Sü he al. Pa asi es Vec o s (2021) 14:451
h ps://doi.o g/10.1186/s13071-021-04959-0
SHORT REPORT
Bac e icidal ac i i y o a ian complemen :
acon ibu ion ounde s and a ian-hos opism
o Lyme bo eliae
Vale ie Sü h1, Isabel Lopes de Ca alho2,3, Ma ia So ia Núncio2,3, Ana Cláudia No e2,4*† and Pe e K aiczy1*†
Abs ac
Complemen has been conside ed as an impo an ac o impac ing he hos –pa hogen associa ion o spi oche es
belonging o he Bo elia bu gdo e i sensu la o complex, and may play a ole in he spi oche e’s ecology. Bi ds a e
known o be impo an hos s o icks and in he main enance o bo eliae. Recen ield su eys and labo a o y ans-
mission s udies indica ed ha ce ain a ian species ac as ese oi hos s o di e en Bo elia species. Ne e heless,
ou cu en unde s anding o he molecula mechanisms de e mining hos opism o Bo elia is s ill in i s ledgling
s age. Conce ning he ole o complemen in a ian-hos opism, only a ew bi d species and Bo elia species ha e
been analysed so a . He e, we pe o med in i o se um bac e icidal assays wi h se um samples collec ed om ou
bi d species including he Eu opean obin E i hacus ubecula, he g ea i Pa us majo , he Eu asian blackbi d Tu dus
me ula, and he acing pigeon Columba li ia, as well as ou Bo elia species (B. a zelii, B. ga inii, B. alaisiana, and B.
bu gdo e i sensu s ic o). F om July o Sep embe 2019, ju enile wild bi ds we e caugh using mis ne s in Po ugal.
Racing pigeons we e sampled in a lo in Oc obe 2019. Independen o he bi d species analysed, all Bo elia species
displayed an in e media e se um- esis an o se um- esis an pheno ype excep o B. a zelii challenged wi h se um
om blackbi ds. This genospecies was e icien ly killed by a ian complemen , sugges ing ha blackbi ds se ed as
dead-end hos s o B. a zelii. In summa y, hese indings sugges ha complemen con ibu es in he a ian–spi o-
che e– ick in ec ion cycle and in Bo elia-hos opism.
Keywo ds: Spi oche es, Bo elia, Tick, Ixodes, Inna e immuni y, Immune e asion, Complemen , A ian, Bi ds, Hos
opism
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Like o he blood-bo ne pa hogens, spi oche es belonging
o he Bo elia bu gdo e i sensu la o (s.l.) complex ci cu-
la e in na u e in a complex ec o -hos ansmission cycle
wi hou causing symp oms in hei ese oi hos s [1]. To
da e, a leas six Bo elia species ha e been unambigu-
ously associa ed wi h human Lyme disease o bo eliosis
including B. ga inii, B. a zelii, B. bu gdo e i sensu s ic o
(s.s.), B. spielmanii, B. ba a iensis, and B. mayonii [2].
Conce ning he human pa hogenic po en ial o B. lusi-
aniae and B. alaisiana, only a ew cases ha e been
epo ed so a [2]. Appa en ly, Bo elia species de el-
oped ce ain means o su i e o p olonged imes in
hei hos s in a “silen ” o un ecognizable manne , like a
T ojan ho se, possibly leading o a long-las ing in ec ion
in hei hos s ha has a conside able impac on Bo elia
ecology. Complemen e asion is conside ed o be one o
he d i ing ac o s de e mining he hos ese oi com-
pe ence o Bo elia [2–4]. In con as o non- ese oi
o dead-end hos s like humans and ce ain e eb a es
Open Access
Pa asi es & Vec o s
*Co espondence: K aicz[email p o ec ed] .de; K aicz[email p o ec ed] .de
†Ana Cláudia No e and Pe e K aiczy ha e con ibu ed equally o his
s udy and sha e senio au ho ship
1 Ins i u e o Medical Mic obiology and In ec ion Con ol, Uni e si y
Hospi al, Goe he Uni e si y F ank u , F ank u , Ge many
2 Cen e o Vec o s and In ec ious Diseases D . F ancisco Cambou nac,
Na ional Ins i u e o Heal h Dou o Rica do Jo ge, Lisbon, Po ugal
Full lis o au ho in o ma ion is a ailable a he end o he a icle
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Sü he al. Pa asi es Vec o s (2021) 14:451
(e.g., dee ), ese oi hos s allow Bo elia o su i e and
mul iply in hei issues and, mo e impo an ly, a e capa-
ble o ansmi ing spi oche es o eeding icks, and hus
main aining he bac e ia in ci cula ion [1].
