Pa hological and se ological insigh s in o Lago i us diseases dynamics in he
Eu opean b own ha e (Lepus eu opaeus): A nine-yea longi udinal s udy
Josep Es uch
a,*
, Pa izia Ca adini
b
, An onio La azza
b
, Lo enzo Capucci
b
, Joana Ab an es
c,d,e
,
Ana M. Lopes
c,d, ,g
, Te eza Almeida
c,d
, Aleksija Neimanis
h,i
, San iago La ín
a
, Ca los Rouco
j
,
Emmanuel Se ano
a
, Rose Vela de
a
a
Wildli e Ecology & Heal h g oup (WE&H) and Se ei d’Ecopa ologia de Fauna Sal a ge (SEFaS). Depa amen de Medicina i Ci u gia Animals, Facul a de Ve e in`
a ia,
Uni e si a Au `
onoma de Ba celona (UAB), Bella e a 08193, Spain
b
Vi ology Uni , WOAH Re e ence Labo a o y o Rabbi Haemo hagic Disease (RHD), Is i u o Zoop o ila ico Spe imen ale della Lomba dia e dell’Emilia Romagna
(IZSLER), B escia 25124, I aly
c
CIBIO, Cen o de In es igaç˜
ao em Biodi e sidade e Recu sos Gen´
e icos, InBIO Labo a ´
o io Associado, Campus de Vai ˜
ao, Uni e sidade do Po o, Vai ˜
ao 4485-661,
Po ugal
d
BIOPOLIS P og am in Genomics, Biodi e si y and Land Planning, CIBIO, Campus de Vai ˜
ao, Vai ˜
ao 4485-661, Po ugal
e
Depa amen o de Biologia, Faculdade de Ciˆ
encias, Uni e sidade do Po o, Po o 4099-002, Po ugal
UMIB-Uni o Mul idisciplina y Resea ch in Biomedicine, ICBAS-School o Medicine and Biomedical Sciences, Uni e si y o Po o, Po o, Po ugal
g
ITR, Labo a o y o In eg a i e and T ansla ional Resea ch in Popula ion Heal h, Po o, Po ugal
h
Depa men o Pa hology and Wildli e Diseases, Swedish Ve e ina y Agency, Uppsala SE-751 89, Sweden
i
Depa men o Animal Biosciences, Swedish Uni e si y o Ag icul u al Sciences, Uppsala SE-751 89, Sweden
j
Depa amen o Biología Vege al y Ecología, ´
A ea de Ecología, Uni e sidad de Se illa, Se illa 41012, Spain
ARTICLE INFO
Keywo ds:
Eu opean b own ha e synd ome
Epidemics
Hos -shi
Mo ali y
Popula ion
Rabbi haemo hagic disease
Wildli e disease su eillance
ABSTRACT
The Eu opean b own ha e synd ome i us (EBHSV; GII.1) and abbi haemo hagic disease i us 2 (RHDV2;
GI.2) a e pa hogenic lago i uses a ec ing he Eu opean b own ha e (Lepus eu opaeus). EBHSV/GII.1 causes
pe iodic epidemics, while RHDV2/GI.2 in ec ions eme ge om spillo e e en s in a eas whe e ha es a e sym-
pa ic wi h Eu opean abbi s (O yc olagus cuniculus). In he no heas o he Ibe ian Peninsula, he o e lap o
hese species p o ides a unique oppo uni y o in es iga e how he epidemiology o hese i uses co ela es wi h
disease cou se. We analysed he p esence o lago i uses in 113 Eu opean b own ha e ca casses eco e ed in
Ca alonia (NE Spain) be ween 2015 and 2024. Animals we e nec opsied, and issue and se um samples we e
collec ed o his opa hology, i ological in es iga ion, and se ology. Se a om hun ed ha es appa en ly heal hy
(n =89, 2015–2023) we e also included in he s udy. PCR on li e samples (n =58) and i ological ELISA on
posi i e se a (n =52) con i med 28 EBHSV/GII.1 and 24 RHDV2/GI.2 cases. A e he i s EBHSV/GII.1
de ec ion in 2016, an ibody i es dec eased p og essi ely un il 2020–2021, coinciding wi h an ou b eak. No
conclusi e se oposi i i y o RHDV2/GI.2 was obse ed du ing he s udy. Pa hology e ealed mo e acu e lesions
in RHDV2/GI.2-in ec ed ha es compa ed o EBHSV/GII.1. These lesions, esul ing in sudden dea h due o a
de icien immune esponse, may explain his dis inc epidemiological scena io. Despi e a decade o ci cula ion,
RHDV2/GI.2 has no ully adap ed o ha es. Howe e , ongoing moni o ing is essen ial, as mu a ions o ecom-
bina ion e en s could inc ease i s epizoo ic po en ial. The co-ci cula ion o bo h lago i uses, combined wi h
o he co- ac o s, migh jeopa dise he iabili y o Eu opean b own ha e popula ions a he sou he n limi o hei
ange.
* Co esponding au ho .
E-mail add esses: [email p o ec ed] (J. Es uch), [email p o ec ed] (P. Ca adini), [email p o ec ed] (A. La azza), [email p o ec ed]
(L. Capucci), [email p o ec ed] (J. Ab an es), [email p o ec ed] (A.M. Lopes), [email p o ec ed] (T. Almeida), [email p o ec ed]
(A. Neimanis), [email p o ec ed] (S. La ín), [email p o ec ed] (C. Rouco), [email p o ec ed] (E. Se ano), [email p o ec ed] (R. Vela de).
Con en s lis s a ailable a ScienceDi ec
Ve e ina y Mic obiology
jou nal homepage: www.else ie .com/loca e/ e mic
h ps://doi.o g/10.1016/j. e mic.2025.110478
Recei ed 31 Decembe 2024; Recei ed in e ised o m 10 Ma ch 2025; Accep ed 12 Ma ch 2025
Ve e ina y Mic obiology 304 (2025) 110478
A ailable online 22 Ma ch 2025
0378-1135/© 2025 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (
h p://c ea i ecommons.o g/licenses/by/4.0/ ).
1. In oduc ion
The Eu opean b own ha e synd ome i us (EBHSV; Lago i us eu o-
paeus/GII.1) and abbi haemo hagic disease i us 2 (RHDV2; Lago i us
eu opaeus/GI.2), he ea e called by bo h he common name and he
geno ype name (Le Pendu e al. 2017), a e wo small, non-en eloped
RNA i uses belonging o he genus Lago i us, wi hin he amily Cal-
ici i idae, causing he Eu opean b own ha e synd ome (EBHS) and abbi
haemo hagic disease (RHD), espec i ely (Capucci e al., 2019).
EBHSV/GII.1, since i s desc ibed in Sweden in 1980 (Ga ie and
M¨
o ne , 1989; La azza and Vecchi, 1989), has been esponsible o
epidemics in he Eu opean b own ha e (Lepus eu opaeus) in se e al Eu-
opean coun ies whe e now i is conside ed endemic (Du and Ga -
ie -Wid´
en, 2012). This i al in ec ion causes se e e nec o ising hepa i is
and is a al o 40–70 % o in ec ed ha es when in oduced in o a naï e
popula ion (Cammi e al., 2003; Du and Ga ie -Wid´
en, 2012). The
i us also a ec s o he lepo id species li ing in p oximi y o Eu opean
b own ha e popula ions, including he moun ain ha e (Lepus imidus),
he I alian ha e (Lepus co sicanus) and he eas e n co on ail (Syl ilagus
lo idanus) (Ga ie -Wid´
en and M¨
o ne , 1993; La azza e al., 2015;
Domanico e al., 2023). Howe e , ansmission o Eu opean abbi s
(O yc olagus cuniculus) has nei he been obse ed in he wild no
expe imen ally demons a ed (La azza e al., 1996).
Disease occu ence is densi y-dependen , being in luenced by pop-
ula ion size, dis ibu ion, immune s a us and a ailabili y o new sus-
cep ible ec ui s (Sokos e al., 2018). In places whe e he densi y o ha es
is low, ou b eaks wi h high mo ali y may cyclically eme ge, coinciding
wi h popula ion enewal and a high numbe o suscep ible indi iduals.
