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Ticks and Tick-borne Diseases 5 (2014) 213– 218

Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases

jo ur nal homepage: www.elsev ier .com/ locate / t tbd is

hort communication

potted fever group rickettsiae in ticks in Turkey

mer Orkun ∗, Zafer Karaer, Ays e C akmak, Serpil Nalbantogluepartment of Parasitology, Faculty of Veterinary Medicine, Ankara University, Ankara 06110, Turkey

r t i c l e i n f o

rticle history:eceived 12 September 2012eceived in revised form 8 November 2012ccepted 14 November 2012vailable online 25 November 2013

a b s t r a c t

One hundred twenty-six ticks belonging to 12 tick species were collected from humans, domestic andwild animals, and from the ground as unfed (questing ticks) from distinct localities in Turkey in 2011.Ticks were individually tested by polymerase chain reaction (PCR) for Rickettsia spp., amplifying citratesynthase (gltA), and outer membrane protein (ompA) genes. Twenty-five ticks (19.8%) were found to beinfected with Rickettsia species. Five SFG rickettsiae were identified, including 4 pathogens: Ri. aeschli-

eywords:potted fever group rickettsiaeicksurkeyolecular detection

hylogenetic analysis

mannii in Hyalomma marginatum, Hy. aegyptium, Hyalomma sp. (nymph), and Rhipicephalus turanicus; Ri.africae in Hy. excavatum, Hy. aegyptium, and Hyalomma sp. (nymph); Ri. slovaca and Ri. raoultii in Der-macentor marginatus; and one species with unknown pathogenicity, Ri. hoogstraalii, in Haemaphysalisparva. Rickettsia slovaca and Ri. hoogstraalii were reported for the first time from Turkey. In addition,Ri. hoogstraalii and Ri. africae were detected for the first time in Ha. parva and Hy. excavatum ticks,respectively.

ntroduction

Spotted fever group (SFG) rickettsioses are caused by obligatentracellular bacteria belonging to the genus Rickettsia within theamily Rickettsiaceae in the order Rickettsiales (Raoult and Roux,997). The genus Rickettsia has been divided into 3 groups: the

spotted fever group’ (SFG), the ‘typhus group’ (TG), and the ‘scrubyphus group’ (STG). It has been known so far that 31 rickettsialpecies were recognized, and many more species are to be iden-ified (Merhej and Raoult, 2011). These bacteria transmitted byrthropods, mainly ticks, may cause disease in vertebrate hosts,uch as humans, domestic animals, birds, and wildlife (Parola et al.,005). Some of ticks can transmit the rickettsiae both transstadiallynd transovarially; therefore, ticks are known to be the main reser-oirs and vectors of SFG rickettsiae in nature (Socolovschi et al.,009). This infection can cause spotted fever, and the clinical signs

nclude fever, headache, rash, muscle pain, local lymphadenopathy,nd sometimes a characteristic eschar (tache noire) at the side ofick bite in humans (Raoult and Roux, 1997; Parola et al., 2005).

In Turkey, ixodid ticks belong to the genera Hyalomma, Der-acentor, Rhipicephalus, Haemaphysalis, and Ixodes; argasid ticks

elong to the genera Argas, Ornithodoros and Otobius. These ticksften take blood from animals and humans (Estrada-Pena et al.,

004; Aydin and Bakirci, 2007; Karaer et al., 2011). Data regardingccurrence of rickettsiae in ticks and in humans are vague inurkey. Ri. monacensis, Ri. helvetica, Ri. aeschlimannii, Ri. conorii

∗ Corresponding author. Tel.: +90 312 317 0315; fax: +90 312 316 4472.E-mail address: omerorkun@yahoo.com.tr (Ö. Orkun).

877-959X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.ttbdis.2012.11.018

© 2013 Elsevier GmbH. All rights reserved.

subsp. conorii, Ri. africae, Ri. raoultii, and Ri. felis were detected inticks (Christova et al., 2003; Gargili et al., 2012), whilst Ri. conoriiwas detected in patients in Turkey (Kuloglu et al., 2004). Thepresent study aimed to identify and characterize species withinthe SFG rickettsiae using sequence and phylogenetic analysis ofticks collected from humans, domestic and wild animals, and unfed(questing) ticks from the ground in distinct localities in Turkey.