Conce ning a ian species, ese oi compe ence
has been expe imen ally p o en in se e al ansmis-
sion s udies [5–10] as well as in ield s udies in which
eeding la ae we e collec ed om a ian hos s [5, 11,
12]. Unde he assump ion ha anso a ial ansmis-
sion o B. bu gdo e i s.l. is e y a e in ixodid icks
[13], in ec ions in ed la ae collec ed om bi ds cap-
u ed in he ield ha e been used as a p oxy o ese -
oi hos compe ence. In addi ion, se um suscep ibili y
may also p o ide in o ma ion on po en ial e eb a e
ese oi hos s. S udies aiming a de e mining se um
suscep ibili y ha e been pe o med o se e al e e-
b a es including a ian, ep ilian, oden , and uminan
hos s ( e iewed in [2]). In e es ingly, da a ga he ed
om se um suscep ibili y s udies wi h pheasan s (Pha-
sianus colchicus), blackbi d (Tu dus me ula), seabi d
(unknown species), and quail (Co u nix sp.) indica ed
ha (i) B. alaisiana and B. ga inii a e mainly esis an
o a ian complemen , (ii) B. bu gdo e i s.s. displayed
an in e media e se um esis ance pheno ype, and (iii)
B. a zelii, B. ba a iensis, and B. lusi aniae we e la gely
suscep ible o a ian se a ( e iewed in [2] and [3]). These
indings e ealed a s ong co ela ion be ween he abil-
i y o a pa icula Bo elia species o esis comple-
men -media ed killing and i s capabili y o in ec and
su i e in he hos om whe e he se um o igina es.
Thus, oden -associa ed genospecies a e killed by a ian
se um whe eby a ian-associa ed genospecies a e highly
suscep ible o oden se um. Mo eo e , Passe i o mes
and gallinaceous bi d species appea ed o be capable o
se ing as ese oi s o B. ga inii, B. alaisiana, and B.
bu gdo e i s.s. (he ea e e e ed o as B. bu gdo e i),
bu no o B. a zelii, B. lusi aniae, B. japonica, and B.
ba a iensis.
The speci ic in ec ion pa e n suppo s he no ion ha
complemen is a d i ing ac o o hos opism o Lyme
bo eliae. To gain u he insigh in o he complemen -
hos associa ion pa e n, we pe o med se um-bac e-
icidal assays wi h a ian se a collec ed om ju eniles
o ou bi d species cap u ed in Po ugal: Eu opean
obin E i hacus ubecula (he ea e obin), g ea i
Pa us majo , Eu asian blackbi d Tu dus me ula (he e-
a e blackbi d), and acing pigeon Columba li ia and
ou Bo elia species (B. a zelii, B. ga inii, B. alaisiana,
and B. bu gdo e i). Ou pilo s udy also aimed o be -
e unde s and he indings on he ese oi compe ence
o a ian species o Lyme bo eliae [6, 7, 14], in pa icu-
la o hose Bo elia genospecies commonly ound in
Eu ope.
Wild obins (n = 14), g ea i s (n = 16), blackbi ds
(n = 11), and acing pigeons (n = 9) we e cap u ed wi h
mis ne s om July o Sep embe 2019 in Vale Soei o
(40°19′ N, 8°24′ W), Po ugal. We sampled blackbi d,
g ea i , and obin ju enile bi ds (ha ched in he p e i-
ous b eeding season). The sampled bi ds we e inged and
ca e ully inspec ed o eeding icks. Racing pigeons we e
sampled om a lo in An anhol (40°09′ N, 8°27′ W),
Coimb a, Po ugal. None o he bi ds we e in es ed wi h
eeding icks a he ime o cap u e. In addi ion, ca le
and goa se um we e included as con ols.
