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P e alence and dis ibu ion o Babesia and Theile ia species in oe dee om
Spain
Susana Remesa
a
, Pablo Díaz
a,∗
, Albe o P ie o
a
, Flo encio Ma kina
b
, Jose Manuel Díaz Cao
a
,
Gonzalo López-Lo enzo
a
, Gonzalo Fe nández
a
, Ce e ino M. López
a
, Rosa io Panade o
a
,
Pablo Díez-Baños
a
, Pa ocinio Mo ondo
a
a
In es igación en Sanidad Animal: Galicia (G upo INVESAGA), Facul ade de Ve e ina ia, Uni e sidade de San iago de Compos ela, Lugo, Spain
b
Asociación del Co zo Español (ACE), Spain
ARTICLE INFO
Keywo ds:
Babesia
Theile ia
PCR
Roe dee
Spain
ABSTRACT
Babesiosis and Theile iosis a e impo an wo ldwide-dis ibu ed ick-bo ne diseases o human and animals.
Thei p esence in a pa icula a ea depends on he p esence o sui able ick- ec o and hos species as well as
compe en ese oi s such as oe dee , one o he mos abundan wild ce ids in Spain.
Spleen samples om 174 oe dee hun ed in Spain we e analysed o de e mine he p e alence o Babesia and
Theile ia species. DNA o bo h pi oplasms was fi s ly de ec ed using a comme cial qPCR. Then, posi i e samples
we e molecula ly cha ac e ized a he 18S RNA and ITS1 genes o Babesia spp. and Theile ia spp. The possible
influence o some ac o s such as ecological a ea, age and sex was also assessed.
O e all, 89.7% o oe dee we e posi i e o any o he wo pi oplasms. Theile ia spp. was mo e p e alen
(60.9%) han Babesia spp. (19.0%); species iden ifica ion could no be achie ed in 17.3% o posi i e samples.
Babesia p e alence was significan ly highe in young animals and in oe dee om Oceanic egions, in con as o
Theile ia spp. Fi e species we e iden ified: Theile ia sp. OT3 (60.3%), Babesia cap eoli (15.5%), Babesia ena o um
(2.9%), Theile ia sp. 3185/02 (0.6%) and Babesia bigemina (0.6%). The coin ec ion B. cap eoli/T. sp. OT3 was he
mos common (4.6%) ollowed by B. ena o um/T. sp. OT3 (0.6%) and B. bigemina/T. sp. OT3 (0.6%).
Ou esul s e eal ha Theile ia spp. and Babesia spp. a e p e alen pi oplasms in oe dee om Spain. These
ce ids can ac as ese oi s o se e al Babesia and Theile ia species, including he zoono ic B. ena o um. This
s udy ep esen s he fi s desc ip ion o B. ena o um and B. bigemina in oe dee om Spain.
1. In oduc ion
Pi oplasmoses a e impo an wo ldwide-dis ibu ed ick-bo ne dis-
eases o bo h domes ic and wild animals caused by apicomplexan he-
mopa asi es o he genus Babesia and Theile ia (Zane e al., 2014). The
impo ance o hese pa hogens in he no he n hemisphe e has in-
c eased in ecen yea s since some o hem can cause diseases con-
side ed eme ging zoonosis (Hildeb and e al., 2013).
The dis ibu ion o bo h Babesia and Theile ia in ec ions depends on
se e al ac o s, mainly he p esence o p ope ick ec o species as well
as sui able hos s and ese oi s (Es ada-Peña and de la Fuen e, 2014).
Al hough Babesia spp. can be ansmi ed by a wide ange o ick gene a
including Rhipicephalus,Haemaphysalis,Hyalomma and De macen o
(Uilenbe g, 2006;Requena-Ga cía e al., 2017), i is wo h no ing ha
he mos dis ibu ed ick in Eu ope, Ixodes icinus, has been iden ified as
a ec o o some species such as B. di e gens, B. mic o i, B. o is and B.
ena o um (Pé ez e al., 2012;Rizzoli e al., 2014). I has also been
epo ed ha some species o Rhipicephalus,Hyalomma,Amblyomma and
Haemaphysalis can ansmi Theile ia spp. (Vise as e al., 1999;Mans
e al., 2015).
