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survey of ixodid tick species and molecular identification ofick-borne pathogens�
unir Aktas ∗
epartment of Parasitology, Faculty of Veterinary Medicine, University of Firat, 23119, Elazig, Turkey
a r t i c l e i n f o
rticle history:eceived 27 June 2013eceived in revised form 7 December 2013ccepted 11 December 2013
eywords:xodid ticksick-borne pathogensCRLB
a b s t r a c t
This study was undertaken in two different climatic areas of Turkey to determine thepresence of tick-borne pathogens of medical and veterinary importance. The ticks wereremoved from humans, pooled according to species and developmental stages, and ana-lyzed by PCR, reverse line blot (RLB) and sequencing. Of the 2333 removed ticks from10 species, 1238 (53.06%) were obtained from the arid cold zone, and the remaining1095 (46.93%) were obtained from the humid zone. The removed ticks were identifiedas Hyalomma marginatum, Hyalomma detritum, Hyalomma excavatum, Rhipicephalus bursa,Rhipicephalus turanicus, Rhipicephalus sanguineus, Dermacentor marginatus, Haemaphysalispunctata, Haemaphysalis sulcata, Ixodes ricinus, Haemaphysalis and Ixodes spp. nymphs. Thedominant species was I. ricinus (61.27%) in the humid zone, whereas the Haemaphysalisspp. nymph dominated (30.29%) in the arid zone. Infection rates were calculated as themaximum likelihood estimation (MLE) with 95% confidence intervals (CI). Of the 169 poolstested, 49 (28.99%) were found to be infected with the pathogens, and the overall MLEof the infection rate was calculated as 2.44% (CI 1.88–3.17). The MLE of the infection var-ied among tick species, ranging from 0.85% (CI 0.23–2.34) in Haemaphysalis spp. nymphto 17.93% (CI 6.94–37.91) in D. marginatus. Pathogens identified in ticks included Theile-ria annulata, Babesia ovis, Babesia crassa, Anaplasma/Ehrlichia spp., Anaplasma ovis, Ehrlichia
canis, Anaplasma phagocytophilum, Hepatozoon canis and Hepatozoon felis. Most tick poolswere infected with a single pathogen. However, four pools infected with H. canis displayedinfections with B. crassa, A. phagocytophilum and E. canis. The sequencing indicated thatAnaplasma/Ehrlichia spp. was 100% identical to the sequence of Ehrlichia sp. Firat 2 and 3
ed from
previously identifi
. Introduction
Ticks are ectoparasites of domestic and wild animals, asell as humans. They transmit a wide variety of pathogens
ncluding protozoa, bacteria, fungi and viruses that infectomestic livestock and wild animals in most regions ofhe world, causing diseases of zoonotic and veterinary
� Note: Nucleotide sequence data reported in this paper are availablen GenBank, EMBL and DDBJ databases under accession numbers fromF034779 to KF034789.∗ Tel.: +90 424 237 0000; fax: +90 424 238 8173.
importance (de La Fuente et al., 2008). For the devel-opment and implementation of control strategies, it isimportant to identify the vector ticks and their transmis-sion pattern of the pathogens in the target geographicalregion.
Piroplasmosis caused by Theileria and Babesia speciesleads to clinical infections in domestic and wild animalswith high mortality and morbidity (Friedhoff, 1997). Tropi-cal theileriosis, caused by Theileria annulata, is an importanttick-borne disease of cattle in tropical and sub-tropical
regions (Dumanli et al., 2005). The parasite is transmit-ted from cattle to cattle by ticks of the genus Hyalomma.Ovine babesiosis, caused by Babesia ovis, Babesia motasi andBabesia crassa, is the most important tick-borne disease
of small ruminants (Uilenberg, 2001; Aktas et al., 2006a).Ovine babesiosis is transmitted by ixodid ticks to causingfever, anemia, hemoglobinuria and icterus in sheep andgoats (Uilenberg, 2001).