By con as , in high-densi y a eas, mo ali y may be lowe due o an
inc eased likelihood o ha es de eloping p o ec i e he d immuni y (Paci
e al., 2011; Chia i e al., 2014; Sal ioli e al., 2017). The disease has no
been obse ed in le e e s younge han 40–50 days. Le e e s aged be-
ween wo and h ee mon hs may become in ec ed bu ypically do no
de elop clinical symp oms (Paci e al., 2011). The non-pa hogenic ha e
calici i us (HaCV/GII.2), a lago i us gene ically ela ed o EBHSV/-
GII.1, has also been desc ibed in he Eu opean b own ha e in Eu ope and
Aus alia. This i us causes a subclinical in ec ion in he small in es ine
wi hou inducing no iceable clinical signs o signi ican pa hological
lesions (Maha e al., 2019; D oilla d e al., 2020; Ca adini e al., 2021).
RHDV2/GI.2 eme ged in 2010 in Eu opean wild abbi popula ions
in F ance (Le Gall-Recul´
e e al., 2011; Le Gall-Recul´
e e al., 2013), and
subsequen ly sp ead apidly ac oss he globe (Rouco e al., 2019). This
no el i us is now conside ed endemic in Eu ope and has displaced he
classical s ains (RHDV/GI.1, i s ly desc ibed in China in 1984 (Liu
e al., 1984)), almos wo ldwide (Le Gall-Recul´
e e al., 2013; Cal e e
e al., 2014; Dal on e al., 2014; Lopes e al., 2014; Maha e al., 2018).
In e es ingly, a no able cha ac e is ic o RHDV2/GI.2 is i s high
ecombina ion a e — a mechanism inhe en o all calici i uses — ha
main ains he same capsid p o ein while ha ing di e en dono s o he
non-s uc u al p o eins (Lopes e al., 2015; Ab an es e al., 2020). The
capsid p o ein de e mines he i us’ abili y o in ec and induce a al
disease in se e al species wi hin he Lepus genus (Maha e al., 2021;
Asin e al., 2024), including he Eu opean b own ha e (Hall e al. 2017;
Le Gall-Recul´
e e al. 2017; Vela de e al. 2017; Ca adini e al., 2024).
Like EBHSV/GII.1 and RHDV/GI.1, RHDV2/GI.2 p ima ily a ec s
he li e o i s hos , causing ulminan hepa i is (Neimanis e al., 2018b).
Lesions caused by RHDV2/GI.2 may be indis inguishable om
EBHSV/GII.1 in ec ion in ha es i labo a o y con i ma ion and yping
a e no pe o med (Vela de e al., 2017; Neimanis e al., 2018a). Da a
ega ding he epidemiology o RHDV2/GI.2 on Lepus species is limi ed,
as well as he consequences on ha e popula ions (By ne e al., 2022). The
mos accep ed hypo hesis sugges s ha in ec ion may occu h ough
spillo e e en s om in ec ed abbi s coexis ing in he same habi a
(Hall e al., 2017; Le Gall-Recul´
e e al., 2017; Vela de e al., 2017; Maha
e al., 2018; Neimanis e al., 2018a), al hough ansmission be ween
ha es canno be excluded (Le Gall-Recul´
e e al., 2017; Neimanis e al.,
2018a).
The Eu opean b own ha e is a lagomo ph wi h a b oad dis ibu ion
ac oss Eu asia, se ing as a p ima y p ey o nume ous ca ni o es and
ap o s (Vi iano e al., 2021). Mo eo e , i is one o he mos ele an
small game species in i s his o ical dis ibu ion ange (Tsokana e al.,
2020). O e he pas ew decades, a gene alised p og essi e popula ion
decline has been obse ed ac oss Eu ope (Edwa ds e al., 2000; Hack-
lande and Schai-B aun, 2019). This dec ease could be a ibu ed o
a ious causes, including ag icul u al in ensi ica ion, p eda ion and
hun ing p essu e, clima e change o ansmissible in ec ious diseases
(Smi h e al., 2005; Du and Ga ie -Wid´
en, 2012; La azza and Cooke,
2018; Schai-B aun e al., 2019). In he no h-eas e n Ibe ian Peninsula,
whe e abbi s and ha es cohabi (Go aza e al., 2009; Ruiz-Olmo and
Camps, 2023), bo h diseases ha e been desc ibed in he Eu opean b own
ha e, wi h he i s RHDV2/GI.2 in ec ion cases obse ed in Feb ua y
2014 (Vela de e al., 2017; Almeida e al., 2024).
The biological easons behind he epidemiological di e ences be-
ween pa hogenic lago i uses in he Eu opean b own ha e emain un-
clea . Many unce ain ies s ill exis ega ding RHDV2/GI.2-hos
in e ac ions ha could in luence i al i ness in his species (By ne e al.,
2022), which likely equi es expe imen al in ec ions o add ess
(Ca adini e al., 2024). Rega dless, conside ing ha es as spillo e hos s
o RHDV2/GI.2, i is possible ha hese di e ences could be a ibu ed
o a ying suscep ibili y o each i us in ec ion, measu ed by he
se e i y and du a ion o he disease. In ac , in many in ec ious diseases,
high i ulence can esul om a hos shi (Longdon e al., 2015). In his
s udy, we le e aged he concu en p esence o bo h lago i uses in he
Eu opean b own ha e popula ion o Ca alonia (no h-eas e n Spain) o
explo e his hypo hesis. Ou main objec i es we e o compa e
EBHSV/GII.1 and RHDV2/GI.2 epidemiology h ough a wide se osu ey
in he same ha e popula ion and o in es iga e whe he pa hological
di e ences could explain he dis inc disease dynamics o hese i uses.
2. Ma e ials and me hods
2.1. Nec opsy, sampling and his opa hology
Be ween Oc obe 2015 and Ma ch 2024, a o al o 113 ca casses o
ee- anging Eu opean b own ha es oppo unis ically ound dead in he
ield by hun e s and Ru al Agen s ( o es y ange s o he Ca alan go -
e nmen ) we e anspo ed o he Ve e ina y Facul y o he Au onomous
Uni e si y o Ba celona (UAB), Spain. This was pa o he passi e su -
eillance p og am o game species in Ca alonia. Ha es we e con i med
as Eu opean b own ha es based on pheno ypic mo phome ic cha ac-
e is ics (Palacios, 1989) and sexed (Female=64; Male=48; No
de e mined=1), weighed and aged (Adul s=77; Ju eniles=36) by using
adium-ulna ossi ica ion c i e ion (B oekhuizen and Maaskamp, 1981).
A sys ema ic nec opsy was pe o med o each animal. Li e samples
(n =113) we e e ie ed and p ese ed a −20 ºC, and blood om he
hea o ho acic luid (n =110) was collec ed in o s e ile ubes. When
he ca cass decomposi ion s a us allowed, samples o b ain (n =40),
lung (n =43), achea (n =45), hea (n =43), li e (n =44), spleen
(n =40) and kidney (n =40) we e collec ed and ixed in 4 %
neu al-bu e ed o malin. Subsequen ly, issue samples we e immed
and embedded in pa a in, and 3–4
μ
m hick sec ions we e s ained wi h
Maye ’s haema oxylin and eosin o mic oscopic e alua ion.
Addi ionally, blood samples collec ed om 89 appa en ly heal hy
Eu opean b own ha es sho by hun e s du ing egula game ac i i ies
be ween Decembe 2015 and Ma ch 2023 we e also included in he
s udy. These animals we e examined in he ield and iden i ied as Eu-
opean b own ha es based on he geog aphical dis ibu ion a ea whe e
hey we e ha es ed (Go aza e al. 2009). They we e sexed
(Female=36; Male=35; No de e mined=18) and, when possible, clas-
si ied in o wo age ca ego ies (Adul s=49; Ju eniles=9; No
de e mined=31) using mo phological and sexual ma u i y cha ac e is-
ics. Howe e , since hun e s did he classi ica ion, de ails — pa icula ly
J. Es uch e al.
Ve e ina y Mic obiology 304 (2025) 110478
2
abou age — we e no always eco ded. Blood samples collec ed om
hun ed and ound dead indi iduals we e cen i uged a 3500 g o
15 min o sepa a e he se um, which was hen s o ed a −20 ºC o u u e
analysis.
