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A cross-sectional study was conducted to assess the circulation and risk factors associated with West Nile virus (WNV) exposure in equine and wild bird populations following the largest epidemic outbreak ever reported in Spain. A total of 305 equids and 171 wild birds were sampled between November 2020 and June 2021. IgG antibodies against flaviviruses were detected by blocking enzyme-linked immunosorbent assay (bELISA) in 44.9% (109/243) and 87.1% (54/62) of unvaccinated and vaccinated equids, respectively. The individual seroprevalence in unvaccinated individuals (calculated on animals seropositive by both bELISA and virus microneutralization test [VNT]) was 38.3% (95%CI: 33.1-43.4). No IgM antibodies were detected in animals tested (0/243; 0.0%; 95%CI: 0.0-1.5) by capture-ELISA. The main risk factors associated with WNV exposure in equids were age (adult and geriatric), breed (crossbred) and the absence of a disinsection programme on the facilities. In wild birds, IgG antibodies against flaviviruses were found in 32.7% (56/171; 95%CI: 26.8-38.6) using bELISA, giving an individual WNV seroprevalence of 19.3% (95%CI: 14.3-24.3) after VNT. Seropositivity was found in 37.8% of the 37 species analysed. Species group (raptors), age (>1-year old) and size (large) were the main risk factors related to WNV seropositivity in wild birds. Our results indicate high exposure and widespread distribution of WNV in equid and wild bird populations in Spain after the epidemic outbreak in 2020. The present study highlights the need to continue and improve active surveillance programmes for the detection of WNV in Spain, particularly in those areas at greatest risk of virus circulation., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

The 2018 outbreak of myxomatosis in the Iberian hare (Lepus granatensis) has been hypothesized to originate from a species jump of the rabbit-associated myxoma virus (MYXV), after natural recombination with an unknown poxvirus. Iberian hares were long considered resistant to myxomatosis as no prior outbreaks were reported. To provide insights into the emergence of this recombinant virus (ha-MYXV), we investigated serum samples from 451 Iberian hares collected over two time periods almost two decades apart, 1994-1999 and 2017-2019 for the presence of antibodies and MYXV-DNA. First, we screened all serum samples using a rabbit commercial indirect ELISA (iELISA) and then tested a subset of these samples in parallel using indirect immunofluorescence test (IFT), competitive ELISA (cELISA) and qPCR targeting M000.5L/R gene conserved in MYXV and ha-MYXV. The cut-off of iELISA relative index 10 = 6.1 was selected from a semiparametric finite mixture analysis aiming to minimize the probability of false positive results. Overall, MYXV related-antibodies were detected in 57 hares (12.6%) including 38 apparently healthy hares (n = 10, sampled in 1994-1999, none MYXV-DNA positive, and n = 28 sampled in 2017-2019 of which four were also ha-MYXV-DNA positive) and 19 found-dead and ha-MYXV-DNA-positive sampled in 2018-2019. Interestingly, four seronegative hares sampled in 1997 were MYXV-DNA positive by qPCR, the result being confirmed by sequencing of three of them. For the Iberian hares hunted or live trapped (both apparently health), seroprevalence was significantly higher in 2017-2019 (13.0%, CI95% 9.2-18.2%) than in 1994-1999 (5.4%, CI95% 3.0-9.6%) (p = .009). Within the second period, seroprevalence was significantly higher in 2019 compared to 2017 (24.7 vs 1.7% considering all the sample, p = .007), and lower during the winter than the autumn (p < .001). While our molecular and serological results show that Iberian hares have been in contact with MYXV or an antigenically similar virus at least since 1996, they also show an increase in seroprevalence in 2018-2019. The remote contact with MYXV may have occurred with strains that circulated in rabbits, or with unnoticed strains already circulating in Iberian hare populations. This work strongly suggests the infection of Iberian hares with MYXV or an antigenically related virus, at least 20 years before the severe virus outbreaks were registered in 2018., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