Blood was collec ed om he b achial ein in o ubes
con aining clo ac i a o (BD Mic o aine ubes, Bec on
Dickinson, Spain, ca alogue numbe 365967). The blood
was immedia ely placed in a cool box un il anspo o
he labo a o y whe e i was cen i uged o 10min a
14,000×g and 4°C wi hin a maximum o 9h om collec-
ion. The se um was ans e ed in o a new ial and o-
zen immedia ely a −80°C un il analyses.
Bo elia s ains B. a zelii FEM1 (skin isola e, Ge many),
B. ga inii G1 (CSF, Ge many), B. alaisiana VS116 ( ype
s ain, ick isola e, Swi ze land), B. bu gdo e i B31
(ATCC
® 35210, ick isola e, USA), and B. lusi aniae
PoHL (skin isola e, Po ugal) we e cul u ed un il mid-
exponen ial phase (5 × 107 spi oche es pe ml) a 33°C in
modi ied Ba bou -S oenne -Kelly (BSK-H) medium (Bio
& Sell GmbH, Feuch , Ge many).
The Bo elia s ains used in his s udy ha e been
selec ed o he ollowing easons: (i) hey ep esen he
mos equen ly isola ed genospecies in ixodid icks in
Eu ope [5, 7, 11, 15], and (ii) he suscep ibili y/ esis -
ance pa e n o human se um did no change in epea ed
s udies. Fo ins ance, B. a zelii FEM1 always displayed a
se um- esis an pheno ype, B. ga inii G1, B. alaisiana
VS116, and B. lusi aniae PoHL we e s ongly se um-
suscep ible, and B. bu gdo e i B31 could be ca ego ized
as in e media e se um- esis an [2]. In addi ion, he
mo ili y, iabili y, and mo phology o each s ain was
inspec ed by da k- ield mic oscopy o be su e ha only
highly iable spi oche es we e used in he expe imen s.
Fo sc eening o an i-Bo elia IgG in he collec ed
bi d samples, a highly sensi i e ELISA was used (An i-
Bo elia Plus VlsE ELISA IgG, EUROIMMUN, Lübeck,
Ge many) con aining whole cell ex ac s o B. a zelii, B.
ga inii, and B. bu gdo e i as well as ecombinan OspC
and VlsE. The p o ocol was pe o med acco ding o he
manu ac u e ’s ecommenda ion excep ha se a we e
dilu ed 1:20 p io es ing. Calib a o s, s anda ds, and
he se um samples we e added o he wells, and mic o-
i e pla es we e incuba ed o 30min a oom empe a-
u e. A e washing, an igen–an ibody complexes we e
de ec ed a e an incuba ion o 30min by using ho se-
adish pe oxidase (HRP)-conjuga ed goa an i-bi d IgG
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Sü he al. Pa asi es Vec o s (2021) 14:451
an ibody (abcam, Camb idge, UK) (dilu ion 1:50,000).
A e wa ds, he eady- o-use subs a e was added, and
he eac ions we e e mina ed a e an incuba ion pe iod
o 15min. The abso bance was measu ed a 450nm. The
inal in e p e a ion o he es esul s a e as ollows: al-
ues < 0.8 we e nega i e, alues be ween ≥ 0.8 < 1.1 a e
bo de line, and alues ≥ 1.1 we e conside ed posi i e.
Se um suscep ibili y o spi oche es o bi d se a was
assessed as p e iously desc ibed wi h sligh modi ica-
ions [16]. B ie ly, spi oche es g own a mid-loga i hmic
phase we e sedimen ed by cen i uga ion, esuspended in
500µl BSK medium, and coun ed by da k- ield mic os-
copy. Reac ion mix u es consis ing o 20 µl o highly
iable spi oche es (4 × 107 cells pe ml) and 20 µl o
a ian se um we e incuba ed a 38°C wi h gen le agi a-
ion (350 pm). The pe cen age o iable spi oche es was
de e mined by da k- ield mic oscopy a e 0, 3, and 6h.
