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.

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.

Epizootic haemorrhagic disease virus serotype 8 (EHDV-8) emerged in southern Europe in 2022–2023, but clinical and pathological characterization in free-ranging wildlife remains limited. This study investigated EHDV-8-associated morbidity and mortality in wild ruminants in a 2023 outbreak in Sierras de Cazorla, Segura y Las Villas Natural Park (Jaén, Andalusia, Spain). Moribund animals demonstrated a consistent acute neuro-respiratory syndrome characterized by weakness, ataxia, nystagmus and severe dyspnoea with frothy oral discharge. On the carcasses of 39 red deer, two fallow deer, and one mouflon, necropsy was performed and subsequently histopathology and a real-time polymerase chain reaction (RT-PCR) on the collected samples. Gross lesions included marked pulmonary oedema, tracheal foam and widespread congestion, while histopathology revealed lymphoid depletion, pulmonary haemorrhage, vascular injury and renal tubular necrosis. All animals tested positive for EHDV-8 with low RT-qPCR cycle threshold values, indicating high viral loads. This series provides the first confirmed clinical, pathological, and molecular evidence of EHDV-8 infection in fallow deer and mouflon in Europe. The observations demonstrate that EHDV-8 causes a peracute systemic haemorrhagic disease in susceptible wild ruminants and underline the importance of integrated wildlife surveillance and timely diagnostic sampling during peak vector activity.

Encephalomyocarditis virus (EMCV) causes sporadic and epizootic outbreaks among various domesticated and non-domesticated animal species worldwide. Although outbreaks are mostly reported in domestic pigs, mortality is reported in elephants, ungulates, nonhuman primates (NHPs), and rodents. Rats of the genus Rattus serve as primary reservoirs and vectors, but alternative infection routes have been proposed. Clinical disease is characterized by acute heart failure in most taxonomic groups, often culminating in rapid death. Due to the rapid progression of the disease, diagnostic confirmation is most commonly obtained postmortem. Pathological examination reveals interstitial lymphohistiocytic myocarditis and multiorgan congestion in most cases. EMCV is often demonstrated with RT-PCR or virus isolation techniques, but other methods, e.g., serology and immunohistochemistry, are available. The rapid progression of EMCV precludes effective therapeutic intervention, though agents such as interferon, verapamil, and curcumol have shown potential efficacy. Preventative strategies are crucial, emphasizing biosecurity measures to mitigate rodent contamination of feed and water. Inactivated vaccines have demonstrated protective efficacy in experimental models involving mice, pigs, and elephants, with analogous immunogenic responses observed in various zoological species. Live attenuated vaccines have conferred protection in pigs and NHPs, albeit with variable seroconversion rates in different species.

This study aimed to evaluate daily variations in sperm quality, testosterone levels, cryoresistance, and the relocation of AQP3 in plasma membrane domains in wild and domestic goats. Samples were collected from five Murciano-Granadina bucks and seven Iberian ibex at 12:00 h (noon) and 00:00 h (midnight), during the rutting season. Sperm motility, viability, acrosome integrity, DNA fragmentation, and immunolocalization of AQP3 were assessed in fresh and frozen-thawed semen samples. Serum testosterone concentrations were also determined at both times. In domestic goats, kinematic parameters were greater (p < 0.05) in semen collected at midnight, whereas the cryoresistance ratio was greater (p < 0.05) for these variables in semen recovered at noon. In ibexes, fresh semen collected at midnight was greater (p = 0.05) for VCL, VSL, VAP, and ALH values. After freezing-thawing, a significant increase in AQP3 labelling at the acrosomal region was observed in samples collected at midnight for both species, as well as in samples collected at noon for bucks (p < 0.05). An increase in the proportion of spermatozoa with AQP3 labelling in the mid-piece was detected in bucks in frozen-thawed samples collected at noon (p < 0.05). Testosterone concentrations were higher (p < 0.05) at midnight in bucks but no differences were found in ibexes. The findings reveal daily variations in sperm quality in both domestic and wild goats, which do not exactly coincide with the daily changes in cryoresistance, thus calling for attention when processing semen.

Background: West Nile virus (WNV) is the most widely distributed mosquito‐borne flavivirus. Over the past decade, its spread across Europe has raised significant concerns for both public and animal health. Although WNV exposure has been evidenced in various wild mammal species in Spain, no seroepidemiological studies have been conducted on this flavivirus in wild lagomorphs so far.Aim: This study aimed to assess WNV exposure in European wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis) populations inhabiting Spanish Mediterranean ecosystems.Methods: Sera from 540 wild lagomorphs (399 European wild rabbit and 141 Iberian hares), from 106 hunting grounds distributed throughout Andalusia (southern Spain), were collected between the 2018/2019 and 2022/2023 hunting seasons.Results: Antibodies against flavivirus were detected by blocking enzyme‐linked immunosorbent assay (bELISA) in 5.0% (27/540; 95% CI: 3.2–6.8) of the wild lagomorphs. Exposure to WNV was confirmed in 4.8% (19/394; 95% CI: 2.7–6.9) of wild rabbits and 0.7% (1/141; 95% CI: 0.0–2.1) of Iberian hares by virus microneutralisation test. Anti‐WNV antibodies were found in wild lagomorphs sampled from three (2.8%) hunting grounds located in western Andalusia during the seasons 2020–2021 and 2021–2022. Remarkably, this spatiotemporal distribution overlaps with the largest outbreak of WNV in Spain. Antibodies against Usutu virus and Bagaza virus were not detected in the wild lagomorph populations analysed.Conclusions: This study constitutes the first report of WNV exposure in wild rabbit in Spain and in Iberian hare worldwide. While these species seem not play a primary role in the epidemiology of the virus, they could serve as sentinel for monitoring WNV in Iberian Mediterranean ecosystems.