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O iginal a icle
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P e alence o selec ed ick-bo ne pa hogens in wild ungula es and icks in sou he n
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Spain
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Running head: Tick-bo ne pa hogens in wild ungula es and icks in Spain
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José Manuel Díaz-Cao1†, Łukasz Adaszek2†, Bea a Dzięgiel2, Jo ge Paniagua1, Ja ie
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Caballe o-Gómez1,3, S anislaw Winia czyk2, Dagma a Winia czyk2, Da id Cano-Te iza1*,
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Ignacio Ga cía-Bocaneg a1
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1 Animal Heal h and Zoonosis Resea ch G oup (GISAZ), Depa men o Animal Heal h, Facul y
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o Ve e ina y Medicine, Uni e si y o Co doba, Campus de Rabanales, 14014 Co doba,
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Spain.
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2Depa men o Epizoo iology and In ec ious Diseases, Facul y o Ve e ina y Medicine,
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Uni e si y o Li e Sciences in Lublin, 30 Głęboka S . 20-612 Lublin, Poland.
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3In ec ious Diseases Uni , Clinical Vi ology and Zoonoses esea ch g oup, Hospi al
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Uni e si a io Reina So ía de Có doba, Ins i u o Maimonides de In es igación Biomédica de
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Có doba (IMIBIC), Uni e si y o Co doba, 14006 Co doba, Spain.
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†These au ho s con ibu ed equally o his wo k.
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*Co espondence: Co esponding au ho : D . Cano-Te iza. Depa men o Animal Heal h,
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Uni e si y o Co doba, Campus Uni e si a io Rabanales, 14014 Có doba, Spain. Tel.: +34
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957218718; E-mail: da idcano e @gmail.com
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Summa y
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A su ey s udy was ca ied ou o assess he occu ence o selec ed ick-bo ne pa hogens (TBP)
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in wild ungula es in Medi e anean ecosys ems in sou he n Spain. Spleen samples we e
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collec ed om 1,132 wild ungula es, including 578 ed dee , 269 wild boa , 135 mou lon, 121
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allow dee and 29 oe dee , be ween 2009 and 2015. Eigh y-nine icks collec ed om TBP-
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posi i e animals we e also analyzed. Samples we e es ed by PCR and sequenced whene e
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possible. TBP DNA was de ec ed in 127 o 863 wild uminan s (14.7%; 95%CI: 12.4-17.3)
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including: Anaplasma phagocy ophilum (9.2%), Babesia di e gens (2.9%), Theile ia sp. OT3
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(1.7%), Bo elia a zelii (0.7%) and Theile ia cap eoli (0.2%); bu no posi i e samples we e
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de ec ed in wild boa (0/269). All he s ains om mou lon we e iden i ied as Theile ia sp. OT3,
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while B. di e gens and T. cap eoli we e mainly ound in ed dee . Co-in ec ion wi h A.
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phagocy ophilum and B. di e gens, and A. phagocy ophilum and Theile ia spp. we e de ec ed
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in ed dee and mou lon, espec i ely. The isk ac o analysis showed ha he p e alences o
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A. phagocy ophilum and pi oplasms we e species- ela ed. Eigh y-nine ick specimens collec ed
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om ungula es ound o be in ec ed wi h he selec ed TBP we e iden i ied as Hyalomma
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lusi anicum (95.5%) and Ixodes icinus (4.5%). Thi y icks we e posi i e o
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Anaplasma/Eh lichia spp. (33.7%), 25 o Babesia/Theile ia (28.1%) and wo o B.
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bu gdo e i s.l. (2.3%). Ele en specimens showed co-in ec ions wi h Anaplasma/Eh lichia and
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Babesia/Theile ia (10.1%) o Anaplasma/Eh lichia and B. bu gdo e i s.l. (2.3%). The
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es ima ed p e alences ob ained in he p esen s udy sugges he possible con ibu ion o wild
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uminan s o he main enance o some selec ed TBP in Medi e anean ecosys ems in sou he n
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Spain, while he ole o wild boa in he epidemiology o hese pa hogens seems o be limi ed
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in his egion.
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Keywo ds: Vec o -bo ne disease; wild uminan s; public heal h; ick-bo ne pa hogens; Spain
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In oduc ion
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Tick-bo ne diseases (TBD) cons i u e a di e si ied g oup o diseases o inc easing impo ance
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in human and e e ina y medicine (Vayssie -Taussa e al., 2015). Among he high numbe o
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ick-bo ne pa hogens (TBP) o human and animal heal h conce n, in ec ions caused by he
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obliga e in acellula pa asi es Anaplasma/Eh lichia spp., Babesia/Theile ia spp. and Bo elia
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spp. a e especially no ewo hy. They cause se e e economic losses in he li es ock indus y
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(Bock, Jackson, de os, & Jo gensen, 2004; Kocan, de la Fuen e, & Cabezas-C uz, 2015;
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Mo ison, 2015) and include some species ha a e well known zoono ic pa hogens causing
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diseases wi h an inc easing incidence in Eu ope (Kocan e al., 2015; S anek, Wo mse , G ay,
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& S le, 2012).
