pa hogens
Communica ion
Se ological Su ey and Molecula Cha ac e iza ion o
Theile ia annula a in Sicilian Ca le
Vale ia Ga gano 1, Vale ia Blanda 1,*, Delia Gambino 1,* , F ancesco La Russa 1, Sophia Di Ca aldo 2,
An onino Gen ile 1, Gio gia Schi ò1, Alessand a To ina 1, Ja ie Millán3,4,5 and Domenico Vica i 1


Ci a ion: Ga gano, V.; Blanda, V.;
Gambino, D.; La Russa, F.; Di Ca aldo,
S.; Gen ile, A.; Schi ò, G.; To ina, A.;
Millán, J.; Vica i, D. Se ological
Su ey and Molecula
Cha ac e iza ion o Theile ia annula a
in Sicilian Ca le. Pa hogens 2021,10,
101. h ps://doi.o g/10.3390/
pa hogens10020101
Recei ed: 7 Janua y 2021
Accep ed: 20 Janua y 2021
Published: 21 Janua y 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Is i u o Zoop o ila ico Spe imen ale della Sicilia “A. Mi i”, Via Gino Ma inuzzi n. 3, 90129 Pale mo, I aly;
[email p o ec ed] (V.G.); [email p o ec ed] (F.L.R.); [email p o ec ed] (A.G.);
gio gia.schi [email p o ec ed] (G.S.); [email p o ec ed] (A.T.); [email p o ec ed] (D.V.)
2P og ama de Doc o ado en Medicina de la Conse ación, Facul ad de Ciencias de la Vida,
Uni e sidad And es Bello, San iago, Chile; m.dica aldohanna@uand esbello.edu
3Ins i u o Ag oalimen a io de A agón-IA2 (Uni e sidad de Za agoza-CITA), 50013 Za agoza, Spain;
[email p o ec ed]
4Fundación ARAID, 50018 Za agoza, Spain
5Facul ad de Ciencias de la Vida, Uni e sidad And es Bello, San iago, Chile
*Co espondence: [email p o ec ed] (V.B.); [email p o ec ed] (D.G.)
Abs ac :
T opical heile iosis is a ick-bo ne disease caused by hemop o ozoan pa asi es wi h
conside able e e ina y and economic impac wo ldwide. Ticks ansmi ing he disease belong
o he Haemaphysalis,Rhipicephalus, and Hyalomma gene a. The Hyalomma genus is e y common
in Sicily (I aly) and ep esen s he main Theile ia annula a ec o in he island. Da a conce ning
he molecula epidemiology o his pa hogen a e missing in he egion. In 2018–2019, blood and
se um samples we e collec ed om 480 cows in se en Sicilian a ms om ou di e en p o inces.
Se op e alence in he a ms anged om 22% o 71%. Th ee a ms we e selec ed o molecula
analysis consis ing o eal- ime PCR a ge ing he almos comple e 18S ibosomal RNA ( RNA).
Fou amplicons pe a m we e sequenced and phylogene ic analyses we e ca ied ou . The ou
sequences we e iden ical wi hin each a m and showed 92–99% iden i y wi h he o he a ms and
wi h sequences om Genbank. Acco ding o he phylogene ic analysis, hese h ee sequences and
an addi ional one om a labo a o y-cul u ed Theile ia annula a s ain ob ained in 1999 belonged o
a single T. annula a clade wi h good boo s ap suppo wi h o he sequences om I aly, India, and
I an, indica ing limi ed geog aphical and empo al gene ic a iabili y o he pa asi e. This s udy
ep esen s he i s phylogene ic analysis o T. annula a in Sicily, which will be use ul o imp o e he
s a egies o heile iosis con ol and p e en ion.
