E alua ion o d ied blood spo s o se ological su eys o myxoma and
abbi hemo hagic disease i uses in hei wild ese oi
Joana Fe ei a-e-Sil a
a,b,c
, Saúl Jim´
enez-Ruiz
d
, Ma isa Rod igues
a,b,e
, Emídio San os
,
Sab ina Cas o-Schol en
d
, Vi o Lizana
g,h
, Alba Ma í-Ma co
g
, Te eza Almeida
a,b
,
Ana M. Lopes
a,b,i
, Joana Ab an es
a,b,c
, Juan B´
a cena
j
, Es he Blanco
j
, Ca los Rouco
k
,
Ignacio Ga cía-Bocaneg a
d,l
, Paulo C´
elio Al es
a,b,c,e
, Nuno San os
a,b,e,*
a
CIBIO, Cen o de In es igaç˜
ao em Biodi e sidade e Recu sos Gen´
e icos, InBIO Labo a ´
o io Associado, Uni e sidade do Po o, Campus de Vai ˜
ao, Vai ˜
ao 4485-661,
Po ugal
b
BIOPOLIS P og am in Genomics, Biodi e si y and Land Planning, CIBIO, Campus de Vai ˜
ao, Vai ˜
ao 4485-661, Po ugal
c
Depa amen o de Biologia, Faculdade de Ciˆ
encias, Uni e si y o Po o, R. do Campo Aleg e, s/n, Po o 4169-007, Po ugal
d
GISAZ-ENZOEM. Animal Heal h and Zoonoses Resea ch G oup, Compe i i e Resea ch Uni on Zoonoses and Eme ging Diseases, Uni e si y o C´
o doba, C´
o doba
14014, Spain
e
Es aç˜
ao Biol´
ogica de M´
e ola (EBM), CIBIO, P aça Luís de Cam˜
oes, M´
e ola 7750–329, Po ugal
ICNF - Ins i u o de Conse aç˜
ao da Na u eza e das Flo es as, I.P., A . da República, 16, Lisboa 1050-191, Po ugal
g
Se icio de An´
alisis, In es igaci´
on, Ges i´
on de Animales Sil es es (SAIGAS), Facul ad de Ve e ina ia, Uni e sidad Ca denal He e a-CEU, CEU Uni e si ies, Al a a del
Pa ia ca, Valencia 46115, Spain
h
Wildli e Ecology & Heal h G oup (WE&H), Uni e si a Au `
onoma de Ba celona (UAB), Bella e a PC08193, Spain
i
Ins i u o de Ciˆ
encias Biom´
edicas Abel Salaza (ICBAS)/Unidade Mul idisciplina de In es igaç˜
ao Biom´
edica (UMIB), Uni e si y o Po o, R. Jo ge Vi e bo Fe ei a,
228, Po o 4050-313, Po ugal
j
Cen o de In es igaci´
on en Sanidad Animal (CISA-INIA/CSIC), Ca e e a Alge e-El Casa de Talamanca, Km 8.1, Valdeolmos, Mad id 28130, Spain
k
´
A ea de Ecología, Depa amen o Biología Vege al y Ecología, ´
A ea de Ecología, Uni e sidad de Se illa, Se illa 41012, Spain
l
CIBERINFEC, ISCIII-CIBER de En e medades In ecciosas, Ins i u o de Salud Ca los III, Mad id 28029, Spain
ARTICLE INFO
Keywo ds:
Eu opean abbi
O yc olagus cuniculus
Epidemiology
Wildli e
P o ein Sa e ca ds
ABSTRACT
Myxoma (MYXV) and abbi hemo hagic disease (RHDV) i uses a e pa hogens o economic ele ance o
cunicul u e and conse a ion conce n o wild Eu opean abbi s (O yc olagus cuniculus), ecen ly classi ied as
‘Endange ed’ in i s na i e ange. La ge-scale se ological su eys, acili a ed by sample collec ion using d ied
blood spo s (DBS), allow moni o ing se op e alence in he wild ese oi bu equi e e alua ing he echnique o
he hos and pa hogen o in e es . This s udy aimed o e alua e P o ein Sa e 903 DBS o MYXV and RHDV
(geno ype GI.2) se ological su eys in Eu opean abbi s. Pai ed se um and DBS collec ed om 172 abbi s
ha es ed o ound dead in he Ibe ian Peninsula we e es ed o IgG an ibodies speci ic agains MYXV and RHDV
GI.2 using indi ec ELISA. We ound an almos pe ec ag eemen be ween se um and DBS o MYXV (Cohen’s
κ=0.914, CI
95
0.847 – 0.981) and a s ong ag eemen o RHDV GI.2 (Cohen’s κ=0.808, CI
95
=0.722 – 0.893).
