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.

Exposure to Coxiella burnetii in Wild Lagomorphs in Spanish Mediterranean Ecosystems.

Coxiella burnetii is an important zoonotic pathogen of worldwide distribution that can infect a wide range of wild and domestic species. The European wild rabbit (Oryctolagus cuniculus) can play a role as a reservoir for this bacterium in certain epidemiological scenarios, but, to date, a very limited numbers of large-scale serosurveys have been conducted for this species worldwide. Although exposure in hare species has also been described, C. burnetii in Iberian hare (Lepus granatensis) has never been assessed. Here, we aimed to determine the seroprevalence and risk factors associated with C. burnetii exposure in wild lagomorphs in the Mediterranean ecosystems of southern Spain. Between the 2018/2019 and 2021/2022 hunting seasons, blood samples from 638 wild lagomorphs, including 471 wild rabbits and 167 Iberian hares, were collected from 112 hunting grounds distributed across all eight provinces of Andalusia (southern Spain). The overall apparent individual seroprevalence was 8.9% (57/638; 95% CI: 6.8-11.4). Antibodies against C. burnetii were found in 11.3% (53/471; 95% CI: 8.4-14.1) of the wild rabbits and 2.4% (4/167; 95% CI: 0.1-4.7) of the Iberian hares. Seropositive animals were detected for 16 (14.3%; 95% CI: 7.8-20.8) of the 112 hunting grounds tested and in all the hunting seasons sampled. A generalized estimating equations model showed that the geographical area (western Andalusia) and presence of sheep were risk factors potentially associated with C. burnetii exposure in wild lagomorphs. A statistically significant spatial cluster (p < 0.001) was identified in the south-west of Andalusia. Our results provide evidence of moderate, endemic and heterogeneous circulation of C. burnetii in wild lagomorph populations in Spanish Mediterranean ecosystems. Risk-based strategies for integrative surveillance programs should be implemented in these species to reduce the risk of transmission of the bacterium to sympatric species, including humans.

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.

Molecular detection of Ehrlichia spp. in ticks parasitizing wild lagomorphs from Spain: characterization of a novel Ehrlichia species.

BACKGROUND: Several species belonging to the genus Ehrlichia are considered pathogenic for animals and humans. Although wildlife are known to play an important role in the epidemiology of these bacteria, information on the role of wild lagomorphs in their sylvatic cycle is limited. Thus, the objective of the present study was to assess the occurrence of Ehrlichia spp. in ticks collected from wild lagomorphs in Spanish Mediterranean ecosystems. METHODS: A total of 1122 pooled ticks (254 pools) collected from 506 wild rabbits (Oryctolagus cuniculus) and 29 Iberian hares (Lepus granatensis) were analysed using a nested PCR assay targeting the partial groEL gene. Ehrlichia spp.-positive samples were further subjected to a second PCR assay targeting 16S rRNA. RESULTS: Three (1.2%) tick pools comprising Rhipicephalus pusillus collected from nine wild rabbits were positive for Ehrlichia spp. All the Ehrlichia DNA sequences were identical, and use of sequence and phylogenetic analyses allowed us to identify a novel Ehrlichia species. CONCLUSIONS: We provide evidence that a novel Ehrlichia species, named herein as 'Candidatus Ehrlichia andalusi', which may be of concern for animal and public health, is circulating in R. pusillus in Spanish Mediterranean ecosystems. Further studies are warranted to assess the epidemiology, pathogenicity and zoonotic potential of this Ehrlichia species.

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.

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.

Sarcoptic mange in wild ruminants in Spain: solving the epidemiological enigma using microsatellite markers.

BACKGROUND: In Spain, sarcoptic mange was first described in native wildlife in 1987 in Cazorla Natural Park, causing the death of nearly 95% of the local native population of Iberian ibex (Capra pyrenaica). Since then, additional outbreaks have been identified in several populations of ibex and other wild ungulate species throughout the country. Although the first epizootic outbreak in wildlife was attributed to the introduction of an infected herd of domestic goats, the origin and the cause of its persistence remain unclear. The main aims of this study are to understand (i) the number of Sarcoptes scabiei "strains" circulating in wild ruminant populations in Spain, and (ii) the molecular epidemiological relationships between S. scabiei and its hosts. METHODS: Ten Sarcoptes microsatellite markers were used to characterize the genetic structure of 266 mites obtained from skin scrapings of 121 mangy wild ruminants between 2011 and 2019 from 11 areas in Spain. RESULTS: Seventy-three different alleles and 37 private alleles were detected. The results of this study show the existence of three genetic strains of S. scabiei in the wild ruminant populations investigated. While two genetic clusters of S. scabiei were host- and geography-related, one cluster included multi-host mites deriving from geographically distant populations. CONCLUSIONS: The molecular epidemiological study of S. scabiei in wild ruminants in Spain indicates that the spreading and persistence of the parasite may be conditioned by host species community composition and the permissiveness of each host population/community to the circulation of individual "strains," among other factors. Wildlife-livestock interactions and the role of human-driven introduction or trade of wild and domestic animals should be better investigated to prevent further spread of sarcoptic mange in as yet unaffected natural areas of the Iberian Peninsula.

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.

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.