Epizootic Haemorrhagic Disease Virus (EHDV) Infection in Red Deer (Cervus elaphus), Fallow Deer (Dama dama) and Mouflon (Ovis orientalis musimon) in South-Eastern Spain: Implications for Wildlife Health and Ruminant Disease Ecology

TL;DR

A 2023 outbreak of EHDV-8 in wild ruminants in Spain showed rapid, fatal disease in red deer, fallow deer, and mouflon, highlighting the need for wildlife and veterinary monitoring.

Contribution

First confirmed clinical and molecular evidence of EHDV-8 in fallow deer and mouflon in Europe.

Findings

EHDV-8 caused peracute systemic hemorrhagic disease in wild ruminants with high viral loads.

Clinical signs included neuro-respiratory symptoms like ataxia, nystagmus, and severe dyspnea.

Gross and histopathological findings included pulmonary edema, lymphoid depletion, and vascular injury.

Abstract

In 2023, veterinary field teams investigated an outbreak of acute morbidity and mortality in free-ranging wild ruminants in a national park in Andalusia (Spain). Forty-two carcasses were examined. A consistent peracute clinical picture was observed. Affected animals demonstrated marked weakness, ataxia, and eye tremors, as well as difficulty breathing, often with frothy oral discharge. On postmortem examination, blood and key organs (e.g., spleen, lung and lymph nodes) were collected for comprehensive pathological evaluation. Presumptive diagnosis of epizootic haemorrhagic disease virus (EHDV) was established based on observed symptoms, clinical findings, and gross pathology observations. Infection with EHDV was confirmed using a real time-polymerase chain reaction on blood and tissues. Positive cases occurred in red deer (Cervus elaphus), fallow deer (Dama dama), and mouflon (Ovis orientalis musimon). To our knowledge, this series provides the first confirmed EHDV infections in fallow deer and mouflon. These observations demonstrate that EHDV can cause rapidly progressive, fatal disease in wild ruminants following its detection in southern Europe. Practical implications for veterinary and wildlife health services include prompt field triage, safe carcass handling, rapid laboratory testing, and coordinated monitoring of wildlife, livestock and biting midges during late summer and early autumn.

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.

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1. Introduction

Epizootic haemorrhagic disease virus (EHDV), Orbivirus ruminantium, is a vector-borne virus of the genus Orbivirus, family Sedoreoviridae, which affects wild and domestic ruminants [1]. A systematic literature review by the European Food Safety Authority (EFSA) identified EHDV as an emerging orbiviral threat for Europe, synthesizing evidence across hosts, vectors, diagnostics and epidemiology, and highlighting the Mediterranean basin as a gateway for incursions [2]. The virus is transmitted by biting midges of the genus Culicoides, and is closely related to the bluetongue virus (BTV), Orbivirus caerulinguae [3]. In Europe, surveillance summarized by EFSA points to the Culicoides imicola and Culicoides obsoletus/scoticus complexes as key vectors of concern given their distribution and seasonal abundance [2]. Presently, at least eight serotypes (EHDV-1, -2, -4, -5, -6, -7, -8, -10) are recognized worldwide, with a broad global distribution spanning North America, Africa, Asia, and Oceania [4,5]. Genome segment 2, which encodes the outer capsid protein VP2, determines the serotype of EHDV and is therefore commonly targeted for molecular typing [6]. Historically, EHDV has been most extensively described in North America, where it is associated with recurrent outbreaks and high mortality in white-tailed deer (Odocoileus virginianus) [7,8]. This virus was first confirmed in Italy in November 2022, and subsequently in Spain [9]. In Europe, serotype 8 is most prevalent, but reassortment among field strains remains a credible route for future serotype introductions [2].

In cervids, EHDV infection may be frequently associated with high mortality and a peracute to acute clinical course, characterized by marked weakness, ataxia, nystagmus, respiratory distress, excessive salivation or frothy oral discharge, oedema, cyanosis of mucous membranes and sudden death. Recent European outbreaks have been associated with high mortality in susceptible wild ruminants, particularly cervid species such as red deer (Cervus elaphus) [10]. The disease is characterized in deer by marked weakness, ataxia, nystagmus, respiratory distress, excessive salivation or frothy oral discharge, oedema, cyanosis of mucous membranes and sudden death. In contrast, clinical disease in domestic cattle is typically less severe, characterized by transient systemic signs and bluetongue-like manifestations [1,10,11,12].