The diagnos ic sensi i i y o DBS was 95.4 % (CI
95
90.3 – 97.9 %) o MYXV and 82.1 % (CI
95
73.2 – 88.5 %) o
RHDV GI.2. The diagnos ic speci ici y and posi i e p edic i e alue we e 100 % o bo h pa hogens. This s udy
suppo s DBS as a sui able sampling s a egy o se ological su eys o an ibodies speci ic o MYXV and RHDV
GI.2 in Eu opean abbi s, which gene ally ag ees wi h esul s om o he hos s and pa hogens whe e his ech-
nique was e alua ed.
Abb e ia ions: MYXV, myxoma i us; RHDV GI.2, abbi hemo hagic disease i us GI.2; DBS, d ied blood spo s; PS, P o ein Sa e ; IELISA, indi ec enzyme-linked
immunoso ben assay; NAR, no malized abso bance a io.
* Co esponding au ho a : CIBIO, Cen o de In es igaç˜
ao em Biodi e sidade e Recu sos Gen´
e icos, InBIO Labo a ´
o io Associado, Uni e sidade do Po o, Campus de
Vai ˜
ao, Vai ˜
ao 4485-661, Po ugal
E-mail add ess: [email p o ec ed] (N. San os).
Con en s lis s a ailable a ScienceDi ec
P e en i e Ve e ina y Medicine
jou nal homepage: www.else ie .com/loca e/p e e med
h ps://doi.o g/10.1016/j.p e e med.2024.106369
Recei ed 24 Janua y 2024; Recei ed in e ised o m 19 Augus 2024; Accep ed 3 No embe 2024
P e en i e Ve e ina y Medicine 234 (2025) 106369
A ailable online 6 No embe 2024
0167-5877/© 2024 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
La ge-scale se ological su eys in wildli e a e challenging o pe o m
due o he o en-sec e i e na u e o wild animals, emo e loca ions, and
haphaza d oppo uni ies o sampling (Ryse -Degio gis, 2013; Ca doso
e al., 2022). Ob aining se um equi es he e ige a ion o blood sam-
ples and hei p omp cen i uga ion, hus es ic ing he collec ion o
samples o specialized pe sonnel wi h access o app op ia e equipmen
and acili ies (Maceda-Veiga e al., 2015). D ied whole blood samples
collec ed in specialized abso ben ma ices (d ied blood spo s, he ein
DBS) ha e eme ged as a iable al e na i e o se ological su eys in
humans, li es ock, and wildli e (Samsono a e al., 2022). D ied blood
spo s can be collec ed by lay pe sonnel, s o ed a oom empe a u e o
se e al weeks, and sa ely shipped o dedica ed labo a o ies (Manak
e al., 2018; Samsono a e al., 2022). They allow inco po a ing ci izen
science app oaches in he su eillance o wildli e diseases, inc easing
he geog aphical co e age o sampling in a cos -e icien way (Peng
e al., 2023). Ne e heless, hey equi e he e alua ion o he echnique
o he hos (s) and pa hogen(s) unde s udy (Samsono a e al., 2022).
Myxoma osis and abbi hemo hagic disease a e impo an i al
diseases o he Eu opean abbi (O yc olagus cuniculus) in he wild and
cunicul u e (Rosell e al., 2019; Delibes-Ma eos e al., 2021). While
hese pa hogens a e p ima ily con olled h ough accina ion and s ic
biosecu i y measu es in cunicul u e, he p esence o a widesp ead wild
ese oi con inually a o s hei eme gence (Rosell e al., 2019). Eu-
opean abbi s we e ecen ly classi ied as endange ed in hei na i e
ange mainly because o he demog aphic impac o hese i al diseases
(Villa ue e & Delibes-Ma eos, 2019).