Ehrlichia and Anaplasma species are obligate intracellu-lar microorganisms that multiply in vertebrate reservoirsand tick vectors (Dumler, 2005). The pathogens are dividedinto various distinct genogroups. The genus Anaplasmacomprises six species: Anaplasma centrale, A. marginale, A.bovis, A. ovis, A. phagocytophilum and A. platys. The genusEhrlichia consists of five species, which include Ehrlichiacanis, E. chaffeensis, E. muris, E. ewingii and E. ruminantum(Dumler, 2005). Species from both genera are transmittedby ticks of the family Ixodidae (Friedhoff, 1997).
Hepatozoon spp. are protozoan parasites that infect awide range of domestic and wild carnivores, birds, reptiles,and amphibians and are transmitted by ingestion of ixodidticks harboring the pathogen (Baneth, 2011). Hepatozooncomprises more than 300 species, 46 known to infect mam-mals. Hepatozoon canis and Hepatozoon americanum causehepatozoonosis in canids. H. canis has long been recog-nized to infect and cause disease in dogs in Asia, Europe,Africa, and Latin America (Baneth and Vincent-Johnson,2005). The main vector of the pathogen is the brown dogtick, Rhipicephalus sanguineus, although several tick specieshave been imputed to be potential vectors (Giannelli et al.,2013a).
Although tick-borne pathogens, such as, Theileria,Babesia, Anaplasma and Hepatozoon have been documentedin domestic animals and tick vectors in some parts ofTurkey (Altay et al., 2008; Aktas et al., 2010, 2011), there islimited information on the frequency of ixodid tick speciesand the prevalence of tick-borne bacteria and haemopro-tozoan parasites in most areas of the country (Aktas et al.,2006b). The objective of this study was to investigate thepresence of Theileria, Babesia, Anaplasma, Ehrlichia and Hep-atozoon species in ixodid ticks from two different climaticareas of Turkey; PCR, reverse line blot (RLB) hybridizationand sequencing were used for detection. Tick-transmittedpathogens are passed on either from parent to offspring tick(transovarial transmission), or ticks acquire the infectionas larvae or nymphs and subsequently transmit it to thenext stage (trans-stadial transmission). The infection ratefor most of the pathogens was significantly higher in feed-ing than in questing ticks, suggesting that a number of thesepathogens originated from the hosts blood ingested beforetick collection rather than from transstadially maintainedinfections acquired during earlier blood meals. Therefore,the detection of pathogens in feeding ticks cannot estab-lish vector competence, whereas infected unfed ticks haveat least maintained the pathogen transstadially. However,additional experiments should be supported to this idea.The ticks used in this study were removed from humans ashort time after they bite, not collected from vegetation.
2. Materials and methods
2.1. Study area and collection of tick samples
Tick sampling was conducted from March 2007 toDecember 2007 in ten provinces of Turkey: Giresun,
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Trabzon, Rize, Elazıg, Bingol, Mus, Malatya, Erzurum, Erz-incan and Tunceli. The study area covered two climaticzones: (i) humid, in the Black Sea coastal sea region, whichexperiences frequent rainfall and mild temperatures (Gire-sun, Trabzon, Rize), the main tick species recorded beingIxodes ricinus (Aktas et al., 2010, 2012); and (ii) arid, in anarea of eastern Turkey with warmer summers and colderwinters (Elazıg, Bingol, Mus, Malatya, Erzurum, Erzincan,Tunceli), with mainly Hyalomma spp., Rhipicephalus spp.,and Haemaphysalis spp. (Aktas et al., 2004).
At the request of The Turkish Ministry of Health, ticksrecovered from humans in the provinces were sent to ourlaboratory (Department of Parasitology, Faculty of Veteri-nary Medicine, Firat University, Elazig, Turkey). Adult andnymphal ticks were removed from patients by medicalstaff. The ticks were placed into 1.5 ml tubes filled with70% ethanol or isopropanol and submitted to us along withdocumentation. Although the samples included immatureticks, only the adults were identified to the species levelusing standard taxonomic keys (Estrada-Pena et al., 2004).