Following the detection of EHDV in southern Europe, the Andalusian Wildlife Epidemiological Surveillance Programme intensified passive surveillance for EHDV-compatible morbidity and mortality events in free-ranging wildlife. Within this framework, the present study constitutes part of the coordinated surveillance response in the Jaén Province of Andalusia, Spain. The Andalusian Hunting Reserve of Sierras de Cazorla and Segura (AHRCS) is located within the Cazorla, Segura y Las Villas Natural Park in south-eastern Spain and represents the geographic focus of the investigations reported in this study. The programme conducts passive, event-based surveillance of a wide range of infectious and parasitic diseases affecting wildlife and game species, including pathogens of zoonotic relevance. It operates through standardized reporting of morbidity and mortality events, followed by field-based veterinary assessment, necropsy and targeted diagnostic investigations. Disease-specific surveillance protocols are implemented in response to regional epidemiological priorities, including highly pathogenic avian influenza, sarcoptic mange, and myxomatosis in Iberian hare (Lepus granatensis), among other emerging or re-emerging wildlife diseases, thereby supporting early detection and risk assessment at the wildlife–livestock interface [13,14,15].

The AHRCS hosts one of the most diverse wild ungulate populations in the region, with an approximate size estimate of around 1500 wild boars (Sus scrofa), 2900 red deer, 3500 fallow deer (Dama dama), 2000 Iberian ibex (Capra pyrenaica) and 3500 mouflons (Ovis orientalis musimon). The estimated sex ratio for red deer, fallow deer, and mouflon currently ranges between approximately 1.3–2.5 in favor of females. In addition, it is estimated that around 70% of females of breeding age contribute offspring to the population each year.

Following its introduction, EHDV-8 spread rapidly within southern Spain. While early reports from 2022 suggested limited involvement of wildlife, the summer of 2023 was characterized by a marked increase in red deer mortality across Andalusia, indicating intensified transmission in wild ruminant populations [10]. Over subsequent months, EHDV-8 spread northwards throughout the country and into neighboring Portugal and France [12]. The virus was likely introduced in Italy and Spain via wind-borne Culicoides midges, originating from North Africa [13]. Supporting this hypothesis, Tunisia reported over 200 confirmed cases of EHDV-8 in bovine populations during the 2021–2022 vector season, as well as in local wildlife [14]. Although Culicoides midges typically travel short distances (around 2 km), wind-assisted dispersal over long distances, especially over open water, has been documented [15].

The arrival of EHDV-8 to Andalusia during 2023 affected multiple ungulate species. In view of the above, this study reports the clinical presentation, diagnostic approach and pathological lesions of EHDV-8 infection in wild ruminants encountered during the 2023 outbreak. In addition, we provide the first molecular and pathological confirmation of EHDV infection in fallow deer and mouflon in Europe. This case represents an extension of the recognized host-range in Europe, with direct relevance for wildlife health, game management, monitoring programs and veterinary diagnostics. These observations have direct implications for livestock health, wildlife disease surveillance, and the management of game species in EHDV-emergent zones.

2. Materials and Methods

2.1. Animals, Clinical Observations and Sampling Methods

A total of 42 wild ruminants were examined between July and October 2023 in the AHRCS, located within the Sierras de Cazorla, Segura y Las Villas Natural Park, Jaén Province, southern-eastern Spain (Figure 1) (software for mapping: ArcGIS Desktop ArcMap 10.8.2).

Fourteen animals were found dead, 28 were found moribund and were subsequently euthanized on welfare grounds. Affected ruminants consisted of red deer (n = 39; 92.9%), fallow deer (n = 2; 4.7%), and mouflon (n = 1; 2.4%). Whenever possible, the age and sex of the animals were recorded. Sex was determined by direct observation of external genitalia and secondary sexual characteristics. Age estimation was based on tooth eruption and wear patterns [16,17,18]. Animals were categorized into three age classes: ‘young’ (<1 year), ‘subadult’ (1–6 years), and ‘adult’ (>6 years). In some cases, age and/or sex data were unavailable, particularly for animals detected by game reserve rangers or other observers where such information was not recorded; these cases were classified as ‘no data’ (Table 1 and Table 2).