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T ansmission o TBP depends upon a complex a angemen o ac o s including he p esence
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and abundance o compe en ec o s, densi y o compe en hos s, clima ic ac o s ( empe a u e,
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humidi y and ain all) and landscape s uc u e (Ma ei e al., 2019; P ä le, Li win, Mude s, &
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Pe ney, 2013). Wild ungula es ha e been shown o be impo an con ibu o s o he main enance
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o TBP by hos ing icks and a o ing hei abundance (Ho mees e e al., 2017; Kilpa ick,
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Labon e & S a o d III, 2014), as well as ac ing as na u al ese oi s o some o hese pa hogens
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(Kau man e al., 2017; Kazimí o á e al., 2018). Thei impo ance in he epidemiology o TBD
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is cu en ly ising due o he inc easing densi y and abundance o hese species in some
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Eu opean egions in ecen decades, pa icula ly ed dee (Ce us elaphus), oe dee (Cap eolus
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cap eolus) and wild boa (Sus sc o a) (Apollonio, Ande sen, & Pu man; 2010; Ma in,
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Chamaillé-Jammes, & Walle , 2020; Massei e al., 2015). This has also led o he equen
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sha ing o na u al esou ces wi h li es ock and humans in ce ain a eas, which enhances he isk
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o in e species ansmission o TBP (Go áza , Ace edo, Ruiz-Fons, & Vicen e, 2006).
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In ec ions wi h TBP species ha e been epo ed in di e en egions in Spain, in bo h domes ic
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(Calleja-Bueno e al., 2017; Habela e al., 1999; Nago e e al., 2004; Ros-Ga cía e al., 2013)
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and wild ungula es (de la Fuen e e al., 2005; Ga cía-Pé ez e al., 2016; Ga cía-Sanma ín e al.,
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2007; Remesa e al., 2019) wi h ou b eaks o clinical disease and economic loss associa ed
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wi h TBP obse ed in li es ock (Ga cía-Pé ez e al., 2003; Hu ado e al., 2015; Lacas a e al.,
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2020). Fu he mo e, B. bu gdo e i s.l. is endemic in humans in no he n Spain (Po illo,
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San ibáñez & O eo, 2014; Vázquez e al., 2015) and human cases o anaplasmosis and
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babesiosis ha e also been epo ed in his coun y (Ga cía, Núñez, Po illo & O eo, 2015;
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Gue e o-Espejo, Muñoz-Pa ada & Tomás-Dols, 2017).
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The Medi e anean ecosys ems o sou he n Spain p esen speci ic cha ac e is ics ha may
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in luence he epidemiology o ick-bo ne disease (TBD). Fi s , he e is a a ie y and abundance
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o hose popula ions o wild ungula e species, such as ed dee , allow dee (Dama dama), oe
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dee , mou lon (O is a ies musimon) and wild boa (Palomo, Gisbe , & Blanco, 2007), ha can
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ac as po en ial hos s o di e en TBP. Second, hese wildli e species equen ly sha e habi a s
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wi h ce ain ick species and li es ock, which is a o ed by he li es ock p oduc ion sys em in
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sou he n Spain, cha ac e ized by he p e alence o ex ensi e a ming (Eu opean Pa liamen ,
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2016). Finally, he clima ic condi ions o his egion, wi h low p ecipi a ion and d y summe s,
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in luence he composi ion o ick popula ions and hence he epidemiology o TBD. In his
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ega d, he coun ies o he Medi e anean Basin lie along he limi s o he dis ibu ion ange o
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a numbe o ick species such as I. icinus which a e compe en ec o s o di e en TBP and
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a e highly p e alen in no he n Spain and se e al Eu opean egions (EFSA, 2010). In addi ion,
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hese egions a e conside ed o be especially sensi i e o clima e change (Lionello & Sca ascia,
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2018), which may a ec u u e ends in he epidemiology o TBD (Semenza & Suk, 2018).
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Despi e his, he a ailable in o ma ion abou he ole o wild ungula es in he main enance o
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TBP in Spanish Medi e anean ecosys ems is limi ed, and mainly ocused on cen al and
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no he n egions (Ga cía-Pé ez e al., 2016; Ga cía-Sanma ín e al., 2007). The lack o epo s
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is an impo an limi a ion o TBP such as Anaplasma/Eh lichia spp., Babesia/Theile ia spp.
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and B. bu gdo e i sensu la o (s.l.), since high a es o in ec ion wi h some o hese ha e been
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epo ed in bo h domes ic and wild ungula es in neighbo ing a eas o no he n Spain and
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Po ugal (Ga cía-Pé ez, Ba andika, Opo o, Po edano, & Jus e, 2003; Ga cía-Pé ez e al., 2016;
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Ga cía-Sanma ín e al., 2007; Nago e, Ga cía-Sanma ín, Ga cía-Pé ez, Jus e & Hu ado,
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2004; Na anjo e al., 2006; Pe ei a e al., 2016; Sil a, Ma ques & Oli a, 2010). The e o e, he
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aims o he p esen s udy we e: 1) o de e mine he occu ence o selec ed TBP
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(Anaplasma/Eh lichia spp., Babesia/Theile ia spp. and B. bu gdo e i s.l.) in wild ungula es,
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and 2) o assess he p esence o hese pa hogens in icks emo ed om TPB-in ec ed animals
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in Medi e anean ecosys ems in sou he n Spain.