2.2. Se ological and i ological su eillance o Lago i us de ec ion
Se um samples ozen a −20 ºC (n =199) we e sen o he WOAH
Re e ence labo a o y o RHD a Is i u o Zoop o ila ico Spe imen ale
della Lomba dia e dell’Emilia Romagna (IZSLER) (B escia, I aly) whe e
hey we e es ed in wo cELISAs o de ec speci ic an ibodies o EBHSV/
GII.1 and RHDV2/GI.2. The wo me hods a e iden ical wi h he excep-
ions o he speci ic immunological eagen s owa ds EBHSV/GII.1 and
RHDV2/GI.2 (Wo ld O ganisa ion o Animal Heal h, 2024). B ie ly, a
ha e an i-EBHSV/GII.1 o a abbi an i-RHDV2/GI.2 hype immune
se um was adso bed o he ELISA pla es (Nunc Maxiso b) in s anda d
ca bona e bu e . Then, each se um es ed a ou dilu ions s a ing om
1/10, 1/40, 1/160 and 1/640, was incuba ed wi h he speci ic i us
used a a dilu ion gi ing 1.0–1.2 OD492. Finally, a speci ic
an i-EBHSV/GII.1 o an i-RHDV2/GI.2 HRP-conjuga ed MAb
semi-quan i ied he i us bound o he solid phase. A se um sample was
conside ed nega i e i i s OD alue a he 1/10 dilu ion was highe han
85 % o he OD alue a he same dilu ion o he nega i e con ol se um.
As samples we e coming om ca casses, se um was conside ed doub ul
(inconclusi e esul ) i i s OD alue a he 1/10 dilu ion was equal o o
highe han 40 % o he OD alue a he same dilu ion o he nega i e
con ol se um. A se um sample was conside ed posi i e i i s OD alue a
he 1/10 dilu ion was lowe han 40 % o he OD alue a he same
dilu ion o he nega i e con ol se um. The i e o a posi i e se um
sample co esponded o he dilu ion causing a 40–60 % educ ion o he
OD alue o he nega i e con ol se um. Since he cELISA se ological es
also wo ks as a i ological es , he i us (an igen) concen a ion used is
he limi ing ac o in he eac ion o achie e he highes possible
analy ical sensi i i y. Consequen ly, when a blood o se um sample om
an animal wi h acu e RHD o EBHS is analysed using cELISA, he la ge
amoun o i us in he sample adds o he an igen in he eac ion, o en
causing he OD o exceed 2.0. The e o e, all blood o se um samples ha
showed an OD alue g ea e han 1.4 a he ini ial 1/10 dilu ion (n =52)
in cELISA we e subsequen ly subjec ed o a i ological sandwich ELISA
o con i m o ule ou he p esence o he i us (Wo ld O ganisa ion o
Animal Heal h, 2024).
2.3. Lago i us molecula con i ma ion
Li e samples om animals showing lesions consis en wi h a Lago-
i us in ec ion o ound dead du ing he EBHSV/GII.1 ou b eak om
Decembe 2020 o Sep embe 2021 (n =58) (Almeida e al., 2024) we e
sen o he Resea ch Cen e in Biodi e si y and Gene ic Resou ces
(CIBIO-InBIO) (Po o, Po ugal) o con i m Lago i us in ec ion by mo-
lecula analyses. A po ion o he li e (app oxima ely 30 mg) was
homogenised o each sample using a o o -s a o homogenise (Mixe
Mill MM400, Re sch) a 30 Hz o 7 min. To al RNA was ex ac ed wi h
he GeneJET RNA Pu i ica ion ki (The mo Scien i ic). Vi al cDNA was
hen syn hesised using he NZY i s -s and cDNA syn hesis ki (Nzy ech)
acco ding o he manu ac u e ’s ins uc ions.
EBHSV/GII.1 and RHDV2/GI.2 sc eening was pe o med wi h wo
PCRs o each genog oup, as a u he con i ma ion o he p esence o
lago i uses. The PCRs we e ca ied ou using he Phusion Flash High-
Fideli y PCR Mas e Mix (The mo Scien i ic), wo pmol o each
p ime , one
μ
L o cDNA and ul a-pu e wa e o a inal PCR olume o
10
μ
L. Cycling condi ions consis ed o 98ºC o h ee min, ollowed by 40
cycles o 30 sec a 98ºC, 30 sec a he annealing empe a u e and
ex ension a 72 ºC (ex ension imes indica ed below). A inal ex ension o
5 min a 72 ºC e mina ed he eac ion. Fo EBHSV/GII.1, he p ime s
used we e 5
′
-ATGGAGGGTAAGCCWCGGGCTGA-3
′
(nucleo ide posi-
ions 5275–5297) and 5
′
-GACATAGGAATATCCAGTGGTGGC-3
′
(nucleo ide posi ions 6979–7002) wi h an annealing empe a u e o
54ºC and 1 min. 30 sec. o ex ension and p ime s 5
′
-GACA-
GACCTCATTGACGTG-3
′
(nucleo ide posi ions 6903–6921) and 5
′
-
CAAAYCGCTAGGCGTTACTC-3
′
(nucleo ide posi ions 7330–7349), wi h
annealing empe a u e o 54ºC and 30 sec. o ex ension. Fo RHDV2/
GI.2, he p ime s used we e 5
′
-GTGAAAGTTATGGCGGCTATGTCG-3
′
(nucleo ide posi ions 1–24) and 5
′
-GCCACATTTGTCACATGTCTCCAG-3
′
(nucleo ide posi ions 178–201), wi h annealing empe a u e o 52ºC and
20 s o ex ension, and p ime s 5
′
-CAGCGGGCACTGCTACCACAGCATC-3
′
(nucleo ide posi ions 5342–5366) and 5
′
-CCAGCCCAACCAGCYTACAT-
3
′
(nucleo ide posi ions 5629–5648), wi h annealing empe a u e o
55 ºC and 15 s o ex ension. A e pu i ica ion, posi i e esul s we e
con i med by Sange sequencing on an au oma ic sequence ABI PRISM
3500xL Gene ic Analyze (PE Applied Biosys ems).
2.4. Da a analyses
Di e ences be ween EBHS and RHD mac oscopic and mic oscopic
lesions we e es ed using a mul iple chi-squa ed es o independence.
We p epa ed a con ingency able o display he equency coun s and
compu ed he chi-squa ed s a is ic. Signi icance was ini ially se a
α
=0.05 bu la e co ec ed using a Bon e oni co ec ion o con ol ype 1
e o a e. Addi i e analysis was conduc ed using he package “mgc ”
1.9–1 e sion (Wood, 2010) o he R s a is ical so wa e 4.4.2 e sion (R
Co e Team, 2023).
3. Resul s
A o al o 202 Eu opean b own ha es we e analysed o his s udy.
Among hem, 52 indi iduals we e diagnosed wi h a lago i us in ec ion
(EBHSV/GII.1, n =28; RHDV2/GI.2, n =24, wi h no co-in ec ions
de ec ed). Speci ically, 50 ou o 58 ha es es ed posi i e h ough mo-
lecula PCR analyses, wi h 42 yielding posi i e esul s in he i ological
sandwich ELISA pe o med on se um samples. Addi ionally, wo se a
samples om hun ed ha es, o which issue samples we e no collec ed,
es ed posi i e o RHDV2/GI.2 using he i ological sandwich ELISA
(Supplemen a y able S1).
Be ween 2015 and 2024, RHDV2/GI.2 cases in Eu opean b own
ha es we e egis e ed annually, excep o 2023, wi h a cumula i e o al
o 24 cases. In con as , EBHSV/GII.1 was ini ially documen ed as a
single case in 2016 and la e as an epidemic ou b eak be ween 2020 and
2021 (n =27) (Fig. 1). Seasonal dis ibu ion was simila o bo h dis-
eases, wi h mos cases occu ing du ing he cold seasons, om Oc obe
o Ma ch (n =17 o EBHSV/GII.1; n =21 o RHDV2/GI.2), whe eas in
sp ing and summe , Lago i us occu ence was lowe (EBHSV/GII.1,
n =11; RHDV2/GI.2, n =13). Rega ding geog aphic dis ibu ion,
EBHSV/GII.1 cases we e homogenously obse ed in he no he n hal o
he egion, whe eas RHDV2/GI.2 cases we e ound in cen al, wes e n
and no h-eas e n Ca alonia (Fig. 1).