A total of 2333 ticks (1667 adults and 666 nymphs)were screened for the presence of tick-borne haemoproto-zoan parasites and bacteria. The ticks were washed in 70%ethanol, rinsed three times in sterile phosphate-bufferedsaline, and dried on filter paper. They were separatedby location, species, life stage, sex, and bioclimatic zoneinto 169 pooled samples consisting of 143 adult (2–25per pool) and 26 nymphal (10–44 per pool) pools andstored at −80 ◦C until DNA extraction. Detailed informationregarding the tick samples used in this study is presentedin Table 1.
The tick pools were crushed as described by Aktas et al.(2010). DNA was extracted from crushed ticks using a DNAtissue kit (Qiagen, Hilden, Germany) according to the man-ufacturer’s instructions.
2.2. Amplification of tick-borne pathogen DNA
For the amplification of Hepatozoon DNA, standard poly-merase chain reaction (PCR) was performed in a reactionvolume of 25 �l containing 2.5 �l of the DNA samplewith a pair of genus-specific primers. The forward primerHepF (5′-ATACATGAGCAAAATCTCAAC-3′) and the reverseprimer HepR (5′-CTTATTATTCCATGCTGCAG-3′) were usedto amplify a fragment of approximately 660 base pairs (bp)of the 18S rRNA gene of Hepatozoon spp., as described byInokuma et al. (2002). Cycling conditions were as describedby Aktas et al. (2013). Positive control DNA previouslyisolated from a dog naturally infected with H. canis (Gen-Bank accession no. JQ867390) and negative control DNA(non-infected canine blood DNA and distilled water) wereincluded in each PCR test.
The PCR for the amplification of Babesia and Theile-ria species was performed as previously described (Aktaset al., 2011). Genus-specific primers, RLBF2/RLBR2, wereused to amplify a fragment of 460–540 base pairs (bp) ofthe 18S SSU rRNA gene of the V4 region of Theileria and
Babesia species (Georges et al., 2001). For the identifica-tion of Anaplasma and Ehrlichia species, the primers 16S8FEand BGA1B were used to amplify a fragment of approx-imately 500 bp of the 16S rRNA gene of the V1 region
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Table 1Distribution of ixodid tick species and infection rates of tick-borne pathogens detected by RLB and PCR in two different climatic zones of Turkey.
Climatic zone Tick species A/B/C (%)* MLE (% 95CI)
Test
RLB(RLBF2/RLBR2)
PCR(HepF/HepR)
T. annulata B. ovis B. crassa Anaplasma/Ehrlichia spp.
of Anaplasma and Ehrlichia spp., as described by Schoulset al. (1999). The PCR reactions were performed in a PCRSprint (Thermo Electron Corporation, USA), using a touch-down PCR program. Positive control DNA from T. annulata,Babesia bigemina, B. ovis, Babesia canis, A. marginale andE. canis (previously isolated from naturally infected hosts,Gen-Bank accession nos. HM176661, EU622822, JQ867387,KF038318, GU201518, KF034789, respectively) were usedin the PCR and for the subsequent RLB hybridization. Sterile,deionized water was used as a negative control.
2.3. Reverse line blot hybridization
To detect Theileria, Babesia, Anaplasma and Ehrlichiaspecies, a reverse line blot (RLB) hybridization was per-formed on PCR products, as described previously (Aktaset al., 2011). Briefly, genus-specific oligonucleotides werediluted in 500 mM NaHCO3 (pH 8.4) at concentrations of300 pmol/150 �l for the Theileria/Babesia catch-all probeand 100 pmol/150 �l for the Anaplasma/Ehrlichia catch-allprobes. Twenty microliters of PCR product was dilutedto a final volume of 150 �l in 2× SSPE/0.1% SDS for RLBhybridization. Thirty-three different specific probes wereused to identify common tick-borne protozoa and bacte-ria of veterinary importance in Turkey. All of the probeswere obtained from The Midland Certified Reagent (Texas,USA). The preparation, hybridization, and stripping of theRLB membrane were performed as described by Aktas et al.(2012).
2.4. Sequence analyses
To confirm the results of the PCR and RLB and to identifypathogens at species levels, all of the positive PCR productswere purified with a PCR purification kit (Qiagen, Hilden,Germany) and sequenced. DNA sequences obtained wereevaluated with Chromas Lite software, version 2.01 (Tech-nelysium Pty Ltd) and compared for similarity to sequencesdeposited in GenBank.