Spi oche es in nine mic oscopy ields we e coun ed o
each ime poin pe s ain and se um sample using Glass-
ic slides 10 (KOVA In e na ional Inc., CA, USA). Due
o he small amoun o blood collec ed om he indi id-
ual bi d, each Bo elia species was es ed agains se um
samples o h ee di e en indi iduals o each bi d spe-
cies (excep blackbi d, n = 2). O no e, i e ou o se en
se um samples collec ed om blackbi ds showed hae-
molysis which leads o an agglu ina ion o spi oche es
immedia ely a e adding o he eac ion mix u e (da a
no shown). The espec i e se um esis ance pa e n was
de ined acco ding o he spi oche es’ mo ili y and classi-
ied as ollows: se um- esis an pheno ype, 75–100% o
iable spi oche es; in e media e se um- esis an pheno-
ype, 30–75% o iable spi oche es; se um-sensi i e phe-
no ype, 0–30% o iable spi oche es [17]. The pe cen ages
o iable spi oche es we e calcula ed by se ing he num-
be o mo ile bac e ia a 0h o incuba ion o 100%. Da a
we e isualized by using G aphPad P ism e sion 7.04.
Means a e p esen ed wi h s anda d de ia ion (SD).
To de e mine complemen ac i i y o he collec ed
se um samples, he ep ile-associa ed genospecies B.
lusi aniae was employed. Due o he limi ed numbe o
bi d se a, only a single se um sample om P. majo was
analysed. As u he con ols, all Bo elia s ains in es i-
ga ed we e also exposed o ca le and goa se a, and ia-
ble spi oche es we e coun ed a e 3 and 6h as desc ibed
abo e.
To u he assess o ole o complemen as a ac o
in ol ed in a ian-hos associa ion o Lyme bo eliae, we
in es iga ed blood samples om ju enile bi ds, includ-
ing wo indi idual blackbi ds, 12 g ea i s, 12 obins, and
six acing pigeons. In addi ion, all se um samples in es i-
ga ed we e sc eened o he p esence o an i-Bo elia IgG
an ibodies. None o he se um samples es ed we e an i-
Bo elia-posi i e excep wo se um samples collec ed
om blackbi ds, bo h o which we e conside ed bo de -
line (0.83 and 0.99, espec i ely).
Se um suscep ibili y es ing was conduc ed by coun -
ing iable spi oche es by da k- ield mic oscopy a e an
incuba ion pe iod o 0, 3, and 6h, as p e iously desc ibed
[16]. As demons a ed in Fig.1a,B.ga inii G1, B. alaisi-
ana VS116, and B. bu gdo e i B31 displayed a esis an
o an in e media e se um- esis an pheno ype o all bi d
species in es iga ed (mean pe cen age o iable spi o-
che es a e 6h o incuba ion: 75 ± 15%, ange = 42–97%).
The da a show ha B. a zelii FEM1 esis s complemen -
media ed killing when indi idual se um samples om
g ea i s and obins we e employed (mean pe cen age
o mo ile spi oche es a e 6h o incuba ion wi h g ea
i and obin se a: 84 ± 13%). By con as , when B. a zelii
FEM1 was exposed o se um samples o blackbi ds, an
a e age o 66 ± 7% spi oche es had been killed a e 3h
o incuba ion, and only 16 ± 9.6% o spi oche es su i ed
se um ea men a e 6h (Fig.1b).
O no e, all ou Bo elia species su i ed in he p es-
ence o se um collec ed om acing pigeons (85 ± 6.5%
o mo ile spi oche es a e 6h o incuba ion). The high-
es su i al a es a e 6h o incuba ion was obse ed o
B. ga inii G1 ea ed wi h blackbi d se um (94 ± 2.9%)
and B. a zelii FEM1 incuba ed wi h se um o he g ea i
(93 ± 8.8%) (Fig. 1b). Employing da k- ield mic oscopy,
only 6.5% o he cells o B. lusi aniae PoHL su i ed,
while 94% o he cells o B. bu gdo e i B31 esis com-
plemen -media ed killing a e 6h o incuba ion in he
p esence o g ea i se um (da a no shown). This ind-
ing indica es ha a ian complemen was s ill ac i e a e
blood collec ion and long- e m s o age. Fu he mo e, all
Bo elia s ains challenged wi h ca le and goa se um
we e e icien ly killed a e 6h o incuba ion (Fig.2) as
p e iously desc ibed [18, 19].