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Ma e ials and Me hods
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Sampling and da a collec ion
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A o al o 1,132 spleen samples om wild ungula e species, including ed dee (n = 578), wild
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boa (n = 269), mou lon (n = 135), allow dee ) (n = 121) and oe dee (n = 29) we e collec ed
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on 53 hun ing es a es in sou he n Spain (Figu e 1). The animals we e legally hun ed du ing he
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hun ing seasons (Oc obe o Ma ch) om 2009 o 2015. Ha es ed animals we e collec ed
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oge he a he same loca ion on each o he hun ing es a es whe e isual inspec ion and
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sampling we e ca ied ou . Da a on species, sex age, hun ing es a e sampled and sampling yea
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we e eco ded o each animal, whene e possible. De e mina ion o age (yea lings: < 1 yea
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old; sub-adul s: be ween 1 and 3 yea s old; adul s: > 3 yea s old) was pe o med acco ding o
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he me hod desc ibed by Sáenz de Bu uaga, Lucio-Cale o & Pu oy-I aizoz (2001). Sampled
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animals we e inspec ed in si u o he p esence o icks. A ho ough examina ion o he skin o
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he animals was ca ied ou , paying special a en ion o he p e e ed eeding si es o icks ound
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he ea s, head, neck and en al su ace o he sampled animals. Ticks we e kep in plas ic ubes
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pe meable o ai un il hei a i al in he labo a o y. Spleen samples and icks we e s o ed a -
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20 ºC un il u he analysis. The icks we e iden i ied on he basis o mo phology using
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axonomic keys (Es ada-Peña, Boua ou , Camicas & Walke , 2004). The eeding s a us o
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collec ed icks was unknown. An epidemiological ques ionnai e was comple ed ia on-si e
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in e iews wi h gamekeepe s in each hun ing es a e o assess he in luence o di e en
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explana o y a iables including p esence o ences, p esence o li es ock (goa s, ca le, sheep,
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ho ses and swine), p esence o i e , s agnan wa e s, epopula ions o wild ungula e species in
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he las wo yea s, p esence o Medi e anean o es , pinewood and moun ain pas u es,
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es ima ed densi y o ed dee and wild uminan s (high: > 30 indi iduals/km2; medium: be ween
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20 and 30 indi iduals/km2 and low: < 20 indi iduals/km2). Densi y cu -o s we e se in
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acco dance wi h he op imal densi y alues ecommended o he Medi e anean ecosys ems o
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sou he n Spain o ensu e sus ainabili y o game species and conse a ion o biological di e si y
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(Pe ea, Gi a dello & San Miguel, 2014). In addi ion, clima ological a iables (con inuous
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a iable) including a e age empe a u e (ca ego ized using Jenk’s na u al b eaks), ela i e
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humidi y (ca ego ized using Jenks na u al b eaks) and ain all (ca ego ized by e ciles) o he
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season in which each sampling was ca ied ou we e e ie ed om public da abases (RIA,
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2020). The se o a iables collec ed is showed in Supplemen a y Table 1.
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DNA ex ac ion and PCR ampli ica ion
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DNA om spleen samples was ex ac ed o de e mine he p esence o TBP. Ticks collec ed
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om TBP-posi i e animals we e also analyzed o s udy he ela ionship be ween TBP s a us in
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hos and po en ial ec o s. DNA was ex ac ed using he comme cial DNA Genomic ki (A&A
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Bio echnology Gdańsk, Poland), ollowing he manu ac u e ’s ins uc ions. PCR analyses we e
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pe o med o de ec in ec ion wi h Anaplasma/Eh lichia spp., Babesia/Theile ia spp. and B.
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bu gdo e i s.l. The PCR p o ocols we e ca ied ou as desc ibed p e iously (Supplemen a y
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Table 2). A. phagocy ophilum and B. bu gdo e i s.l. DNA ob ained om he Na ional
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Re e ence Cen e o Bo elia o he Max on Pe enko e Ins i u e, as well as Babesia canis
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EU622792 DNA and Babesia/Theile ia DNA ob ained om a p e ious s udy (Adaszek e al.,
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2012) we e used as posi i e con ols. S e ile nuclease- ee wa e was used as a nega i e con ol.
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The size o each PCR p oduc was analyzed by elec opho esis in 1.5 % aga ose gels s ained
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wi h e hidium b omide.
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TBP species iden i ica ion was based on an analysis o he nucleo ide sequences o he ob ained
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amplicons. DNA pu i ica ion was pe o med using he comme cial QIAquick PCR pu i ica ion
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ki (Qiagen, Hilden, Ge many). In he case o ick samples, he ec opa asi es we e washed wice
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in s e ile PBS solu ion o 5 min, shaken slowly, and hen kep o e nigh a 4 °C. Each ick was
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manually cu in o ou pieces wi h a s e ile lance and hen suspended in 100 μl o T is, 500 μl
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o lysis bu e , and 20 μL o P o einase K p o ided by he comme cial ki . The emaining s eps
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o he pu i ica ion o samples om wild ungula es and icks we e pe o med ollowing he
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manu ac u e ’s ecommenda ions. Finally, DNA ob ained om wild ungula es and ick samples
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was elu ed in 50 μl o T is10 mM, pH 7.6 and 10 μL o elu ion bu e espec i ely and s o ed
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a -20 °C o subsequen analysis. A olume o 5 μL o pu i ied DNA was used in PCR
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eac ions.