Wildli e animals a e impo an hos s o icks, playing a c i ical ole
in hei li e-cycle as well as in he ansmission o se e al ick-bo ne
pa hogens such as pi oplasms (Medlock e al., 2013). Thus, a wide
a ie y o ee anging hos s such as oe dee (Cap eolus cap eolus), ed
dee (Ce us elaphus), allow dee (Dama dama), Spanish ibex (Cap a
py enaica), alpine chamois (Rupicap a upicap a) and wild boa (Sus
h ps://doi.o g/10.1016/j.ijppaw.2019.05.005
Recei ed 17 Janua y 2019; Recei ed in e ised o m 16 May 2019; Accep ed 17 May 2019
∗
Co esponding au ho . Facul ad de Ve e ina ia, Pabellón I, Plan a Baja. Campus Uni e si a io s/n, 27002, Lugo, Spain.
E-mail add esses: [email p o ec ed] (S. Remesa ), [email p o ec ed] (P. Díaz), [email p o ec ed] (A. P ie o),
[email p o ec ed] (F. Ma kina), [email p o ec ed] (J.M. Díaz Cao), [email p o ec ed] (G. López-Lo enzo),
[email p o ec ed] (G. Fe nández), [email p o ec ed] (C.M. López), [email p o ec ed] (R. Panade o), [email p o ec ed] (P. Díez-Baños),
[email p o ec ed] (P. Mo ondo).
IJP: Pa asi es and Wildli e 9 (2019) 195–201
2213-2244/ © 2019 The Au ho s. Published by Else ie L d on behal o Aus alian Socie y o Pa asi ology. This is an open access a icle unde he CC
BY-NC-ND license (h p://c ea i ecommons.o g/licenses/BY-NC-ND/4.0/).
T
sc o a) ha e been iden ified as suscep ible o Theile ia and/o Babesia
in ec ions in Eu ope (Fe e e al., 1998;Hoby e al., 2007;Tampie i
e al., 2008;Bas ian e al., 2012). Since ha wildli e a ely de elop
clinical disease, hese wild animals ac as asymp oma ic ca ie s o
ese oi s o hese pi oplasm species (Bas ian e al., 2012;Zane e al.,
2014). Up- o-now, se e al Babesia and Theile ia species and geno ypes
ha e been iden ified in wild ungula es, including Babesia bigemina,
Babesia cap eoli, Babesia di e gens, Babesia mic o i, Babesia odocoilei,
Babesia o is, Babesia sp. MO1, Babesia ena o um ( o me ly Babesia sp.
EU1), Theile ia sp. 3185/02, Theile ia sp. OT3 and Theile ia sp. ZS TO4
(Ga cía-SanMa in e al., 2007;Hoby e al., 2007;Zin l e al., 2011;
Fueh e e al., 2013;Michel e al., 2014;Zane e al., 2014;Ebani e al.,
2016). I is wo h no ing ha some o hese pi oplasms ha e zoono ic
po en ial; hus, he common bo ine pi oplasm B. di e gens is he mos
equen agen o human babesiosis in Eu ope, and o he species such as
B. ena o um and B. mic o i ha e also been ound in ec ing humans
(Olmeda e al., 1997;Maland in e al., 2010). In con as , no zoono ic
Theile ia species ha e been cu en ly iden ified (Yabsley and Shock,
2012).
Roe dee is one o he mos abundan wild ce ids in Spain, espe-
cially in no he n closed o es s and b oad meadows. This ungula e may
play an impo an ole as a ese oi o se e al Babesia species wi h
nega i e implica ions in animal and public heal h such as B. cap eoli,
which can cause a al in ec ions in Alpine chamois (Hoby e al., 2007,
2009). In addi ion, he oe dee -associa ed B. ena o um (Zin l e al.,
2011) has been ela ed o human babesiosis cases in Eu ope (He wald
e al., 2003). Despi e o he inc ease o oe dee popula ions and hei
dis ibu ion o e Eu ope in ecen yea s (Fandos and Bu on, 2013;
Valen e e al., 2014), epidemiological s udies abou ick-bo ne pa ho-
gens in his ce id hos a e s ill limi ed in Spain. Fo hose easons, he
main objec i e o his s udy was o iden i y he Babesia and Theile ia
species in oe dee hun ed in Spain using molecula echniques and o
de e mine hei p e alence; he possible influence o se e al in insic
(age, sex) and ex insic (ecological a ea o hun ing) ac o s on he
p e alence o he diffe en pi oplasm species was also assessed. These
esul s will be use ul o un a el he ole o oe dee as a ese oi o
pi oplasm species on human and animal heal h in Spain.
2. Ma e ials and me hods
2.1. Sample collec ion and p ese a ion
A o al numbe o 174 oe dee hun ed in 14 Spanish p o inces
du ing fi e yea s (2013–2017) we e examined in his s udy. Samples
we e collec ed by hun e s o he Spanish Roe Dee Associa ion
(Asociación del Co zo Español; ACE); scien ific collabo a o s membe s
o ACE supe ised he collec ion o samples in o de o ensu e a co ec
sampling echnique and a oid c oss-con amina ions.