Da a om he se ological su ey on Eu opean b own ha es a e
summa ised in Fig. 2. Doub ul and nega i e esul s we e g ouped, as he
quan i y and quali y o he se a ob ained om blood samples collec ed
in he ield o ob ained as su oga es om ca casses (hea clo / ho acic
luid) a e o en subop imal, leading o an inc eased p opo ion o un-
ce ain esul s. Addi ionally, con i med lago i us in ec ion cases we e
excluded om da a analysis and se op e alence calcula ions. EBHSV/
GII.1 se op e alence emained ela i ely s able, wi h peaks in 2015
(n =25, 84 %) and 2021 (n =12, 66.7 %) (Fig. 2, le panel). The lowes
se op e alences we e eco ded in 2019 (n =9, 0 %), and 2020 (n =12,
33.3 %) (Fig. 2, le panel). The esul s showed sligh a ia ions when
examining EBHSV/GII.1 an ibody i es. Al hough i es we e gene ally
low, anging be ween 1/10 (26 %) and 1/20 (31 %) (Fig. 2, igh panel),
a i s peak was obse ed in 2016–2017 (Fig. 2, le panel). Following a
pe iod o decline, i es peaked again in 2020–2021, jus a e he la ge
obse ed ou b eak. Du ing his yea , mos se a showed i es be ween 1/
40 and 1/80, wi h occasional cases eaching highe le els o 1/160, 1/
J. Es uch e al.
Ve e ina y Mic obiology 304 (2025) 110478
3
320 o 1/640 (Fig. 2, igh panel).
Conce ning RHDV2/GI.2, se op e alence and i e dis ibu ion we e
like EBHSV/GII.1 se ology bu wi h mo e nega i e/doub ul cases
(Fig. 2, igh panel). Only one sample (1 %) showed i es >1/80. To
be e unde s and he se ological da a and in e he possible p esence o
RHDV2/GI.2 an ibodies while disca ding c oss- eac i i y, we used he
app oach p oposed by Vela de e al. (2017) and also ollowed in o he
s udies (S i e e al., 2020; Faehnd ich e al., 2023), calcula ing he a io
(R ) be ween he EBHSV/GII.1 and RHDV2/GI.2 i es. Mos o he
se um samples had i es consis en ly highe o he espec i e speci ic
i al an igen (R >2), excep o 18 cases, in which he a io was 1
(n =16) o 0.5 (n =2). Thus, none o he es ed se um samples could be
Fig. 1. Geog aphical (le ) and empo al ( igh ) dis ibu ion o he 52 Lago i us-posi i e cases, caused by ei he Eu opean b own ha e synd ome i us (EBHSV/GII.1;
in blue) o abbi haemo hagic disease i us 2 (RHDV2/ GI.2; in ed). Yellow ba s and do s indica e o he ha es ound dead wi h a diagnosis di e en om Lago i us
disease o appa en ly heal hy hun ed indi iduals wi h no g oss lesions obse ed.
Fig. 2. Se op e alence (le Y axis) and mean an ibody i e ( igh Y axis) o Eu opean b own ha e synd ome i us (EBHSV/GII.1; le ) and an ibody i es e-
quencies (%) agains bo h lago i uses, EBHSV/GII.1 and abbi haemo hagic disease i us (RHDV2/GI.2), in he s udy a ea be ween 2015 and 2024 ( igh ).
Se oposi i e indi iduals include hose wi h an an ibody i e o 1/20 o highe .
J. Es uch e al.
Ve e ina y Mic obiology 304 (2025) 110478
4
de ini i ely classi ied as posi i e o RHDV2/GI.2 an ibodies, excep in
hose cases wi h R =1 o 0.5. In such ins ances, he esul s a e incon-
clusi e; hus, in ec ion canno be uled ou .
O he 52 Lago i us-in ec ed cases, 49 we e sui able o mac oscopic
and mic oscopic pos -mo em e alua ion. Lesions consis en wi h
Lago i us synd ome we e obse ed in 47 Eu opean b own ha es p e i-
ously con i med as molecula ly posi i e (EBHSV/GII.1, n =25; RHDV2/
GI.2, n =22). In he emaining wo EBHSV/GII.1-in ec ed indi iduals
wi hou consis en lesions o Lago i us in ec ion, he cause o dea h was
auma. The mac oscopic and mic oscopic pa hological ea u es o bo h
diseases, EBHS and RHD, a e de ailed in Tables 1 and 2 and Figs. 3 and 4.
In mos cases, he ana omopa hological al e a ions we e gene ally
simila o bo h diseases. Ha es ypically exhibi ed pale li e s wi h a
e icula pa e n, indica i e o ex ensi e zonal hepa ocellula loss.
Mic oscopically, his was cha ac e ised by pe ipo al o massi e (i.e., in
he en i e lobules) coagula i e nec osis/apop osis, occasional sinusoidal
haemo hages, po al mononuclea in lamma o y in il a e, small
mul i ocal a eas o ly ic nec osis, ac i a ed Kup e cells, and spo adic
mi ochond ial mine alisa ion o he hepa ocy es. Splenomegaly, asso-
cia ed wi h conges ion and ib inoid deposi ion in he ed pulp, espi-
a o y conges ion and oedema, and enal ubula inju y we e o he
pa hologic indings obse ed. In e es ingly, in bo h diseases, wo ha es
showed mic o h ombi in he glome ula capilla ies. Despi e hese sim-
ila i ies, he s a is ical analysis e ealed some dis inc ions upon in-dep h
compa ison (Tables 1 and 2).
On ex e nal examina ion, EBHSV/GII.1-in ec ed ha es showed
poo e body condi ion, as e idenced by diminished pe i- enal and sub-
cu aneous a s o age, han hose in ec ed wi h RHDV2/GI.2 ( espec-
i ely 10/28 and 1/20; p =0.03, bu p =0.05 a e Bon e oni
co ec ion). Nasal epis axis and conjunc i al and espi a o y conges ion
we e mo e p onounced in RHDV2/GI.2 cases, bu only he o me was
s a is ically signi ican (20/20 and 14/28 espec i ely; p=0.01).
Con e sely, subcu aneous jaundice, al hough no consis en ly p esen ,
was mo e equen ly obse ed in EBHSV/GII.1-in ec ed indi iduals han
in hose wi h RHDV2/GI.2 (11/28 and 1/20 espec i ely; p=0.02).
His opa hologically, he hepa ocellula nec osis pa e n was always
massi e in RHDV2/GI.2 cases (19/19) wi h absen o minimal mono-
nuclea in lamma o y eac ion in po al a eas (RHDV2/GI.2 =14/14;
EBHSV/GII:1 =9/15; p=0.02). On he o he hand, in some EBHSV/
GII.1-in ec ed ha es, he dis ibu ion o he hepa ic cell damage was
mo e es ic ed o pe ipo al o midzonal a eas (EBHSV/GII.1 =7/22;
RHDV2/GI.2 =0/14; p=0.01), wi h a mo e ma ked in lamma o y e-
ac ion composed o mononuclea and some polymo phonuclea cells
(EBHSV/GII.1 =6/15; RHDV2/GI.2 =0/14; p=0.03), and an ex ensi e
deg ee o lipid- ype hepa ocy ic acuolisa ion (EBHSV/GII.1 =11/22;
RHDV2/GI.1 =0/15, p=0.01). In kidneys, mul i ocal degene a ion o
he enal ubula epi helium, equen ly associa ed wi h haemoglobin
cas s (haemoglobinu ia), was also mo e egula ly and signi ican ly
ound in EBHSV2/GII.1 in ec ion cases a he han RHDV2/GI.2 (15/18
and 6/14 espec i ely; p=0.04, bu p=0.05 a e Bon e oni
co ec ion).
4. Discussion
This s udy p o ides new insigh s in o he epidemiology and pa hol-
ogy o lago i uses in he Eu opean b own ha e popula ion o he Ibe ian
Peninsula, enhancing ou unde s anding o he associa ed i al diseases.
Fu he mo e, he epidemiology o RHDV2/GI.2 in he Eu opean b own
ha e was in es iga ed using an in eg a i e app oach ha combined
molecula diagnosis, se ology and pa hology.
The empo al dis ibu ion o he posi i e cases e ealed wo di e en
epidemiological scena ios: a cons an incidence o RHDV2/GI.2 and a
pe iodic ou b eak-like pa e n o EBHSV/GII.1. In ac , while EBHSV/
GII.1 se oposi i i y dynamics o he popula ions in he s udy a ea sug-
ges a ela i ely s able disease ci cula ion, luc ua ion in an ibody i es
aligns wi h he empo al dis ibu ion o cases, p o iding a clea e un-
de s anding o wha occu s in he ield. EBHSV/GII.1 i es we e
gene ally low (<1/40). S ill, wo peaks o highe i es, p eceded by low
i es in p io yea s, we e egis e ed in 2016–2017 and, especially in
2020–2021, indica ing ecen in ec ion (Scicluna e al., 1994; D ews
e al., 2011). These indings sugges ha EBHSV/GII.1 was likely p esen
and ci cula ing silen ly in he ha e popula ion h oughou he obse ed
pe iod, mani es ing a cyclical ou b eak pa e n as de ec ed h ough
passi e heal h moni o ing. This epidemiological pa e n is di ec ly
ela ed o ha e popula ion densi y (Paci e al. 2011; Chia i e al. 2014;
Sal ioli e al. 2017), pa icula ly in a eas wi h low mean densi ies such
as Ca alonia, whe e alues ange om 0.5 o 3 ha es/km
2
(Ruiz-Olmo
and Camps, 2023).