A standardized clinical triage was applied in the field to all live animals prior to sampling or postmortem examination. This triage consisted of a structured veterinary assessment of general condition, neurological status and respiratory function, with particular attention to the severity and progression of clinical signs. Animals presenting with severe neurological impairment (including marked ataxia, recumbency, nystagmus or inability to stand), advanced respiratory distress (including dyspnoea, open-mouth breathing or profuse frothy oral discharge), profound weakness, or a moribund state with a poor prognosis were considered unlikely to recover and to be experiencing significant suffering. In such cases, euthanasia was performed on animal-welfare grounds. Specimens of game species found sick or moribund within the reserve were euthanized by firearm in accordance with the approved technical hunting plan and applicable regional legislation, including Law 8/2003 of 28 October on wild flora and fauna in Andalusia and subsequent regulatory updates. Firearm euthanasia was selected as the most rapid and humane option under field conditions, allowing immediate loss of consciousness, minimizing animal suffering and ensuring operator safety in free-ranging wildlife where capture or chemical euthanasia is not logistically feasible.

In accordance with the official EHDV outbreak response protocol implemented by the Spanish veterinary authorities, laboratory confirmation by RT-qPCR was performed on a single index animal per defined sampling area (Figure 2). Consequently, the 42 animals included in the present study do not correspond to 42 distinct sampling areas. Index cases from each area were submitted for molecular testing and confirmed as EHDV-positive. Given the high incidence of cases during the outbreak, subsequent animals found dead within the same sampling areas and temporal window, as well as exhibiting comparable epidemiological and clinicopathological features, were considered to be epidemiologically linked to the confirmed index cases. Those animals were therefore classified as outbreak cases without systematic additional individual laboratory testing.

For index cases, both blood and spleen samples were collected and analyzed separately by RT-qPCR (AgPath-ID™ One-Step RT-PCR Reagents, Applied BioSystems, Whaltman, MA, USA); samples were not pooled. Blood and spleen samples from red deer and mouflon were submitted to the Laboratorio Central de Veterinaria (Ministerio de Agricultura, Pesca y Alimentación, Algete, Madrid, Spain), while blood and spleen samples originating from fallow deer were analyzed at the regional laboratory in Sevilla. Following laboratory confirmation of EHDV infection within a defined sampling area, subsequent sampling focused on animals originating from different areas or different species, in accordance with the official outbreak response protocol.

For diagnostic purposes, blood samples were collected during necropsy from the cavernous sinus and, when this was not feasible, directly from the heart [19]. Samples included serum, whole blood collected in EDTA, and spleen. In addition, selected organ samples were collected for histopathological examination and fixed in 10% neutral-buffered formalin. Sample collection was performed in the field by trained veterinary staff of the Natural Park and by authorized game reserve rangers, in accordance with established surveillance and handling protocols. Samples collected were transported under refrigerated conditions (4 °C) in accordance with the protocols established by the Spanish national animal health authorities. Blood samples were allowed to clot and were subsequently centrifuged at 2500 revolutions per minute for 10 min to obtain serum (Eppendorf Centrifuge 5810R, Eppendorf SE, Hamburg, Germany) which was then used for serological testing.

2.2. Gross Pathology and Histopathology

Complete postmortem examinations were performed on all carcasses following a standardized protocol. Pathological and histopathological examinations were performed at the Servicio de Anatomía Patológica, Hospital Veterinario, Universidad de León (León, Spain). Representative samples were collected from each animal for histological analysis, and included superficial lymph nodes (submandibular, parotid, pre-scapular, inguinal, mammary and popliteal), and intracavitary lymph nodes (mediastinal, mesenteric, hepatic, renal), as well as skeletal muscle, heart, lungs (apical, middle and caudal lobes), liver, kidney, small intestine (ileum), and large intestine. All samples were fixed in 10% neutral-buffered formalin, routinely processed and paraffin-embedded, sectioned at 2.5 µm using a rotation microtome (Thermo Shandon Finesse®, Thermo Scientific, Waltham, MA, USA), mounted on electrocharged adhesive gelatin-coated microscope slides (Thermo Scientific, Waltham, MA, USA), deparaffinized, rehydrated, and stained with hematoxylin and eosin for histological analysis. Histological examinations were performed using a Nikon Eclipse E600 research microscope (Nikon Corporation, Tokyo, Japan) equipped with a Nikon Digital Sight DS-Fi1 camera (Nikon Instruments Inc., Tokyo, Japan).