Myxoma osis is caused by a pox i us (myxoma i us - MYXV)
ansmi ed by mechanical ec o s o di ec ly om in ec ed o suscep-
ible hos s (Ke e al., 2015). I is cha ac e ized by umou -like lesions
(myxomas) and espi a o y signs (Be agnoli and Ma chandeau, 2015).
Al hough causing only mild symp oms in i s na u al hos (Syl ilagus spp.
lagomo phs), in Eu opean abbi s he disease can be se e e (Be agnoli
and Ma chandeau, 2015). Myxoma i us is cu en ly endemic in wild
Eu opean abbi s in he Ibe ian Peninsula (Villa ue e e al., 2017), bu
i s case a ali y a e d as ically dec eased since i s eme gence in he
1950s (Camacho-Sille o e al., 2022; Pacheco e al., 2022).
Rabbi hemo hagic disease is caused by a calici i us ( abbi hem-
o hagic disease i us - RHDV) ansmi ed by di ec o indi ec con ac
o mechanical ec o s (Ab an es e al., 2012; Lopes e al., 2023). The
disease is cha ac e ized by nec o izing hepa i is and dissemina ed
in a ascula coagula ion (Neimanis e al., 2019). The cu en ly ci cu-
la ing RHDV GI.2 eme ged in F ance in 2010 (Le Gall-Recul´
e, 2013) and
quickly dissemina ed ac oss Eu ope (Aguayo-Ad´
an e al., 2022). Rabbi
hemo hagic disease i us GI.2 is endemic bu no homogeneously
dis ibu ed in he Ibe ian Peninsula, and i s case a ali y a e can be high
in na u al ou b eaks (Rouco e al., 2018; Camacho-Sille o e al., 2019;
Jim´
enez-Ruiz e al., 2023).
La ge-scale da a on he se op e alence o MYXV and RHDV GI.2 in
wild Eu opean abbi popula ions a e in aluable o in o m managemen
o he domes ic and wild compa men s o hei epidemiological cycles
(Villa ue e e al., 2017; Ramsey e al., 2023). Despi e wild Eu opean
abbi s being commonly ha es ed, in o ma ion on se op e alence is
haphaza d h oughou much o i s ange due o he di icul ies in pe -
o ming la ge-scale wildli e epidemiological su eys. This s udy aimed
o e alua e a p o ocol o se ological su eys o myxoma and abbi
hemo hagic disease i uses in wild Eu opean abbi s, using DBS o
acili a e la ge-scale and long- e m moni o ing.
2. Me hods
2.1. Blood collec ion and d ied blood spo elu ion
Whole blood (1–2 ml) was collec ed pos -mo em wi h a 5 ml sy inge
om he ho acic o abdominal ca i ies o wild Eu opean abbi s legally
ha es ed o ec ea ional pu poses in Po ugal (n=95) and Spain
(n=70). Addi ional samples we e collec ed upon s anda d nec opsy o
ca casses ound in he ield and ozen a −20◦C o 2–7 mon hs be o e
nec opsy (n=7). Whole blood was collec ed, e ige a ed un il p ocess-
ing a he labo a o y 2–24 h a e blood collec ion, cen i uged a 1500 g
o 10 min, and he se um collec ed and s o ed ozen a −20◦C un il
analysis.
D ied blood spo s we e ob ained om he same abbi s by placing
P o ein Sa e (PS) 903 ca ds (Wha man, Maids one, U.K.) in con ac
wi h blood in he ho acic o abdominal ca i ies. Whole blood collec ed
in he PS ca ds was le o d y a oom empe a u e o 2–6 weeks,
p o ec ed om di ec sunligh . I was hen ozen a −20◦C un il elu ion,
1–12 mon hs la e . One 5 mm diame e DBS punch, which, acco ding o
he manu ac u e , should hold 11.6–12.4 µl whole blood, was ex ac ed
om each DBS and incuba ed wi h 50 µl o phospha e bu e ed saline
(pH=7.4), a e a sho spin. The elua e was collec ed a e o e nigh
incuba ion a 4 ◦C ollowed by ano he sho spin and kep ozen a
−20◦C un il analysis.