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Nucleo ide sequences we e assembled and edi ed using SeqMan (DNAS a , Lase gene, USA)
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and MegAlign (DNAS a , Lase gene, USA) wi h alignmen s o ep esen a i e sequences o 16S
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RNA gene o Anaplasma spp. and Bo elia spp. and 18S RNA gene o Theile ia spp. and
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Babesia di e gens. Phylogene ic ees we e cons uc ed using he Neighbo -Joining me hod
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(boo s ap analysis o 1000 eplica es) wi h MEGA so wa e e sion 7 (Kuma , S eche &
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Tamu a, 2016). The ees a e d awn o scale wi h b anch leng hs measu ed as he numbe o
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subs i u ions pe si e. Only boo s ap alues ≥ 70 a e shown.
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S a is ical analysis
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The p e alence o in ec ions wi h A. phagocy ophilum, pi oplasms (Babesia/Theile ia spp.) and
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B. bu gdo e i s.l. was es ima ed om he p opo ion o posi i es o he o al numbe o
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samples, wi h exac binomial con idence in e als o 95% (95%CI). Sample collec ion was
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di ided in o h ee consecu i e pe iods: 2009-2011, 2011-2013 and 2013-2015. Di e ences
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be ween he p e alence o TBP in ec ion and explana o y a iables we e analyzed using a
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Pea son’s chi-squa e es o Fishe ’s exac es , as app op ia e. We e alua ed wo dependen
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a iables: p e alence o Anaplasma spp. and p e alence o Babesia/Theile ia spp. P e alence
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o B. bu gdo e i s.l. was no inally assessed because o he numbe o posi i es de ec ed was
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low. All he explana o y a iables (Supplemen a y Table 1) we e analyzed and hose wi h P-
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alue < 0.20 we e selec ed as po en ial isk ac o s. C ame ’s V coe icien be ween pai s o
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a iables was compu ed o assess collinea i y. When collinea i y (V > 0.6) occu ed, only he
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a iable mos clea ly linked o in ec ion was e ained. The selec ed a iables we e included in
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penalized maximum likelihood mul iple logis ic eg ession models o de ec s a is ical
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associa ions con olling o po en ial issues due o sepa a ion. An ini ial model was ob ained
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using all he selec ed explana o y a iables and a iables wi h a non-signi ican P- alue we e
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emo ed sequen ially. Model selec ion was assessed using he Aikake’s in o ma ion c i e ion
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(AIC). Va iables ha al e ed he coe icien s o he independen a iables o in e es by 30% o
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mo e when emo ed om he model we e classi ied as con ounding ac o s. The model was e-
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un un il all emaining a iables p esen ed s a is ically signi ican alues (P < 0.05) and he
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be e AIC. A ecei e ope a ing cha ac e is ic (ROC) cu e was also used o e alua e he
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accu acy o he inal models. S a is ical analysis was pe o med using S a a 14.0 so wa e
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(S a aCo p, Texas, USA).
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Resul s
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In ec ion wi h he selec ed TBP was de ec ed in 127 o 863 wild uminan s (14.7%; 95%CI:
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12.4-17.3) bu no in wild boa (0/269). A o al o 79 wild uminan s we e posi i e o
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Anaplasma/Eh lichia spp. (9.2%; 95%CI: 7.3-11.3), 42 o Babesia/Theile ia spp. (4.9%;
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95%CI: 3.5-6.5) and six o B. bu gdo e i s.l. (0.7%; 95%CI: 0.3-1.5) (Supplemen a y Table
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1). The equencies o in ec ions and iden i ica ion wi h he selec ed pa hogens a e shown in
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Table 1. A leas one in ec ed animal was de ec ed in 33 (66.0%) o he 50 sampled hun ing
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es a es (Figu e 1, Supplemen a y Tables 3-6).
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All he s ains o Anaplasma/Eh lichia spp. de ec ed we e iden i ied as A. phagocy ophilum,
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wi h 99.0–100 % sequence iden i y wi h he e e ence s ain. The pe cen age o sequence
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iden i y was lowe o o he Anaplasma species: 98.0% o A. pla ys, 97.0% o A. cen ale and
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A. ma ginale, and 96.0% o A. o is (Supplemen a y Figu e 1). Wi h espec o he pi oplasms,
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Babesia di e gens in ec ion was iden i ied in 25 animals (24 ed dee and one allow dee ) wi h
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98.0-99.0% sequence iden i y wi h lowe pe cen ages o iden i y wi h o he pi oplasms (78.0%
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wi h T. cap eoli, 78.0% wi h Theile ia sp. OT3, 79.0% wi h T. equi, and 77.0% wi h T. o is).
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Theile ia cap eoli was only ound in wo ed dee (99.5% sequence iden i y). Theile ia
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sequences highly iden ical (97.1 o 99.7%) o Theile ia spp. OT3 AY533145 we e ound in 15
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samples om mou lons and p esen ed lowe iden i y wi h o he pi oplasms (91.0% wi h T.
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cap eoli and T. o is, 86.0% wi h T. equi, and 74.0% wi h B. di e gens) (Table 1, Supplemen a y
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Figu e 2). In he six B. bu gdo e i s.l.-posi i e ce ids ( i e ed dee and one allow dee ), all
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hese samples we e iden i ied as Bo elia a zelii wi h sequence iden i y alues ha anged om
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99.4 o 100%. The sequences o A. phagocy ophilum, Babesia spp., Theile ia spp. and B. a zelii
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ob ained in he p esen s udy (n = 127) we e deposi ed in he GenBank da abase (GenBank
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Accession Numbe s: MT614368-MT614446, MT622544-MT622568, MT622569-MT622584
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and MW054193, and MW054157-MW054162 espec i ely). Thei phylogene ic ela ionships
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wi h e e ence s ains a e shown in he Supplemen a y Figu es 2-4. Co-in ec ions wi h A.
