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Emergence of epizootic hemorrhagic disease in red deer (Cervus elaphus),

Spain, 2022

Article in Veterinary Microbiology · March 2024

DOI: 10.1016/j.vetmic.2024.110069

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 Veterinary Microbiology 292 (2024) 110069

Contents lists available at ScienceDirect

Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Emergence of epizootic hemorrhagic disease in red deer (Cervus elaphus),

Spain, 2022
Francisco Ruiz-Fons a, b, *, 1, Ignacio García-Bocanegra b, c, 1, Marta Valero d, Raúl Cuadrado-
Matías a, David Relimpio a, Remigio Martínez c, Sara Baz-Flores a, Moisés Gonzálvez c, e,
David Cano-Terriza b, c, José Antonio Ortiz f, Christian Gortázar a, María A. Risalde b, g, **, 1
a
SaBio (Health and Biotechnology) group, Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ciudad Real, Spain
b
CIBERINFEC, ISCIII - CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid, Spain
c
Departamento de Sanidad Animal, Grupo de Investigación en Sanidad Animal y Zoonosis (GISAZ), UIC Zoonosis y Enfermedades Emergentes ENZOEM, Universidad de
Córdoba, Córdoba, Spain
d
Laboratorio Central de Veterinaria (LCV), Ministerio de Agricultura y Pesca, Alimentación y Medio Ambiente, Madrid, Spain
e
Departamento de Sanidad Animal, Facultad de Veterinaria, Campus de Excelencia Internacional Regional “Campus Mare Nostrum”, Universidad de Murcia, Murcia,
Spain
f
Medianilla S.A., Benalup-Casas Viejas, Spain
g
Departamento de Anatomía y Anatomía Patológica Comparadas y Toxicología, Grupo de Investigación GISAZ, UIC Zoonosis y Enfermedades Emergentes ENZOEM,
Universidad de Córdoba, Córdoba, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Epizootic hemorrhagic disease (EHD) virus serotype 8 (EHDV-8) emerged in Spain in autumn 2022. In this study,
Culicoides we aimed to (1) characterize the clinical and lesional presentation of EHDV infection in European red deer
Emerging disease (Cervus elaphus), and (2) study the spatial spread of the virus in wild ruminants in Spain after its introduction, in
Orbivirus
2022/2023. We confirmed EHDV infection in two clinically compatible sick red deer by PCR and detection of
Serotype
Vector-borne disease
anti-EHDV specific antibodies. EHDV infection occurred in red deer with hyperacute to acute clinical signs and
Wildlife lesions associated to vascular changes leading to death of the animals. Partial sequences of variable segment 2
(VP2) and segment 5 (NS1) genes of the detected viruses had >99% nucleotide identity with EHDV-8 sequences
from Tunisia and Italy. In a cross-sectional serological study of EHDV in 592 wild ruminants, mainly red deer
(n=578), in southwestern Spain, we detected anti-EHDV antibodies in 37 of 592 samples (6.3%; 95% confidence
interval: 4.3–8.2), all from red deer and from the localities where clinical cases of EHD were confirmed in red
deer. We conclude that EHDV-8 infection causes severe EHD in European red deer. The serosurvey revealed a
limited spread of EHDV-8 in Spanish wild ruminant populations in the first year of virus detection in Spain.

1. Introduction States between 2013 and 2014 (USDA, 2016). EHDV can also affect
other wild ruminants such as mule deer (Odocoileus hemionus), prong­
Epizootic hemorrhagic disease (EHD) is a reportable vector-borne horn antelope (Antilocapra americana), elk (Cervus canadensis) (USDA,
disease of wild and domestic ruminants caused by an Orbivirus related 2016) and Barbary deer (C. elaphus barbarus) (Thabet et al., 2023).
to bluetongue virus (BTV). Seven EHD virus (EHDV) serotypes are In 2006, an epidemic of EHDV serotype 6 (EHDV-6) spread across
known, distributed over five continents (Jiménez-Cabello et al., 2023) Morocco, Algeria, and Tunisia, and entered the European continent in
and with a variable pathogenic potential (EFSA, 2009). Variants of se­ 2007 with the notification of EHD cases in cattle in Turkey (EFSA, 2009).
rotypes 1, 2 and 6, that are enzootic in North America, have caused However, in contrast to what it was observed with BTV serotypes 1 and 4
severe EHD outbreaks with high mortality in white-tailed deer (Odo­ after their introduction into Spain in 2004 and 2007, respectively
coileus virginianus) and the loss of 1.7% of farmed cervids in the United (Ruiz-Fons et al., 2014) and with BTV serotype 8 in west-central Europe