A long-term active epidemiological surveillance programme was conducted to determine seroprevalence to myxoma virus (MYXV), infection prevalence and spatiotemporal patterns and factors associated with MYXV circulation in wild rabbits (Oryctolagus cuniculus) in Spanish Mediterranean ecosystems. A total of 2376 animals were sampled over four study periods: 2009-2012 (P1), 2012-2015 (P2), 2015-2018 (P3) and 2018-2021 (P4). Antibodies against MYXV were detected by a commercial indirect ELISA in 59.9% (1424/2376; 95% CI: 58.0-61.9) of wild rabbits. At least one seropositive animal was detected on 131 (96.3%) of 136 game estates sampled. MYXV infection was confirmed by PCR in 94 of 1063 (8.8%; 95% CI: 7.3-10.7) wild rabbits. Circulation of the novel recombinant MYXV (ha-MYXV) was not found in wild rabbits analysed during P4. Five statistically significant spatiotemporal clusters of high MYXV seroprevalence were identified using a Bernoulli model: one in P2 and four in P3. A generalized linear mixed model (GLMM) analysis identified sampling season (autumn), age (adult and juvenile), outbreaks of myxomatosis in the month prior to sampling, mean annual temperature, humidity and seropositivity to rabbit haemorrhagic disease virus as factors potentially linked with MYXV seropositivity. GLMM analysis identified outbreaks of myxomatosis in the month prior to sampling, MYXV seropositivity and presence of lesions compatible with myxomatosis as factors associated with MYXV infection. The results indicate high exposure, widespread but non-homogeneous distribution, and endemic circulation of MYXV in wild rabbit populations in southern Spain during the last decade. Prevalence of antibodies against MYXV showed fluctuations both within the year and over the study periods, revealing variations in the immunity of wild rabbit populations in Mediterranean ecosystems that could increase the risk of MYXV re-emergence in immunologically naïve populations. The present study highlights the importance of long-term surveillance to better understand the epidemiology of MYXV in wild lagomorphs., (© 2022 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.)

Hepatitis E virus (HEV, family Hepeviridae) is an important emerging and zoonotic pathogen. In recent decades, the number of human cases of zoonotic hepatitis E has increased considerably in industrialized countries and HEV has been detected in an expanding range of mammal species. Although domestic pigs and wild boar are considered the main reservoirs of zoonotic HEV genotypes, the role of other susceptible animals in the epidemiology of the virus is still poorly understood. A large-scale, long-term study was carried out (1) to assess HEV exposure in captive zoo animals in Spain and (2) to determine the dynamics of seropositivity in individuals that were sampled longitudinally during the study period. Between 2007 and 2021, serum samples from 425 zoo animals belonging to 109 animal species (including artiodactyls, carnivores, perissodactyls, proboscideans and rodents) were collected from 11 different zoological parks in Spain. Forty-six of these animals at seven of these zoos were also longitudinally sampled. Anti-HEV antibodies were detected in 36 (8.5%; 95% CI: 5.8-11.1) of 425 sampled zoo animals. Specific antibodies against HEV-3 and HEV-C1 antigens were confirmed in ELISA-positive animals using western blot assay. Two of 46 longitudinally surveyed animals seroconverted during the study period. Seropositivity was significantly higher in carnivores and perissodactyls than in artiodactyls, and also during the period 2012-2016 compared with 2007-2011. HEV RNA was not detected in any of the 262 animals that could be tested by RT-PCR. To the best of the author's knowledge, this is the first large-scale, long-term surveillance on HEV in different orders of zoo mammals. Our results indicate exposure to HEV-3 and HEV-C1 in zoo animals in Spain and confirm a widespread but not homogeneous spatiotemporal circulation of HEV in captive species in this country. Further studies are required to determine the role of zoo species, particularly carnivores and perissodactyls, in the epidemiology of HEV and to clarify the origins of infection in zoological parks., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

The aim of our study was to evaluate HEV antibody kinetics in HIV/HCV-coinfected patients with cirrhosis. A longitudinal retrospective study was designed. Patients were followed up every 6 months; anti-HEV IgG and IgM antibodies levels and HEV-RNA by qPCR were analysed. The prevalence and incidence of every HEV infection marker were calculated. The kinetics of anti-HEV IgG and IgM during the follow-up were evaluated. Seventy-five patients comprised the study population. The seroprevalence observed was 17.3%. None showed IgM antibodies or HEV-RNA at baseline. None showed detectable HEV viral load during the study period. After a median follow-up of 5.1 years, two of 62 seronegative patients (3.2%) seroconverted to IgG antibody. The incidence for IgM was 2.7%. Of the 13 patients with IgG seropositivity at baseline, five (38.5%) seroreverted. Meanwhile, of the two patients who exhibited IgM positivity during the study, one (50%) showed intermittent positivity. We found that HEV seropositivity is common in HIV/HCV-coinfected cirrhotic patients. A remarkable rate of IgG seroreversions and IgM intermittence was found, limiting the use of antibodies for the diagnosis of HEV infection in this population., (© 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.)