Materials and methods

In 2011, ticks were collected from humans, domestic and wildanimals, and from the ground as unfed (questing) ticks in 12 dif-ferent provinces of Turkey: Agrı, Ankara, Artvin, Bolu, C ankırı,C orum, Erzurum, Giresun, Kırs ehir, Kocaeli, Mardin, and Yozgat.Tick species, hosts, and tick localities are shown in Table 1. Host-seeking (questing) ticks were collected by making some vibrationson the ground. Ticks were identified according to the taxonomickeys of Apanaskevich (2003) and Estrada-Pena et al. (2004).

Each tick was first washed in 70% alcohol, then rinsed in ster-ile water, and dried on sterile filter paper. Ticks were individuallyhomogenized using liquid nitrogen, and DNA was individuallyextracted by using the Qiagen DNeasy® blood and tissue kit (Qiagen,Hilden, Germany) according to the manufacturer’s instructions.Rickettsial DNA was detected by PCR using the primers Rp CS.409dand Rp CS.1258n, which amplify the citrate synthase gene (gltA) ofRickettsia spp. (Roux et al., 1997). Each tick positive for gltA was

also tested for the ompA gene of Rickettsia spp. using the primersRr. 190.70 and Rr. 190.701 (Fournier et al., 1998). DNase-RNase-freewater was used as a negative control, and a positive control (DNAfrom Ri. montanensis) was included in all reactions. Successfully

214 Ö. Orkun et al. / Ticks and Tick-borne Diseases 5 (2014) 213– 218

Table 1Tested tick species collected from different localities in Turkey: their hosts, PCR positivity, and targeted gene.

Tick species (no. of tested specimens) Provinces and numbers of the ticks collected Host No. of PCR-positive ticks

Hyalomma marginatum (31) Ankara (15) Human (6M,2F) 1MCattle (4F, 1M) 0Sheep (1M) 0Questing tick (1M) 0

Kırs ehir (10) Cattle (8M, 1F) 2MQuesting tick (1F) 0

Bolu (3) Cattle (3M) 0Corum (1) Human (1M) 0Cankırı (1) Human (1M) 0Erzurum (1) Cattle (1F) 0

Hyalomma aegyptium (23) Ankara (18) Human (3M) 0Tortoise (5M, 5F) 1M, 1FQuesting ticks (3F, 2M)a 1M, 1F

Yozgat (5) Tortoise (4M, 1F) 0Kırs ehir (1) Questing tick (1F) 0

Hyalomma excavatum (7) Ankara (7) Human (3M, 2F) 1MCattle (2M) 0

Hyalomma scupense (2) (syn. Hy. detritum) Ankara (2) Cattle (2M) 0Hyalomma spp. (22) Ankara (22) Human (20N) 3N

Buzzard (2N) 0Dermacentor marginatus (10) Ankara (8) Cattle (4M, 1F) 4M, 1F

Human (3M) 3MBolu (2) Cattle (1M) 1M

Questing tick (1M) 0Haemaphysalis parva (11) Ankara (11) Human (8M, 3F) 2F, 2MRhipicephalus sanguineus (2) Ankara (2) Buzzard (1F, 1M) 0Rhipicephalus turanicus (5) Ankara (2) Sheep (1F) 1F

Cattle (1F) 0Kırs ehir(3) Cattle (2F, 1M) 0

Rhipicephalus bursa (1) Ankara (1) Cattle (1M) 0Ixodes ricinus (4) Kocaeli (1) Human (1F) 0

Giresun (1) Human (1F) 0Artvin (1) Human (1F) 0Yozgat (1) Vole (1F) 0

Argas persicus (2) Mardin (2) Chicken (2F) 0Argas spp. (5) Mardin (5) Chicken (5N) 0Otobius megnini (1) Agrı (1) Human (1F) 0

