Surveillance of Sarcoptic Mange in Iberian Ibexes (Capra pyrenaica) and Domestic Goats (Capra hircus) in Southern Spain.

Sarcoptic mange is a highly contagious skin disease caused by Sarcoptes scabiei. Sera were collected from 411 Iberian ibexes, comprising 157 individuals with sarcoptic mange skin lesions and 254 clinically healthy animals, in 13 population nuclei across Andalusia (southern Spain) between 2015 and 2021. Skin samples from 88 of the 157 animals with mange-compatible lesions were also obtained. Moreover, 392 serum samples from domestic goats (Capra hircus) were collected in the same region and study period. Antibodies against S. scabiei were tested using an in-house indirect ELISA, while the presence of mites of S. scabiei was evaluated in the skin samples by potassium hydroxide digestion. Seropositivity was found in eight (3.1%) of the clinically healthy ibexes and in 104 (66.2%) of the animals with mange-compatible lesions. The presence of S. scabiei was confirmed in 57 (64.8%) out of the 88 skin samples analysed and anti-S. scabiei antibodies were found in 49 (86.0%) of these 57 mite-positive individuals. Seropositive animals were detected in population nuclei with previous records of sarcoptic mange, where S. scabiei mites were detected by potassium hydroxide digestion in individuals with sarcoptic mange-compatible external lesions. However, seropositivity was not observed in population nuclei that were historically free of this disease. None of the 392 domestic goats had antibodies against S. scabiei, suggesting an independent epidemiological cycle of sarcoptic mange in Iberian ibex populations in the study area, and a limited or null role of domestic goats in the transmission of the parasite to this wild species. Overall, our findings underscore the importance of maintaining and/or implementing integrated surveillance programs and control strategies in wildlife and livestock, to limit the risk of S. scabiei circulation in Iberian ibex populations.

Serosurveillance of Trichinella sp. in wild boar and Iberian domestic suids in Mediterranean ecosystems of southwestern Spain.

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.

Mapping the risk of exposure to Crimean-Congo haemorrhagic fever virus in the Iberian Peninsula using Eurasian wild boar (Sus scrofa) as a model.

Crimean-Congo haemorrhagic fever (CCHF) virus (CCHFV) is a tick-borne zoonotic pathogen that can cause a lethal haemorrhagic disease in humans. Although the virus appears to be endemically established in the Iberian Peninsula, CCHF is an emerging disease in Spain. Clinical signs of CCHFV infection are mainly manifested in humans, but the virus replicates in several animal species. Understanding the determinants of CCHFV exposure risk from animal models is essential to predicting high-risk exposure hotspots for public health action. With this objective in mind, we designed a cross-sectional study of Eurasian wild boar (Sus scrofa) in Spain and Portugal. The study analysed 5,291 sera collected between 2006 and 2022 from 90 wild boar populations with a specific double-antigen ELISA to estimate CCHFV serum prevalence and identify the main determinants of exposure probability. To do so, we statistically modelled exposure risk with host- and environment-related predictors and spatially projected it at a 10 × 10 km square resolution at the scale of the Iberian Peninsula to map foci of infection risk. Fifty-seven (63.3 %) of the 90 populations had at least one seropositive animal, with seroprevalence ranging from 0.0 to 88.2 %. Anti-CCHFV antibodies were found in 1,026 of 5,291 wild boar (19.4 %; 95 % confidence interval: 18.3-20.5 %), with highest exposure rates in southwestern Iberia. The most relevant predictors of virus exposure risk were wild boar abundance, local rainfall regime, shrub cover, winter air temperature and soil temperature variation. The spatial projection of the best-fit model identified high-risk foci as occurring in most of western and southwestern Iberia and identified recently confirmed risk foci in eastern Spain. The results of the study demonstrate that serological surveys of CCHFV vector hosts are a powerful, robust and highly informative tool for public health authorities to take action to prevent human cases of CCHF in enzootic and emergency settings.

Equilibration time improves the sperm variables of wild ruminant ejaculated and epididymal sperm cryopreserved by ultra-rapid freezing.

This work examines the effect of equilibration time with extender on ultra-rapidly frozen-thawed wild ruminant epididymal (origin: Iberian ibex) and ejaculated (origin: mouflon) sperm variables. Sperm samples were prepared either without prior equilibration, or equilibrated for 30 min before freezing. Higher quality (p < 0.05) frozen-thawed spermatozoa were obtained when equilibration was allowed, for ejaculated sperm in terms of sperm motility, acrosome apical ridge integrity, sperm viability, and percentage of normal cells, and for epididymal sperm in terms of linearity and straightness of sperm movement. The sperm head area, head perimeter, head length and head width were smaller (p < 0.01) in the equilibrated than non-equilibrated frozen-thawed epididymal sperm; no such dimensional changes were recorded for ejaculated sperm. In conclusion, equilibration prior to ultra-rapid freezing improves the cryoresistance of sperm cells, although viable sperm cells can be obtained without equilibration. The epididymal sperm showed greater cryoresistance, supporting the idea that it is more resistant to freeze-thawing than ejaculated sperm.