Keywo ds: Pi oplasmida; phylogene ic analysis; ick-bo ne pa hogens; 18S RNA; Sicily
1. In oduc ion
Pa asi es o he Theile ia genus (o de Pi oplasmida, amily Theile idae) in ec a wide
spec um o domes ic and wild animals and a e ansmi ed by Ixodidae icks belonging o
he Haemaphysalis,Hyalomma, and Rhipicephalus gene a [
1
]. In ecen yea s, he e has been a
g owing in e es in he diseases caused by Theile ia spp., also due o he inc eased accu acy
o he diagnos ic es s [
2
]. T opical heile iosis in ca le is caused by Theile ia annula a,
and i is cha ac e ized by leukocy e p oli e a ion ollowing pa hogen in ec ion [
3
]. O he
symp oms include e e , lymphadenopa hy, and anemia. I no ea ed, animals die wi hin
3–4 weeks o in ec ion [
4
]. This pa hology also causes a signi ican educ ion in animal
e ili y. In Sicily, heile iosis is one o he mos common ick-bo ne diseases wi h se e al
ou b eaks no iced each yea [
5
]. The cu en con ol measu es include he use o aca icides
o ec o con ol, he apy wi h bupa aquone, and accina ion [
6
]. Se e al pa hogen
molecules able o induce he hos immune esponses a e cu en ly unde in es iga ion as
candida e accine an igens [7]. In I aly, accine p ophylaxis agains opical heile iosis is
Pa hogens 2021,10, 101. h ps://doi.o g/10.3390/pa hogens10020101 h ps://www.mdpi.com/jou nal/pa hogens
Pa hogens 2021,10, 101 2 o 7
no ca ied ou since he cu en ly a ailable li e-a enua ed accine is no comme cialized
in Eu ope [
8
]. The success o he he apy depends on he imeliness o he execu ion o he
pha macological ea men [
9
]. Fo his eason, molecula biology ep esen s a e y e icien
diagnos ic app oach o con as he disease, since molecula me hods, such as eal- ime
PCR, allow apidly de ec ing he pa hogen and p omp ly ini ia ing con ol ac ions [10].
Mo eo e , he ad ances in sequence da a analysis allowed an imp o emen in pa hogen
iden i ica ion and cha ac e iza ion. Ribosomal RNA ( RNA) is he mos used gene ic
ma ke o phylogene ic analysis in euka yo ic o ganisms due o he conse ed unc ion
and s uc u e o he 18S RNA molecule. This gene has been sequenced om se e al
o ganisms, esul ing in a la ge da abase o sequence compa isons.
The ma ke 18S RNA gene is widely used o de e mining e olu iona y pa e ns and
simila i y among he T. annula a species due o he p esence o a conse ed and hype a i-
able egion (V4). Indeed, he molecule also possesses phylogene ically in o ma i e a iable
egions ha a e use ul o de e mining ela ionships among s ains [11].
T. annula a emains one o he mos common ick-bo ne pa hogens a ec ing domes ic
uminan s no only in I aly [
5
] bu in se e al coun ies o Eu ope [
12
,
13
]. In Sicily, Hyalomma
lusi anicum has been p oposed as he main T. annula a ec o in ca le, and his ick species
is ela i ely p e alen in ex ensi e ca le b eeding [
14
,
15
]. Al hough he p esence o T.
annula a in Sicily is well known, phylogene ic s udies conce ning T. annula a ha e been
ca ied ou in o he Eu opean coun ies bu no in I aly [16–18].
This s udy was aimed a he phylogene ic cha ac e iza ion o T. annula a s ains p esen
in ca le in Sicily o be e unde s and he epidemiology o he pa asi e on he island.
2. Resul s
O e all, se ological p e alence in he Sicilian ca le was 26.0% (95% con idence in e -
als = 31.7–41.5). A he a m le el, obse ed p e alence alues anged om 22% o 71%
(Table 1).
Table 1. Resul s o se ological analysis ob ained in his s udy. ID, iden i ie .
Fa m ID P o ince Sampled Animals Posi i e Animals P e alence
1 Ragusa 216 48 22%
2 Ragusa 53 52 23%
3 Si acusa * 101 51 50%
4 Ragusa 49 18 37%
5 Ragusa * 21 15 71%
6 Enna * 17 10 59%
7 Pale mo 24 8 33%
* Fa ms selec ed o he molecula s udy.