2.5. Statistical analysis
Infection rates in tick pools were calculated usingmaximum likelihood estimation (MLE) methods with95% confidence intervals (CI) for unequal pool sizes andexpressed as MLE of infection rate per 100 ticks. The calcu-lation of MLE for the different pool sizes required numericaliterations and computer implementation. We used Pooled-InfRate estimation software (version 4.0) as an add-in toMicrosoft Excel (Biggerstaff, 2009). In addition, a Pearsonchi-square test was used, and P values of ≤0.05 were con-sidered statistically significant.
3. Results
3.1. Tick species and distribution
The occurrence and abundance of identified tick species,the number of removed ticks, the number of analyzed andpooled ticks, and infection rates for tick-borne protozoaand bacteria for each bioclimatic area are shown in Table 1.
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f the 2333 removed ticks, 1238 (53.07%) were obtainedrom the arid cold zone, and the remaining 1095 (46.93%)ere obtained from the humid zone. The removed ticksere identified as adult Hyalomma marginatum, Hyalomma
etritum, Hyalomma excavatum, Rhipicepalus bursa, Rhipi-epalus turanicus, Rh. sanguineus, Dermacentor marginatus,aemaphysalis punctata, Haemaphysalis sulcata, I. ricinus,aemaphysalis spp. nymphs and Ixodes spp. nymphs. Thereere 9 species of ticks collected from the arid cold zone,hereas 6 species were identified from the humid zone.
he dominant species was I. ricinus (61.27%) in the humidone, whereas the H. marginatum dominated (25.20%) inhe arid zone. All ticks from the genus Ixodes were from theumid bioclimatic zone, whereas Hy. detritum, Hy. excava-um, Rh. turanicus and Rh. sanguineus were from the aridioclimatic one.
.2. Detection of tick-borne protozoan and bacterialathogens
A total of 169 pools, representing 2333 ticks belongingo 10 species, were screened for common tick-borne proto-oan and bacterial pathogens by PCR and RLB with a panelf probes. Of these 169 tick pools, 49 (28.99%) were foundo be positive for the tick-borne pathogens, and the over-ll MLE of the infection rate was calculated as 2.44% (CI.88–3.17) (Table 1). The pathogens were detected in 23f 75 (30.66%) pools in the arid bioclimatic zone (2.12%, CI.44–3.09), whereas 26 of 94 (27.65%) pools tested posi-ive in the humid zone (2.91%, CI 1.97–4.20). The statisticalnalysis showed that tick-borne pathogen prevalence wasot statistically significant between the arid and the humidones (P > 0.05).
No tick-borne pathogen DNA was detected in Hy.arginatum, Rh. turanicus and Hae. punctata. Most tickools were infected with a single pathogen. However, fourools (3 from Hae. sulcata, 1 Rh. sanguineus) infected with H.anis displayed infections with B. crassa, A. phagocytophilumnd E. canis (Table 1).
.3. Sequencing
The sequence analysis of representative samples fromach species confirmed the presence of T. annulata, B. ovis,. crassa, A. ovis, E. canis and A. phagocytophilum. Threeick pools comprising of one each from Hy. excavatum,h. bursa, and D. marginatus showed positive signals tonaplasma/Ehrlichia catch-all probe but did not show aignal with the Anaplasma and Ehrlichia species-specificrobes on the RLB membrane. At the species level, sequencenalysis showed these samples to be 100% identical tohe sequence of Ehrlichia sp. Firat 2 and 3 (accessionos. EU191228, EU191229), previously identified fromyalomma anatolicum in Turkey (Aktas et al., 2009).
To confirm results and identify species, all Hepato-oon spp. positive PCR products detected in tick poolsere directly sequenced. Results showed four amplicons
0.17%, CI 0.06–0.41) to be 100% identical to the sequenceor Hepatozoon felis (Tateno et al., 2013). The remainingequences (1.51%, CI 1.06–2.09) shared 99% similarity withhe H. canis isolate DD11 previously identified in unfed
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Rh. sanguineus from Turkey (Aktas et al., 2013). The par-tial sequences of the isolates detected in this study havebeen deposited in GenBank under the following accessionnumbers: KF034779–KF034789.