Hepatitis E virus (HEV) is an emerging zoonotic pathogen in Europe. In the Iberian Peninsula, wild boar (Sus scrofa) is considered the main wildlife reservoir of HEV. This wild ungulate shares habitat and resources with other potential HEV carriers in Iberian Mediterranean ecosystems, although information about the role of such sympatric species in the HEV epidemiological cycle is still very limited. The aims of the present large-scale, long-term study were: (1) to determine the seroprevalence and prevalence of HEV in both free-living and captive populations of the Iberian lynx (Lynx pardinus), the most endangered felid in the world; (2) to determine potential risk factors associated with HEV exposure in this species and (3) to evaluate the dynamics of seropositivity in longitudinally sampled animals during the study period. Between 2010 and 2021, serum samples from 275 Iberian lynxes were collected in free-ranging and captive populations across the Iberian Peninsula. Forty-four of the 275 lynxes were also longitudinally sampled during the study period. A double-antigen sandwich ELISA was used to test for the presence of antibodies against HEV. A subset of seropositive samples was analysed by Western blot (WB) assay to confirm exposure to HEV. In addition, serum, liver and/or faecal samples from 367 individuals were tested for orthohepevirus RNA by RT-PCR. A total of 50 (18.2%; 95% CI: 14.1-23.2) of the 275 animals analysed had anti-HEV antibodies by ELISA. Exposure to HEV was confirmed by WB in most of the ELISA-positive Iberian lynxes analysed. Significantly higher seroprevalence was found in captive (33.6%) compared to free-ranging (7.4%) individuals. Within captive population, the GEE model identified 'age' (senile, adult and subadult) as risk a factor potentially associated with HEV exposure in the Iberian lynx. Thirteen (29.5%) of 44 longitudinally surveyed individuals seroconverted against HEV during the study period. HEV RNA was detected in the faeces of one (1/364; 0.3%; 95% CI: 0.0-0.8) free-ranging adult animal sampled in 2021. Phylogenetic analysis showed that the sequenced strain belongs to HEV-3f subtype and shared a high nucleotide sequence identity (97-99.6%) with human HEV-3f sequences from Spain and France. To the best of the authors' knowledge, this is the first survey study on HEV in the Iberian lynx and the first molecular report of HEV-A infection in free-ranging felines. Our results indicate high exposure to HEV-3 in Iberian lynx populations, particularly those kept in captivity. The serological results suggest widespread but not homogeneous circulation of HEV in Iberian lynx populations. Further studies are required to assess the epidemiological role of this endangered species as a potential spillover host of HEV., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Enteropathogenic parasites can infect a wide range of mammals, including humans, supposing an important zoonotic risk. Hepatitis E virus (HEV) is an emerging foodborne pathogen of increasing public health relevance, affecting both humans and animal populations. Because both microorganisms share faecal-oral transmission route they may constitute an excellent model to evaluate the interplay between them. Thus, we aim to evaluate the viral-parasite interactions at the enteric interface in swine. We included pigs of two different breeds farming in South Spain under different production systems. We compared the HEV prevalence by the presence of Giardia duodenalis, Cryptosporidium spp., Balantioides coli, Blastocystis sp. and Enterocytozoon bieneusi in faecal samples. The HEV prevalence was 13.1 (62 out 475, 95% CI: 10.2-16.4). Those pigs infected with Cryptosporidium spp. showed a higher prevalence of HEV (30.8 vs. 12%; p = .012). In the same way, animals bearing E. bieneusi seem to have a higher rate of HEV infection (24.2 vs. 12.2%; p = .06). According to their location in the gut, animals bearing intracellular enteroparasites showed a higher HEV prevalence than those uninfected (29.6 vs. 12.7%; p = .038), meanwhile those carrying extracellular enteroparasites had a lower likelihood to be infected by HEV than those uninfected (12.1 vs. 23.1%; p = .071). Those animals bearing both types of enteroparasites showed a similar prevalence of HEV infection than those exhibiting negative for both (20.8 vs. 26.1%; p = .763). Our study provides evidence that intracellular and extracellular enteroparasites modulate the susceptibility to HEV infection in pigs. Meanwhile, the presence of extracellular enteroparasites shows a protective effect on the risk of HEV acquisition in swine, whereas intracellular enteroparasites seems to have the opposite effect, favouring the HEV infection., (© 2021 Wiley-VCH GmbH.)