Acco ding o he se ological esul s, he h ee a ms wi h he highes p e alence alues
and showing a leas 10 animals se ologically posi i e o T. annula a we e selec ed o
molecula s udies (Fa ms 3, 5, and 6; Table 1).
Fo each o he h ee a ms, ou animals we e selec ed o he sequencing o he
almos comple e 18S RNA gene. Sequences ob ained om he animals o he same a m
we e iden ical o each o he and we e conside ed as a single oligonucleo ide sequence
(Sequences 2–4; Table 2). These h ee sequences showed 98–100% iden i y.
Mo eo e , a u he 18S RNA sequence, named sequence 1 (Seq1), was ob ained
om he T. annula a s ain, isola ed se e al yea s ea lie a ou ins i u e and main ained in
cul u e as desc ibed abo e. The ou ob ained sequences sha ed be ween 99.8% and 100%
nucleo ide sequence iden i y wi h sequences p e iously epo ed in GenBank (AY508463
and MN944852, espec i ely). Alignmen s o mul iple 18S RNA gene sequences e ealed
ou haplo ypes o T. annula a RNA. The phylogene ic analysis (Figu e 1) indica ed ha
Pa hogens 2021,10, 101 3 o 7
ou sequences we e classi ied in a single T. annula a clade wi h good boo s ap suppo
wi h o he sequences om I aly (FJ426369) and India and I an (MF287931 and KF429799).
Only sequence 3 oo ed apa om he o he sequences, indica ing a likely di e en o igin
om he o he s. This inding calls o u he in es iga ion on he pa icula i ies o his
a m.
Figu e 1. Phylogene ic ee ob ained in his s udy using he 18S ibosomal RNA ( RNA) gene sequences.
Table 2. De ails o Theile ia annula a sequences ob ained in his s udy.
Sequence
ID
GenBank Accession
Numbe
(This S udy)
P o enience P o ince
Numbe o
Sequences om
Posi i e Animals
GenBank Accession
Numbe o he
Closes Sequence
Sequence 1 MN944852 Cul u ed S ain Pale mo
(Sicily) 1 KF429799
Sequence 2 MT341815 Field S ain Ragusa
(Sicily) 4 MK849885
Sequence 3 MT341857 Field S ain Sy acuse
(Sicily) 4 KT367871
Sequence 4 MT341858 Field S ain Enna
(Sicily) 4 KT367868
3. Discussion
T. annula a emains one o he mos common ick-bo ne pa hogens a ec ing domes ic
uminan s in se e al Eu opean coun ies [
5
,
12
,
13
]. The obse ed se op e alences a e lowe
han hose epo ed in o he Medi e anean coun ies such as Tunisia o in o he coun ies
such as Sudan o I aq [
18
–
22
]. No wi hs anding his, he pa asi e is ac i ely ci cula ing in
he Sicilian ca le a ms. In his s udy, he 18S RNA gene pa ial sequences we e used o
e alua e he phylogene ic ela ionships o Sicilian T. annula a s ains wi h o he T. annula a
s ains, as well as wi h di e en Theile ia species. Al hough he p esence o T. annula a in
Sicily is well known, phylogene ic s udies conce ning T. annula a ha e been ca ied ou in
o he Eu opean coun ies bu no in I aly [14–18].
In his s udy, Sicilian sequences o med a unique clade close o sequences om
I aly, India, and I an, con i ming a molecula s udy conduc ed on a accine s ain in
I an [
23
]. In ha s udy, he 18S RNA gene sequence o he T. annula a I an accine
Pa hogens 2021,10, 101 4 o 7
s ain showed om 97.9% o 99.9% iden i y wi h s ains om China, Spain, and I aly.