Total 126 63M (50%), 36F (28.5%), 27N (21.4%) 25 (19.8%)

F, female; M, male; N, nymph.g itive f

ained

a(mUA

BwamvbtdJ

R

f(ses(b

ltA, citrate synthase A gene; ompA, outer membrane protein A gene. Only ticks posa Four ticks collected from humans and one tick collected from buzzard were obt

mplified product was purified using the QIAquick® Extraction KitQiagen GmbH). Purified DNA was sequenced using BigDye® Ter-

inator V3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City,SA). Automated fluorescence sequencing was performed with anBI PRISM® 3100 Genetic Analyzer (Applied Biosystems).

Nucleotide sequences were processed using nucleotideLAST (National Center for Biotechnology Information,ww.ncbi.nlmn.nih.gov/BLAST). Sequences were edited and

ligned by using BioEdit software (Hall, 1999). Phylogenetic andolecular evolutionary analyses were performed by using MEGA

ersion 4 (Tamura et al., 2007). The phylogenetic tree was producedy applying the Bootstrap Test of Phylogeny, Neighbor-Joiningechnique. The sequence data obtained in this study have beeneposited in GenBank under the accession numbers JQ691710 to

Q691734.

esults

A total of 126 ticks representing 12 species, 7 genera, and 2amilies were collected. Eight (6.3%) were argasid ticks, and 11893.7%) were ixodid ticks. Collected ticks belonged to the followingpecies: Hyalomma marginatum (n = 31), Hy. aegyptium (n = 23), Hy.

xcavatum (n = 7), Hy. scupense (syn. Hy. detritum) (n = 2), Hyalommapp. (n = 22), Dermacentor marginatus (n = 10), Haemaphysalis parvan = 11), Rhipicephalus sanguineus (n = 2), Rh. turanicus (n = 5), Rh.ursa (n = 1), Ixodes ricinus (n = 4), Argas persicus (n = 2), Argas spp.

or the gltA gene were tested for the ompA gene. as engorged nymph and were then allowed to molt to the adult stage.

(n = 5), and Otobius megnini (n = 1). Ticks were collected fromhumans (n = 56), from wild and domestic animals, including cat-tle (Bos taurus) (n = 26), sheep (Ovis aries) (n = 2), a vole (Microtussp.) (n = 1), tortoises (Testudo graeca) (n = 5), buzzards (Buteo rufi-nus) (n = 2), chickens (Gallus gallus domesticus) (n = 2), and unfedticks from the ground. The DNA of Rickettsia spp. was found in 25ticks by using PCR and primers specific for the gltA gene. Twenty-five ticks positive for the gltA gene were tested for the ompA gene,and 4 ticks were found negative while 21 ticks were found positive.Twenty-one ticks yielded amplicons for both the gltA and the ompAgene; however, a PCR product of the ompA gene was not obtained in4 Ha. parva species. Further information is summarized in Table 1.OmpA gene sequence analyses indicated that Ri. aeschlimannii wasdetected in 8 tick individuals (32%): 3 Hy. marginatum (collectedfrom cattle and humans), 2 Hy. aegyptium (collected from humansas engorged nymphs; later the nymphs molted to the adult stageunder the suitable conditions), 2 Hyalomma spp. (nymphs collectedfrom humans), and 1 Rh. turanicus (from sheep). Ri. africae wasfound in 4 ticks (16%): 2 Hy. aegyptium (from tortoise), 1 Hy. exca-vatum (from human), and 1 Hyalomma sp. (nymph from human).Whilst 8 D. marginatus (32%) (from cattle and humans) were foundinfected with Ri. slovaca, only one D. marginatus (4%) (from cattle)

was found infected with R. raoultii. GltA gene sequence analysesindicated that Ri. hoogstraalii occurred in 4 Ha. parva (16%) (fromhumans). None of the Hy. scupense (syn. Hy. detritum), Rh. sanguin-eus, Rh. bursa, I. ricinus, Ar. persicus, Argas spp., or O. megnini ticks

Ö. Orkun et al. / Ticks and Tick-borne Diseases 5 (2014) 213– 218 215

Table 2Turkish rickettsial strains found in this study and their level of nucleotide similarity with other rickettsial strains.