* Corresponding author at: Instituto de Investigación en Recursos Cinegéticos (IREC), CSIC-UCLM-JCCM, Ronda de Toledo 12, Ciudad Real 13005, Spain.
** Corresponding author at: Universidad de Córdoba. Edificio de Sanidad Animal, Campus de Rabanales s/n, Córdoba 14014, Spain.
E-mail addresses: [email protected] (F. Ruiz-Fons), [email protected] (M.A. Risalde).
1
Equal contributors

https://doi.org/10.1016/j.vetmic.2024.110069
Received 9 January 2024; Received in revised form 27 March 2024; Accepted 30 March 2024
Available online 31 March 2024
0378-1135/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
F. Ruiz-Fons et al. Veterinary Microbiology 292 (2024) 110069

in 2008 (van Rijn et al., 2012), EHDV-6 did not spread from North Africa 2. Methods
and Turkey to neighboring European countries. In 2021, more than 200
EHD outbreaks were notified in cattle in Tunisia caused by EHDV 2.1. Clinical cases in red deer
serotype 8 (EHDV-8) (Sghaier et al., 2023). In autumn 2022, the first
EHDV-8 cases were reported in cattle in Italy (Sicily and Sardinia; Lor­ In mid-November 2022, we were notified of an unusual outbreak of
usso et al., 2023) and in southwestern Spain (Jiménez-Cabello et al., mortality in a red deer farm in southwestern Spain (Fig. 1). On
2023). Almost simultaneously with the reporting of EHD outbreaks in November 19, 2022, we performed a necropsy on a 1.5-year-old male
cattle in autumn 2022, passive wildlife disease surveillance detected in deer from that farm (Case 1) which had been observed with depression,
southwestern Spain outbreaks of unusual mortality and isolated clinical anorexia, weakness, ataxia, and severe respiratory distress three days
cases in native red deer (Cervus elaphus). Our objectives were (1) to before being found dead. On November 28, 2022, during a rutinary visit
confirm whether the fatal cases observed in red deer were EHD and (2) to an integrated wildlife monitoring site 180 km North of Case 1, we
to determine the spread of the virus among wild ruminants during its found a moribund stag presenting severe dyspnea and weakness (Case
first incursion into Spain. 2). The animal died within an hour of detection and was necropsied.
Organ samples (lung, spleen, kidney, liver, heart) were fixed in 10%
phosphate-buffered formalin, embedded in paraffin, sectioned at 4 µm

Fig. 1. Map of southwestern Spain (bottom left) and its location within the western Palearctic (top right) showing the spatial distribution of the 14 wild ruminant
populations studied within provincial boundaries (black lines) and serological results (blue circle: seronegative; red circle: seropositive). Positive animal samples in
relation to those tested (positive/sample size) and seroprevalence (in %) per population are shown. The red stars indicate the countries/localities where outbreaks of
epizootic hemorrhagic disease in cattle were reported in 2022 (MAPA, 2023), while the black stars show the location of the two clinical cases in red deer with
specification of the case number.

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F. Ruiz-Fons et al. Veterinary Microbiology 292 (2024) 110069