Emphasizing he po en ial impac o complemen as
an impo an ac o de e mining hos opism o Lyme
bo eliae, he e we analysed he se um suscep ibili y o
he ou main Bo elia genospecies in Eu ope o se a
collec ed om ju enile wild bi ds. Fo compa ison, and
apa om blackbi ds [3], we included g ea i s and ob-
ins, wo wild ubiqui ous bi d species o en in es ed wi h
ixodid icks [11, 20], and he acing pigeon as po en ial
hos o Lyme bo eliae. The compa a i e analyses o
ou pilo s udy la gely ei e a e p io indings ha a ian-
associa ed Bo elia species such as B. ga inii and B. al-
aisiana esis complemen -media ed killing o all se um
samples analysed [3, 4, 21]. B. bu gdo e i has p e iously
been shown o esis complemen -media ed killing by
a ian se um collec ed om pheasan s and quail, bo h
o which a e u ilized as model o ganisms o elucida -
ing hos associa ion o Lyme bo eliae [8, 21, 22, 23]. As
expec ed, g ow h o B. a zelii FEM1 was s ongly a ec ed
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Fig. 1 Se um suscep ibili y o Lyme bo eliae exposed o bi d se um samples. a Each genospecies and he espec i e a ian species was
independen ly challenged wi h se um samples om h ee indi idual bi ds, excep o he se a o Eu asian blackbi ds (Tu dus me ula, n = 2). b
Pe cen age o iable spi oche es a e 6 h o incuba ion wi h a ian se a. The da a ep esen s h ee independen expe imen s o each Bo elia
genospecies/bi d species combina ion
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Sü he al. Pa asi es Vec o s (2021) 14:451
by blackbi d se um as also demons a ed in p e ious
s udies in which pheasan s (Phasianus colchicus), black-
bi ds, and a seabi d we e examined [3]. In con as o
p e ious indings, ou da a e ealed o he i s ime ha
B. a zelii appea s o su i e in he p esence o se um o
obins, g ea i s, and acing pigeons, sugges ing a spe-
cies-speci ic hos associa ion/ opism o ce ain Bo elia
species as discussed mo e ecen ly o B. bu gdo e i and
B. ba a iensis [2, 24].
Al hough nume ous ield s udies and ansmission
expe imen s sugges ed ha bi ds including g ea i s and
obins play a limi ed o e en a negligible ole in main ain-
ing B. a zelii in ci cula ion [5, 6, 25], p e ious indings o
B. a zelii-in ec ed la ae and nymphs ha ed on bi ds
e ealed ha ce ain bi d species, including g ea i s
[11, 26], obins [14], dunnocks, and song h ushes [6],
migh se e as po en ial ese oi hos s o ansmi e s
o ce ain B. a zelii s ains and a ian s, espec i ely
[14, 15]. B. a zelii-in ec ed la ae could esul om co-
eeding o incomple e blood meal on ano he in ec ed
e eb a e hos [15]. In addi ion, Heylen e al. [6] ound
ha pa hogen- ee la al Ixodes icinus icks co- eeding
wi h B. a zelii-in ec ed I. icinus nymphal icks on nai e
blackbi ds did no become in ec ed wi h B. a zelii, bu
a low pe cen age o in ec ed la ae we e de ec ed when
his expe imen was pe o med in naï e g ea i s. These
Fig. 2 Bac e icidal e ec o con ol se a on he iabili y o Lyme bo eliae. Each Bo elia species was ea ed wi h se um samples collec ed om
ca le (n = 1) and goa (n = 1). Th ee independen echnical eplica es (E1 o E3) we e pe o med

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Sü he al. Pa asi es Vec o s (2021) 14:451
indings sugges ha a ian blood o naï e g ea i s and
blackbi ds was bac e icidal o pa icula B. a zelii s ains
o a ian s and, hus, p o ide addi ional e idence o a
s ain-speci ic hos associa ion. I is emp ing o specu-
la e ha he concep o a es ic ed genospecies-speci ic
hos associa ion o Lyme bo eliae (i.e., di ision in o
oden -associa ed, a ian-associa ed, and ep ile-associ-
a ed genospecies) has o be eadjus ed, in pa icula due
o ecen obse a ions e ealing mo e di e se ela ion-
ships o Lyme bo eliae wi h hei hos s in na u e as p e-
iously hough .