Mo eo e , a p e ious s udy conce ning Theile ia species in small uminan s in I aly also
epo ed a close associa ion be ween he I alian Theile ia o is s ain and Theile ia spp. om
Namibia and I an [
11
]. These esul s may be he consequence o mo emen s o animals
and icks be ween hese coun ies, a hypo hesis u he s eng hened by he usual p ac ice
o ancien peoples o mig a ing om hese a eas in he pas , b inging wi h hem some a m
animals [
24
]. Fu he mo e, ou da a show ha he sequences ob ained om ield samples
a e no subs an ially di e en om he sequence ob ained om he s ain isola ed almos
20 yea s ago and cul u ed in ou labo a o y, sugges ing a low a e o gene ic a iabili y o
he pa hogens.
4. Ma e ials and Me hods
4.1. Field Sample Collec ion
F om Sep embe 2018 o Ma ch 2019, se en a ms we e in es iga ed o T. annula a
p esence. Fou a ms we e in he p o ince o Ragusa, one in he p o ince o Sy acuse, and
one each in he p o inces o Enna and Pale mo. All he selec ed a ms we e di ec ed o
he p oduc ion o milk and mea . Animals o each a m, acco ding o he egula ions in
o ce, a e housed and canno come in o con ac wi h he animals o o he a ms. E hylene-
diamine e aace ic acid (EDTA)- ea ed and un ea ed blood samples we e ob ained om
each animal. The se um was sepa a ed by cen i uga ion and s o ed wi h EDTA- ea ed
blood samples a −20◦C.
4.2. In Vi o Cul i a ion o T. annula a Schizon s
Pe iphe al blood mononuclea cells (PBMCs) we e isola ed om hepa inized blood
samples as desc ibed elsewhe e [
25
]. T. annula a schizon s om a posi i e blood sample
ob ained in he yea 1999 we e isola ed and main ained in ou labo a o y in Roswell
Pa k Memo ial Ins i u e (RPMI) 1640 medium added wi h 2 mM L-glu amine, 1% peni-
cillin/s ep omycin, and 10–20% e al bo ine se um (pH a 7.3) and incuba ed a 37
◦
C in
5% CO
2
. Once he cell concen a ion o 5–9
×
10
5
cells/mL was eached, he cul u e was
p opaga ed in esh soil.
4.3. Se ological Analysis
Samples aken om animals housed on he se en a ms we e subjec ed o se ological
analysis. The p esence o an ibodies agains T. annula a was assessed by immuno luo es-
cence using a p o ocol modi ied om Bu idge and Kimbe [26].
4.4. Molecula Analysis and Phylogene ic S udies
Fo molecula analysis, samples belonging o he h ee a ms wi h he highes p e a-
lence alues and showing a leas 10 animals se ologically posi i e o T. annula a we e
selec ed.
DNA was ex ac ed using he Pu eLink Genomic DNA ki (The mo Fishe Scien i ic,
Wal ham, MA, USA) acco ding o he manu ac u e ’s ins uc ions. DNA was ex ac ed
om he blood o ield samples and PBMCs in ec ed by T. annula a, and 200 ng o each
pu i ied genomic DNA was analyzed on 1% aga ose gel (da a no shown). Fu he mo e,
he A206/A280 a io was e alua ed o e i y he quali y o he ex ac ed DNA. Each sample
was ound sui able o u he analysis wi h an A206/A280 a io close o 2.0. T. annula a
DNA was de ec ed h ough eal- ime PCR [
27
]. Fo each posi i e a m, ou samples, wi h
a C alue below 28, we e selec ed o ca y ou he phylogene ic analysis.