Hun ing loca ions we e classified in ou diffe en ecological a eas
p e iously desc ibed (Mo ondo e al., 2017): Oceanic (n= 67),
Moun ain (n= 28), Con inen al (n= 34) and Medi e anean (n= 45)
(Fig. 1). In addi ion, mos oe dee included in his s udy we e male
(n= 123) and only 51 we e emales since he hun ing pe iod is longe
o males han o emales. Age was es ima ed on he basis o ee h
analysis (Høye, 2006); hus, animals we e di ided in 2 age-g oups,
younge han 2 yea s (n= 40) and adul s olde han 2 yea s (n= 132).
In o ma ion om wo animals was incomple e so hey we e no age-
classified.
In o de o de ec Babesia and Theile ia DNA, he whole spleen o
each oe dee was collec ed du ing field e isce a ion, indi idually
bagged up, iden ified and kep e ige a ed. In he labo a o y, he
spleen was fi s ly insed in s e ile PBS, and hen dissec ed wi h a s e ile
scalpel; small pieces o spleen we e aken and s o ed a −20 °C un il
DNA ex ac ion.
2.2. DNA ex ac ion and polime ase chain eac ion
DNA was ex ac ed om 200 μg o splenic issue using a comme cial
ki (High Pu e PCR Templa e P epa a ion Ki
®
, Roche Diagnos ics
GmbH, Mannheim, Ge many) ollowing he manu ac u e ’ins uc ions
o DNA ex ac ion om issue, and hen s o ed a −20 °C un il used.
In o de o de ec Babesia and Theile ia DNA, a comme cial eal ime
PCR de ec ing bo h pi oplasms (EXOone Pi oplasm
®
, Exopol, Za agoza,
Spain) was pe o med ollowing he manu ac u e ’ins uc ions. All
qPCR posi i e samples we e hen selec ed and molecula ly cha ac e -
ized using a con en ional PCR assay a ge ing he 18S RNA gene o
Babesia spp. and Theile ia spp. using p e iously epo ed p ime s
(Table 1) and p o ocols (Zahle e al., 2000;da Sil ei a e al., 2011). In
o de o confi m species iden ifica ion, a subse o samples o each
pi oplasm species de ec ed a he 18S RNA gene was selec ed and
es ed indi idually using a second PCR assay a ge ing he in e nal
ansc ibed space 1 (ITS1) o Babesia spp. and Theile ia spp. as p e-
iously desc ibed (Blaschi z e al., 2008;Baje e al., 2014). In o de o
de ec Babesia-Theile ia coin ec ions, all posi i e samples we e u he
analysed using wo 18S RNA PCR specific o Babesia and Theile ia,
espec i ely (Bi kenheue e al., 2003;Heida pou -Bami e al., 2009).
In each amplifica ion eac ion, nega i e con ols as well as posi i e
con ols ob ained om blood o Babesia-posi i e dogs and spleen o
Theile ia-posi i e oe dee we e included.
2.3. Sequence analysis
All p oduc s ob ained using con en ional PCR assays we e pu ified
and subsequen ly sequenced in bo h senses on an ABI 3730xl
®
(Applied
Biosys ems, Fos e Ci y, CA, USA) using a Big dye Te mina o 3.1 cycle
sequencing ki
®
(Applied Biosys ems, Fos e Ci y, CA, USA) a he
Sequencing and F agmen Analysis Uni o he San iago de Compos ela
Uni e si y (Spain). Sequences we e aligned and edi ed using
Ch omasP o
®
(Technelysium, B isbane, Aus alia), and consensus se-
quences we e scanned agains he GenBank da abase using he Basic
Local Alignmen Sea ch Tool (BLAST; h p://blas .ncbi.nlm.nih.go /
Blas .cgi). Unique pa ial sequences iden ified in his s udy we e de-
posi ed in GenBank unde accession numbe s MH522427-MH522439
and MK318268-MK318270.
2.4. S a is ical analysis
All s a is ical analyses we e pe o med using he s a is ical so wa e
R(R Co e Team, 2018). The possible influence o in insic (age, sex)
and ex insic (ecological a ea) ac o s on he p e alence o in ec ion by
Babesia spp. and Theile ia spp. was analysed using a logis ic eg ession.