Conce ning B. ga inii and B. alaisiana, bo h geno-
species ha e p edomina ely been iden i ied in icks col-
lec ed om g ound-dwelling and mig a ing songbi ds
such as ee pipi s, blackbi ds, g ea i s, obins, and song
h ushes, e ealing a s ong associa ion o hese Bo elia
genospecies wi h dis inc a ian hos s [15]. In line wi h
hese indings, we showed ha B. ga inii and B. alaisi-
ana exhibi ed an in e media e se um- esis an o se um-
esis an pheno ype o he se a o all ou wild bi ds
es ed in ou s udy (Fig.1).
In con as o he Uni ed S a es whe e B. bu gdo e i
is he main Bo elia species causing Lyme disease [27,
28, 30, 31], he compe ence o bi ds as ese oi hos s
has no ully been elucida ed in Eu ope [29]. He e, we
showed ha B. bu gdo e i displayed ei he an in e me-
dia e se um- esis an ( obin) o a se um- esis an (black-
bi d, g ea i , and acing pigeon) pheno ype o a ian
se a (Fig.1). These indings a e also in close ag eemen
o he da a ob ained om p e ious se um bac e icidal
assays demons a ing ha B. bu gdo e i was pa ially
esis an o blackbi d, pheasan , and seabi d se a [3, 4]. In
summa y, ou da a sugges ha Bo elia-speci ic ac o s
along wi h he a ian inna e immune sys em con ibu es
o hos specia ion and compe ence as p oposed ecen ly
[2].
The acing pigeon is hough o ha e a negligible o
e en a mino ole in he B. bu gdo e i s.l. ansmission
cycle, as hose bi ds a e la gely main ained in cap i i y.
Ne e heless, ee-li ing pigeons a e known o ca y se -
e al zoono ic pa hogens [32] and, hus, may con ibu e
o he ci cula ion o ce ain pa hogens. Fu he in es i-
ga ions aiming a elucida ing he ese oi compe ence
o ee-li ing pigeons will undoub edly complemen ou
indings indica ing ha se um o pigeons did no exhibi
a bo eliacidal e ec on spi oche es; hus a po en ial ole
o his bi d species in B. bu gdo e i s.l. ansmission can-
no be comple ely discoun ed.
All se um samples analysed de i ed om bi ds ha
we e cap u ed in he wild, excep o he se a collec ed
om acing pigeons. Al hough none o he bi ds we e
in es ed wi h icks a he ime o cap u ing, we canno
comple ely ule ou ha hey ha e had p e ious con ac
wi h Bo elia-in ec ed icks. Howe e , hose we e ju e-
nile bi ds, bo n in he p e ious sp ing, and he ick ac i -
i y in his egion is ela i ely low du ing wa m and d y
summe mon hs [20], educing he p obabili y o con ac
o hese bi ds wi h ques ing icks. In addi ion, se ological
es s con i med ha none o he se a in es iga ed we e
posi i e o an i-Bo elia IgG an ibodies excep he wo
samples collec ed om blackbi ds showing a bo de line
esul . O no e, B. a zelii FEM1 also displayed a se um-
sensi i e pheno ype when an an i-Bo elia-nega i e
blackbi d sample was used o he bac e icidal assays
(da a no shown). This inding sugges ed ha B. a zelii
FEM1 was p ima ily killed by he ac i a ion o he al e -
na i e pa hway, bu no h ough an an ibody-dependen
complemen ac i a ion ia he classical pa hway. Also,
he da a o he se um bac e icidal assays p esen ed he ein
e ealed a high consis ency when di e en indi idual
se a we e es ed wi h a gi en Bo elia species, sugges ing
ha hose bi ds we e likely naï e.