In con as , RHDV2/GI.2 in ec ion in he Eu opean b own ha e may
ollow epidemiological dynamics ha a e dis inc and po en ially
opposi e o EBHSV/GII.1. The obse ed RHDV2/GI.2 an ibody i es a e
likely a ibu ed o c oss- eac i i y (EBHSV/GII.1 p e iously in ec ed
ha es) o , in case o low i es, o non-speci ic ac o s such as sample
quali y o , pe haps, in ec ions wi h non-pa hogenic calici i uses, as i
may occu wi h RHDV/GI.1 (McPhee e al., 2009; Vela de e al., 2017).
Based on hese indings, RHDV2/GI.2 se oposi i i y in he ha e popu-
la ion appea s o be ei he minimal o absen . This sugges s ha
RHDV2/GI.2 does no ci cula e endemically in ha es and ha ongoing
in ec ions likely esul om spillo e e en s om he abbi popula ion,
as p e iously p oposed (Hall e al., 2017; Le Gall-Recul´
e e al., 2017;
Vela de e al., 2017), causing a high case a ali y a e wi h ew se o-
posi i e su i o s. Al hough o mal spa ial analyses ha e no been
conduc ed, he geog aphical dis ibu ion o lagomo ph species in Ca a-
lonia sugges s a s ong co ela ion be ween RHDV2/GI.2 cases in ha es
Table 1
Summa y o he g oss pa hology lesions in Eu opean b own ha es in ec ed ei he wi h Eu opean b own ha e synd ome i us (EBHSV/GII.1), o abbi haemo hagic
disease i us 2 (RHDV2/GI.2). The numbe o cases assessed o each ca ego y a ies due o issue a ailabili y and deg ee o pos -mo em a e ac .
O gan/Tissue G oss Pa hology n/To al
χ
2
p- alue Bon e oni adjus ed p- alue
GII.1 GI.2
Body condi ion Poo (absence o a )
Fai
Good o e y good
10/28
13/28
5/28
1/20
8/20
11/20
4.61
-
5.67
0.03*
-
0.02*
0.05
-
0.02
Ex e nal
Nose Nasal Epis axis 14/28 20/20 11.80 0.0006*0.01
Eye conjunc i a Conges ion/haemo hage 12/28 14/20 2.45 0.11 -
In e nal
Subcu is Ic e us 11/28 1/20 5.60 0.02*0.02
T achea Mucosal conges ion 21/28 19/20 2.07 0.15 -
Lung Conges ion/haemo hage 12/28 19/20 0.31 0.57 -
Li e Pale
Re icula Pa e n
22/28
19/28
20/20
18/20
3.13
2.11
0.08
0.15
-
Spleen Enla ged 22/28 13/20 0.51 0.48 -
*
S a is ically signi ican
J. Es uch e al.
Ve e ina y Mic obiology 304 (2025) 110478
5
and ag icul u al a eas in wes e n, cen al, and eas e n Ca alonia, whe e
abbi popula ions a e abundan (Fig. 1) (Ruiz-Olmo and Camps, 2023).
This sympa ic con ex be ween species likely inc eases he p obabili y
o in ec ion o ha es in hese egions.
His o ically, RHDV/GI and EBHSV/GII genog oups we e ega ded as
“ abbi ” and “ha e” i uses, espec i ely (G een e al., 2000). The
eme gence o RHDV2/GI.2 (Le Gall-Recul´
e e al., 2011) ollowed by
spillo e e en s leading o in ec ion and mo ali y in a ious ha e spe-
cies, including he Eu opean b own ha e (Hall e al., 2017; Le
Gall-Recul´
e e al., 2017; Vela de e al., 2017), has blu ed he pa ho-
logical dis inc ion be ween he wo diseases, as bo h lago i uses can
now in ec ha es. Ou esul s sugges ha ha es in ec ed wi h
RHDV2/GI.2 may consis en ly expe ience a mo e acu e clinical o m o
Lago i us synd ome, leading o mo e apid dea h. In con as , ha es
in ec ed wi h EBHSV/GII.1 usually exhibi ed a mo e p olonged disease
cou se. This is i s ly obse ed in he body condi ion o ha es. E en i
nu i ional s a us may depend on ac o s such as sex, age, o b eeding
pe iod (Bus os e al., 1998), EBHSV/GII.1-in ec ed ha es we e consis-
en ly in poo e body condi ion compa ed o hose in ec ed wi h
RHDV2/GI.2.
The hallma k lesion o Lago i us diseases, pe ipo al o massi e
coagula i e hepa ocellula nec osis (Du and Ga ie -Wid´
en, 2012),
p esen di e en ly in he wo in ec ions. In RHDV2/GI.2, nec osis ends
o be massi e, whe eas in EBHSV/GII.1, hepa ocellula damage may
occasionally be con ined o pe ipo al a eas. In he li e a u e, he
massi e nec osis pa e n in EBHS has o en been associa ed wi h he
acu e o m o he disease, ypically a ec ing indi iduals wi h weake
immune sys em (Zanni e al., 1993; Ga ie -Wid´
en, 1994; D ews e al.,
2011). Howe e , mos EBHSV/GII.1-in ec ed ha es also exhibi ed
changes indica i e o longe disease p og ession, such as acuola he-
pa ocy e degene a ion, a sligh ly heigh ened in lamma o y esponse,
and subcu aneous jaundice. These indings sugges a less agg essi e
clinical o m and a mo e p olonged disease cou se, despi e he ul i-
ma ely le hal ou come (Ga ie -Wid´
en, 1994; Sy j¨
al¨
a e al., 2005; F ¨
olich
and La azza, 2008).
Renal degene a ion was signi ican ly mo e p onounced in EBHSV/
GII.1 han in RHDV2/GI.2 in ec ions. Despi e ubula degene a ion
being commonly epo ed, li le is known abou he speci ic enal pa-
hology associa ed wi h lago i uses in ec ion in ha es (Poli e al., 1991;
F ¨
olich e al., 2001; Vela de e al., 2017). In abbi s in ec ed wi h
RHDV/GI.1, enal degene a ion appea s o be linked o he p og ession
o li e ailu e (Alonso e al., 1998; Chen e al., 2008), a co ela ion also
well-es ablished in human medicine (Moo e e al., 2013). Chen e al.
(2008) and Plassia e al. (1992) obse ed ini ial enal ubula lesions in
abbi s expe imen ally in ec ed wi h RHDV only a e 30- and 54-hou s
pos -in ec ion, espec i ely, while o he s succumbed o he disease
be o e hese changes could de elop. Ex apola ing cau iously o ha es,
hese indings ag ee wi h longe clinical p og ession in EBHSV/GII.1
in ec ion a he han in RHDV2/GI.2 in ec ion.
The pa hological p esen a ion o hese in ec ions may pa ially
explain he s iking di e ences in he epidemiology o he wo lago i-
uses. Acu e and subacu e EBHSV/GII.1 cases we e iden i ied h ough
passi e su eillance, along wi h e idence o indi iduals su i ing he
in ec ion, as indica ed by an ibody de ec ion in hei se a. In con as , no
e idence was ound o ha es su i ing RHDV2/GI.2 in ec ion. This may
also esul om he apid clinical p og ession o he disease, which
p e en s animals om moun ing an e ec i e immune esponse.