Fac o s we e elimina ed om he ini ial model using a backwa d and
o wa d condi ional me hod based in AIC alue (Akaike In o ma ion
C i e ion) un il he bes model was buil . Nex , all pai wise in e ac ions
we e e alua ed. Odds a io we e compu ed by aising “e” o he powe
o he logis ic coefficien o e he fi s ca ego y o each ac o ( e-
e ence ca ego y), no o e he las . The logis ic analyses and he AIC
selec ion we e pe o med wi h glm () and s ep () unc ions in he R
s a is ical package (R Co e Team, 2018).
3. Resul s
Using qPCR, 156 ou o 174 (89.7%) spleen samples yielded Babesia
and/o Theile ia DNA. All qPCR posi i e samples also amplified a he
18S RNA gene, bu sequencing was only success ul in 129 (82.7%)
samples; wen y-se en samples (17.3%) had unde lying signals in he
elec ophe og am ha p e en ed he accu a e eadou o sequences.
A e sequence analysis, he o al p e alence o Theile ia spp. and
Babesia spp. We e 60.9% and 19.0%, espec i ely. Mixed Babesia/
Theile ia in ec ions we e iden ified in 10 (5.7%) animals. When he
possible effec o he sex o he animals on he p e alence was analysed
S. Remesa , e al. IJP: Pa asi es and Wildli e 9 (2019) 195–201
196
(Table 2), bo h sexes showed simila pe cen ages o in ec ion by Babesia
spp. In con as he p e alence o Theile ia spp. was highe in males
han in emales. Ne e heless, hose diffe ences we e no significan in
any case (Table 3). In addi ion, Babesia p e alence was significan ly
highe in young han in adul animals in con as o ha obse ed o
Theile ia spp. (Table 2); ne e heless, logis ic eg ession esul s showed
ha diffe ences in Theile ia p e alence we e no significan (Table 3).
The pe cen age o Babesia-in ec ed oe dee was significan ly highe in
he Oceanic a ea han in Con inen al and Moun ain a eas (Tables 2 and
3). In con as , he p e alence o Theile ia spp. was significan ly lowes
in animals om he Oceanic a ea when compa ed o hose om he es
o a eas (Tables 2 and 3).
Fi e pi oplasms species/geno ypes we e iden ified: Theile ia sp. OT3
was he mos equen ly iden ified pi oplasm (105/174; 60.3%), ol-
lowed by Babesia cap eoli (27/174; 15.5%) and Babesia ena o um (5/
174; 2.9%); Theile ia sp. 3185/02 (0.6%) and Babesia bigemina (0.6%)
we e only ound in a single animal each. The mos common coin ec ion
was B. cap eoli/T. sp. OT3 (4.6%) ollowed by B. ena o um/T. sp. OT3
(0.6%) and B. bigemina/T. sp. OT3 (0.6%).
Mos T. sp. OT3 sequences a he 18s RNA we e iden ical o showed
a single nucleo ide polymo phism (SNP) when compa ed o he T. sp.
OT3 e e ence isola e KF470868 (Supplemen a y da a 1); mo e han
se en nucleo ide diffe ences we e ound wi h o he Theile ia species
such as T. cap eoli o Theile ia luwenshuni. A he ITS1, in con as , ou T.
sp. OT3 sequences only showed a 97% (459/475 base pai s) simila i y
wi h he deposi ed sequences o T. sp. OT3 ob ained om sheep in
China (KF470865-KF470867); i is no ewo hy ha mos diffe ences
we e due o dele ions in poly-adenine epea egions. Mos B. ena o um
isola es showed one o h ee SNP's when compa ed o KM289158 a he
18S RNA gene, whe eas a 100% iden i y wi h he e e ence sequence
HM113372 was obse ed a he ITS1. Babesia cap eoli sequences a he
18S RNA gene we e iden ical o showed a single SNP when compa ed
o KX839234; hey we e also showed a 99% homology wi h B. di e gens
sequences (KP742785). Al hough no sequences o B. cap eoli a he ITS1
gene a e cu en ly deposi ed in GenBank, ou B. cap eoli ITS1 isola es
showed a 94% simila i y (512/543 bp) wi h he ITS1 gene o B. di e -
gens (LK935835) and o e lapped a 23% o a 18S RNA sequence co -
esponding o B. cap eoli (KP742785), showing a 99% iden i y (127/
128).