All p ocedu es we e pe o med in compliance wi h ele an laws and
ins i u ional guidelines. No abbi s we e killed o he pu pose o his
s udy, and no li e animal expe imen a ion was pe o med.
2.2. Se ological assays
Se um and DBS elua es we e analyzed o IgG speci ic o myxoma
and RHD GI.2 i uses by indi ec enzyme-linked immune se um assays
(iELISA). The in-house iELISA a ge ing RHDV GI.2-speci ic IgG was
pe o med as desc ibed in Pacheco e al. (2022), based on B´
a cena e al.
(2015). B ie ly, 100 ng/well o GI.2-de i ed i us-like pa icles pu i ied
as desc ibed in Almanza e al. (2008) dilu ed in ca bona e/bica bona e
bu e (pH=9.5) we e abso bed o Nunc Maxiso p 96-well ELISA pla es
and incuba ed o e nigh a 4◦C. The pla es we e blocked wi h PBS–5 %
skim milk solu ion, washed h ee imes wi h PBS–0.05 % Tween 20, and
he se a assayed a 1/200 dilu ion in PBS-5 % skim milk solu ion. As he
concen a ion o IgG was expec ed o be highe in se um han in DBS
elua es, he la e we e es ed a highe concen a ions: 1/67, 1/50, and
1/40 dilu ions. We aimed o es ablish which DBS elua e dilu ion ach-
ie ed he bes diagnos ic pe o mance using he same cu -o h eshold
as se um es ed a 1/200 dilu ion. This app oach has he ad an age o
no equi ing es ablishing independen cu -o h esholds o each o he
DBS elua e dilu ion e alua ed.
A e incuba ion o 1 h, he conjuga e goa an i- abbi IgG / HRP
(BioRad, Po ugal) was added a 1/4000 dilu ion, incuba ed o 1 h, and
100 µl o he subs a e 3,3
′
,5,5’- e ame hylbenzidine (Abcam, U.K.) was
added. Reac ions we e s opped a e 4±1 min wi h 65 µl o 1 M phos-
pho ic acid, and he op ical densi y a 450 nm (OD
450 nm
) was eco ded
wi hin 15 minu es. Posi i e con ols consis ed o pooled se a om ab-
bi s wi h high iELISA op ical densi ies (Pacheco e al., 2022), and
nega i e con ols we e pooled se a om un accina ed domes ic Eu o-
pean abbi s wi hou a his o y o clinical disease and kep in-house. A
comme cial ki (Ingezim 17.MIX.K1, Eu o ins Technologies Ingenasa,
Spain) a ge ing MYXV-speci ic IgG was employed acco ding o he
manu ac u e ’s ins uc ions. Se a we e assayed by MYXV iELISA a
1/200, and elua es a 1/67, 1/50, and 1/40 dilu ions, as o RHDV GI.2.
The MYXV and RHDV GI.2 assays we e conside ed alid i he
a e age OD
450 nm
o he wo eplica es o he posi i e con ol was >5
imes he a e age OD
450 nm
o he wo eplica es o he nega i e con ol.
The esul s o bo h assays we e s anda dized as no malized abso bance
a ios (NAR) (Ramanakuma e al., 2010) acco ding o Eq. (1):
NAR =a e age OD450 sample
2× (a e age OD450 nega i e con ol)(1)
The dicho omous se ological s a us (se oposi i e/nega i e) o each
sample was a ibu ed based on he cu -o h esholds p e iously es i-
ma ed by ini e mix u e models o se a es ed a 1/200 dilu ion, being
NAR=2.0 o RHDV GI.2 and NAR=2.4 o MYXV. Unde hese
J. Fe ei a-e-Sil a e al.
P e en i e Ve e ina y Medicine 234 (2025) 106369
2
condi ions, bo h iELISA es s we e shown o achie e 100 % sensi i i y
and speci ici y when es ing wild abbi se a a 1/200 dilu ion (Pacheco
e al., 2022).