A survey study was carried out to identify tick species parasitizing wild lagomorphs in Mediterranean ecosystems in southern Spain and to determine the occurrence of Rickettsia species present in these ticks in this region. A total of 1304 European wild rabbits (Oryctolagus cuniculus) and 58 Iberian hares (Lepus granatensis) were individually examined for the presence of ticks. Ticks were found in 42.9% and 50% of the wild rabbits and hares sampled, respectively. A total of 1122 ticks were collected and five species, including Rhipicephalus pusillus, Hyalomma lusitanicum, Haemaphysalis hispanica, Ixodes ventalloi and Rhipicephalus sanguineus sensu lato (s.l.), were microscopically and molecularly identified at the 16S rRNA gene. This is the first study on Ixodidae parasitizing Iberian hares. The presence of Rickettsia DNA was assessed in 254 tick pools (according to hunting states, lagomorph species, tick species and tick development stage) using PCR assays targeting the rOmpA, rOmpB and gltA. Twenty-seven pools (10.6%) were positive to Rickettsia DNA. Five zoonotic Rickettsia species were identified, being Rickettsia massiliae the most frequent (4.7%), followed by Rickettsia sibirica subsp. mongolitimonae (2.8%), Rickettsia slovaca (2.0%), Rickettsia aeschlimannii (0.8%) and Rickettsia africae (0.4%). The results suggest that wild rabbits and Iberian hares are parasitized by a wide range of tick species and that these lagomorphs may play an important role in the sylvatic cycle of some zoonotic Rickettsia species in Mediterranean ecosystems. Our data represent the first report of R. massiliae, R. aeschlimannii, R. slovaca and R africae in ticks collected in wild lagomorphs in Europe, and the first report of not imported R. africae in this continent. Since R. slovaca and R. africae DNA was detected in tick species different to their main vectors, further studies are warranted to unravel the role of wild lagomorphs in the epidemiology of these vector-borne pathogens., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Numerous protist species are shared between humans and pigs. Among those, Giardia duodenalis, Cryptosporidium spp. and Balantioides coli have a clear public and animal health significance. For others such as Enterocytozoon bieneusi and Blastocystis sp., their impact on animal health has not been fully established. Little information is currently available on the molecular diversity of these protists in swine populations. To fill this gap, we molecularly assessed G. duodenalis, Cryptosporidium spp., B. coli, Blastocystis sp. and E. bieneusi in faecal samples from Iberian and Large White pigs raised under different (intensive and/or extensive) management systems in southern Spain. A total of 151 extensively raised Iberian pigs, 140 intensively raised Iberian pigs, and 184 intensively raised Large White pigs were investigated. Blastocystis sp. was the agent most prevalently found (47.8%), followed by B. coli (45.5%), G. duodenalis (10.7%), E. bieneusi (6.9%), and Cryptosporidium spp. (5.5%). Blastocystis sp. was significantly less prevalent in intensively raised Iberian pigs (22.9%) than in their extensively raised counterparts (51.0%) or in intensively raised Large White pigs (64.1%). A significantly higher prevalence was found for G. duodenalis, Cryptosporidium spp., and E. bieneusi in Large White pigs than Iberian pigs. Balantioides coli was similarly distributed (40.0-51.1%) in all three investigated swine populations. Sequence analyses revealed the presence of G. duodenalis assemblage E, two Cryptosporidium species (Cryptosporidium scrofarum and Cryptosporidium suis), B. coli (genotypes A and B), Blastocystis sp. (ST1, ST3, and ST5), and E. bieneusi (EbpA, EbpC, EbpD, O, and a novel genotype named PigSpEb2). Novel genotype PigSpEb2 was found alone or in combination with EbpA. Data suggest a widespread exposure to protist enteroparasites in domestic pig populations irrespectively of breed and raising management system. Many of the species/genotypes identified have a zoonotic potential and might represent a public health concern., (© 2021 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.)