The T. sp. 3185/02 sequence showed a single SNP when compa ed
o he e e ence sequence DQ866842, and mo e han 5 nucleo ide
disc epancies when compa ed o o he Theile ia species such as Theile ia
o is,Theile ia pa a o Theile ia annula a. As o B. cap eoli, no ITS1
sequences o T. sp. 3185/02 a e cu en ly deposi ed in GenBank, and
he mos simila sequence was a agmen o he ITS1 gene o T. sp. OT3
(KF470867) showing an homology o 76% (340/445 bp). Finally, a
Fig. 1. Map o Spain (modified om Mo ondo e al., 2017) showing he ou ecological a eas. Do s ep esen he p esence o Babesia spp. and/o Theile ia spp. in
each egion.
Table 1
P ime s used in he ou con en ional PCR assays used o de ec ing Babesia and Theile ia DNA.
O de P ime Nucleo ide sequence (5′-3′) P oduc size Re e ence
Fi s PCR analysis:
18S RNA o Babesia and/o Theile ia spp.
RIB-19 CGG GAT CCA ACC TGG TTG ATC CTG C 430 bp (Zahle e al., 2000;da Sil ei a e al., 2011)
RIB-20 CCG AAT TCC TTG TTA CGA CTT CTC
BAB- umF ACC TCA CCA GGT CCA GAC AG
BAB- umR GTA CAA AGG GCA GGG ACG TA
Second PCR analysis:
ITS1 o Babesia and/o Theile ia spp.
BAITS1-F CGA GTG ATC CGG TGA ATT ATT C 600 bp (Blaschi z e al., 2008;Baje e al., 2014)
BAITS1-R CCT TCA TCG TTG TGT GAG CC
Thi d PCR analysis: 18S RNA o Babesia spp. 5–22F GTT GAT CCT GCC AGT AGT 293-338 bp Bi kenheue e al. (2003)
1661R AAC CTT GTT ACG ACT TCT C
455–479F GTC TTG TAA TTG GAA TGA TGG TGA C
793-772R ATG CCC CCA ACC GTT CCT ATT A
Fou h PCR analysis:
18S RNA o Theile ia spp.
TheiF1 AAC CTG GTT GAT CCT GCC AG ≈1420 bp Heida pou -Bami e al. (2009)
TheiR 1 AAA CCT TGT TAC GAC TTC TC
TheiF2 TGA TGT TCG TTT YTA CAT GG
TheiR 2 CTA GGC ATT CCT CGT TCA CG
S. Remesa , e al. IJP: Pa asi es and Wildli e 9 (2019) 195–201
197
single isola e showed a 99% simila i y wi h he B. bigemina e e ence
sequence (KU206297) since a single SNP was de ec ed; mo e han ou
disc epancies we e de ec ed when compa ed o o he Babesia species
such as Babesia o a a (LC125457) and Babesia sp. Sichuan (AY603403).
Al hough amplifica ion a he ITS1 was obse ed, he band was ain
and could no be sequenced.
4. Discussion
Ou esul s e eal ha pi oplasms a e p e alen pa asi es in oe dee
om Spain since mo e han 80% o hese wild ungula es es ed posi i e.
These esul s a e a highe han hose p e iously epo ed in oe dee
om no he n a eas o Spain (62.3%) (Ga cía-SanMa ín e al., 2007)o
om o he Eu opean coun ies such as I aly o Ge many (Tampie i
e al., 2008;Bas ian e al., 2012;Fueh e e al., 2013; Kauffmann e al.,
2017) whe e p e alence a es anged om 12.6% o 62.7%.
Mul i a ia e analysis showed conside able diffe ences in Babesia
and Theile ia p e alences when conside ing he ecological a eas. In his
way, mos o he iden ified Babesia species we e ound in oe dee om
oceanic a eas loca ed in he no hwes o he coun y, whe eas Theile ia
species we e significan ly mo e p e alen in he es o Spain. These
esul s a e consis en wi h a p e ious in es iga ion pe o med in oe
dee om no h and no heas egions o Spain (Ga cía-SanMa ín e al.,
2007) whe e 53.62% o samples we e posi i e o Theile ia spp. and only
8.70% o Babesia spp. which is p obably ela ed o he p esence o
sui able ick ec o s as well as ese oi s and amplifica ion hos s
(Es ada-Peña and de la Fuen e, 2014). In his espec , I. icinus, he
main ec o o B. ena o um and a compe en ec o o B. cap eoli
(Maland in e al., 2010), is he mos common ick in no he n Spain,
especially in he no hwes egion, whe e i s p esence is a ou ed by
high humidi y le els (Ba andika e al., 2011;Espí e al., 2017;Remesa
e al., 2019). Since I. icinus is also he mos common ick in no he n
and cen al Eu ope, ou esul s ag ee wi h o he s udies ca ied ou in
oe dee om o he Eu opean coun ies showing highe p e alences o
Babesia spp. han hose o Theile ia spp. (Duh e al., 2005;Tampie i
e al., 2008;Michel e al., 2014). In con as , ansmission o Theile ia
spp. is mainly ela ed o icks species belonging o Rhipicephalus,De -
macen o ,Hyalomma and Haemaphysalis gene a (Vise as e al., 1999;
Mans e al., 2015); hose ixodids a e mo e p e alen han I. icinus in
cen al and sou he n Spain (Má quez, 2008;Fe nández de Me a e al.,
2013;Requena-Ga cía e al., 2017), coinciding wi h he p edominance
o Theile ia spp. in oe dee om hose a eas.