2.3. D ied blood spo e alua ion
The ag eemen be ween he dicho omous se ological esul s,
exp essed as posi i e/nega i e acco ding o he abo emen ioned cu -o
h esholds, was assessed by Cohen’s unweigh ed Kappa (κ) (Cohen,
1960), es ima ed using he package "DescTools" (Signo ell e al., 2022).
The co ela ion be ween he semi-quan i a i e se ological esul s
(P echl, 2021) o se a (NAR
se um
) and DBS elua es (NAR
elua e
) was
assessed by he coe icien o de e mina ion (R
2
) o he eg ession be-
ween he NAR
elua e
and NAR
se um
. The sensi i i y, speci ici y, nega i e
and posi i e p edic i e alues o DBS elua es we e es ima ed by com-
pa ison wi h se um, he e assumed as he e e ence ma ix. S a is ical
analyses we e pe o med using R 4.2.1 (R De elopmen Co e Team,
2023).
3. Resul s
The summa y o he es esul s ob ained ac oss biological ma ices
and es dilu ions is p esen ed in Table 1.
Cohen’s Kappa (κ) showed an almos pe ec ag eemen be ween he
binomial esul s o se um and DBS elua es o MYXV when es ed a bo h
1/40 dilu ion (0.942, CI
95
0.886 – 0.998) and 1/50 dilu ion (0.914, CI
95
0.847 – 0.981), and a s ong ag eemen o RHDV GI.2 when es ed a 1/
50 dilu ion (0.808, CI
95
0.722 – 0.893) (Table 2).
The coe icien o de e mina ion (R
2
) showed s ong linea co ela-
ions be ween NAR
se um
and NAR
elua e
a 1/50 dilu ion o MYXV and
RHDV GI.2 (Table 2 and Fig. 1 A and 1B). The ela ionship be ween
NAR
se um
and NAR
elua e
o RHDV GI.2 a 1/50 was be e desc ibed as
quad a ic (R
2
=0.795, Fig. 1 C). The di e ences in he pa ame e s κ and
R
2
be ween he DBS elua e’s es dilu ions 1/40 and 1/50 we e small
(Table 2).
Assuming se um as he e e ence ma ix o de ec pa hogen-speci ic
IgG, he diagnos ic sensi i i y o DBS elua es es ed a 1/50 dilu ion was
82.1 % (CI
95
73.2 – 88.5 %) o RHDV GI.2 and 95.4 % (CI
95
90.3 –
97.9 %) o MYXV. The diagnos ic speci ici y o DBS elua es es ed a 1/
50 dilu ion o bo h i uses was 100 % (Table 3). The se op e alence o
MYXV in se um es ed a 1/200 was 74.3 % (CI
95
67.2 – 80.6 %) and
RHDV GI.2 54.3 % (CI
95
46.6 – 62.8 %). Unde hese p e alences, he
nega i e p edic i e alue o iELISA pe o med on DBS elua es a 1/50
dilu ion was >80 % and he posi i e p edic i e alue was 100 % o
MYXV and RHDV GI.2.
4. Discussion
He e we demons a e he sui abili y o DBS elua es as al e na i e o
se a o se ological su eys o MYXV and RHDV GI.2 in wild Eu opean
abbi s. P e ious s udies used his echnique in o he hos (s) and pa h-
ogen(s), gene ally inding i an easy, a o dable, and eliable sample
collec ion me hod (Samsono a e al., 2022). Duncombe e al. (2013)
assessed PS ca ds in he ca le-B ucella abo us sys em and ound a high
co ela ion be ween an ibody i e s ob ained in pai ed se um and DBS
elua e samples, wi h Pea son’s co ela ion being 0.975 o in ec ed and
0.758 o non-in ec ed animals. The du a ion o he s o age o DBS and
he loca ion o he punch wi hin he DBS had negligible in luence on he
es esul s (Duncombe e al., 2013). D ied blood spo s in PS ca ds we e
also shown o ha e good sensi i i y (96.1 %, CI
95
91.2–98.7 %) and
speci ici y (98.6 %, CI
95
95.1–99.8 %) compa ed o se a o SARS-CoV-2
IgG in human samples (Meye s e al., 2021). Good pe o mance o PS
DBS was also shown in se ological es s o animal ube culosis in wild
boa (San os e al., 2018) and Toxoplasma gondii exposu e in wild un-
gula es (As on e al., 2014).