The aetiology and epidemiology of vector borne apicomplexan Babesia and Hepatozoon and kinetoplastid Leishmania infantum infections in wildlife have not been explored in wide areas of southern Spain. We investigated these infections in 151 wild carnivores, including foxes, badgers, beech martens, hedgehogs, wild cats, Egyptian mongooses, otters, genets and racoons. Overall, Hepatozoon, Babesia and L. infantum infections were detected in 68%, 48% and 23% of the wild animals surveyed, respectively. L. infantum-infected wildlife were more likely to be also infected with the apicomplexan Hepatozoon and Babesia spp. compared to the non-infected counterparts (p < .05). We report for the first time Hepatozoon martis in badgers and wild cats and H. canis in beech martens, and a Babesia sp. in wild cats from Spain. Our results also indicate the widespread distribution of H. canis in foxes (91%) and beech martens (13%), H. martis in beech martens (81%), wild cats (20%) and badgers (13%), H. felis in wild cats (60%), B. vulpes in foxes (64%), Babesia sp. type A closely related to B. vulpes, in badgers (58%) and Babesia sp. in wild cats (20%). Moreover, L. infantum infection was found in foxes (29%), beech martens (13%), badgers (8%) and 1/3 Egyptian mongooses. We also detected Cytauxzoon sp. in a wild cat and the first Sarcocystis sp. in a genet. This study provided evidence of increased risk of L. infantum infection in wildlife animals co-infected with Babesia spp. or Hepatozoon spp. and indicated that these infections are widespread in wild carnivores from Spanish Mediterranean ecosystems., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Bluetongue (BT) is an emerging and re-emerging communicable vector-borne disease of animal health concern. A serosurvey was performed to assess exposure to BT virus (BTV) in zoo animals in Spain and to determine the dynamics of seropositivity in longitudinally sampled individuals during the study period. Serum samples were collected from 241 zoo animals belonging to 71 different species in five urban zoos (A-E) in Spain between 2007 and 2019. Twenty-four of these animals were longitudinally surveyed at three of the sampled zoos (zoos B, C and E) during the study period. Anti-BTV antibodies were found in 46 (19.1%; 95% CI: 14.1-24.1) of the 241 captive animals analysed by commercial ELISA. A virus neutralization test confirmed specific antibodies against BTV-1 and BTV-4 in 25 (10.7%; 95% CI: 6.7-14.6) and five (3.0%; 95% CI: 0.3-4.0) animals, respectively. Two of the 24 longitudinally sampled individuals (one African elephant (Loxodanta africana) and one aoudad (Ammotragus lervia)) showed anti-BTV antibodies at all samplings, whereas seroconversions were detected in one mouflon (Ovis aries musimon) in 2016, and one Asian elephant (Elephas maximus) in 2019. To the best of the authors' knowledge, this is the first large-scale survey on BTV conducted in both artiodactyl and non-artiodactyl zoo species worldwide. The results confirm BTV exposure in urban zoo parks in Spain, which could be of animal health and conservation concern. Circulation of BTV was detected in yearling animals in years when there were no reports of BTV outbreaks in livestock. Surveillance in artiodactyl and non-artiodactyl zoo species could be a valuable tool for epidemiological monitoring of BTV., (© 2021 Wiley-VCH GmbH.)

A survey study was carried out to assess the occurrence of selected tick-borne pathogens (TBP) in wild ungulates in Mediterranean ecosystems in southern Spain. Spleen samples were collected from 1,132 wild ungulates, including 578 red deer, 269 wild boar, 135 mouflon, 121 fallow deer and 29 roe deer, between 2009 and 2015. Eighty-nine ticks collected from TBP-positive animals were also analysed. Samples were tested by PCR and sequenced whenever possible. TBP DNA was detected in 127 of 863 wild ruminants (14.7%; 95% CI: 12.4-17.3) including the following: Anaplasma phagocytophilum (9.2%), Babesia divergens (2.9%), Theileria sp. OT3 (1.7%), Borrelia afzelii (0.7%) and Theileria capreoli (0.2%), but no positive samples were detected in wild boar (0/269). All the strains from mouflon were identified as Theileria sp. OT3, while B. divergens and T. capreoli were mainly found in red deer. Co-infection with A. phagocytophilum and B. divergens, and A. phagocytophilum and Theileria spp. was detected in red deer and mouflon, respectively. The risk factor analysis showed that the prevalences of A. phagocytophilum and piroplasms were species-related. Eighty-nine tick specimens collected from ungulates found to be infected with the selected TBP were identified as Hyalomma lusitanicum (95.5%) and Ixodes ricinus (4.5%). Thirty ticks were positive for Anaplasma/Ehrlichia spp. (33.7%), 25 for Babesia/Theileria (28.1%) and two for B. burgdorferi s.l. (2.3%). Eleven specimens showed co-infections with Anaplasma/Ehrlichia and Babesia/Theileria (10.1%) or Anaplasma/Ehrlichia and B. burgdorferi s.l. (2.3%). The estimated prevalences obtained in the present study suggest the possible contribution of wild ruminants to the maintenance of some selected TBP in Mediterranean ecosystems in southern Spain, while the role of wild boar in the epidemiology of these pathogens seems to be limited in this region., (© 2021 Wiley-VCH GmbH.)