Wi h ega d o hos - ela ed a iables, he age o oe dee only had a
significan impac on he p e alence o Babesia. Thus, a significan in-
e se ela ion be ween Babesia p e alence and he age o animals was
ound in his s udy. Since some Babesia species, such as Babesia cap eoli,
ha e been epo ed as a al o oe dee (Hinaidy, 1987), ou esul s
migh be a consequence o pa hogenic Babesia in ec ions nega i ely
influencing he su i al o young oe dee and hus a oiding eaching
adul hood. Ne e heless, se e al in es iga ions epo ha Babesia spp.
a e low i ulen o oe dee , showing li le clinical signs o disease
(Hinaidy, 1987;Ga cía-SanMa ín e al., 2007;Bas ian e al., 2012;
Zane e al., 2014). These disc epan opinions indica e ha his issue
should be u he in es iga ed, analysing mo e samples, especially om
young oe dee , o ob ain mo e obus conclusions. In addi ion, i e-
gula sample dis ibu ion should be conside ed, since mos young oe
dee o igina ed om Oceanic a eas.
A e sequence analysis, fi e species/geno ypes we e iden ified.
Theile ia sp. OT3, T. sp. 3185/02 and B. cap eoli had been p e iously
epo ed in Spain (Ga cía-SanMa ín e al., 2007). Howe e , his is he
fi s epo o B. ena o um and B. bigemina in oe dee om his
coun y. All hese species/geno ypes a e widely dis ibu ed in oe dee
om Eu ope, since B. ena o um and B. cap eoli we e de ec ed in Ge -
many (Kauffmann e al., 2017), I aly (Tampie i e al., 2008;Zane e al.,
2014)and F ance (Bas ian e al., 2012), B. bigemina and T. sp. OT3 in
I aly (Zane e al., 2014) and T. sp. 3185/02 was also iden ified in
Table 2
P e alence o pi oplasm species in oe dee om Spain when conside ing diffe en in insic and ex insic ac o s.
Hun ing loca ion Sex Age
Oceanic (n = 67)
(95% CI)
Moun ain (n = 28)
(95% CI)
Con inen al (n = 34)
(95% CI)
Medi e anean (n = 45) (95% CI) Female (n = 51)
(95% CI)
Male (n = 123)
(95% CI)
Young (n = 40)
(95% CI)
Adul (n = 132)
(95% CI)
B. bigemina 1.49% (0.08–9.14) 0 (0.00–15.02) 0 (0.00–12.64) 0 (0.00–9.80) 0 (0.00–8.73) 0.81% (0.43–5.11) ND ND
B. ena o um 5.97% (1.93–15.35) 3.57% (0.19–20.24) 0 (0.00–12.64) 0 (0.00–9.80) 5.88% (1.53–17.23) 1.63% (0.28–6.34) 10.00% (3.25–24.60) 0.76% (0.04–4.77)
B. cap eoli 35.82% (24.74–48.53) 0 (0.00–15.02) 8.82% (2.31–24.81) 0 (0.00–9.80) 13.73% (6.15–26.87) 16.26% (10.45–24.24) 27.50% (15.14–44.14) 12.12% (7.30–19.22)
To al Babesia 43.28% (31.42–55.92) 3.57% (0.19–20.24) 8.82% (2.31–24.81) 0 (0.00–9.80) 19.61% (10.29–33.55) 18.70% (12.46–26.95) 37.50% (23.17–54.19) 12.88% (7.90–20.09)
T. sp. 3185/02 1.49% (0.08–9.14) 0 (0.00–15.02) 0 (0.00–12.64) 0 (0.00–9.80) 0 (0.00–8.73) 0.81% (0.43–5.11) 0 (0.00–10.91) 0.76% (0.04–4.77)
T. sp. OT3 25.37% (15.88–37.73) 75.00% (54.78–88.87) 85.29% (68.17–94.46) 84.44% (69.94–93.01) 47.06% (33.16–61.40) 65.84% (56.69–74.02) 50.00% (35.20–64.80) 63.64% (54.77–71.70)
To al Theile ia 26.87% (17.10–39.31) 75.00% (54.78–88.87) 85.29% (68.17–94.46) 84.44% (69.94–93.01) 47.06% (33.16–61.40) 66.67% (57.52–74.75) 50.00% (35.20–64.80) 64.39% (55.54–72.40)
No iden ified 19.40% (11.13–31.25) 25.00% (11.43–45.22) 5.88% (1.03–21.06) 11.11% (4.16–24.85) 25.49% (14.77–39.91) 11.38% (6.59–18.68) 12.5% (4.70–27.60) 16.67% (10.96–24.37)
ND: No age in o ma ion was collec ed om his animal.