Po ejoie e al. (2009) assessed he use o DBS collec ed in a di e en
ca d (Wha man 3D55) in lagomo phs o de ec an ibodies agains Eu-
opean b own ha e synd ome calici i us in domes ic and wild ha es. The
speci ici y o DBS was 100 % and he speci ici y 85 % when compa ed o
se um bu sensi i i y was lowe in samples wi h low i e s. O e all, he
ag eemen be ween he esul s om se um and DBS was good (Po ejoie
e al., 2009).
We analyzed he ag eemen o he dicho omous (posi i e/nega i e)
es esul s be ween pai ed se um and DBS elua es om Eu opean ab-
bi s. Ou s a egy was o adap he DBS elua e es dilu ions o achie e a
diagnos ic pe o mance as simila as possible o se um, employing he
cu -o h esholds p e iously es ablished by ou eam o he la e ma ix
(Pacheco e al., 2022). These cu -o h esholds we e es ablished by
applying ini e mix u e models o se ological da a om o he pop-
ula ions o wild Eu opean abbi s. Fini e mix u e models allow o
cha ac e ize he dis ibu ions o he quan i a i e es esul s o he
se oposi i e and se onega i e subg oups wi hin da ase s, wi hou
equi ing samples o known se ological s a us. These models hus es i-
ma e he p obabili y o any gi en sample being posi i e o nega i e o a
diagnos ic es (Meye e al., 2018). Ou indings suppo PS DBS elua e
as a iable al e na i e o se um o es ima ing se op e alence o MYXV
and RHDV GI.2 in wild abbi popula ions (Tables 2 and 3), which
gene ally ag ees wi h esea ch pe o med on o he pa hogens
(Samsono a e al., 2022).
We also in es iga ed he co ela ion be ween he semi-quan i a i e
iELISA s anda dized esul s (NAR) o se um and DBS elua es. The dilu-
ion ac o s needed o achie e b oadly simila NAR wi h DBS elua es (1/
50) we e lowe when compa ed o se um (1/200), sugges ing a lowe
concen a ion o IgG in he o me ma ix wi h he elu ion p o ocol
applied. We p opose es ing DBS elua es a 1/50 dilu ion in u u e su -
eys o RHDV GI.2 and MYXV, as his dilu ion showed highe co-
e icien s o de e mina ion o bo h pa hogens and a simila ag eemen
o he 1/40 dilu ion.
The co ela ion be ween he NAR o DBS elua es and se um o
MYXV co esponds o a linea ela ionship. In e es ingly, he same did
no apply o RHDV GI.2, whe e a quad a ic ela ionship be e i ed he
da a (Fig. 1 C). Samples wi h high RHDV GI.2 esul s in se um
(NAR
se um
≥9) ended o show much highe NAR
elua e
, up o 15.4
(Fig. 1B). The easons behind his unexpec ed appa en concen a ion o
RHDV GI.2-speci ic IgG in DBS elua es a e unknown bu could be biased
by he sca ce numbe (n=4) o samples wi h NAR
se um
and NAR
elua e
≥9.
Ne e heless, cau ion is wa an ed when in e p e ing semi-quan i a i e
se ological esul s o DBS elua es showing high RHDV GI.2 NAR.
Table 1
Summa y o he es esul s. Dicho omous esul s (posi i e/nega i e) ob ained om se um es ed a 1/200 and d ied blood spo elua es es ed a 1/67, 1/50, and 1/40
dilu ions.