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phagocy ophilum and B. di e gens, and A. phagocy ophilum and Theile ia spp. we e de ec ed
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in one ed dee and ou mou lons, espec i ely.
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Mul iple eg ession models showed ha equency o in ec ion wi h A. phagocy ophilum and
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pi oplasms was associa ed only wi h species o in ec ed hos s. Anaplasma phagocy ophilum
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in ec ion was signi ican ly lowe in ed dee , oe dee and wild boa han in mou lon, al hough
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Su eillance p og ams should also be implemen ed o cha ac e ize he isk o TBD ansmission
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in his coun y. This may also be impo an in coun ies in he Medi e anean Basin ha p esen
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simila ecological a ia ions. In addi ion o his, u he esea ch is wa an ed o assess he
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p e alence o o he TBP no included in he p esen s udy in wild ungula es in sou he n Spain.
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Acknowledgemen s
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This wo k has bene i ed om he inancial aid o esea ch g an s unded by Spanish Minis y
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o Economy and Compe i i eness (AGL2013-49159-C2-2-R). We also wan o hank he
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collabo a ion o all in ol ed hun ing s a es and game ese es and he dedica ed assis ance o
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hei game wa dens, as well as o many colleagues and ellow s uden s who pa icipa ed in he
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ield sampling.
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Con lic o in e es
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The au ho s decla e ha hey ha e no compe ing in e es s. The au ho s ce i y ha hey ha e
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no a ilia ion wi h o inancial in ol emen in any o ganiza ion o en i y wi h a di ec inancial
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in e es in he subjec ma e o ma e ials discussed in he manusc ip .
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E hical app o al
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No animals we e killed speci ically o his s udy. Samples used in his s udy we e collec ed
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om animals legally hun ed in comple e ag eemen wi h Spanish and Eu opean egula ions. No
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e hical app o al by an Ins i u ional Animal Ca e and Use Commi ee was deemed necessa y.
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P o ocols, amendmen s and o he esou ces we e used acco ding o he guidelines app o ed by
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each Au onomous go e nmen ollowing he R.D.1201/2005 o he Spanish Minis y o
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P esidency.
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Da a A ailabili y S a emen
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The da a ha suppo he indings o his s udy a e a ailable om he au ho s upon easonable
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eques
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Re e ences
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Jou nal o T opical Medicine and Hygiene, 81, 67–74.
628
h ps://doi.o g/10.4269/aj mh.2009.81.67
629
24
Valcá cel, F., González, J., González, M. G., & Sánchez, M. (2020). Compa a i e Ecology o
630
Hyalomma lusi anicum and Hyalomma ma gina um Koch, 1844 (Aca ina : Ixodidae).
631
Insec s, 11, 303–321. h ps://doi.o g/10.3390/insec s11050303
632
Vayssie -Taussa , M., Cosson, J. F., Degeilh, B., Eloi , M., Fon ane , A., Mou aille , S., …
633
Zylbe mann, P. (2015). How a mul idisciplina y “One Heal h” app oach can comba he
634
ick-bo ne pa hogen h ea in Eu ope. Fu u e Mic obiology, 10, 809–818.
635
h ps://doi.o g/10.2217/ mb.15.15
636
Vázquez, M.E., Pego-Reigosa, R., Díez-Mo ondo, C., Cas o-Gago, M., Díaz, P., Fe nández,
637
G., & Mo ondo, P. (2015). Epidemiology o Lyme disease in a heal hca e a ea in no h-
638
wes Spain, Gace a Sani a ia, 29, 213-216.
639
h ps://doi.o g/10.1016/j.gace a.2015.01.008. [A icle in Spanish]
640
Zane , S., T isciuoglio, A., Bo e o, E., Fe nández-De-Me a, I. G., Go aza , C., Ca pignano,
641
M. G., & Fe oglio, E. (2014). Pi oplasmosis in wildli e: Babesia and Theile ia a ec ing
642
ee- anging ungula es and ca ni o es in he I alian Alps. Pa asi es and Vec o s, 7, 1–
643
7. h ps://doi.o g/10.1186/1756-3305-7-70
644
Zeman, P., & Pecha, M. (2008). Seg ega ion o gene ic a ian s o Anaplasma phagocy ophilum
645
ci cula ing among wild uminan s wi hin a Bohemian o es (Czech Republic).
646
In e na ional Jou nal o Medical Mic obiology, 298, 203–210.
647
h ps://doi.o g/10.1016/j.ijmm.2008.03.003
648
649
650
651
25
Table legends
652
Table 1. P e alence o selec ed ick-bo ne pa hogens in wild ungula es (n = 1,132) in sou he n
653
Spain.
654
Table 2. Resul s o he penalized eg ession models o he associa ion wi h he p esence o
655
DNA o Anaplasma spp. (Anaplasma phagocy ophillum) and pi oplasms (Babesia/Theile ia
656
spp.) in wild ungula es.
