of known BTV infection status and field samples. When control sera were tested, the double recognition ELISA (DR-ELISA) showed 100 % sensitivity (Se) and specificity (Sp), while the competitive ELISA (C-ELISA) had 86.4 % Se and 97.1 % Sp. Using field samples, the selected latent-class analysis model showed 95.7 % Se and 85.9 % Sp for DR-ELISA, 58.2 % Se and 95.8 % Sp for C-ELISA and 84.2 % Se for the serum neutralization test (SNT). Our results indicate that the DR-ELISA may be a useful diagnostic method to assess BTV circulation in endemic areas, while the C-ELISA should be selected when free-areas are surveyed. The discrepancy between control and field samples point out that the inclusion of field samples is required to assess the accuracy of commercial ELISAs for the serological diagnosis of BTV in wild ruminants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Pigs reared under extensive farming conditions are currently in high commercial demand because they are associated with high-quality products. Nevertheless, the risk of contact with different pathogens of animal and public health concern is also higher in extensive production systems. Toxoplasma gondii is a widely prevalent zoonotic pathogen and transmission by contaminated pork is likely one of the main routes of human toxoplasmosis. The aim of this study was to determine the seroprevalence, risk factors and spatial distribution of T. gondii on extensive Iberian pig herds in Spain. Sera from 2245 Iberian pigs from 114 herds were collected between 2015 and 2017 and analyzed using a commercial ELISA. The apparent individual prevalence of antibodies against T. gondii was 24.1 % (542/2245) and the estimated true seroprevalence was 24.3 % (CI95 %: 22.5-26.1). Seropositivity was detected in 86.0 % (98/114; CI95 %: 77.4-91.1) of 114 herds analyzed. A multi-level logistic regression model showed that T. gondii infection was significantly more frequent in sows than in fattening pigs (OR: 2.6; CI95 %: 1.5-4.8) and in herds with more than three cats compared to no cats (OR: 2.9; CI95 %: 1.1-8.7). Our results indicate a widespread but heterogenous distribution of T. gondii in extensively reared Iberian pig herds, which may have important implications for public health through the consumption of undercooked or improperly cured pork products., Competing Interests: Declaration of Competing Interest None of the authors of this study has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the manuscript., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Wild boar (Sus scrofa) is considered as the main wildlife reservoir of zoonotic hepatitis E virus (HEV) genotypes. The aim of this study was to evaluate the circulation of HEV in free-ranging wild boar in the Doñana National Park (DNP), Spain. Blood samples were collected from 99 wild boar in the DNP during 2015. Sera were analysed in parallel using indirect ELISA and real-time RT-PCR. A total of 57 of the 99 tested animals (57.6%; 95%CI: 47.8%-67.3%) had anti-HEV antibodies, indicating that this virus is widespread in wild boar in the DNP. HEV RNA was detected in one animal and phylogenetic analysis showed that the sequence isolated belonged to subtype 3r. The results suggest a potential risk of zoonotic transmission of this novel HEV-3 subtype, which could be of public health concern. Further studies are required to assess the role of wild boar in the epidemiology of HEV-3r and to determine the infectivity of this emergent HEV subtype in other species, including humans., (© 2019 Blackwell Verlag GmbH.)

Schmallenberg disease (SBD) is an emerging disease transmitted mainly among ruminant species by biting midges of the genus Culicoides. Since the Schmallenberg virus (SBV) was first identified in Germany in late 2011, it rapidly spread to other European countries. The aims of the present study were to describe the first SBD outbreak in Spain and to assess the spread and risk factors associated with SBV infection in domestic ruminants from nearby farms during the following year. In March 2012, one malformed stillborn lamb from a sheep farm located in Cordoba province (Southern Spain) was subjected to necropsy. Pathological compatible lesions and molecular analyses confirmed the first SBV infection in Spain. Afterwards, serum samples from 505 extensively reared domestic ruminants from 29 farms were analysed using both blocking ELISA and virus neutralization test against SBV. The overall seroprevalence was 54.4% (CI 95% : 50.0-58.7). Antibodies were detected in 70.6%, 46.0% and 34.8% of cattle, sheep and goats, respectively. A generalized estimating equation model indicated that the main risk factors associated with SBV infection were: species (cattle), age (adult), and absence of animal insecticide treatment. Pathological and molecular results confirmed the presence of SBV in Spain few months after it was firstly identified in Germany. The seroprevalence detected indicates a widespread circulation of SBV in nearby domestic ruminant farms one year after this first outbreak was reported in Spain. Further studies are warranted to determine the spatio-temporal trend of SBV in domestic ruminants in this country., (Copyright © 2019 Elsevier Ltd. All rights reserved.)