Rickettsia species Sequenced gene Tick species Nucleotide identity (in %) GenBank accession no.

Rickettsia aeschlimannii ompA Hyalomma marginatum 99.8a JQ691714Hyalomma marginatum 99.8a JQ691717Hyalomma marginatum 99.8a JQ691734Hyalomma aegyptium 98.4a JQ691727Hyalomma aegyptium 98.9a JQ691728Hyalomma sp. (nymph) 99.0a JQ691729Hyalomma sp. (nymph) 97.7a JQ691732Rhipicephalus turanicus 99.5a JQ691733

Rickettsia africae ompA Hyalomma excavatum 99.1b JQ691718Hyalomma aegyptium 98.9c JQ691722Hyalomma aegyptium 99.1b JQ691723Hyalomma sp. (nymph) 99.4b JQ691730

Rickettsia slovaca ompA Dermacentor marginatus 100d JQ691715Dermacentor marginatus 99.6e JQ691716Dermacentor marginatus 98.9d JQ691719Dermacentor marginatus 99.8e JQ691720Dermacentor marginatus 99.6d JQ691721Dermacentor marginatus 99.8e JQ691724Dermacentor marginatus 100e JQ691725Dermacentor marginatus 99.8d JQ691726

Rickettsia raoultii ompA Dermacentor marginatus 100f JQ691731Rickettsia hoogstraalii gltA Haemaphysalis parva 99.2g JQ691710

Haemaphysalis parva 99.6g JQ691711Haemaphysalis parva 99.5g JQ691712Haemaphysalis parva 98.7g JQ691713

a Rickettsia aeschlimannii strain EgyRickHimp-El-Arish-18, accession no. HQ335159.b Rickettsia africae strain EgyRickHd-Qalet El-Nakhl-9, accession no. HQ335137.c Rickettsia africae ESF-5 accession no. CP001612.d

wopwPg

D

bptehdR

bIm(Hatm2etsidatR

Rickettsia slovaca strain WB3/Dm Pavullo accession no. HM161776.e Rickettsia slovaca 13-B accession no. CP002428.f Rickettsia raoultii strain WB16/Dm Monterenzio accession no. HM161789.g Rickettsia hoogstraalii accession no. FJ767737.

as infected with Rickettsia spp. Detailed information about thebtained rickettsial isolates is given in Table 2. Additionally, thehylogenetic relationships of the rickettsiae found in this studyith other rickettsial species are demonstrated in Figs. 1 and 2.

hylogenetic trees were constructed separately by using ompA andltA genes, respectively (Figs. 1 and 2).

iscussion

Tick-borne rickettsioses are among the oldest known vector-orne zoonotic diseases (Parola et al., 2005), and they cause majorroblems in public health. Ticks play a very important role as vec-ors of rickettsial disease, including SFG rickettsiae (Socolovschit al., 2009) transmitting their Rickettsia infection to vertebrateosts. Distribution and spread of tick-borne rickettsioses are largelyetermined by the occurrence of infected vector ticks (Parola andaoult, 2001).

Ri. aeschlimannii, a pathogenic rickettsia, is transmitted mainlyy Hyalomma ticks (including Hy. marginatum) (Beati et al., 1997).

n Turkey, Ri. aeschlimannii was found in 5 Hy. aegyptium, in 2 Hy.arginatum, and in one Rh. bursa collected from humans in Istanbul