Pathological changes and viral antigen distribution in tissues of Iberian hare (Lepus granatensis) naturally infected with the emerging recombinant myxoma virus (ha-MYXV).

BACKGROUND: A cross-species jump was confirmed in 2018, when a novel recombinant myxoma virus (MYXV) (ha-MYXV) caused high mortality in Iberian hare (Lepus granatensis) in the Iberian Peninsula. METHOD: The aim of this study was to evaluate the main lesions, tissular distribution and target cells of ha-MYXV in Iberian hare. Gross postmortem examinations and histological and immunohistochemical studies to detect ha-MYXV were carried out in 28 animals that were confirmed as ha-MYXV positive by PCR. RESULTS: The main macroscopic lesions were bilateral blepharoconjunctivitis, epistaxis, intense congestion and oedema in several organs and some internal haemorrhages. Visible myxomas were not found. Histopathological examination revealed hyperplastic epidermis with predominant hyperkeratosis and myxoid matrix in the dermis. ha-MYXV-positive keratinocytes showed hydropic degeneration and cytoplasmic inclusion bodies. Alveolar oedema, interstitial pneumonia, dramatic lymphoid depletion in the spleen and necrosis in the liver and testis were observed. ha-MYXV was mainly detected in epithelial and myxoma cells in the skin, and also in macrophages, lymphocytes, fibroblasts and endothelial cells in several organs, as well as in hepatocytes and Leydig cells. LIMITATIONS: A non-homogeneous number of samples were included in all the animals. Future experimental studies with controlled variables are necessary. CONCLUSION: These findings correspond to an unusual form of myxomatosis, characterised by an acute or hyperacute presentation.

Spatiotemporal monitoring of myxomatosis in European wild rabbit (Oryctolagus cuniculus) in Spanish Mediterranean ecosystems.

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.

Red deer reveal spatial risks of Crimean-Congo haemorrhagic fever virus infection.

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.

Spatiotemporal monitoring of Cysticercus pisiformis in European wild rabbit (Oryctolagus cuniculus) in Mediterranean ecosystems in southern Spain.

Cysticercosis in wild lagomorphs is caused by Cysticercus pisiformis, the larval stage of Taenia pisiformis. Although previous studies have reported the presence of T. pisiformis in different wild carnivore species, information about the prevalence of C. pisiformis in their intermediate hosts is still very scarce. An epidemiological surveillance program was carried out to determine the prevalence and spatiotemporal patterns of cysticercosis in wild rabbits (Oryctolagus cuniculus) from Spanish Mediterranean ecosystems. A total of 2,923 animals were sampled in 164 hunting estates from Andalusia (southern Spain) during four study periods: 2009-2012 (P1), 2012-2015 (P2), 2015-2018 (P3) and 2018-2020 (P4). The presence of cysticerci was assessed by macroscopical examination and a subset of the collected parasites were molecularly identified by conventional PCR targeting the ITS-1 and 12S rRNA partial genes of Taenia spp. Risk factors associated with cysticercus infection were assessed by generalized estimating equation (GEE) analysis. A spatial statistical analysis was carried out using a Bernoulli model to identify statistically significant spatial clusters. Cysticercus infection was confirmed in 81 (2.8 %; 95 % CI: 2.2-3.4) rabbits. Cysticerci from 18 infected animals were molecularly identified as T. pisiformis. The GEE model showed the study period as the only risk factor associated with C. pisiformis infection in wild rabbits. Significantly higher prevalence was found in P2 (6.1 %; 95 % CI: 4.4-8.4) compared to the rest of the periods. At least one cysticerci-positive animal was detected in 41 (25.0 %; 95 % CI: 18.4-31.6) out of the 164 hunting estates. No statistically significant spatial clusters of high cysticercus prevalence were identified. Our results indicate an endemic circulation of C. pisiformis in wild rabbits in southern Spain. The spatial results highlight a widespread distribution of this parasite in their populations. Further studies should focus in determining which sympatric species may act as definitive hosts for T. pisiformis and the relevance of other potential intermediate host species (e.g. hares and rodents), as the relevance of wild rabbits in the sylvatic cycle of this cestode in Mediterranean ecosystems seems to be low.

Seroepidemiology of Toxoplasma gondii in wild ruminants in Spain.

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.

Serosurveillance of Schmallenberg virus in wild ruminants in Spain.

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.

Monitoring of emerging myxoma virus epidemics in Iberian hares (Lepus granatensis) in Spain, 2018-2020.

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.

Re-emergence of bluetongue virus serotype 4 in Iberian ibex (Capra pyrenaica) and sympatric livestock in Spain, 2018-2019.

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.

Distribution of Pestivirus exposure in wild ruminants in Spain.

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.

Zoonotic Microsporidia in Wild Lagomorphs in Southern Spain.

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.

Spatiotemporal monitoring of selected pathogens in Iberian ibex (Capra pyrenaica).

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.

Absence of Hepatitis E virus circulation in wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis) in Mediterranean ecosystems in Spain.

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.