No ably, li le is known abou he immune esponse o ha es o RHDV2/
GI.2 in ec ion. By compa ison, in abbi s in ec ed wi h RHDV/GI.1 ia
o al in ec ion ansmission, IgM and IgA esponses begin app oxima ely
72 hou s pos -in ec ion, while IgG p oduc ion s a s a ound 5–7 days
pos -in ec ion (Cooke e al., 2000; Mülle e al., 2021). A simila imeline
has been obse ed in abbi s accina ed o passi ely immunised agains
RHDV2/GI.2 (Mülle e al., 2019; Hall e al., 2021). The e o e, i may be
hypo hesised ha RHDV2/GI.2 in ec ion in he Eu opean b own ha e is
spo adic, as hey a e no well-adap ed hos s o his i us and ypically
ha e a limi ed chance o su i e once in ec ed. Howe e , he ac o s
p edisposing some ha es o in ec ion and disease while o he s emain
unin ec ed a e ye o be disclosed. These may include a iables such as
in ec ion dose and in ec ion p essu e (e.g., exposu e o high o epea ed
doses o i ions). In he Uni ed S a es, RHDV2/GI.2 ou b eaks a e
a ec ing la ge numbe s o indi iduals om Lepus species, as well as om
Syl ilagus and o he lepo ids (Ringenbe g e al., 2024). The e a e s ill
many gaps in knowledge ega ding ansmission dynamics, bu in-
e ac ions wi h domes ic abbi s may con inue o be he po en ial sou ce
o in ec ion (Asin e al., 2022). Va ia ions in suscep ibili y among Lepus
species may also con ibu e o la ge-scale ou b eaks, as obse ed in he
Sa dinian Cape ha e (Lepus capensis medi e aneus) (Puggioni e al.,
2013). Unde s anding he mechanisms unde lying suscep ibili y, ans-
mission and in luencing ac o s, po en ially linked o he i us’s gene ic
s uc u e (Ca adini e al., 2024), will equi e u he in es iga ion.
Sampling was pa ially ca ied ou h ough passi e su eillance,
which is o en subjec ed o bias such as unde epo ing, delayed de ec-
ion, o limi ed geog aphic co e age (Ryse -Degio gis, 2013; Tomaselli,
Table 2
Summa y o he his ological lesions in Eu opean b own ha es in ec ed ei he wi h Eu opean b own ha e synd ome i us (EBHSV/GII.1), o abbi haemo hagic disease
i us 2 (RHDV2/GI.2). The numbe o cases assessed o each ca ego y a ies due o issue a ailabili y and deg ee o pos -mo em a e ac .
O gan/Tissue His opa hology n/To al
χ
2
p- alue Bon e oni adjus ed p- alue
GII.1 GI.2
T achea Mucosal conges ion 20/25 20/20 2.70 0.10 -
Lung Oedema/Conges ion
Haemo hages
22/24
6/15
19/19
2/6
0.31
1.60e−31
0.56
1
-
-
Li e Nec osis pa e n
Massi e
Pe ipo al
Ly ic nec osis a eas
In lamma ion
15/22
7/22
22/22
19/19
0/19
15/15
3.30
5.22
-
0.07
0.02*
-
-
0.01
-
Minimum
Mild-mode a e
Vacuola degene a ion
Mine alisa ion
9/15
6/15
11/22
9/23
14/14
0/14
0/15
14/21
4.83
4.83
8.41
2.32
0.03*
0.03*
0.004*
0.12
0.02
0.02
0.01
-
Spleen Lymphoid deple ion
Lymphocy olysis
Fib inoid deposi s
19/20
12/19
12/20
8/9
2/8
5/9
4.58e−31
1.93
1.44e−31
1
0.16
1
-
-
-
Kidney Tubula degene a ion
Haemoglobin cas s
DIC
15/18
16/23
2/21
6/14
6/17
2/15
4.07
3.36
4.58e−31
0.04*
0.07
1
0.05
-
-
DIC, dissemina ed
*
S a is ically signi ican
J. Es uch e al.
Ve e ina y Mic obiology 304 (2025) 110478
6
2022). Ac i e su eillance was suppo ed by olun ee hun e s; how-
e e , hei hun ing ac i i ies we e nei he e enly dis ibu ed ac oss he
e i o y no consis en in in ensi y each yea , leading o a ia ion in
sample size. Addi ionally, no all he indi iduals we e es ed using
molecula analyses, which ep esen he mos sensi i e and speci ic
labo a o y echnique (Ab an es and Lopes, 2021). Consequen ly, some
in ec ed ha es, hose nega i e o an ibodies o in he ea ly s ages o
in ec ion wi hou lesions, may ha e been misclassi ied as unin ec ed. Fo
hun ed indi iduals, collec ing issue samples was no easible, so
misdiagnosis could also be possible. None heless, any alse nega i es,
likely limi ed in numbe , would no signi ican ly al e ou s udy’s con-
clusions. Despi e hese limi a ions, 44 ou o 50 se ological samples om
Lago i us-in ec ed indi iduals con i med by molecula analyses also
es ed posi i e using i ological ELISA. This likely e lec s he high i al
load du ing he i emic phase, indica ing ha his se ological echnique
p o ides eliable esul s. Hence, hese me hods could se e as a aluable
al e na i e o complemen a y diagnos ic ool o epidemiological ield
s udies.
5. Conclusions
The pa hological indings o his s udy e eal signi ican di e ences
in he clinical cou se o Lago i us in ec ions in ha es. EBHSV/GII.1 ap-
pea s o be well-adap ed o he Eu opean b own ha e, allowing o mo e
p olonged su i al and po en ially con ibu ing o i s pe sis ence wi hin
ha e popula ions. In con as , he apid and se e e p og ession o
RHDV2/GI.2 in ec ions and low se oposi i i y a es suppo he hy-
po hesis ha ha es ac as spillo e hos s o RHDV2/GI.2 om abbi s.
Despi e a decade o po en ial co-e olu ion be ween ha es and RHDV2/
GI.2, he e is no e idence o adap a ion in ha es. They con inue o
exhibi acu e disease, limi ed immune esponse, wi h p obable ine i-
cien ansmission be ween ha es, making i unlikely ha he Eu opean
b own ha e cu en ly ac s as a ese oi o RHDV2/GI.2. Howe e , in
a eas whe e ha es and abbi s a e sympa ic, ha es could s ill play a ole
in e- ansmi ing he i us o abbi s.
None heless, ongoing su eillance is c ucial, as mu a ions o
ecombina ion e en s could o e come exis ing hos -speci ic ba ie s,
inc easing he isk o an epizoo ic. These dis inc ions in pa hology and
epidemiology u he highligh he impo ance o a ge ed su eillance
Fig. 3. Eu opean b own ha e (Lepus eu opaeus), a ious animals. A) Bila e al nasal epis axis in an indi idual in ec ed wi h abbi haemo hagic disease i us 2
(RHDV2/GI.2). B) Ic e us o he subcu aneous issue in a ha e wi h Eu opean b own ha e synd ome (EBHS) caused by EBHSV/GII.1. C) Li e pallo wi h di use
e icula pa e n and mul i ocal lung haemo hages, ea u es obse ed in an EBHS-a ec ed ha e bu also isible in cases in ec ed wi h RHDV2/GI.2. D) Ma ked
splenomegaly in a ha e a ec ed by EBHS. E) Mul i ocal haemo hages in he lungs in a ha e in ec ed wi h RHDV2/GI.2. F) Good body condi ion, as e idenced by he
p esence o a a ound he kidneys, in an indi idual in ec ed wi h RHDV2/GI.2.
J. Es uch e al.
Ve e ina y Mic obiology 304 (2025) 110478
7
and managemen s a egies o mi iga e he impac o Lago i us in ec ions
in ha e popula ions. The no h-eas e n Spain cons i u es he sou he n-
mos limi o he Eu opean b own ha e, whe e in o ma ion ega ding he
popula ion s a us is limi ed. The co-ci cula ion o bo h lago i uses, oc-
casional local su ges in Eu opean abbi popula ions wi hin ha e habi-
a s, and in ensi e ag icul u al p ac ices may h ea en he iabili y o
Eu opean b own ha e popula ions a he sou he n edge o hei dis i-
bu ion in Eu ope.
Funding
This wo k has bene i ed om he inancial aid o esea ch g an s
unded by he Spanish Minis y o Science and Inno a ion [Ibe -
LagoHeal h p ojec , PID2023–151954NB-I00, LagoHeal h p ojec ,
REF: PID2019–111080RB-C21, and LAGMED p ojec PRIMAS2–11-
PCI2019–103698]. The passi e su eillance p og amme is suppo ed by
he Subdi ecci´
o Gene al d’Ac i i a s Cineg`
e iques i Pesca Con inen al
om he Depa men o Ag icul u e, Li es ock, Fishe ies, and Food
(DARPA) o he Go e nmen o Ca alonia. Fundaç˜
ao pa a a Ciˆ
encia e
Tecnologia (FCT, Po ugal) suppo ed he Junio Resea che g an o
Ana M. Lopes [CEECIND/01388/2017] and he LAGMED p ojec [REF:
PRIMA/0003/2018].