2.3. Serology (c-ELISA and Virus Neutralisation Test)

Sera were screened for EHDV antibodies using a VP7-based competitive ELISA (c-ELISA) (ID Screen^®^ EHDV Competition reference EHDVC-5P, Innovative Diagnostics, Grabels, France). The VP7-based c-ELISA used in this study has been previously validated for the detection of EHDV antibodies in both domestic and wild ruminant species [20]. Samples tested positive by c-ELISA were subsequently analyzed for EHDV serotype 8 using a virus neutralization test. Neutralization assays were performed in duplicate on serial two-fold serum dilutions beginning at 1:5 using 100 TCID_50_ of EHDV-8 per well, as recommended by the World Organisation for Animal Health (WOAH) [3]. Neutralizing antibody titers were expressed as the reciprocal of the highest serum dilution that produced complete inhibition of cytopathic effects. Endpoint titers were calculated according to the Spearman–Kärber method [21]. Diagnostic procedures and interpretation criteria followed the WOAH Terrestrial Manual and the European Union Reference Laboratory Reference Protocol GL-LCV-09 Rev.02 for serological confirmation of EHDV infection [3,22].

2.4. Molecular Detection (RT-qPCR)

During the initial phase of the outbreak in July, when the aetiology was unknown, complete necropsy and targeted sampling were performed on the first confirmed index cases, which are included among the 42 animals examined in the present case series, to determine the cause of death. A broad differential diagnostic approach was applied, allowing exclusion of non-infectious causes such as toxicosis and parasitic disease, as well as bacterial causes compatible with the observed lesions and clinical presentation. In parallel, molecular testing was performed to exclude other infectious agents associated with acute or peracute disease in ruminants, including bluetongue virus, Schmallenberg virus and border disease virus. These investigations were negative, whereas EHDV was detected. Following confirmation of the aetiology, a national outbreak management protocol was implemented by the Spanish authorities, whereby Andalusia was divided into defined surveillance areas. Subsequent deaths within confirmed areas were reported to the authorities but not systematically re-sampled, as they were considered outbreak-related.

Whole EDTA blood and spleen samples were used for nucleic-acid extraction (BioSprint 96 robotic system, Qiagen, Valencia, CA, USA). Total RNA was extracted from 200 µL of sample using the BioSprint 96 robotic system (Qiagen, Valencia, CA, USA) with the IndiMag Pathogen Kit^®^ (Indical Bioscience, Sachsen, Germany), according to the manufacturer’s protocol. RNA was eluted in 100 µL of nuclease-free water and stored at 4 °C for up to 2 h, or −20 °C for longer storage prior to RT-qPCR analysis. RNA was eluted in nuclease-free water to ensure optimal compatibility with downstream RT-qPCR assays and to minimize the risk of enzymatic inhibition. Positive controls (dilutions of an EHDV reference strain in nuclease-free water) and negative controls (nuclease-free water) were included in each 96-well extraction plate to verify extraction performance and to exclude cross-contamination.

Detection of EHDV RNA was performed using the duplex pan-EHDV RT-qPCR targeting segment 9 of the viral genome in combination with a ruminant β-actin internal control, as described by Viarouge et al. [23]. Amplifications were performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA).

To establish the serotype, all samples that tested positive on the duplex pan-EHDV RT-qPCR were subsequently analyzed using an EHDV-8-specific RT-qPCR targeting segment 2 (VP2), as reported by Lorusso et al. [9].

3. Results

3.1. Clinical Observations

The first dead animal was detected in July 2023, and this concerned a subadult female red deer found in the municipality of Santiago-Pontones, within the AHRCS.

All live animals were moribund upon initial observation and presented an acute neuro-respiratory syndrome. Clinical signs included weakness, nystagmus and ataxia consistent with cerebellar and/or vestibular involvement, plus severe respiratory distress with frothy oral discharge. No notable clinical signs, increased mortality or relevant health disorders were observed in other wildlife species within the Sierras de Cazorla, Segura y Las Villas Natural Park during the study period.

3.2. Pathological Observations

3.2.1. Gross Pathology

Gross pathological observations are based on field necropsies conducted under wildlife surveillance conditions. All examined carcasses presented similar postmortem lesions, including poor body condition, generalized or multi-organ congestion, cyanosis of the oral mucosa and tongue, severe pulmonary oedema, foam in the trachea, and petechial to ecchymotic haemorrhages of the pleura and lungs.