First outbreak of myxomatosis in Iberian hares (Lepus granatensis).

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.

Role of wild ruminants in the epidemiology of bluetongue virus serotypes 1, 4 and 8 in Spain.

Although the importance of wild ruminants as potential reservoirs of bluetongue virus (BTV) has been suggested, the role played by these species in the epidemiology of BT in Europe is still unclear. We carried out a serologic and virologic survey to assess the role of wild ruminants in the transmission and maintenance of BTV in Andalusia (southern Spain) between 2006 and 2010.A total of 473 out of 1339 (35.3%) wild ruminants analyzed showed antibodies against BTV by both ELISA and serum neutralization test (SNT). The presence of neutralizing antibodies to BTV-1 and BTV-4 were detected in the four species analyzed (red deer, roe deer, fallow deer and mouflon), while seropositivity against BTV-8 was found in red deer, fallow deer and mouflon but not in roe deer. Statistically significant differences were found among species, ages and sampling regions. BTV RNA was detected in twenty-one out of 1013 wild ruminants (2.1%) tested. BTV-1 and BTV-4 RNA were confirmed in red deer and mouflon by specific rRT-PCR.BTV-1 and BTV-4 seropositive and RNA positive wild ruminants, including juveniles and sub-adults, were detected years after the last outbreak was reported in livestock. In addition, between the 2008/2009 and the 2010/2011 hunting seasons, the seroprevalence against BTV-1, BTV-4 and BTV-8 increased in the majority of provinces, and these serotypes were detected in many areas where BTV outbreaks were not reported in domestic ruminants. The results indicate that wild ruminants seem to be implicated in the dissemination and persistence of BTV in Spain.

Epidemiological surveillance of bluetongue virus serotypes 1, 4 and 8 in Spanish ibex (Capra pyrenaica hispanica) in southern Spain.

A cross-sectional study was carried out to assess the prevalence and circulation of bluetongue virus (BTV) in Spanish ibexes (Capra pyrenaica hispanica). A total of 770 sera samples, 380 blood samples and 34 spleen samples were collected between 2006 and 2009 in Andalusia (southern Spain), a region and time period with a wide circulation of BTV in livestock. Thirty-one out of 770 (4.0%; CI(95%): 2.6-5.4) sera samples analyzed by ELISA showed antibodies against BTV. Twenty-four out of 31 seropositive samples were tested against BTV serotypes 1, 4 and 8 by serum neutralization test (SNT). Neutralizing antibodies against BTV-1 and BTV-4 were detected in seven and ten animals, respectively, four of them showed neutralizing antibodies to both serotypes. The animals seropositive to BTV-4 were sampled between 2006 and 2008, while BTV-1 circulation was confirmed in ibexes sampled between 2007 and 2009. None of the ibexes presented neutralizing antibodies against BTV-8. Statistically significant differences were found among regions and years, which is in coincidence with what occurred in domestic ruminants. There were no statistically significant differences between sexes, age classes and habitats (captivity vs. free-living). BTV RNA was not found in any of the 380 blood samples analyzed. However, BTV-1 RNA was detected from spleen in one Spanish ibex from Málaga province in August 2008. This finding evidences the presence of BTV-1 in Spanish ibex in a municipality where BT outbreaks were not detected in domestic ruminants during that period. Results of the present study show that Spanish ibexes were exposed and responded serologically to both BTV-1 and BTV-4. The low seroprevalence obtained suggests that Spanish ibex is not a relevant species in the dissemination of BT. However, the detection of BTV-1 RNA and the presence of seropositive ibexes in areas where BT outbreaks were not detected in livestock, could not exclude a significant role in the epidemiology of BTV in certain areas.

Influence of recovery method and microbial contamination on the response to freezing-thawing in ibex (Capra pyrenaica) epididymal spermatozoa.

The method of sperm recovery may influence the initial quality of sperm samples and their response to freezing-thawing. The aim of the present work was to compare two methods for collecting epididymal spermatozoa in order to improve the quality of recovered sperm and reduce possible contamination. Testes were obtained from 23 legally hunted, adult ibex males. The sperm mass of the right epididymis was collected by small longitudinal and transverse cuts made in the cauda epididymidis. The sperm mass of the left epididymis was collected by retrograde flushing from the vas deferens to the cauda epididymidis (using a cannula), employing a Tris, citric acid, glucose, egg yolk-based medium. The flushing method recovered more spermatozoa (P<0.001) than the cutting method. After freezing-thawing, greater acrosomes damage (P<0.001) and more morphological abnormalities (P<0.05) were seen among the sperm cells recovered by the cutting method than among those obtained by retrograde flushing. The method of sperm recovery did not, however, influence the microbial contamination rate. In frozen-thawed samples that were microbially contaminated, motility was significantly reduced (P<0.05) and membrane integrity tended to be poorer (P=0.06). In conclusion, retrograde flushing is recommended for ibex sperm collection since it would appear that microbial contamination is no more of a problem than that encountered with the cutting method, while a larger number of sperm cells more resistant to freezing-thawing can be obtained.