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.)

Hepatitis E virus (HEV) is an emerging zoonotic pathogen that is currently recognized as one of the major causes of acute human hepatitis worldwide. In Europe, the increasing number of hepatitis E cases is mainly associated with the consumption of animal food products or contact with infected animals. Dogs and cats have been suggested as a zoonotic source of HEV infection. The aim of this study was to assess Orthohepevirus circulation, including HEV-A, HEV-B and HEV-C species, in sympatric urban cats and dogs in southern Spain. Between 2017 and 2020, blood samples were collected from 144 stray cats and 152 dogs, both strays and pets. The presence of antibodies against HEV were tested using a double-antigen sandwich ELISA and seropositive samples were further analysed by western blot. A RT-PCR was performed to detect RNA of Orthohepevirus species (HEV-A, HEV-B and HEV-C). A total of 19 (6.4%; 95%CI: 3.6-9.2) of the 296 animals tested showed anti-HEV antibodies by ELISA. Seropositivity was significantly higher in dogs (9.9%; 15/152; 95%CI: 5.1-14.6) than in cats (2.8%; 4/144; 95%CI: 0.1-5.5). Ten of the 18 ELISA-positive animals that could be further analysed by western blot, reacted against HEV-3 and/or HEV-C1 antigens, which suggest circulation of both genotypes in urban cats and dogs in the study area. However, HEV-A, HEV-B and HEV-C RNA were not detected in any of the tested sera. This is the first study to assess HEV circulation in both stray cats and dogs in Europe. Our results provide evidence of HEV exposure in sympatric urban cat and dog populations in southern Spain. Further studies are needed to determine the role of these species in the epidemiology of HEV., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Viruses that affect lagomorphs have decades of reported history of spillover events. One of these viruses is the causative agent of the so-called rabbit or 'lagomorph' haemorrhagic disease (e.g. Lagovirus europaeus/GI.1 and L. europaeus/GI.2). In particular, L. europaeus/GI.2 has shown a great capacity to recombine with existing lagoviruses. In fact, it has replaced the former GI.1 genotype in the wild, and recently, an increase on spillover events has been detected among several lagomorph species including European and North American species of hares. In this study, we report for the first time the infection of a wild Iberian hare with GI.2 (RHDV2/b), potential shedding and associated histopathological alterations. We identify the recombinant GI.4P-GI.2 as causative of the infection and discuss plausible causes regarding the origin of the spillover event and its potential consequences for the Iberian hare wild populations, which is an endemic species of the Iberian Peninsula as well as an important game and prey species for many predators, including endangered species., (© 2021 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.)

Schmallenberg virus (SBV) is an emerging Culicoides-borne Orthobunyavirus that affects ruminant species. Between 2011 and 2013, it was responsible for a large-scale epidemic in Europe. In the present study, we aimed to determine the seroprevalence, spatial distribution and risk factors associated with SBV exposure in sheep and goats in the region where the first Schmallenberg disease outbreak in Spain was reported. Blood samples from 1,796 small ruminants from 120 farms were collected in Andalusia (southern Spain) between 2015 and 2017. Antibodies against SBV were detected in 536 of 1,796 animals (29.8%; 95%CI: 27.7-32.0) using a commercial blocking ELISA. The individual seroprevalence according to species was 31.1% (280/900; 95%CI: 28.1-34.1) in sheep and 28.6% (256/896; 95%CI: 25.6-31.5) in goats. The farm prevalence was 76.7% (95%CI: 69.1-84.2). Seropositivity to SBV was confirmed in both sheep and goats in all provinces by virus neutralization test. Two significant (p < .001) spatial clusters of high seroprevalence were identified. The generalized estimating equation analysis showed that management system (extensive), temperature (>14ºC) and altitude (<400 metres above sea level) were risk factors associated with SBV exposure in small ruminants. Our results highlight widespread but not homogeneous circulation of SBV in small ruminant populations in Spain., (© 2020 Wiley-VCH GmbH.)

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.)