3.2.2. Histopathological Observations

Lymphoid tissues, including peripheral and visceral lymph nodes and Peyer’s patches, demonstrated a moderate to marked lymphoid depletion. This was characterized by multifocal loss of follicular germinal centers and their replacement by macrophages, accentuation of the reticular and sinusoidal framework and a generalized reduction in lymphocyte density.

The subcapsular and medullary sinuses were distended by erythrocytes, foamy macrophages demonstrating erythrophagocytosis, lymphocytes, plasma cells, neutrophils, fibrin, oedema and cellular debris (Figure 3).

In the lungs, widespread areas of alveolar and interstitial haemorrhages were accompanied by moderate vascular congestion and focal emphysema. Multifocally, mainly along the pleura, there were aggregates of haemosiderin-laden macrophages (siderophages), reflecting prior intrapulmonary haemorrhages and subsequent erythrocyte breakdown (Figure 4).

In fallow deer, renal alterations were prominent and diffuse. Cortical and medullary tubules were ectatic and lined by attenuated epithelium, with lumina containing bright red hyaline to granular casts, consistent with haemoglobin and/or myoglobin. Less frequently, pale eosinophilic homogeneous proteinaceous material was present in the lumina. Tubular epithelial cells exhibited cytoplasmic vacuolation, nuclear swelling, and karyopyknosis, with intracytoplasmic haemosiderin or lipofuscin granules in numerous cells (Figure 4a,b). In severely affected regions of the kidney, tubulorrhexis was observed. The interstitium was expanded and partially obliterated by fibrin, oedema, and haemorrhages, with scattered aggregates of a low number of lymphocytes and plasma cells. (Figure 4). The adhesion of visceral to parietal epithelium (glomerular synechiae) was observed focally within Bowman’s space. These lesions are consistent with acute tubular necrosis secondary to hypoxic or hemolytic injury. In one of the examined deer specimens, histological changes compatible with lymphoplasmacytic meningoencephalitis were observed (Figure 5). The lesions were characterized by multifocal nodular aggregates of astrocytes and microglial cells (reactive gliosis) within the periventricular white and grey matter. Numerous blood vessels lined by hypertrophic, reactive endothelium were present and surrounded by moderate to marked perivascular cuffs composed of macrophages, lymphocytes, and plasma cells. Multifocally, within the grey matter and subependymal neuroparenchyma, glial nodules were observed. These were occasionally surrounded by swollen or vacuolated neurons demonstrating hypereosinophilic cytoplasm and pyknotic nuclei, consistent with neuronal degeneration (satellitosis). Additionally, multifocal loss of myelin sheaths was observed, with replacement by gitter cells (demyelination), as well as marked dilation of myelin sheaths (spongiosis) containing inflamed axons with hypereosinophilic axoplasm (axonal spheroids). Generalized vascular congestion and vasogenic oedema were also present.

3.3. Molecular Results

RT-qPCR confirmed EHDV infection in index animals sampled from each defined sampling area. All tissues that were subjected to molecular analysis tested positive on the pan-EHDV duplex RT-qPCR (segment 9) and were subsequently confirmed as EHDV-8 by EHDV-8-specific RT-qPCR (segment 2). Cycle threshold Ct values ranged from 24 to 25 across positive samples, indicating high viral loads in the analyzed tissues. All RT-qPCR-positive samples were identified as EHDV-8.

4. Discussion

This investigation describes a peracute outbreak of EHDV-8 in free-ranging wild ruminants within the AHRCS during July–October 2023. The results corroborate the observations of Lorusso et al. and Viarouge et al., confirming the presence of the same serotype circulating in Italy and Spain in 2022–2023 [9,23]. The predominant field presentation in moribund animals was an acute neuro-respiratory syndrome characterized by weakness and ataxia, nystagmus, severe dyspnoea, and frequent foamy oral discharge, with many animals found dead or requiring welfare-based triage and euthanasia. These clinical observations were accompanied by a coherent set of gross and histopathological observations consistent with severe systemic vascular injury and capillary leakage, and by molecular confirmation of EHDV infection with low RT-qPCR Ct values in tested samples. All analyzed samples tested positive for EHDV-8 and compatible clinicopathological observations were documented across multiple species in the reserve, including red deer, fallow deer, and mouflon, supporting multi-species involvement during the outbreak.