S. Remesa , e al. IJP: Pa asi es and Wildli e 9 (2019) 195–201
198
Hunga y (Ho nok e al., 2017).
In his s udy, he mos p e alen pi oplasm was T. sp. OT3, de ec ed
in mo e han a hal o he analysed samples, especially in oe dee om
cen al and sou he n a eas o Spain. This species was also ound in o he
wildli e species such as ed dee , allow dee and chamois om Spain
and o he Eu opean coun ies (Ga cía-SanMa ín e al., 2007;Pe ei a
e al., 2016). In addi ion, se e al in es iga ions ha e iden ified his
species in sheep om I aly (Giangaspe o e al., 2015), Tu key (Bilgic
e al., 2017) and China (Tian e al., 2014); in ac , T. sp. OT3 was he
second mo e p e alen Theile ia species/geno ype in sheep om
no he n Spain (42.2%) ollowing T. sp. OT1 (48.1%) (Nago e e al.,
2004). Fo hose easons, he possible ole o ce ids as asymp oma ic
ese oi s o his species has been sugges ed (Ga cía-SanMa ín e al.,
2007), al hough i s pa hogenici y o li es ock has no been comple ely
demons a ed ye (Uilenbe g, 2006).
Babesia cap eoli was he second mos p e alen pi oplasm in ou
s udy. I is mo phological and se ologically ela ed o B. di e gens bu
showing diffe en hos specifici y (Ga cía-SanMa ín e al., 2007); hus,
B. cap eoli has been ound in oe dee , sika dee , allow dee and ed
dee , al hough i has been sugges ed ha he o me could ep esen he
na u al ese oi o his species (Kauffmann e al., 2017). A p e ious
s udy ca ied ou in oe dee om Spain showed a lowe p e alence o
B. cap eoli (8.7%) han ha ound in ou s udy (Ga cía-SanMa ín e al.,
2007). Ne e heless, i was he main pi oplasm species ound in cha-
mois, ed dee and oe dee om I aly (Tampie i e al., 2008;Zane
e al., 2014), oe dee om Sweden (Ande sson e al., 2016) and
mouflon and oe dee om Ge many (Kauffmann e al., 2017). I seems
ha his species is unable o in ec ca le o sheep e y h ocy es
(Maland in e al., 2010), and no human in ec ions we e cu en ly de-
sc ibed (Ande sson e al., 2016).
The pe cen age o in ec ion by B. ena o um was low in oe dee
om Spain, being consis en wi h o he in es iga ions on oe dee om
I aly, Ge many and Slo enia (Duh e al., 2005;Tampie i e al., 2008;
Zane e al., 2014; Kauffmann e al., 2017); i was also ound in a low
pe cen age o mouflons om Ge many (Kauffmann e al., 2017). Since
B. ena o um has been implica ed in human babesiosis cases in Eu ope
(He wald e al., 2003), i s p esence in oe dee om Spain may in ol e
zoono ic implica ions, especially conside ing ha he popula ion and
dis ibu ion o his wild ungula e has been ecen ly inc eased in Spain
(Fandos and Bu on, 2013;Valen e e al., 2014); in addi ion, oe dee
has been iden ified as a ep oduc ion hos o ick popula ions (Kauff-
mann e al., 2017) which could be po en ially in ec ed wi h his Babesia
species.
Theile ia sp. 3185/02 and Babesia bigemina we e only occasionally
iden ified. The o me has been p e iously iden ified in oe dee and
ed dee om no he n Spain wi h a p e alence o 10.1% and 53.6%
espec i ely (Ga cía-SanMa ín e al., 2007), which is highe han ha
ound in ou s udy. Babesia bigemina is one o he causa i e agen s o
bo ine babesiosis; some wildli e species may ac as ese oi s since i
has been p e iously iden ified in whi e- ailed dee in USA and B azil
(da Sil ei a e al., 2011;Holman e al., 2011).