Gargili et al., 2012). In our study, Ri. aeschlimannii was found in 3y. marginatum collected from 2 cattle and one human in Kırs ehirnd Ankara, respectively. Hy. marginatum, known as a main vec-or of the Crimean-Congo hemorrhagic fever virus (CCHFv), is the

ost widespread Hyalomma species in Turkey (Vatansever et al.,007). Two Hy. aegyptium unfed adults, obtained from 2 humans asngorged nymphs and then molted to the adult stage, were foundo be infected with Ri. aeschlimannii in Ankara. So the bacteriumurvived the tick molt. Hy. aegyptium nymphs have a special affin-ty to humans (Karaer et al., 2011). Additionally, this pathogen was

etermined in 2 Hyalomma spp. nymphs detached from humansnd one Rh. turanicus collected from a sheep in Ankara. Accordingo nucleotide Blast and phylogenetic analysis (ompA gene), Turkishi. aeschlimannii strains are closely related with an Egypt strain

obtained from Hy. impeltatum (97.7–99.8% similarity, accessionnumber HQ335159). The nucleotide similarities of sequences andphylogenetic relationships are shown in detail in Table 2 and Fig. 1,respectively. These results show that Ri. aeschlimannii, frequentlyfound (32%) in Rickettsia-positive ticks, is widespread in Turkey andcan occur in various Hyalomma species and also in non-Hyalommatick species.

Ri. africae, the agent of African tick-bite fever (ATBF), is prevalentmostly in sub-Saharan Africa, where it is transmitted by Ambly-omma species (mainly Am. hebraeum and Am. variegatum). ATBFis a common and widespread disease in Africa and after malaria,the second most frequent cause of systemic febrile illness amongtravelers (Jensenius et al., 2003; Freedman et al., 2006). In Turkey,Ri. africae was found in only one Hy. aegyptium collected from ahuman in Istanbul (Gargili et al., 2012). In our study, Ri. africaewas found in 2 Hy. aegyptium collected from tortoises, in one Hy.excavatum collected from a human, which is the first detectionof Ri. africae in Hy. excavatum, and in one Hyalomma nymph col-lected from a human in Ankara. All the ticks were obtained fromhosts during feeding. According to nucleotide Blast and phyloge-netic analysis (ompA gene), Turkish Ri. africae obtained from oneH. aegyptium was 98.9% similar to Ri. africae ESF-5 (isolated fromAm. variegatum, accession number CP001612), whereas the other 3strains match with an Egypt strain by 99.1–99.4% (obtain from Hy.dromedarii, accession number, HQ335137). More detailed data andthe phylogenetic tree are given in Table 2 and Fig. 1, respectively.Hyalomma ticks may have a significant role in the transmission ofRi. africae in Mediterranean countries as this agent was also foundin Hy. dromedarii, Hy. marginatum, Hy. impeltatum in Egypt (Abdel-Shafy et al., 2011) and in Hy. aegyptium in Turkey (Gargili et al.,2012). Further epidemiological studies have to show whether or

not this hypothesis is correct.

Ri. slovaca, a human pathogenic rickettsia and the etiologicalagent of TIBOLA/DEBONEL, is mainly transmitted by D. marginatusand D. reticulatus ticks. This bacterium is transmitted transstadially

216 Ö. Orkun et al. / Ticks and Tick-borne Diseases 5 (2014) 213– 218

Fig. 1. Phylogenetic tree based on aligned sequences of the rickettsial ompA gene and constructed by using neighbor-joining method in MEGA4 software. GenBank accessionnumbers of sequences and names of lineages are given before bacterial species names.

Ö. Orkun et al. / Ticks and Tick-borne Diseases 5 (2014) 213– 218 217

F consn es.

aasc

ig. 2. Phylogenetic tree based on aligned sequences of the rickettsial gltA gene andumbers of sequences and names of lineages are given before bacterial species nam

nd transovarially in these ticks, which are known as both vectornd reservoir of these bacteria (Parola et al., 2005, 2009). In ourtudy, R. slovaca was detected in 8 D. marginatus obtained fromattle and from humans in Ankara. In the present study, 80% of the

tructed by using neighbor-joining method in MEGA4 software. GenBank accession

ticks infected with Ri. slovaca were Dermacentor ticks. This was thefirst time that Ri. slovaca was detected in Turkey. According to thenucleotide Blast and phylogenetic analysis (ompA gene), TurkishRi. slovaca strains are by 98.9–100% similar to the reference strains

2 -born

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18 Ö. Orkun et al. / Ticks and Tick

Ri. slovaca 13-B, accession no. CP002428, and strain WB3/Dmavullo, accession no. HM161776). More detailed data and ahylogenetic tree are given in the Table 2 and Fig. 1, respec-ively. A high infection prevalence of Ri. slovaca in D. marginatusicks was detected in the present study. Perhaps, Ri. slovaca is aeglected disease agent in Turkey because our previous study indi-ated that D. marginatus was one of the predominant tick specieshat attach to humans (Karaer et al., 2011), and this tick wasound to be common in all regions of Turkey (Aydin and Bakirci,007).