Myxomatosis is an infectious disease caused by the myxoma virus (MYXV), which has very high mortality rates in European wild rabbits (Oryctolagus cuniculus). While sporadic cases of myxomatosis have also been reported in some hare species, these lagomorphs are considered to have a low susceptibility to MYXV infection. In the present study, we describe the spatiotemporal evolution and main epidemiological findings of novel hare MYXV (ha-MYXV or MYXV-Tol) epidemics in Iberian hares (Lepus granatensis) in Spain. In the period 2018-2020, a total of 487 hares from 372 affected areas were confirmed to be MYXV-infected by PCR. ha-MYXV outbreaks were detected in most of the Spanish regions where the Iberian hare is present. The spatial distribution was not homogeneous, with most outbreaks concentrated in the southern and central parts of Spain. Consecutive outbreaks reported in the last two years suggest endemic circulation in Spain of this emerging virus. A retrospective study carried out just after the first epidemic period (2018-2019) revealed that the virus could have been circulating since June 2018. The number of outbreaks started to rise in July, peaked during the first half of August and October and then decreased sharply until January 2019. The apparent mean mortality rate was 55.4% (median: 70%). The results indicated high susceptibility of the Iberian hare to ha-MYXV infection, but apparent resistance in the sympatric hare species present in Spain and less infectivity in European rabbits. The novel ha-MYXV has had significant consequences on the health status of Iberian hare populations in Spain, which is of animal health and conservation concern. The present study contributes to a better understanding of ha-MYXV emergence and will provide valuable information for the development of control strategies. Further research is warranted to assess the impact of this emerging virus on wild lagomorph populations and to elucidate its ecological implications for Iberian Mediterranean ecosystems., (© 2020 Wiley-VCH GmbH.)

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.)

Between early October and mid-December 2018, mortalities were detected in Iberian ibex (Capra pyrenaica) populations in southern Spain. In the same region and period, bluetongue virus (BTV) circulation was also reported in sentinel and clinically affected domestic ruminant herds. Molecular analyses confirmed BTV serotype 4 (BTV-4) infection in eight Iberian ibexes from six hunting areas, and in 46 domestic ruminants from seven herds in close proximity to affected hunting estates. Histopathological analyses revealed vascular changes in several organs, pneumonia, lymphoid depletion, inflammatory mononuclear cell infiltrate and fibrosis as the most frequently observed lesions in the affected Iberian ibexes. Epidemiological and laboratory results indicate that BTV-4 was the main aetiological agent involved in outbreaks detected in Iberian ibex populations during the study period. Sequence analyses indicated that the BTV-4 strain detected in Iberian ibex had high homology (99.4%-100%) with strains isolated in livestock during the same period, and with previous isolates (≥98.9%) from Spain and Mediterranean Basin countries. Further studies are warranted to determine the impact of BTV-4 on the health status of Iberian ibex populations after the outbreaks. The inclusion of this species in the surveillance programme may be useful for early detection of BTV, especially in epidemiological scenarios at the wildlife-livestock interface., (© 2020 Blackwell Verlag GmbH.)

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.)

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.)

An epidemiological surveillance programme was carried out to assess exposure and spatiotemporal patterns of selected pathogens (Brucella spp., Mycobacterium avium subsp. paratuberculosis (MAP), Mycoplasma agalactiae, Pestivirus and bluetongue virus (BTV)) in Iberian ibex (Capra pyrenaica) from Andalusia (southern Spain), the region with the largest population of this species. A total of 602 animals in five distribution areas were sampled during 2010-2012 (P1) and 2013-2015 (P2). The Rose Bengal test (RBT) and complement fixation test (CFT) were used in parallel to detect anti-Brucella spp. antibodies. Commercial ELISAs were used to test for antibodies against the other selected pathogens. Sera positive for BTV and Pestivirus by ELISA were tested by serum neutralization test (SNT) to identify circulating serotypes/genotypes. The overall seroprevalences were as follows: 0.4% for Brucella spp. (2/549; CI 95%: 0.1-1.3) (14/555 positive by RBT; 2/564 by CFT), 0.5% for MAP (3/564; CI 95%: 0.1-1.5), 5.7% for M. agalactiae (30/529; CI 95%: 3.9-8.0), 11.1% for Pestivirus (58/525; CI 95%: 8.5-14.1) and 3.3% for BTV (18/538; CI 95%: 2.0-5.2). Significantly higher seropositivity to both M. agalactiae and BTV was observed in P1 compared with P2. Spatiotemporal clusters of high seroprevalence were also found for M. agalactiae in four of the five sampling areas in 2010, and for BTV in one of five areas in 2012. Specific antibodies against BTV-4, BDV-4 and BVDV-1 were confirmed by SNT. Our results indicate that the Iberian ibex may be considered spillover hosts of Brucella spp. and MAP rather than true reservoirs. The prevalence of antibodies against M. agalactiae and BTV suggests spatiotemporal variation in the circulation of these pathogens, while Pestivirus has a moderately endemic circulation in Iberian ibex populations. Our study highlights the importance of long-term surveillance for a better understanding of the spatiotemporal distribution of shared infectious diseases and providing valuable information to improve control measures at the wildlife-livestock interface., (© 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.)