The rapid clinical course is compatible with severe orbivirus-associated disease described in susceptible cervids, such as in the Virginia white-tailed deer (Odocoileus virginianus), whereas clinical disease in domestic ruminants is often milder and more frequently detected through surveillance [5,8,11]. The predominance of dyspnoea and foamy oral discharge, together with marked pulmonary oedema and/or haemorrhage at necropsy, indicates that acute respiratory failure was likely a major contributor to death. Histopathology further supported systemic involvement, including lymphoid depletion and renal tubular injury, consistent with a severe haemorrhagic/vasculopathic syndrome [1,5,11].

From a diagnostic perspective, this outbreak illustrates the importance of a structured approach to investigating wildlife mortality events. During the initial phase, complete necropsy and histopathology were complemented by molecular testing for other infectious agents associated with acute or peracute disease in ruminants. These differential tests yielded negative results, whereas EHDV was detected, indicating its role as the primary aetiological agent. Subsequent investigations prioritized targeted EHDV testing and typing. Serotype-specific RT-qPCR identified EHDV-8 in all analyzed samples included in the present study. This observation aligns with observations from recent European and North African outbreaks, in which EHDV-8 has been the predominant serotype detected during the 2022–2023 transmission seasons, particularly in Italy and Spain [9,10,12,23,24]. Comparable molecular and epidemiological patterns have also been reported in southern Europe and neighboring regions of North Africa, suggesting widespread circulation of EHDV-8 during this period [5,7]. As serotype-specific assays targeting EHDV-8 were employed, the co-circulation of other EHDV serotypes cannot be excluded; however, the available data support the prominent role of EHDV-8 in the outbreaks described across the region. In the present setting, detections clustered from late July through October, which coincides with the expected seasonal window of the highest Culicoides activity in southern Spain, providing a biologically plausible context for intense transmission and clustering of peracute cases [15,25].

The epidemiological setting of these cases is consistent with the rapid west-Mediterranean expansion of EHDV-8. After first detection in Sardinia in late 2022, EHDV spread across southern Spain and Portugal by mid-2023 and reached south-western France later that year [24]. Serological testing was conducted as part of the active surveillance programme implemented in Andalusia. Anti-EHDV antibodies have been routinely monitored since 2009, with consistently negative results until the first outbreak. Following the initial detection, anti-EHDV antibodies were detected in the screened serum samples. Focusing on the study area, a total of 396 red deer were tested during the outbreak period, of which 48 (12%) were seropositive. During the same period, antibodies against EHDV were detected in 7 out of 58 (12%) tested fallow deer. These serological data were generated through routine surveillance activities and do not derive from the animals included in the present case series; however, they provide epidemiological context supporting the active circulation of EHDV-8 in wild ruminant populations during the outbreak period. In parallel with the investigation of clinically affected animals, serological surveillance was conducted as part of the Andalusian Wildlife Epidemiological Surveillance Programme in apparently healthy wild ruminants. During the study period, 132 serum samples from red deer, fallow deer, and European roe deer (Capreolus capreolus) were screened using a VP7-based c-ELISA, of which 52 (39%) tested positive. Subsequent virus neutralization assays confirmed EHDV-8-specific antibodies in 27 animals. Within Andalusia, the regional surveillance system recorded 90 field reports between November 2022 and June 2024, of which diagnostic samples were obtained in 21 cases. The 21 laboratory-confirmed cases mentioned at the regional level correspond to confirmed EHDV detections across Andalusia within the Wildlife Epidemiological Surveillance Programme and are not equivalent to the 42 wild ruminants examined in the Sierras de Cazorla, Segura y Las Villas Natural Park in the present study. EHDV RNA was confirmed by RT-qPCR in 20 out of those 21 cases (95%), indicating active circulation and effective case capture through passive surveillance. In practice, field detections clustered in late summer and early autumn, which aligns with the known seasonal increase in Culicoides sp. vector activity, and is hence a predictable clinical window for presentation and sampling [1].

Vector data from the French outbreaks complement these clinical observations: EHDV-8 RNA was repeatedly detected in Culicoides imicola and members of the Culicoides obsoletus complex, both recognized vectors in Europe. Cases were reported during periods of high midge trap counts, supporting their operational relevance for transmission at the time animals were becoming ill [24,25,26]. Experimental data indicate that EHDV infection can impair Culicoides sonorensis reproductive output and may be vertically transmitted to offspring, providing a potential mechanism for virus persistence across vector generations [27].