657
Table 3. Dis ibu ion o TBP in icks (n = 89) emo ed om TBP-posi i e wild uminan s (n =
658
31) sampled in sou he n Spain. The numbe o sampled animals wi hin each sampling poin is
659
shown in b acke s.
660
Figu e legend:
661
Figu e 1. Spa ial dis ibu ion o ick-bo ne pa hogens in wild ungula e species in sou he n
662
Spain. The numbe o sampled animals in each loca ion is shown in b acke s.
663
664
32
688
Supplemen a y Table 2. P ime s used in PCR o de ec ion and iden i ica ion o
689
Anaplasma/Eh lichia spp., Bo elia bu gdo e i sensu la o and Babesia/Theile ia spp.
690
691
692
†Fo his PCR p o ocol, we used a concen a ion o p ime o 25 μM ins ead o he 10
693
μM used by hese au ho s.
694
Re e ences
695
Adaszek, Ł., Winia czyk, S., Łukaszewska, J., (2009). A i s case o eh lichiosis in a ho se in
696
Poland. Deu sche Tie ä z liche Wochensch i . 116, 330-334
697
Adaszek, Ł., Ga cía-Bocaneg a, I., A enas-Mon es, A., Ca bone o, A., A enas, A., &
698
Winia czyk, S. (2012). Iden i ica ion o pi oplasms isola ed om asymp oma ic equine
699
species om sou he n Spain. Be line und Münchene Tie ä z liche Wochensch i , 125,
700
509-512.
701
Al ay, K., Aydin, M.F., Dumanli, N., & Ak as, M. (2008). Molecula de ec ion o Theile ia and
702
Babesia in ec ions in ca le. Ve e ina y Pa asi ology, 158, 295–301.
703
h ps://doi.o g/10.1016/j. e pa .2008.09.025.
704
705
Pa hogen
P ime s
Ta ge gene
Amplicon size
Re e ence
Anaplasma/Eh lichia spp.
EHR 521: (5′-TGT AGG
CGG TTC GGT AAG TTA
AAG-3′)
EHR 747: (5′-GCA CTC
ATC GTT TAC AGC
GTG-3′)
16S
247 bp
Adaszek,
Winia czyk &
Lukaszewska,
(2009);
Hodzic e al.,
(1998)
Bo elia bu gdo e i s. l.
M1: (5′-ACG ATG CAC
ACT TGG TGT TAA-3′)
M2: (5′-TCC GAC TTA
TCA CCG GCA GTC A-3′)
16S
357 bp
Lee e al.,
(2019)†
Babesia/Theile ia spp.
RLB R2: (5’-CTA AGA
ATT TCA CCT CTG
ACAGT-3’)
RLB F2 (5’- GAC ACA
GGG AGG TAG TGA
CAAG-3’)
hype a iable
V4 egion o
he 18S
390-430 bp
Adaszek e al.,
(2012);
Al ay, Aydin,
Dumanli &
Ak as, (2008)

33
Hodzic, E., Fish, D., Ma e zki, C.M., De Sil a, A.M., Feng, S., & Ba hold, S.W. (1998).
706
Acquisi ion and ansmission o he agen o human g anulocy ic eh lichiosis by Ixodes
707
scapula is icks. Clinical Jou nal o Mic obiology, 36, 3574–3578.
708
Lee S.H., Healy J.E., & Lambe J.S. (2019). Single Co e Genome Sequencing o De ec ion o
709
bo h Bo elia bu gdo e i Sensu La o and Relapsing Fe e Bo elia Species.
710
In e na ional Jou nal o En i onmen al Resea ch and Public Heal h. 16, 1779
711
712
713
34
Supplemen a y Table 3. Dis ibu ion o wild ungula es posi i e o Anaplasma
714
phagocy ophilum in ec ion in sou he n Spain.
715
ID
Species
Hun ing
es a e
P o ince
Sampling
hun ing season
Sequence
g oups†
GenBank Accession
numbe
1061
Fallow dee
1
Cadiz
2009-2010
A
MT614368
1062
Fallow dee
1
Cadiz
2009-2010
A
MT614369
1074
Fallow dee
1
Cadiz
2010-2011
A
MT614370
1063
Mou lon
1
Cadiz
2009-2010
B
MT614371
1064
Mou lon
1
Cadiz
2009-2010
B
MT614372
1075
Mou lon
1
Cadiz
2010-2011
B
MT614373
1076
Mou lon
1
Cadiz
2010-2011
B
MT614374
1077
Fallow dee
2
Cadiz
2010-2011
I
MT614427
1065
Roe dee
3
Cadiz
2009-2010
C
MT614407
1070
Red dee
4
Co doba
2009-2010
C
MT614381
1071
Red dee
4
Co doba
2009-2010
C
MT614382