and stained with hematoxylin and eosin. Both showed slight pulmonary congestion and presence of liquid and
Samples of spleen and blood taken at necropsy were subjected to froth in the trachea and bronchi, compatible with a severe alveolar
RNA extraction (NucleoSpin RNA, Macherey-Nagel, Germany). The edema (Fig. 2B). One of them also presented a moderate number of
presence of EHDV in all tissue samples was tested using a real-time RT- petechial and ecchymotic hemorrhages in the parietal pleura (Case 1),
PCR duplex pan-EHDV protocol, which targets the segment 9 gene of while the other animal showed an ulcerative lesion on one ear.
EHDV coupled with a real-time RT-PCR that amplifies ruminant beta- Microscopically, the examination of lungs revealed severe alveolar
actin RNA (Viarouge et al., 2015), in a CFX96 Touch Deep Well™ and interstitial edema along with light congestion (Fig. 2C). Lympho­
Real-Time PCR Detection System (Bio-Rad, CA, USA). To further char­ plasmacytic vasculitis on medium to large vessels was observed in lung,
acterize the virus, we amplified and sequenced a 558 bp gene fragment heart, and liver (Fig. 2D, E). Glomerular capillaries of the kidneys were
of the variable segment 2 (VP2) and a fragment of the segment 5 (NS1), congested. Lymphoid follicles of the spleen showed depletion of lym­
both highly expressed and conserved viral regulatory proteins. VP2 and phocytes (Fig. 2F). Thrombi were not seen.
NS1 genes were partially amplified by RT-PCR in positive samples using We detected EHDV in all the tissues and blood collected from both
in-house primers selected based on the published sequence of EHDV-8 deer at Ct values ranging from 22 to 29, and confirmed EHDV-8 infection
from Tunisia (Sghaier et al., 2023) and a previously described proto­ in both cases. Ct values were lower in the blood than in the liver of both
col (Aradaib et al., 1994), respectively. For NS1, we amplified and animals. The BLAST analysis showed high nucleotide identity for both
sequenced a 225 bp fragment in Case 1 and a 348 bp fragment in Case 2. VP2 (>99.5% among the two sequences and with available EHDV-8
The obtained sequences were deposited in the GenBank database and sequences from Tunisia and Italy; Lorusso et al., 2023; Sghaier et al.,
compared with other previously reported EHDV sequences (Accession 2023; Table 1) and NS1 gene segments (100% among the two sequences
numbers are shown in Table 1). and >99.6% with reported EHDV-8 sequences; Lorusso et al., 2023;
Sghaier et al., 2023).
2.2. Cross-sectional serological survey
3.2. Serological survey
Between November 2022 and February 2023, we collected blood
samples from red deer (n=578), fallow deer - Dama dama (n=3), and We detected anti-EHDV antibodies in 37 of 592 (6.3%; 95% Confi­
mouflon - Ovis orientalis musimon (n=11) in 14 localities of southwestern dence Interval: 4.3–8.2) wild ruminants, including Cases 1 and 2, but
Spain, both in areas of confirmed EHD cattle cases (MAPA, 2023) and in only in red deer and in the locations where Cases 1 and 2 occurred
neighboring areas (Fig. 1). Samples were obtained from animals shot by (Fig. 1). None of the wild ruminant samples collected at localities with
rangers and hunters during population control and commercial hunting confirmed EHD cattle cases (MAPA, 2023) were seropositive except Case
events, respectively. Cases 1 and 2 were also included in the serosurvey. 2 (Fig. 1).
We searched for specific antibodies against EHDV VP7 in sera obtained
from blood samples using a commercial multi-species competition ELISA 4. Discussion and conclusions
(ID Screen® EHDV Competition, ID.vet, France; Bréard et al., 2020).
Our findings confirm that EHDV-8 can cause clinical disease and
3. Results mortality in European red deer. The two reported cases suggest a
hyperacute-to-acute clinical presentation. The lesions observed were
3.1. Clinical cases associated to vascular changes such as congestion, edema, and vascu­
litis. However, other typical histologic lesions caused by EHDV-2 and
On external inspection, Cases 1 and 2 presented poor body condition, EHDV-6 in white-tailed deer (Rodrigues et al., 2022) or by EHDV-8 in
severe congestion and cyanosis of the oral mucosa and tongue (Fig. 2A). Barbary deer (Thabet et al., 2023), such as endothelial swelling,
thrombosis, fibrinoid necrosis and multiple hemorrhages, were not
observed.
Table 1
The serosurvey reveals a limited spread of EHDV-8 in wild ruminant
Sequence data of variable segment 2 (VP2) and segment 5 (NS1) of epizootic
hemorrhagic disease (EHD) virus serotype 8 obtained from two clinical cases of populations in the first year of virus detection in Spain, even in locations
EHD in European red deer (Cervus elaphus) confirmed in 2022 in Spain. where EHD was confirmed in cattle. Higher seroprevalence was
observed in the southernmost of the two seropositive locations, sup­
Segment Reference GenBank EHDV-8 sequences Identity
Accession compared (Accession (%) porting the presumed introduction of EHDV from North Africa. How­
No No) ever, in summer 2023, EHDV-8 underwent a notable expansion in Spain
VP2 Case 1 OR188849 OP381191, OP897266, 100
causing numerous outbreaks with red deer mortality, and it appeared in
OP897276, OP897501, Portugal and France (MAPA, 2023). The impact of the emergence of
OP897511, OP897521, EHD on red deer population dynamics is unknown. The outbreak on the
OP897531, OP897541, farm where we report Case 1 caused mortalities approaching 4,1%, with
OP897551, OP897561,
10% mortality in the most affected batches of the farm. We believe that
OP937332, OP971118,
OP971128, OP971138 the impact of EHD emergence will be highly variable among red deer
Case 2 OR188850 OP381191, OP897266, 99.5 populations depending on local conditions, such as the structure of
OP897276, OP897501, EHDV hosts and vector communities. Future studies should be carried
OP897511, OP897521, out to fill this knowledge gap.
OP897531, OP897541,
OP897551, OP897561,
The wide range of competent Culicoides spp. vectors for EHDV is
OP937332, OP971118, likely to favor further spread of EHDV in wild ruminants in Iberia and in
OP971128, OP971138 other European countries in the near future. Integrated wildlife and
NS1 Case 1 OR188846 OP971141, OP971131, 100 vector population monitoring, and disease surveillance (Barroso et al.,
OP971121, OP937335,
Case 2 OR188847 2022) will aid to assess the spread and impact of this emerging Orbivirus.
OP897564, OP897554,
OP897544, OP897534,
OP897524, OP897514, Funding
OP897504, OP897279,
OP897269, OP381194 The study was partly funded by the project TED2021-132599B-C21
OP971151 99.6
of Spanish Ministry for the Science and Innovation (MCIN) through the