4.5. Phylogene ic S udy
4.5.1. 18S RNA Gene Ampli ica ion and Sequencing
A po ion o he 18S RNA gene was ob ained wi h speci ic p ime s 18S TBF and
18S TBR as desc ibed elsewhe e [
28
]. The ampli ied p oduc o app oxima ely 1500 bp
was used o sequencing analysis. DNA sequences we e de e mined using he dideoxy
Pa hogens 2021,10, 101 5 o 7
chain e mina ion me hod wi h a comme cial DNA sequencing ki (BigDye
™
Te mina o
3.1 Cycle Sequencing Ki , Applied Biosys ems
™
(The mo Fishe Scien i ic, Wal ham,
MA, USA) acco ding o he manu ac u e ’s ins uc ions. The ob ained sequences we e
analyzed o nucleo ide sequence iden i y by compa ing hem wi h e e ence s ains in
he GenBank da abase using he Basic Local Alignmen Sea ch Tool (BLAST). Mul iple
sequence alignmen s we e pe o med using he Clus alW algo i hm. Simila i y ma ices
we e hen cons uc ed om aligned sequence da a by single dis ance using he Jukes and
Can o o he Kimu a wo-pa ame e model [
29
]. Phylogene ic ees we e cons uc ed
using he neighbo -joining and he maximum pa simony me hods as implemen ed in
he Mega 2.1 package, dele ing all he gap si es. Toxoplasma gondii was included as an
ou g oup [
30
]. The s abili y o accu acy o in e ed opologies was assessed ia boo s ap
analysis o 1000 eplica es.
4.5.2. Sequence Accession Numbe s
The GenBank accession numbe s o he gene sequences ob ained in his s udy a e
MN944852, MT341815, MT341857, and MT341858.
5. Conclusions
As o o he ick-bo ne pa hogens, he dis ibu ion o Theile ia spp. in ec ions is
ela ed o se e al ac o s, including he p esence o he ick ec o species, he hos , and he
ese oi . Ou da a showed he p esence o T. annula a in sampled animals, con i ming ha
his pa asi e is p e alen in in ensi e ca le b eeding.
Fu he mo e, ou da a show ha he sequences ob ained om ield samples a e no
subs an ially di e en om he sequence ob ained om he s ain isola ed almos 20 yea s
ago and cul u ed in ou labo a o y, sugges ing a low a e o gene ic a iabili y o he
pa hogen.
Se ological in es iga ions and cha ac e iza ion o T. annula a s ains ci cula ing in an
a ea a e use ul o iden i y possible ou es o en y o new s ains in o a e i o y, as well as
iden i y he mos app op ia e he apeu ic app oaches, including accina ion s a egies, o
he p e en ion and con ol o he pa hogen.
Au ho Con ibu ions:
Concep ualiza ion, D.V. and V.G.; me hodology, V.G.; so wa e, S.D.C.;
alida ion, V.G.; in es iga ion, V.G., V.B., A.G., D.G., F.L.R., and G.S.; esou ces, D.V.; da a cu a ion,
V.G., V.B., and S.D.C.; w i ing—o iginal d a p epa a ion, V.G. and V.B.; w i ing— e iew and edi ing,
V.B. and J.M.; isualiza ion, D.V.; supe ision, A.T. and J.M.; p ojec adminis a ion, D.V.; unding
acquisi ion, D.V. All au ho s ead and ag eed o he published e sion o he manusc ip .
Funding: This esea ch was unded by he I alian Minis y o Heal h RC IZSSI 20/16 and 08/19.
Ins i u ional Re iew Boa d S a emen :
The s udy did no in ol e any animal expe imen s. Blood
samples we e aken om ca le ha we e na u ally in ec ed o in which ick-bo ne disease was
suspec ed. Blood sampling was necessa y in o de o pe o m labo a o y analysis and did no in ol e
any su e ing o he animals sampled.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen :
10–20% GenBank accession numbe s o he sequences ob ained in his
s udy a e MN944852, MT341815, MT341857, and MT341858, a ailable a h ps://www.ncbi.nlm.nih.
go /genbank/.
Acknowledgmen s:
The au ho s would like o hank Rosalia D’Agos ino o he suppo in he cell
cul u e labo a o y.
Con lic s o In e es : The au ho s decla e no con lic o in e es .

Pa hogens 2021,10, 101 6 o 7
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