4. Discussion
Ixodid ticks are vectors for diseases that affect bothhumans and animals, especially in tropical and subtropi-cal regions (de La Fuente et al., 2008; Ozdarendeli et al.,2008, 2010). They are also responsible for direct damageby reducing live weight and milk production, and they cancause severe toxic conditions such as paralysis, irritationand allergies. Most ticks have a preference for feeding ona wide variety of domestic and wild animals, while someare host-specific. However, a large number of tick specieshave adapted to feed on humans. More than 20 ixodid tickspecies are reported to be frequently found on humans(Estrada-Pena and Jongejan, 1999). The tick species foundin this study are known to commonly infest humans andlivestock in Turkey (Karaer et al., 2011). The studies alsoshow that humans in the surveyed regions were bitten by10 tick species, and Hy. marginatum and I. ricinus were themain tick species found in the arid cold and humid biocli-matic regions, respectively. These findings are consistentwith previous reports (Aktas et al., 2010, 2012).
Among the tick species examined for common tick-borne protozoan and bacterial pathogens, seven wereinfected with one or more of the pathogens targetedby our protocols (Table 1). Nine tick-borne pathogenswere detected in the arid bioclimatic zone, while threepathogens were present in the humid one. However, ourprevious published work screened the same tick pools fromthe humid zone for the presence of A. phagocytophilum DNAby nested PCR, and 11 tick pools (9 from I. ricinus, 2 Ixodesspp. nymphs) were found to be infected with the pathogen(Aktas et al., 2010). Hence, these results were not includedin the present study.
The results indicated the presence of nine tick-borne pathogens with an overall prevalence of 2.44% (CI1.88–3.17). Among the pathogens detected in ticks, T. annu-lata, B. ovis, B. crassa, Anaplasma/Ehrlichia spp., A. ovis, E.canis, A. phagocytophilum, H. canis and H. felis were identi-fied. Some of these pathogens were expected to be presentbecause they have been previously reported in ixodid ticksand domestic animals in parts of Turkey including thestudy areas (Aktas et al., 2010, 2011, 2012, 2013). How-ever, this is the first time that B. crassa and E. canis wereobserved in ixodid ticks from Turkey, and these resultscontribute greater insight into tick-bone pathogens dis-tribution and phylogenetic diversity. These findings alsoprovide notable information about the vector competenceof these tick species. Detection of the pathogens in unfedticks collected from vegetation is assumed to more likely toserve as potential vectors, however, to confirm this idea itshould be supported by additional experiments. The ticks
used in this study were removed from humans a short timeafter they bite, so because they were not feeding from ananimal at the time of collection, they were treated as unfedticks. The ticks that tested positive for pathogens may have
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been infected as immature stages when feeding on natu-rally infected domestic or wild animals.
In the RLB assay, all positive PCR products showed apositive signal for species-specific probes as well as to thecorresponding catch-all and genus-specific probes. How-ever, 3 of them gave positive signals for the catch-allAnaplasma/Ehrlichia spp. probe, but did not show any sig-nal to the species-specific probes tested. This could indicatethat there were not sufficient amplicons in the samples togive a species-specific signal, but it could also signify thepresence of a novel species or a variant of a known species.This possibility is supported by the recent identification ofa number of new pathogens and genotypes (Bekker et al.,2002). The partial sequencing indicated that those ampli-cons were 100% identical to the unnamed Ehrlichia sp. Firat2 and 3 (accession nos. EU191228, EU191229) previouslyidentified from Hy. anatolicum in Turkey (Aktas et al., 2009).