CRediT au ho ship con ibu ion s a emen
Lopes Ana Ma ga ida: W i ing – e iew & edi ing, Valida ion, Su-
pe ision, Me hodology, In es iga ion. Neimanis Aleksija: W i ing –
e iew & edi ing, Valida ion. Almeida Te eza: W i ing – e iew &
edi ing, Valida ion, Supe ision, Me hodology, In es iga ion. Rouco
Ca los: W i ing – e iew & edi ing, Valida ion, Supe ision, Funding
acquisi ion. La ín San iago: W i ing – e iew & edi ing, Valida ion,
P ojec adminis a ion. Vela de Rose : W i ing – e iew & edi ing,
Valida ion, Supe ision, Resou ces, Me hodology, In es iga ion,
Concep ualiza ion. Es uch Mo en e Josep: W i ing – o iginal d a ,
Resou ces, Me hodology, In es iga ion, Concep ualiza ion. Se ano
Emmanuel: W i ing – e iew & edi ing, Valida ion, Supe ision,
Me hodology, Concep ualiza ion. La azza An onio: W i ing – e iew &
edi ing, Valida ion, Supe ision, Me hodology, In es iga ion. Ca adini
Pa izia: Valida ion, Supe ision, Me hodology, In es iga ion.
Ab an es Joana: W i ing – e iew & edi ing, Valida ion, Supe ision,
Me hodology, In es iga ion, Funding acquisi ion. Capucci Lo enzo:
W i ing – e iew & edi ing, Valida ion, Supe ision, Me hodology,
In es iga ion.
Fig. 4. Eu opean b own ha e (Lepus eu opaeus), a ious animals. A) Li e . Ha e in ec ed wi h Eu opean b own ha e synd ome i us (EBHSV/GII.1). Pe ipo al o
midzonal a eas o coagula i e nec osis (�), wi h mononuclea in lamma ion and ma ked acuola degene a ion o he emnan hepa ocy es (THV, e minal hepa ic
ein; PT, po al ac ). B) Li e . Ha e in ec ed wi h abbi haemo hagic disease i us 2 (RHDV2/GI.2). Massi e coagula i e nec osis (�), sinusoidal haemo hages
and occasional mine alisa ion o he hepa ocy e’s mi ochond ia (a owheads). C) Lung. Ha e in ec ed wi h RHDV2/GI.2. Mul i ocal al eola haemo hages. D)
Kidney. Ha e in ec ed wi h EBHSV/GII.1. Di use a eas o ubula degene a ion wi h occasional mul i ocal haemoglobin cas s wi hin he lumen (a owheads). E)
Spleen. Ha e in ec ed wi h EBHSV/GII.1. Fib inoid-like ma e ial deposi ed on he ed pulp (*). F) Kidney. Ha e in ec ed wi h RHDV2/GI.2. Occasional mic o h ombi
in he lumen o glome ula capilla ies (*) and haemoglobin cas s wi hin he lumen o he ubules (a owhead).
J. Es uch e al.
Ve e ina y Mic obiology 304 (2025) 110478
8
Decla a ion o Gene a i e AI and AI-assis ed echnologies in he
w i ing p ocess
Du ing he p epa a ion o his wo k, he au ho s used OpenAI -
Cha GPT o imp o e eadabili y and language. A e using his ool, he
au ho s e iewed and edi ed he con en as needed and ook ull e-
sponsibili y o he con en o he published a icle.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgemen s
We hank he Ru al Range s o he Cos d’Agen s Ru als om he
Ca alonia go e nmen and hun e s o hei in aluable collabo a ion in
he wildli e passi e su eillance p og amme in Ca alonia and o
epo ing and b inging cases o ou acili ies a he e e ina y acul y
(SEFaS). We also hank, in pa icula , he collabo a ion o Ma ia Josep
Va gas, Mon se a Rubi´
o and Albe Sanz, echnical manage s o he
Ca alan au ho i ies linked o his su eillance p og amme. Se ology was
pe o med a he WOAH Re e ence Labo a o y o abbi haemo hagic
disease (IZSLER, I aly), using labo a o y echnical wo k om M s.
Alessand a P e idi and Sil ia B odini.
Appendix A. Suppo ing in o ma ion
Supplemen a y da a associa ed wi h his a icle can be ound in he
online e sion a doi:10.1016/j. e mic.2025.110478.
Re e ences
Ab an es, J., D oilla d, C., Lopes, A.M., Lemai e, E., Lucas, P., Blancha d, Y.,
Ma chandeau, S., Es e es, P.J., Le Gall-Recul´
e, G., 2020. Recombina ion a he
eme gence o he pa hogenic abbi haemo hagic disease i us Lago i us eu opaeus/
GI.2. Sci. Rep. 10, 1–11. h ps://doi.o g/10.1038/s41598-020-71303-4.
Ab an es, J., Lopes, A.M., 2021. A e iew on he me hods used o he de ec ion and
diagnosis o abbi hemo hagic disease i us (RHDV). Mic oo ganisms 9, 972.
h ps://doi.o g/10.3390/mic oo ganisms9050972.
Almeida, T., Lopes, A.M., Es uch, J., Rouco, C., Ca adini, P., Neimanis, A., Ga ie -
Wid´
en, D., Le Gall-Recul´
e, G., Vela de, R., Ab an es, J., 2024. A new HaCV-EBHSV
ecombinan lago i us ci cula ing in Eu opean b own ha es (Lepus eu opaeus) om
Ca alonia, Spain. Sci. Rep. 14, 2872. h ps://doi.o g/10.1038/s41598-024-53201-1.
Alonso, C., O iedo, J.M., Ma ín-Alonso, J.M., Díaz, E., Boga, J.A., Pa a, F., 1998.
P og ammed cell dea h in he pa hogenesis o abbi hemo hagic disease. A ch.
Vi ol. 143, 321–332. h ps://doi.o g/10.1007/s007050050289.
Asin, J., Cal e e, C., Uzal, F.A., C ossley, B.M., Dua e, M.D., Hende son, E.E., Abade dos
San os, F., 2024. Rabbi hemo hagic disease i us 2, 2010–2023: a e iew o global
de ec ions and a ec ed species. J. Ve . Diagn. In es ig. 36, 617–637. h ps://doi.
o g/10.1177/10406387241260281.
Asin, J., Rejmanek, D., Cli o d, D.L., Mikolon, A.B., Hende son, E.E., Nyaoke, A.C.,
Macías-Rioseco, M., S ei enbe ge , N., Beingesse , J., Woods, L.W., La azza, A.,
Capucci, L., C ossley, B., Uzal, F.A., 2022. Ea ly ci cula ion o abbi haemo hagic
disease i us ype 2 in domes ic and wild lagomo phs in sou he n Cali o nia, USA
(2020–2021). T ansbound. Eme g. Dis. 69, e394–e405. h ps://doi.o g/10.1111/
TBED.14315.
B oekhuizen, S., Maaskamp, F., 1981. Annual p oduc ion o young in Eu opean ha es
(Lepus eu opaeus) in he Ne he lands. J. Zool. 193, 499–516. h ps://doi.o g/
10.1111/j.1469-7998.1981. b01500.x.
Bus os, Jos´
e C., Bonino, N., Bus os, J.C., 1998. Kidney mass and kidney a index in he
Eu opean ha e inhabi ing no hwes e n Pa agonia. Mas ozool. Neo op. 5, 81–85.
By ne, A.W., Ma nell, F., Ba e , D., Reid, N., Hanna, R.E.B., McEl oy, M.C., Casey, M.,
2022. Rabbi Haemo hagic Disease Vi us 2 (RHDV2; GI.2) in I eland ocusing on
wild I ish ha es (Lepus imidus hibe nicus): an o e iew o he i s ou b eaks and
con ex ual e iew. Pa hogens 11, 288. h ps://doi.o g/10.3390/
PATHOGENS11030288.
Cal e e, C., Sa o, P., Cal o, A.J., Mon oy, F., Cal o, J.H., 2014. Could he new abbi
haemo hagic disease i us a ian (RHDVb) be ully eplacing classical RHD s ains
in he Ibe ian Peninsula? Wo ld Rabbi Sci. 22, 91. h ps://doi.o g/10.4995/
w s.2014.1715.
Cammi, G., Capucci, L., Be nini, F., La azza, A., 2003. Indagine sulla di usione
dell’EBHS nella popolazione di lep i p esen e nel e i o io della p o incia di
piacenza nel 1997. J. M . Ecol. Suppl. 7, 165–174.
Capucci, L., Ca adini, P., La azza, A., 2019. Rabbi hemo hagic disease i us and
Eu opean b own ha e synd ome i us (Calici i idae). In: Bam o d, D.H.,
Zucke man, M. (Eds.), Encyclopedia o Vi ology, ou h ed. Else ie L d,
Ne he lands, pp. 724–729. h ps://doi.o g/10.1016/b978-0-12-809633-8.20998-9.