Al hough se um complemen is a limi ing ac o
a ec ing Bo elia su i al in he ick and di ec ly a e
ansmission o he e eb a e hos , he in e ences on
e eb a e ese oi compe ence should ake in o con-
side a ion addi ional ac o s such as he he e ogenei y o
p o eins in e ac ing wi h he hos immune sys em among
indi idual Bo elia s ains as well as hos -speci ic ac-
o s like he physiological s a e and age o he bi ds ha
also may a ec he hos ’s compe ence. Mo eo e , gene ic
polymo phism among a ian hos popula ions canno be
excluded because co-e olu ion be ween hos and spi o-
che es migh also con ibu e o a s ain-speci ic se um
suscep ibili y pa e n. In summa y, he da a p esen ed
he ein no only con i m p e ious indings bu add new
da a on he complexi y o a complemen -d i en hos o-
pism among Bo elia in na u e.
Acknowledgemen s
We hank Luís Pascoal da Sil a, Miguel A aújo, Hugo Ba bosa, and Miguel
Mendonça o hei suppo wi h sample collec ion, and Ma ia Te esa Luz o
he suppo wi h an ibody es ing. We also hank Dania Rich e o p o iding
ca le and goa se um.
Au ho s’ con ibu ions
VS designed and pe o med expe imen s and analysed he da a ega ding
he se um bac e icidal assays. ILC designed he s udy, pe o med labo a o y
analyses, and analysed he da a. MSN pe o med labo a o y analyses and
analysed he da a. ACN designed he s udy, coo dina ed he esea ch, col-
lec ed bi d se um, analysed he da a, and w o e he o iginal d a o he pape .
PK designed and coo dina ed he esea ch, analysed da a, and w o e he main
ex . All au ho s ead and app o ed he inal manusc ip .
Funding
Open Access unding enabled and o ganized by P ojek DEAL. This s udy
ecei ed inancial suppo om Fundação pa a a Ciência e a Tecnologia by he
s a egic p og am o MARE (MARE—UID/MAR/04292/2020) and he ansi o y
no m con ac DL57/2016/CP1370/CT89 o ACN, and he Po uguese Na ional
Ins i u e o Heal h (INSA).
Page 7 o 7
Sü he al. Pa asi es Vec o s (2021) 14:451
A ailabili y o da a and ma e ials
The da ase s gene a ed du ing and/o analysed du ing he cu en s udy a e
a ailable om he co esponding au ho on easonable eques .
Decla a ions
E hics app o al and consen o pa icipa e
This s udy complied wi h he ARRIVE guidelines and was ca ied ou in acco d-
ance wi h he UK Animals (Scien i ic P ocedu es) Ac 1986 and associa ed
guidelines, EU Di ec i e 2010/63/EU o animal expe imen s. Samples we e
collec ed unde licence om compe en au ho i ies (ICNF, no. 4/2019).
Consen o publica ion
No applicable.
Compe ing in e es s
The au ho s decla e no compe ing in e es s.
Au ho de ails
1 Ins i u e o Medical Mic obiology and In ec ion Con ol, Uni e si y Hospi al,
Goe he Uni e si y F ank u , F ank u , Ge many. 2 Cen e o Vec o s and In ec-
ious Diseases D . F ancisco Cambou nac, Na ional Ins i u e o Heal h Dou o
Rica do Jo ge, Lisbon, Po ugal. 3 Ins i u e o En i onmen al Heal h (ISAMB), Lis-
bon, Po ugal. 4 Depa men o Li e Sciences, Facul y o Sciences and Technol-
ogy, MARE-Ma ine and En i onmen al Sciences Cen e, Uni e si y o Coimb a,
Calçada Ma im de F ei as, 3000-456 Coimb a, Po ugal.
Recei ed: 10 May 2021 Accep ed: 17 Augus 2021
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Publishe ’s No e
Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in pub-
lished maps and ins i u ional a ilia ions.