Suspected EHDV cases should be prioritized for sampling during periods of high Culicoides activity, because transmission potential is temperature-dependent and peaks when conditions favor midge survival and vector competence [28]. Genomic evidence from the first European detections of EHDV-8 supports a North African origin (near-identity to Tunisian 2021 strains), consistent with recent Mediterranean phylogeographic syntheses [9,13]. Windborne dispersal of infected Culicoides is considered to be a plausible mechanism for short-range transboundary spread and rapid within-season dissemination, and has been modelled as a realistic pathway during recent European EHDV emergence scenarios [13,29]. More broadly, recent reviews link orbivirus re-emergence (EHDV/BTV) to climate-driven shifts in vector ecology and to animal movement/trade [30].

At wildlife–livestock interfaces, multi-host involvement is evident, but the extent to which wild ruminants act as maintenance hosts versus spill-over recipients remains a key data gap [13,28]. Post-wave serology in France also indicates heterogeneous (regionally variable) exposure patterns in cattle, which may affect onward transmission risk locally [31].

Several commercial vaccines against epizootic haemorrhagic disease have become available on the European market since 2024, including Hepizovac^®^ (CZ Vaccines S.A.U., A Relva s/n, Torneiros, 36410 O Porriño, Pontevedra, Spain) and Syvac^®^ EH (Laboratorios Syva S.A., Calle Marqués de la Ensenada, 28004 Madrid, Spain). Nevertheless, their immunogenicity and cross-protection in wild ruminants remain untested. Given the antigenic diversity of the VP2 protein among orbiviruses, cross-protective immunity between serotypes is likely to be limited. In addition, immune responses to EHDV vaccination may vary between host animal species, which could further influence field efficacy, particularly in wild ruminants [4,11]. Controlled vaccination trials in sentinel wildlife populations could help determine whether herd immunity in livestock indirectly protects sympatric wild ungulates through vector reduction.

Several methodological considerations should be acknowledged. In line with the official EHDV outbreak response protocol implemented by the Spanish veterinary authorities, molecular confirmation by RT-qPCR was performed on a single index animal per defined sampling area. Consequently, not all animals included in the present case series underwent individual molecular testing and subsequent cases were classified based on epidemiological linkage and compatible clinicopathological findings. Although this approach reflects standard practice during large-scale outbreak investigations, it may have limited the assessment of within-area molecular variability or the detection of additional co-circulating pathogens.

5. Future Directions

Despite rapid progress in documenting EHDV-8 emergence in Europe, key gaps remain in disease ecology, host competence and longer-term epidemiological impact. Addressing these gaps requires integrated, cross-sector surveillance and response across wildlife, livestock, and vectors, consistent with the One Health framework [30]. Priority research should (i) clarify likely introduction and spread pathways (including wind-borne dispersal of infected Culicoides from North Africa) [13], (ii) quantify the vector competence and transmission efficiency of European Culicoides taxa, and (iii) strengthen integrated surveillance through standardized necropsy, targeted serology/RT-qPCR and concurrent entomological monitoring across livestock and free-ranging ungulates. Expanded whole-genome sequencing across hosts is also needed to monitor viral evolution and reassortment, and to support predictive risk modeling alongside climatic and ecological drivers [25]. Finally, while vaccination is primarily livestock-focused, evidence from experimental cattle studies supports the high efficacy of inactivated EHDV-8 vaccines; whether similar protection can be achieved (directly or indirectly) in mixed wildlife–livestock systems remains to be determined [32].

6. Conclusions

The confirmation of EHDV-8 infection with compatible clinicopathological observations in fallow deer and mouflon in south-eastern Spain supports multi-species involvement during the 2023 outbreak and demonstrates that fatal disease can occur beyond red deer. Our observations highlight the value of wildlife surveillance networks, alongside livestock monitoring, to enable timely detection and diagnostic confirmation of emerging orbiviral outbreaks.

EHDV should be considered in the differential diagnosis of haemorrhagic disease, respiratory distress, neurological symptoms and sudden death in wild ungulates during periods of vector activity. Diagnostic confirmation should rely on appropriate molecular testing (e.g., RT-qPCR), supported by serology and histopathology where available. The continued detection of EHDV-8 in southern Europe reinforces the importance of coordinated surveillance and communication between veterinary services, wildlife managers and diagnostic laboratories to strengthen preparedness and outbreak response.