1082
Red dee
5
Co doba
2011-2012
C
MT614383
1083
Red dee
5
Co doba
2011-2012
C
MT614384
1069
Red dee
6
Co doba
2009-2010
D
MT614408
1092
Red dee
9
Co doba
2011-2012
F
MT614412
1093
Red dee
9
Co doba
2011-2012
F
MT614413
1121
Red dee
10
Co doba
2014-2015
C
MT614385
1120
Red dee
10
Co doba
2014-2015
C
MT614406
1085
Red dee
11
Co doba
2011-2012
C
MT614386
1119
Red dee
12
Co doba
2014-2015
C
MT614387
1098
Mou lon
14
Co doba
2012-2013
B
MT614375
1099
Mou lon
14
Co doba
2012-2013
B
MT614376
1100
Mou lon
14
Co doba
2012-2013
B
MT614377
1088
Red dee
14
Co doba
2011-2012
C
MT614388
1089
Red dee
14
Co doba
2011-2012
C
MT614389
1090
Red dee
14
Co doba
2011-2012
C
MT614390
1091
Red dee
14
Co doba
2011-2012
C
MT614391
1094
Red dee
15
Co doba
2012-2013
C
MT614392
1125
Red dee
15
Co doba
2014-2015
C
MT614393
1073
Red dee
16
Co doba
2010-2011
C
MT614394
1123
Red dee
16
Co doba
2014-2015
C
MT614395
1124
Red dee
16
Co doba
2014-2015
C
MT614396
1095
Mou lon
17
Co doba
2012-2013
B
MT614378
1096
Mou lon
17
Co doba
2012-2013
B
MT614379
1097
Mou lon
17
Co doba
2012-2013
B
MT614380
1108
Red dee
17
Co doba
2013-2014
G
MT614414
1116
Mou lon
17
Co doba
2013-2014
G
MT614415
1110
Mou lon
17
Co doba
2013-2014
G
MT614416
1111
Mou lon
17
Co doba
2013-2014
G
MT614417
1112
Mou lon
17
Co doba
2013-2014
G
MT614418
1113
Mou lon
17
Co doba
2013-2014
C
MT614419
35
1114
Mou lon
17
Co doba
2013-2014
G
MT614420
1115
Mou lon
17
Co doba
2013-2014
G
MT614421
1117
Mou lon
17
Co doba
2013-2014
G
MT614422
1118
Mou lon
17
Co doba
2013-2014
G
MT614423
1109
Mou lon
17
Co doba
2013-2014
H
MT614424
1128
Red dee
17
Co doba
2014-2015
H
MT614425
1129
Red dee
17
Co doba
2014-2015
H
MT614426
1068
Red dee
18
Co doba
2009-2010
D
MT614442
1078
Red dee
19
Co doba
2011-2012
C
MT614397
1079
Red dee
19
Co doba
2011-2012
C
MT614398
1130
Red dee
20
Co doba
2014-2015
C
MT614399
1066
Red dee
22
Co doba
2009-2010
I
MT614429
1067
Red dee
22
Co doba
2009-2010
I
MT614430
1126
Red dee
23
Co doba
2014-2015
C
MT614401
1084
Red dee
24
Co doba
2011-2012
C
MT614402
1127
Red dee
25
Co doba
2014-2015
I
MT614431
1086
Red dee
26
Co doba
2011-2012
C
MT614403
1087
Red dee
26
Co doba
2011-2012
C
MT614404
1106
Red dee
26
Co doba
2013-2014
I
MT614432
1107
Red dee
26
Co doba
2013-2014
I
MT614433
1056
Red dee
27
Co doba
2009-2010
C
MT614405
1101
Red dee
27
Co doba
2013-2014
E
MT614409
1102
Fallow dee
27
Co doba
2013-2014
I
MT614434
1103
Fallow dee
27
Co doba
2013-2014
I
MT614435
1104
Fallow dee
27
Co doba
2013-2014
I
MT614436
1105
Fallow dee
27
Co doba
2013-2014
I
MT614437
1057
Fallow dee
27
Co doba
2009-2010
J
MT614438
1058
Fallow dee
27
Co doba
2009-2010
J
MT614439
1059
Fallow dee
27
Co doba
2009-2010
J
MT614440
1060
Fallow dee
27
Co doba
2009-2010
J
MT614441
1122
Fallow dee
27
Co doba
2014-2015
E
MT614444
1131
Fallow dee
27
Co doba
2014-2015
E
MT614445
1132
Fallow dee
27
Co doba
2014-2015
E
MT614446
1080
Red dee
28
Huel a
2011-2012
F
MT614410
1081
Red dee
28
Huel a
2011-2012
F
MT614411
1072
Red dee
29
Jaen
2009-2010
D
MT614443
1054
Red dee
31
Se illa
2009-2010
C
MT614400
1055
Red dee
31
Se illa
2009-2010
I
MT614428
†Sequences wi h 100% nucleo ide iden i y we e ga he ed in he same g oup (A-J).