Ri. raoultii, a human pathogenic rickettsia, was recently found inermacentor ticks collected in Russia and France. This bacterium is

ransmitted by Dermacentor ticks, including mainly D. marginatus,. reticulatus, D. niveus, D. nuttallii, and D. silvarum. The infectionaused by Ri. raoultii in humans is very similar to Ri. slovaca infec-ion in terms of the clinical signs, and both bacteria are transmittedy common tick species. As in Ri. slovaca infection, clinical signsf TIBOLA/DEBONEL can also develop in patients infected with. raoultii (Mediannikov et al., 2008). In Turkey, R. raoultii wasetected in one D. marginatus collected from a human in IstanbulGargili et al., 2012). In our study, R. raoultii was detected in one. marginatus collected from cattle in Bolu. According to the ompAene sequence, Ri. raoultii from Turkey was 100% similar to thetalian R. raoultii obtained from D. marginatus (strain WB16/Dm

onterenzio, accession no. HM161789, data are given in Table 2nd Fig. 1). Some studies indicated that R. raoultii seems to beore prevalent than Ri. slovaca in Dermacentor ticks in some Euro-

ean countries like Germany, The Netherlands, Portugal, and Spainreviewed in Parola et al., 2009). However, in this study, Ri. slovacaas found to be more prevalent than R. raoultii. According to our

esults, both Ri. slovaca and Ri. raoultii are present in Turkey; there-ore, TIBOLA/DEBONEL should be taken into consideration in thisountry.

Ri. hoogstraalii, a rickettsia with unknown pathogenicity, wassolated from Ha. sulcata and Carios capensis ticks (Duh et al., 2010).n our study, Ri. hoogstraalii has been detected from 4 Ha. parva ticksollected from humans in Ankara. We could not obtain a PCR prod-ct from the ompA gene. As a result of the gltA sequence, Turkishi. hoogstraalii strains have 98.7–99.6% similarity with the refer-nce strain (Ri. hoogstraalii accession no. FJ767737, data are given inable 2). A phylogenetic tree of the gltA gene is shown in Fig. 2. Thiss the first report about the existence of Ri. hoogstraalii in Turkey.urthermore, Ri. hoogstraalii was detected for the first time in Ha.arva ticks in this study.

Several Rickettsia species not detected in our study were alsoeported from Turkey. Ri. conorii was reported in both ticks (B. annu-atus, D. marginatus, and Rh. bursa) and patients (Christova et al.,003; Kuloglu et al., 2004; Gargili et al., 2012). Moreover, Ri. mona-ensis (in I. ricinus), Ri. Helvetica [in D. marginatus, Hy. plumbeumHy. marginatum?), I. ricinus, and Rh. bursa], and Ri. felis (in Rh. bursa)ave also been reported to occur in ticks in Turkey (Christova et al.,003; Gargili et al., 2012).

In conclusion, the results of this study indicate that tick-borneickettsiae, including the pathogenic species Ri. aeschlimannii, Ri.fricae, Ri. slovaca, and R. raoultii, have a remarkably high preva-ence in Turkish ticks. Although the involved vector tick species

re widely distributed and quite common, we could find only aew individual reports about tick-borne rickettsiosis in humansn Turkey, so it is our impression that tick-borne rickettsiosesre neglected diseases in this region. We recommend that SFG

e Diseases 5 (2014) 213– 218

rickettsioses should be taken into consideration in patients whohad a tick bite in Turkey and in neighboring countries.

Acknowledgments

We thank Didier Raoult and Cristina Socolovschi for providingpositive DNA of Ri. montanensis.

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