Ca adini, P., Molina i, S., Me zoni, F., Visma a, A., Posau z, A., Alzaga Gil, V.,
Chia i, M., Giannini, F., Capucci, L., La azza, A., 2021. Widesp ead occu ence o he
non-pa hogenic ha e calici i us (HaCV Lago i us GII.2) in cap i e- ea ed and ee-
li ing wild ha es in Eu ope. T ansbound. Eme g. Dis. 68, 509–518. h ps://doi.o g/
10.1111/ bed.13706.
Ca adini, P., T ogu, T., Vela de, R., La azza, A., Capucci, L., 2024. Recombina ion
be ween non-s uc u al and s uc u al genes as a mechanism o selec ion in
lago i uses: he e olu iona y dead-end o an RHDV2 isola ed om Eu opean ha e.
Vi us Res 339, 199257. h ps://doi.o g/10.1016/j. i us es.2023.199257.
Chen, S.Y., Chou, C.C., Liu, C.I., Shien, J.H., 2008. Impai men o enal unc ion and
elec oly e balance in abbi hemo hagic disease. J. Ve . Med. Sci. 70, 951–958.
h ps://doi.o g/10.1292/j ms.70.951.
Chia i, M., Fe a i, N., Gia diello, D., A isani, D., Zanoni, M., Albo ali, G.L.,
Lan anchi, P., Gube i, V., Lo enzo, C., An onio, L., 2014. Tempo al dynamics o
Eu opean b own ha e synd ome in ec ion in No he n I alian b own ha es (Lepus
eu opaeus). Eu . J. Wildl. Res. 60, 891–896. h ps://doi.o g/10.1007/s10344-014-
0856-6.
Cooke, B.D., Robinson, A.J., Me chan , J.C., Na din, A., Capucci, L., 2000. Use o ELISAs
in ield s udies o abbi haemo hagic disease (RHD) in Aus alia. Epidemiol. In ec .
124, 563. h ps://doi.o g/10.1017/S0950268899003994.
Dal on, K.P., Nicieza, I., Ab an es, J., Es e es, P.J., Pa a, F., 2014. Sp ead o new a ian
RHDV in domes ic abbi s on he Ibe ian Peninsula. Ve . Mic obiol. 169, 67–73.
h ps://doi.o g/10.1016/j. e mic.2013.12.015.
Domanico, M., Ca adini, P., Na dini, R., Cecca, D., Mas and ea, G., Eleni, C.,
Galie a, V., A ili, L., Pizza elli, A., Ono a i, R., Amo uso, C., S illi, D.,
Pacchia o i, G., Me zoni, F., Cap ioli, A., Ricci, I., Ba is i, A., La azza, A.,
Scicluna, M.T., 2023. Pa hological and i ological insigh s om an ou b eak o
Eu opean b own ha e synd ome in he I alian ha e (Lepus co sicanus). F on .
Mic obiol. 14. h ps://doi.o g/10.3389/ micb.2023.1250787.
D ews, B., Szen iks, C.A., Roellig, K., Fickel, J., Sch oede , K., Du , J.P., La azza, A.,
Hildeb and , T.B., Goe i z, F., 2011. Epidemiology, con ol and managemen o an
EBHS ou b eak in cap i e ha es. Ve . Mic obiol. 154, 37–48. h ps://doi.o g/
10.1016/j. e mic.2011.06.021.
D oilla d, C., Lemai e, E., Cha el, M., Qu´
em´
ene , A., B iand, F.X., Gui on, J.S.,
Ma chandeau, S., Le Pendu, J., E e adossi, N., Le Gall-Recul´
e, G., 2020. Gene ic
di e si y and e olu ion o Ha e Calici i us (HaCV), a ecen ly iden i ied lago i us
om Lepus eu opaeus. In ec . Gene . E ol. 82, 104310. h ps://doi.o g/10.1016/j.
meegid.2020.104310.
Du , J.P., Ga ie -Wid´
en, D., 2012. Calici i us In ec ions. In: Du , J.P., Ga ie -Wid´
en, D.,
Me edi h, A. (Eds.), In ec ious Diseases o Wild Mammals and Bi ds in Eu ope, i s
ed. Wiley, UK, pp. 73–85. h ps://doi.o g/10.1002/9781118342442.ch5.
Edwa ds, P.J., Fle che , M.R., Be ny, P., 2000. Re iew o he ac o s a ec ing he decline
o he Eu opean b own ha e, Lepus eu opaeus (Pallas; 1778) and he use o wildli e
inciden da a o e alua e he signi icance o pa aqua . Ag ic. Ecosys . En i on. 79,
95–103. h ps://doi.o g/10.1016/S0167-8809(99)00153-X.
Faehnd ich, M., Klink, J.C., Rolle , M., Wohlsein, P., Raue, K., S ube, C., P enge -
Be ningho , E., Ewe s, C., Capucci, L., La azza, A., Tomaso, H., Schni zle , J.G.,
Siebe , U., 2023. S a us o in ec ious diseases in ee- anging Eu opean b own ha es
(Lepus eu opaeus) ound dead be ween 2017 and 2020 in Schleswig-Hols ein,
Ge many. Pa hogens 12, 239. h ps://doi.o g/10.3390/PATHOGENS12020239/S1.
F ¨
olich, K., Hae e , G., Baccia ini, L., Jano sky, M., Rudolph, M., Giacome i, M., 2001.
Eu opean b own ha e synd ome in ee- anging Eu opean b own and moun ain
ha es om Swi ze land. J. Wildl. Dis. 37, 803–807. h ps://doi.o g/10.7589/0090-
3558-37.4.803.
F ¨
olich, K., La azza, A., 2008. Eu opean b own ha e synd ome. In: Al es, P.C.,
Fe and, N., Hackl¨
ande , K. (Eds.), Lagomo ph Biology. Sp inge , Be lin, Heidelbe g,
pp. 253–261. h ps://doi.o g/10.1007/978-3-540-72446-9_17.
Ga ie , D., M¨
o ne , T., 1989. The Eu opean b own ha e synd ome in Sweden. In:
P oceedings o he In e na iona-Len Symposium. pp. 261–264..
Ga ie -Wid´
en, D., 1994. Mo phologic and immunohis ochemical cha ac e iza ion o he
hepa ic lesions associa ed wi h Eu opean b own ha e synd ome. Ve . Pa hol. 31,
327–334. h ps://doi.o g/10.1177/030098589403100305.
Ga ie -Wid´
en, D., M¨
o ne , T., 1993. Desc ip i e epizoo iological s udy o Eu opean
b own ha e synd ome in Sweden. J. Wildl. Dis. 29, 15–20. h ps://doi.o g/10.7589/
0090-3558-29.1.15.
Go aza , C., Milla, J., Ace edo, P., Escude o, M.A., Ma co, J., 2009. A la ge-scale su ey
o b own ha e Lepus eu opaeus and Ibe ian Ha e L. g ana ensis popula ions a he limi
o hei anges. Wildl. Biol. 13, 244–250. h ps://doi.o g/10.2981/0909-6396(2007)
13[244:ALSOBH]2.0.CO;2.
G een, K.Y., Ando, T., Balayan, M.S., Be ke, T., Cla ke, I.N., Es es, M.K., Ma son, D.O.,
Naka a, S., Neill, J.D., S udde , M.J., Thiel, H., 2000. Taxonomy o he calici i uses.
J. In ec . Dis. 181, S322–S330. h ps://doi.o g/10.1086/315591.
Hacklande , K., Schai-B aun, S., 2019. Lepus eu opaeus. The IUCN ed lis o h ea ened
species 2019: e.T41280A45187424. 8235. doi:h ps://doi.o g/10.2305/IUCN.
UK.2019- 1.RLTS.T41280A45187424.en.
Hall, R.N., King, T., O’Conno , T.W., Read, A.J., V anko ic, S., Pipe , M., S i e, T., 2021.
Passi e immunisa ion agains RHDV2 induces p o ec ion agains disease bu no
in ec ion. Vaccines 9, 1197. h ps://doi.o g/10.3390/ accines9101197.
Hall, R.N., Peacock, D.E., Ko aliski, J., Maha , J.E., Mou an , R., Pipe , M., S i e, T.,
2017. De ec ion o RHDV2 in Eu opean b own ha es (Lepus eu opaeus) in Aus alia.
Ve . Rec. 180, 121. h ps://doi.o g/10.1136/ .104034.
J. Es uch e al.
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