716
717
718
36
Supplemen a y Table 4. Dis ibu ion o wild ungula es posi i e o Babesia di e gens in ec ion
719
in sou he n Spain
720
ID
Species
Hun ing
es a e
P o ince
Sampling
hun ing season
Sequence
g oups†
GenBank Accession
numbe
233
Red dee
1
Cadiz
2009-2010
A
MT622550
234
Red dee
1
Cadiz
2009-2010
A
MT622551
302
Red dee
1
Cadiz
2010-2011
C
MT622545
238
Red dee
6
Co doba
2009-2010
A
MT622553
788
Red dee
7
Co doba
2014-2015
A
MT622566
236
Red dee
8
Co doba
2009-2010
C
MT622544
1119
Red dee
12
Co doba
2014-2015
C
MT622548
686
Red dee
13
Co doba
2012-2013
C
MT622547
586
Red dee
16
Co doba
2011-2012
B
MT622555
756
Red dee
17
Co doba
2013-2014
B
MT622564
306
Red dee
18
Co doba
2010-2011
B
MT622554
593
Red dee
21
Co doba
2011-2012
A
MT622560
759
Red dee
21
Co doba
2013-2014
A
MT622565
237
Red dee
22
Co doba
2009-2010
A
MT622552
687
Red dee
25
Co doba
2012-2013
A
MT622561
688
Red dee
25
Co doba
2012-2013
A
MT622562
944
Red dee
25
Co doba
2014-2015
A
MT622568
590
Red dee
26
Co doba
2011-2012
B
MT622558
591
Red dee
26
Co doba
2011-2012
B
MT622559
755
Red dee
26
Co doba
2013-2014
B
MT622563
588
Red dee
26
Co doba
2011-2012
B
MT622556
818
Red dee
27
Co doba
2014-2015
A
MT622567
1028
Fallow dee
27
Co doba
2014-2015
B
MT622549
587
Red dee
28
Huel a
2011-2012
C
MT622546
589
Red dee
30
Se ille
2011-2012
A
MT622557
†Sequences wi h 100% nucleo ide iden i y we e ga he ed in he same g oup (A-J).
721
722
723
37
Supplemen a y Table 5. Dis ibu ion o wild ungula es posi i e o Theile ia spp. in ec ion in
724
sou he n Spain
725
ID
Species
Hun ing
es a e
P o ince
Sampling
hun ing season
Sequence
g oups†
GenBank Accession
numbe
235
Mou lon
1
Cadiz
2009-2010
A
MT622570
303
Mou lon
1
Cadiz
2010-2011
A
MT622571
304
Mou lon
2
Cadiz
2010-2011
B
MT622572
305
Mou lon
2
Cadiz
2010-2011
B
MT622573
685
Red dee
13
Co doba
2012-2013
C
MT622569 ‡
681
Red dee
17
Co doba
2012-2013
D
MW054193 ‡
1116
Mou lon
17
Co doba
2013-2014
E
MT622582
1115
Mou lon
17
Co doba
2013-2014
E
MT622581
1117
Mou lon
17
Co doba
2013-2014
E
MT622583
1118
Mou lon
17
Co doba
2013-2014
E
MT622584
682
Mou lon
17
Co doba
2012-2013
E
MT622575
683
Mou lon
17
Co doba
2012-2013
E
MT622576
684
Mou lon
17
Co doba
2012-2013
E
MT622577
757
Mou lon
17
Co doba
2013-2014
E
MT622578
758
Mou lon
17
Co doba
2013-2014
E
MT622579
1012
Mou lon
20
Co doba
2014-2015
E
MT622580
592
Mou lon
26
Co doba
2011-2012
E
MT622574
†Sequences wi h 100% nucleo ide iden i y we e ga he ed in he same g oup (A-J). ‡T. cap eoli
726
s ains.
727
728
729
730

38
Supplemen a y Table 6. Dis ibu ion o wild ungula es posi i e o Bo elia a zelii in ec ion in
731
sou he n Spain
732
ID
Species
Hun ing
es a e
P o ince
Sampling
hun ing season
Sequence
g oups†
GenBank Accession
numbe
275
Fallow dee
1
Cadiz
2010-2011
A
MW054157
868
Red dee
16
Co doba
2014-2015
B
MW054159
1003
Red dee
20
Co doba
2014-2015
A
MW054162
934
Red dee
25
Co doba
2014-2015
A
MW054160
803
Red dee
27
Co doba
2014-2015
B
MW054158
955
Red dee
28
Jaen
2014-2015
B
MW054161
†Sequences wi h 100% nucleo ide iden i y we e ga he ed in he same g oup (A-J).
733
734
39
Supplemen a y Figu e 1. Phylogene ic ee o Anaplasma spp. (based on 245 n o 106
735
sequences) s ains ob ained in he p esen s udy and ep esen a i e sequences a ailable in
736
GenBank. Sequences ob ained o Anaplasma spp. in he p esen s udy (n = 79) a e showed in
737
bold. S ains ob ained om he same hos animal species ha showed 100% homology
738
we e g ouped ( he numbe o sequences wi hin each g oup is shown be ween b acke s). Each
739
s ain o g oup o s ains a e iden i ied by Anaplasma species/GenBank accession
740
numbe (s)/hos /sampling yea (s)/coun y, whene e possible.
741
742
40
Supplemen a y Figu e 2. Phylogene ic ees o Babesia di e gens (A) (n = 25)
743
and Theile ia spp. (B) s ains (n = 17) ob ained in he p esen s udy and ep esen a i e
744
sequences deposi ed in GenBank. Sequences ob ained in he p esen s udy a e in bold. The
745
analysis in ol ed 408 n o 39 sequences and 428 n o 33 sequences o Babesia di e gens
746
and Theile ia spp., espec i ely. Each s ain is iden i ied by pi oplasm
747
species/GenBank accession numbe /hos /sampling yea /coun y, whene e possible.
748
749
750
41
Supplemen a y Figu e 3. Phylogene ic ee o B. a zelii s ains (n = 6) ob ained in he p esen
751
s udy and ep esen a i e sequences deposi ed in GenBank. Sequences ob ained in he p esen
752
s udy a e in bold. The analysis in ol ed 357 n o 31 sequences o Bo elia spp. Each s ain is
753
iden i ied by species/GenBank accession numbe /hos /sampling yea /coun y, whene e
754
possible.
755
756
757