Tropical theileriosis is considered a major threat to thecattle industry in Mediterranean, Middle East, Westernparts of India and China because the causative agent causesmortality and economic losses, particularly in importedand crossbred cattle. T. annulata is very prevalent in Turk-ish cattle, especially in eastern regions (Aktas et al., 2001;Dumanli et al., 2005). It is well known that the main vec-tors of T. annulata are Hy. anatolicum (Sangwan et al., 1989)and Hy. detritum (Samish and Pipono, 1978). Hy. excavatumalso transmits the parasite (Samish and Pipano, 1983). Inthis study, no Hy. anatolicum were examined, but a highMLE of infection rate of T. annulata in Hy. detritum (7.08%,CI 2.34–22.72) and Hy. excavatum (1.52%, CI 0.12–7.84) isconsistent with the results obtained by a previous studycarried out in the same region (Aktas et al., 2004). Theseresults suggest that Hy. detritum and Hy. excavatum mayalso play a role in the transmission of T. annulata infec-tion in Turkey. Interestingly, bovine Babesia species werenot detected in any of the ticks analyzed, especially fromthe humid region, although I. ricinus and Rh. bursa, the vec-tor ticks of the bovine Babesia species, were among thoseanalyzed.
B. ovis is highly pathogenic in sheep and goats, andits case-fatality rate in susceptible hosts ranges from 30%to 50% in field infections, whereas the pathogenicity ofB. crassa is not high, and it is considered non-pathogenicto small ruminants (Hashemi-Fesharki, 1997). B. ovis hasbeen reported to be transmitted by Rh. bursa, Rh. turani-cus, Hy. excavatum, and most likely Rh. evertsi (Friedhoff,1997). Our results showed that 2 out of 13 pools (0.86%,CI 0.16–2.85) derived from Rh. bursa were infected with B.ovis. This finding is consistent with previous studies sug-gesting the development of B. ovis in the salivary glands ofthe vector tick (Shayan et al., 2008; Altay et al., 2008). Thereis no information regarding vector ticks for B. crassa. In ourstudy, it was detected in one tick pool from Hae. sulcata.
A. phagocytophilun is the most common tick-bornezoonotic bacteria of livestock and free-living ungulates inEurope. This pathogen also infects humans, causing gran-ulocytic anaplasmosis. A. ovis is infective in sheep and
goats, and it causes hemolytic anemia and hemoglobin-uria. Recently, the first human case caused by A. ovis wasreported in a young woman with high fever in Cyprus(Chochlakis et al., 2010). Although A. phagocytophilum and
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A. ovis have been detected in ruminants and ixodid ticksin Turkey (Aktas et al., 2010, 2011), there have been noreports of clinical cases in ruminants or humans associatedwith both pathogens. In this study, the MLEs of the infec-tion rate of A. phagocytophilum and A. ovis were detected at2.95% and 0.41%, respectively. Animals and humans can beassumed to be at risk of infection by these two pathogens.
The Anaplasma species may be transmitted mechan-ically by biting flies or blood-contaminated fomites andtrans-stadially by ixodid ticks. A. ovis is transmitted by ticksfrom the genus Dermacentor, Rhipicephalus and Hyalomma(Friedhoff, 1997). I. ricinus is considered the chief vectorof A. phagocytophilum in Europe (Stuen, 2003). Other tickshave been speculated to play an important role in the trans-mission of this agent (Bown et al., 2003). In a previous studyperformed in a semi-arid region of Turkey where I. ricinusis not endemic, A. phagocytophilum was detected in cattle(Aktas et al., 2011). In this study, two tick pools (2.95%, CI0.61–9.44) of Hae. sulcata and one of Rh. bursa (0.41%, CI0.02–2.01) from the arid region were found to be infectedwith A. phagocytophilum and A. ovis, respectively. Thesefindings suggest that Hae. sulcata and Rh. bursa can har-bor A. phagocytophilum and A. ovis, respectively, and maybe potential vectors of these pathogens in some parts ofTurkey.
Ehrlichia species are transmitted by ticks of the fam-ily Ixodidae. Rh. bursa, Rh. sanguineus, D. marginatus andHae. sulcata were found to be infected with E. canis in thisstudy. To the best of our knowledge, this is the first detailedreport demonstrating the presence of E. canis in ixodidticks from Turkey. These findings confirm previous stud-ies that E. canis is typically transmitted by Rh. sanguineus,but other tick species may also serve as potential or alter-native vectors of the pathogen (Stich et al., 2008; Hornoket al., 2013a).