Myxomatosis is an infectious disease caused by myxoma virus (MYXV; genus Leporipoxvirus), which affects the European wild rabbit (Oryctolagus cuniculus) and sporadically brown hares (Lepus europaeus). Here, we describe the first outbreak of myxomatosis in Iberian hares (Lepus granatensis). Between mid-July and the end of September 2018, around 530 dead animals were detected in Iberian hare populations in southern Spain. The apparent mean mortality rate was 56.7%, and the estimated mean case fatality rate was 69.2%. Histopathological and molecular results confirmed MYXV infections in all hares analysed. To the authors' knowledge, this is the first myxomatosis outbreak causing a high mortality in hares and the first detailed characterization of a myxomatosis outbreak in the Iberian hare. The absence of cases in sympatric wild rabbits suggests differences in the susceptibility between both lagomorph species to the virus strain implicated in the outbreak. After the first case, the number of affected areas increased sharply affecting most of the Iberian Peninsula where the Iberian hare is present. Further studies are required to elucidate the origin of the implicated MYXV strain as well as to assess the impact of this outbreak on the Iberian hare populations., (© 2019 Blackwell Verlag GmbH.)

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.)

Hepatitis E virus (HEV) is an emerging zoonotic pathogen that has been detected in different animal species. A survey study was carried out to assess HEV infection in non-human primates (NHPs) housed in zoos in Spain. Anti-HEV antibodies were detected in eight of the 181 NHPs tested (4.4%; 95%CI: 1.4-7.4). At least one seropositive animal was detected in five of the 33 species sampled (15.2%). This is the first report of seropositivity in black-and-white ruffed lemurs (Varecia variegata), common chimpanzees (Pan troglodytes), and Barbary macaques (Macaca sylvanus). Anti-HEV antibodies were found in six of the eight zoos included in the study (75.0%). Seroconversion was detected in one chimpanzee, which confirms HEV circulation in one zoo between 2015 and 2016. Seropositivity was significantly higher in hominids than in other NHP families. HEV RNA was not detected in any of the serum samples tested. The results indicate susceptibility of NHPs to HEV infection. Further studies are required to elucidate the role of these species in the epidemiology of HEV., (© 2019 Blackwell Verlag GmbH.)

Hepatitis E (HE) is an important emerging disease in European countries. To analyse the role of equids as potential reservoirs for HE virus (HEV), we determined the prevalence of HEV infection in 861 equines from 464 herds in Spain. HEV RNA in serum was detected in 0.4% (3/692) of horses, 1.2% (1/86) of donkeys and 3.6% (3/83) of mules. Phylogenetic analysis identified the zoonotic genotype 3 as being closely related to viral human and swine strains. In this first report on HEV in equids in Europe, we confirm the susceptibility of horses, donkeys and mules to HEV infection. The low prevalence detected indicates that equids may be considered spillover hosts rather than true reservoirs., (© 2018 Blackwell Verlag GmbH.)

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., (© 2017 Blackwell Verlag GmbH.)

An epidemiological study was carried out to determine the spatial-temporal trends and risk factors potentially involved in the seropositivity to bluetongue virus (BTV) in hunting areas with presence of red deer (Cervus elaphus). A total of 60 of 98 (61.2%; CI95% : 51.6-70.9) hunting areas sampled presented at least one seropositive red deer. Antibodies against BTV were detected in juvenile animals during the hunting seasons 2007/2008 to 2013/2014 in 15 of 98 (15.3%) hunting areas, which indicates an uninterrupted circulation of BTV in this period. A multivariate logistic regression model showed that the red deer density at hunting area level (>22 individuals/km(2) ), the annual abundance of Culicoides imicola (>1.4 mosquitoes/sampling) and the goat density at municipality level (>24.1 individuals/km(2) ) were factors significantly associated with BTV seropositivity in hunting areas. Control measures against BTV in the studied area include vaccination programmes in wild and domestic ruminants, movement control in areas with high densities and abundance of red deer and C. imicola, respectively. Considering the potential risk of BTV re-emergence, red deer should be included in the BT surveillance programmes in regions where these species share habitats with livestock., (© 2014 Blackwell Verlag GmbH.)