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F. Ruiz-Fons et al. Veterinary Microbiology 292 (2024) 110069

Fig. 2. Lesions consistent with emerging epizootic hemorrhagic disease in European red deer (Cervus elaphus). A) Severe cyanosis of the oral mucosa and tongue. B)
Light congestion and increased size of the lung, observing a lobulillar-like pattern in its surface and presence of liquid and froth in the trachea and bronchi (grey
asterisk, inset). C) Severe acute pulmonary edema with accumulation of fluid inside alveoli (black asterisks) and interstitium (black arrowhead). D) Portal hepatitis
and myocarditis (E) associated to lymphoplasmacytic perivasculitis (white arrowheads). F) Mild depletion of lymphocytes in a lymphoid follicle of the spleen
(white asterisk).

Spanish Research Agency (AEI; DOI: 10.13039/501100011033) and the Conceptualization. Francisco Ruiz-Fons: Writing – review & editing,
Europen Union NextGenerationEU/ PRTR, project PP2F_L1_22 of Writing – original draft, Validation, Supervision, Resources, Project
Research Plan of the University of Cordoba, CIBER - Consorcio Centro de administration, Methodology, Investigation, Funding acquisition,
Investigación Biomédica en Red (CB 2021), Instituto de Salud Carlos III, Formal analysis, Data curation, Conceptualization. Ignacio García-
MCIN and European Union NextGenerationEU and by “Encomienda de Bocanegra: Writing – review & editing, Validation, Investigation,
Gestión” contract from the Spanish Ministry of Agriculture. RC-M and Conceptualization. Marta Valero: Writing – review & editing, Meth­
SB-F acknowledge funding from MCIN/AEI, the European Union/Eu­ odology, Investigation, Data curation. Raúl Cuadrado-Matías: Writing
ropean Social Fund (EU-ESF), the Regional Government of Castilla-La – review & editing, Methodology, Investigation, Formal analysis, Data
Mancha (JCCM) and the University of Castilla-La Mancha (UCLM) curation. David Relimpio: Writing – review & editing, Methodology,
through contracts PRE2018-083801 and PREJCCM2019/11, respec­ Data curation. Remigio Martínez: Writing – review & editing, Writing –
tively. RM acknowledges funding from the “Consejería de Trans­ original draft, Validation, Methodology, Data curation. Sara Baz-
formación Económica, Industria, Conocimiento y Universidades” of the Flores: Writing – review & editing, Methodology, Data curation. Moisés
Regional Government of Andalusia and the University of Córdoba Gonzálvez: Writing – review & editing, Visualization, Methodology.
(05yc77b46). MG was supported by a postdoctoral contract Margarita David Cano-Terriza: Writing – review & editing, Supervision, Data
Salas (University of Murcia) from the Program of Requalification of the curation. José Antonio Ortiz: Writing – review & editing, Visualization,
Spanish University System (Spanish Ministry of Universities) financed Data curation.
by the European Union-NextGenerationEU.

CRediT authorship contribution statement Declaration of Competing Interest

Christian Gortázar: Writing – review & editing, Supervision, The authors declare no competing interests.
Funding acquisition. María Ángeles Risalde: Writing – review & edit­
ing, Writing – original draft, Validation, Supervision, Resources, Meth­ Data availability
odology, Investigation, Funding acquisition, Data curation,
Data are available upon request to the corresponding author.

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F. Ruiz-Fons et al. Veterinary Microbiology 292 (2024) 110069

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veterinarians of the study localities, as well as the veterinarians of the Characterization of a novel reassortant epizootic hemorrhagic disease virus serotype
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