Pa hogen Tes ma ix Elua e 1/67
a
Elua e 1/50 Elua e 1/40
Se um 1/200 Posi i e Nega i e Posi i e Nega i e Posi i e Nega i e
Myxoma i us Posi i e 53 12 124 6 126 4
Nega i e 0 25 0 45 0 45
RHDV Posi i e 51 44 78 17 84 11
Nega i e 1 76 0 77 6 71
a
Only 90 elua es we e a ailable o es o IgG speci ic o Myxoma i us a 1/67 dilu ion
J. Fe ei a-e-Sil a e al.
P e en i e Ve e ina y Medicine 234 (2025) 106369
3
5. Conclusions
This s udy e alua es he sui abili y o DBS elua es as an al e na i e
ool o se ological su eys o MYXV and RHDV GI.2 speci ic an ibodies
in Eu opean abbi s. D ied blood spo s a e an easy and ela i ely low-
cos me hod o sample collec ion ha can be used by non-skilled
pe sonnel. Fu he mo e, i allows o inco po a e ci izen science ap-
p oaches in he se o-epidemiological su eillance. Combined wi h good
diagnos ic pe o mance when es ed o IgG speci ic o MYXV and
RHDV GI.2 a 1/50 dilu ion, hese cha ac e is ics make DBS an op ion
o sampling a la ge geog aphical scale and in emo e loca ions. D ied
blood spo s elua es can become a ool o moni o ing MYXV and RGDV
GI.2 in wild Eu opean abbi popula ions. Such moni o ing is necessa y
o unde s and he epidemiology o hese pa hogens in wildli e and hei
ansmission o comme cial cunicul u e.
CRediT au ho ship con ibu ion s a emen
Ana Ma ga ida Lopes: W i ing – e iew & edi ing, Resou ces,
Me hodology. Joana Ab an es: W i ing – e iew & edi ing, Resou ces,
Me hodology. Juan B´
a cena: W i ing – e iew & edi ing, Supe ision,
Resou ces, Me hodology, Funding acquisi ion. Joana Fe ei a-e-Sil a:
W i ing – e iew & edi ing, W i ing – o iginal d a , In es iga ion,
Fo mal analysis, Da a cu a ion. Es he Blanco: W i ing – e iew &
edi ing, Supe ision, Resou ces, Me hodology, Funding acquisi ion.
Saúl Jim´
enez-Ruiz: W i ing – e iew & edi ing, In es iga ion, Da a
cu a ion. Ca los Rouco: W i ing – e iew & edi ing, Supe ision, Re-
sou ces, Me hodology, Funding acquisi ion. Ma isa Rod igues: W i ing
– e iew & edi ing, Resou ces, In es iga ion. Ignacio Ga cía-Bocane-
g a: W i ing – e iew & edi ing, Supe ision, Resou ces, Me hodology,
Funding acquisi ion. Emídio San os: W i ing – e iew & edi ing, Re-
sou ces, In es iga ion. Paulo C´
elio Al es: W i ing – e iew & edi ing,
Supe ision, Resou ces, P ojec adminis a ion, Funding acquisi ion.
Sab ina Cas o-Schol en: W i ing – e iew & edi ing, Resou ces,
In es iga ion, Da a cu a ion. Nuno San os: W i ing – e iew & edi ing,
W i ing – o iginal d a , Supe ision, P ojec adminis a ion, Me hod-
ology, Funding acquisi ion, Fo mal analysis, Da a cu a ion, Concep u-
aliza ion. Vi o Lizana: W i ing – e iew & edi ing, Resou ces,
In es iga ion, Da a cu a ion. Alba Ma í-Ma co: W i ing – e iew &
edi ing, Resou ces, In es iga ion. Te eza Almeida: W i ing – e iew &
edi ing, Resou ces, Me hodology.
Decla a ion o Compe ing In e es
None.
Table 2
Ag eemen be ween he se ological esul s o he ma ices se um and d ied blood spo elua e. Con idence in e als 95 % a e shown in b acke s.