The vector ticks of Hepatozoon spp. are not fully under-stood. In the present study, Hepatozoon spp. were detectedin Rh. sanguineus, D. marginatus, Hae. sulcata, I. ricinus,Haemaphysalis and Ixodes spp. nymphs. The brown dog tick,Rh. sanguineus, is the most important vector for H. canisin the Mediterranean basin and Balkan countries wherethe tick is endemic (Baneth, 2011). In previous studies oncanine hepatozoonosis in Europe and South America, thepresence of Rh. sanguineus-infested dogs was associatedwith H. canis infection (Dantas-Torres et al., 2012; Aktaset al., 2013). In this study, 3 pools (7.10%, CI 2.42–20.03)of Rh. sanguineus were infected with H. canis. This findingis consistent with report that acquisition of H. canis infec-tion by Rh. sanguineus typically occurs in the nymph andtransmission occurs in adults (Giannelli et al., 2013a). Rh.sanguineus is a 3-host tick. Although the tick feeds on awide variety of mammals, it prefers to feed on dogs in larval,nymphal and adult stages. Among ixodid ticks examined inthis study, the highest infection rate of H. canis was foundin Rh. sanguineus. The results indicate that the tick may bea potential vector for this parasite in Turkey.
The results obtained in this study agree to previ-
ously reported findings that, in addition to Rh. sanguineus,implicated other ixodid tick species as competent vec-tors of H. canis (Murata et al., 2005; Forlano et al., 2005;Gabrielli et al., 2010; De Miranda et al., 2011; Hornok
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t al., 2013b). However, the positive results of PCR do notndicate that the positive ticks are vectors. Some addi-ional experiments are required to confirm this idea. Thus,
recent study revealed that I. ricinus ingested H. canisamonts during the blood meal, as inferred by 3CR-positive specimens, but further development andporogony did not occur (Giannelli et al., 2013b).
Following infection by H. canis of the nymphs, Rh. sang-ineus adults may transmit the parasite to dogs (Aktas et al.,013; Giannelli et al., 2013a). However, the trans-stadialransmission from larvae to the nymph remains in ques-ion. Trans-stadial transmission (from larva to nymph) of H.mericanum in Amblyomma maculatum has been reportedEwing et al., 2002). A recent study also demonstrated thatarvae can become infected with H. canis when feeding onn infected dog (Giannelli et al., 2013a). In this study, nohipicephalus spp. nymphs were examined, but H. canisNA was amplified in 5 pools of Ixodes spp. nymphs and
pools of Haemaphysalis spp. nymphs (3 from arid zone, 1rom humid). The identity of the vertebrate hosts on whichhese nymphal ticks fed in the larval stage was unknown.xodes and Haemaphysalis spp. are three-host ticks, and thearvae and nymphs often infest small mammals such asodents, rabbits, hedgehogs, reptiles, and also birds. Wepeculate that these nymphal ticks acquire the infectionhen feeding on infected hosts as larvae. If this is so, some
mall mammals may play a role in maintaining a source of. canis infection.
Feline hepatozoonosis caused by H. felis has beeneported in domestic cats and wild felids from severalountries worldwide where canine infection is also presentVilhena et al., 2013). However, pathogenesis, transmis-ion, life cycle, and vectors of H. felis are unknown (Baneth,011). Feline hepatozoonosis has never been reported inats in Turkey. In this study, the parasite was detected byCR in two pools of I. ricinus (0.30%, CI 0.05–0.99), one poolf Rh. sanguineus (1.90%, CI 0.13–9.01), and one pool ofea. sulcata (1.90%, CI 0.13–9.01). These findings confirm
previous study by Aktas et al. (2013).In conclusion, various tick-borne pathogens identified
n this study suggest a risk for the emergence of tick-orne diseases in domestic animals and humans in Turkey.owever, further studies involving a larger number of
icks sampled across the country and detailed transmis-ion studies should be conducted to confirm the prevalencef tick-borne pathogens of veterinary and medical impor-ance.
cknowledgements
This work was supported financially by grant 110 O70 from the Scientific and Technical Research Council ofurkey (TUBITAK).
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