I should be poin ed ou ha all he pi oplasm species de ec ed in
his s udy a e no specific o oe dee ; hus, i has been demons a ed
ha oe dee sha e T. sp. OT3,T.sp. 3185/02 and B. cap eoli wi h ed
dee ; Babesia cap eoli, B. o is and T. sp. OT3 wi h chamois; T. sp. OT3
wi h allow dee and B. cap eoli and B. ena o um wi h mouflons
(Ga cía-SanMa ín e al., 2007;Tampie i e al., 2008;Zane e al., 2014;
Kauffmann e al., 2017). Consequen ly, oe dee may play an impo an
ole as ese oi o pa hogenic Babesia and Theile ia species o wild and
domes ic animals and e en humans.
5. Conclusions
Theile ia spp. and Babesia spp. a e p e alen pi oplasms in oe dee
om Spain, showing highe pe cen ages o in ec ion han hose p e-
iously epo ed in o he a eas o Eu ope. Fi e diffe en species we e
ound, including he fi s ci a ion o B. ena o um and B. bigemina in oe
dee om Spain. In his ega d, hese esul s sugges ha oe dee may
ac as a ese oi o pa hogenic Babesia species o human and animals,
playing a ole in he epidemiology o he disease and hus in ol ing
public and animal heal h isk.
Au ho s' con ibu ions
PDB and PM es ablished he final me hods and design. GF, CML and
RP assis ed wi h p elimina y design o he s udy. FM managed he
collec ion o samples. SR and GLL ex ac ed he DNA om spleen
samples. SR, PDF and AP de eloped and pe o med he PCRs. CML and
JMD conduc ed he s a is ical analysis. SR, PD and RP p epa ed he fi s
pape d a . All au ho s ead and app o ed he final manusc ip .
Conflic s o in e es
All au ho s decla e he absence o any financial o pe sonal in e es s
ha could inapp op ia ely influence he cu en wo k. The final a icle
has been app o ed by all au ho s.
Table 3
Logis ic eg ession model o he p e alence o Babesia spp. and Theile ia spp. Fac o s we e emo ed ollowing he Akaike in o ma ion c i e ion alue un il he bes
model was buil .
Es ima e Z- alue P (> | |) OR CI 95%
Babesia spp.
(In e cep ) −0.7019 −2.298 0.022 0.50 0.27–0.89
Adul s –––––
Young 1.2787 2.630 0.009 3.59 1.40–9.56
Oceanic a ea –––––
Con inen al a ea −2.0212 −3.026 0.002 0.13 0.03–0.43
Moun ain a ea −3.1320 −2.944 0.003 0.04 2.34e-03 - 0.23
Medi e anean a ea −19.0800 −0.012 0.990 5.17e-09 6.63e-211- 1.44e25
Theile ia spp.
(In e cep ) −1.1285 −3.253 0.001 0.32 0.16–0.62
Oceanic a ea –––––
Moun ain a ea 3.3257 5.271 1.36e-07 27.81 8.87–109.82
Con inen al a ea 3.2079 4.558 5.16e-06 24.73 7.03–119.30
Medi e anean a ea 1.9758 3.300 0.001 7.21 2.33–24.93
Oceanic:Adul –––––
Medi e anean:Young −2.1972 −1.944 0.052 0.11 0.01–1.12
Con inen al:Young −1.1632 −1.122 0.262 0.31 0.04–2.83
Moun ain:Young 1.0986 0.935 0.350 3.00 0.40–62.51
Oceanic:Young 0.2122 0.357 0.721 1.24 0.37–3.91
S. Remesa , e al. IJP: Pa asi es and Wildli e 9 (2019) 195–201
199
Funding
This esea ch was suppo ed by a p ojec g an (2016-CL018)
awa ded by he Spanish Roe Dee Associa ion [Asociación del Co zo
Español (ACE)], he P og amme o Consolida ing and S uc u ing
Compe i i e Resea ch G oups (GRC2015/003; Xun a de Galicia, Spain)
and he Resea ch P ojec “RUMIGAL: Rede de es udo mul idisciplina
dos uminan es en Galicia”(R2014/005, Xun a de Galicia, Spain).
Decla a ions o in e es
None.
Acknowledgemen s
We would like o hank “O Ve al”Reco e y Cen e o Wild Animals
s affand ACE hun e s o hei ines imable collabo a ion in collec ing
samples.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://
doi.o g/10.1016/j.ijppaw.2019.05.005.
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