Ma ix Rabbi hemo hagic disease i us GI.2 Myxoma i us
Se um
Tes dilu ion
D ied blood spo elua e Tes
dilu ion
Cohen’s Kappa (κ) Coe icien o de e mina ion
(R
2
)
Cohen’s Kappa (κ) Coe icien o de e mina ion
(R
2
)
1/200 1/67 0.503
(0.393–0.613)
0.610
(0.522 – 0.699)
0.711
(0.565 – 0.856)
0.755
(0.670 – 0.840)
1/50 0.808
(0.722 – 0.893)
0.761
(0.700 – 0.822)
0.914
(0.847 – 0.981)
0.931
(0.912–0.950)
1/40 0.801
(0.712 – 0.891)
0.716
(0.646 – 0.786)
0.942
(0.886 – 0.998)
0.926
(0.905 – 0.947)
Fig. 1. Rela ionship be ween he semi-quan i a i e se ological esul s in pai ed samples o se um and d ied blood spo elua es. No malized abso bance a ios o
pai ed se um and d ied blood spo elua es es ed by indi ec ELISA a 1/50 dilu ion: A) myxoma i us; B) abbi hemo hagic disease i us GI.2 (linea ); C) abbi
hemo hagic disease i us GI.2 (quad a ic). Non-conco dan binomial esul s as whi e do s. The cu -o h esholds o posi i i y as dashed g ey lines.
Table 3
Diagnos ic pe o mance o d ied blood spo elua es es ed a 1/50 dilu ion.
Diagnos ic sensi i i y and speci ici y we e es ima ed assuming se um as he
e e ence ma ix.
Pa hogen Diagnos ic pe o mance (%) (95 % con idence in e al)
Sensi i i y Speci ici y Posi i e
p edic i e
alue
Nega i e
p edic i e
alue
Rabbi
hemo hagic
disease i us
GI.2
82.1 (73.2 –
88.5)
100 (95.4 –
100)
100 (95.3 –
100)
82.5 (75.4 –
87.9)
Myxoma i us 95.4 (90.3 –
97.9)
100 (92.1 –
100)
100 (97.0 –
100)
88.2 (77.4 –
94.3)
J. Fe ei a-e-Sil a e al.
P e en i e Ve e ina y Medicine 234 (2025) 106369
4
Acknowledgemen s
This wo k was suppo ed by Fundaç˜
ao pa a a Ciˆ
encia e Tecnologia
(FCT) [g an SFRH/BPD/116596/2016 o N.S., CEECIND/01388/2017
o A.M.L. and CEECIND/00078/2017 o J.A.] and co- unded by he
p ojec NORTE-01–0246-FEDER-000063, suppo ed by No e Po ugal
Regional Ope a ional P og amme (NORTE2020), unde he PORTUGAL
2020 Pa ne ship Ag eemen , h ough he Eu opean Regional De elop-
men Fund (ERDF). This s udy was pa ially unded by p ojec LAGMED
(www.lagmed.eu), suppo ed by FCT (PRIMA/0003/2018), he Spanish
Minis y o Science, Inno a ion and Uni e si y (PRIMA S2–11-
PCI2019–103732 o E.B.) and PRIMA p og amme, an A . 185 ini ia i e
suppo ed and unded unde Ho izon 2020, he Eu opean Union’s
F amewo k P og amme o Resea ch and Inno a ion. This wo k
bene i ed om esea ch g an s unded by he Spanish Minis y o Sci-
ence and Inno a ion (p ojec s Ibe -Lagoheal h; PID2023–151954NB-
100 and LagoHeal h; PID2019–111080RB-C21). I was also pa ially
unded by he Sub-modali y 2.4. "UCOLIDERA" o he "En ique Aguila
Bení ez de Lugo" Resea ch Plan o he Uni e si y o Co doba and CIBER
-Conso cio Cen o de In es igaci´
on Biom´
edica en Red- (CB 2021),
Ins i u o de Salud Ca los III, Minis e io de Ciencia e Inno aci´
on and
Eu opean Union – Nex Gene a ionEU. S.J.R. is suppo ed by a ‘Juan de
la Cie a’ con ac (JDC2022–048850-I) unded by he MCIN/AEI/
10.13039/501100011033 and by he Eu opean Union “Nex Gene -
a ionEU”/PRTR. S.C.S. is suppo ed by an FPU g an om he Spanish
Minis y o Uni e si ies (FPU19/06026). This wo k was suppo ed by
Li e Ibe conejo (LIFE20-GIE ES000731).
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