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Ticks and Tick-borne Diseases 5 (2014) 928–938

Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases

j ourna l ho me pa ge: www.elsev ier .com/ locate / t tbd is

riginal article

istinct Anaplasma phagocytophilum genotypes associated with Ixodesrianguliceps ticks and rodents in Central Europe

ucia Blanarováa, Michal Stankoa,b, Giovanna Carpic,d, Dana Miklisováa,ronislava Víchováa, Ladislav Mosanskya, Martin Bonae, Markéta Derdákováa,b,∗

Institute of Parasitology SAS, Hlinkova 3, 040 01 Kosice, SlovakiaInstitute of Zoology SAS, Dúbravská cesta 9, 845 06 Bratislava, SlovakiaFondazione Edmund Mach, Trento, ItalyYale School of Public Health, Department of Epidemiology of Microbial Diseases, 60 College Street, New Haven, USADepartment of Anatomy, Faculty of Medicine UPJS, Srobárová 2, 041 80 Kosice, Slovakia

r t i c l e i n f o

rticle history:eceived 27 April 2014eceived in revised form 1 July 2014ccepted 15 July 2014vailable online 13 August 2014

eywords:naplasma phagocytophilum genotypes

xodes triangulicepsxodes ricinusodentsenetic loci

a b s t r a c t

Rodents are important reservoir hosts of tick-borne pathogens. Anaplasma phagocytophilum is thecausative agent of granulocytic anaplasmosis of both medical and veterinary importance. In Europe,this pathogen is primarily transmitted by the Ixodes ricinus tick among a wide range of vertebrate hosts.However, to what degree A. phagocytophilum exhibits host specificity and vector association is poorlyunderstood. To assess the extent of vector association of this pathogen and to clarify its ecology in Cen-tral Europe we have analyzed and compared the genetic variability of A. phagocytophilum strains fromquesting and feeding I. ricinus and Ixodes trianguliceps ticks, as well as from rodent’ tissue samples. Tickcollection and rodent trapping were performed during a 2-year study (2011–2012) in ecologically con-trasting setting at four sites in Eastern Slovakia. Genetic variability of this pathogen was studied fromthe collected samples by DNA amplification and sequencing of four loci followed by Bayesian phyloge-netic analyses. A. phagocytophilum was detected in questing I. ricinus ticks (0.7%) from all studied sitesand in host feeding I. trianguliceps ticks (15.2%), as well as in rodent biopsies (ear – 1.6%, spleen – 2.2%),whereas A. phagocytophilum was not detected in rodents from those sites where I. trianguliceps ticks wereabsent. Moreover, Bayesian phylogenetic analyses have shown the presence of two distinct clades, andtree topologies were concordant for all four investigated loci. Importantly, the first clade contained A.phagocytophilum genotypes from questing I. ricinus and feeding I. ricinus from a broad array of hosts (i.e.,:humans, ungulates, birds and dogs). The second clade comprised solely genotypes found in rodents and

feeding I. trianguliceps. In this study we have confirmed that A. phagocytophilum strains display specifichost and vector associations also in Central Europe similarly to A. phagocytophilum’ molecular ecology inUnited Kingdom. This study suggests that A. phagocytophilum genotypes associated with rodents are prob-ably transmitted solely by I. trianguliceps ticks, thus implying that rodent-associated A. phagocytophilumstrains may not pose a risk for humans.

© 2014 Elsevier GmbH. All rights reserved.

∗ Corresponding author at: Institute of Parasitology SAS, Hlinkova 3, 040 01 Kosice,lovakia. Tel.: +421 907977389.

E-mail addresses: blanarova@saske.sk (L. Blanarová), stankom@saske.skM. Stanko), giovanna.carpi@yale.edu (G. Carpi), miklis@saske.skD. Miklisová), vichova@saske.sk (B. Víchová), mosansky@saske.skL. Mosansky), martinbonask@gmail.com (M. Bona),

arketa.derdakova@gmail.com, marketa.derdakova@savba.sk (M. Derdáková).

ttp://dx.doi.org/10.1016/j.ttbdis.2014.07.012877-959X/© 2014 Elsevier GmbH. All rights reserved.

Introduction

Anaplasma phagocytophilum is a gram-negative, intracellular,tick-transmitted bacterium belonging to the Anaplasmataceae fam-ily (Dumler et al., 2001). This causative agent of granulocyticanaplasmosis of both medical and veterinary importance is widelydistributed in North America (USA), Europe and Asia. A. phago-cytophilum is maintained in natural foci by a complex natural

transmission enzootic cycle which involves the vector ticks of theIxodes ricinus complex (Telford et al., 1996; Richter et al., 1996;Ogden et al., 1998; Levin and Fish, 2000; Cao et al., 2003; Eremeevaet al., 2006) and a wide range of vertebrate species as reservoir

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L. Blanarová et al. / Ticks and Ti

osts (Petrovec et al., 2002; de la Fuente et al., 2005; Woldehiwet,006; Stuen, 2007; Carpi et al., 2009), whereas humans are gener-lly incidental hosts. Nidicolous ticks such as Ixodes spinipalpis inSA (Burkot et al., 2001; DeNatale et al., 2002) and Ixodes trian-uliceps in United Kingdom (UK) may also contribute to the naturalnzootic cycle of this bacterium (Bown et al., 2003, 2006, 2008,009).

In the USA, small and medium sized mammals, ungulateswhite-tailed deer) and birds can act as reservoirs (Belongia et al.,997; Magnarelli et al., 1999; Nicholson et al., 1999; Levin et al.,002; Massung et al., 2003; Keesing et al., 2012). Moreover, basedn the 16S rRNA gene, specific pathogen–host associations of twoifferent A. phagocytophilum variants were described: The Ap-1ariant circulates in Ixodes scapularis ticks and free-living ungu-ates, whereas the Ap-ha variant is found in infected humans andts ecology is linked to rodents as reservoir hosts (Levin et al.,002). In contrast to the USA, the role of vertebrate species as nat-ral reservoir of human pathogenic strains of A. phagocytophilum

n Europe and Asia is still poorly understood. In Europe, a higheregree of genetic diversity of A. phagocytophilum strains from dif-erent hosts has been described compared to the USA (de la Fuentet al., 2005; Carpi et al., 2009; Bown et al., 2009; Derdáková et al.,011; Rar and Golovljova, 2011), and wild and domestic ungu-

ates have been suggested as reservoirs (Ogden et al., 1998, 2002;etrovec et al., 2002; Liz et al., 2002; Stuen et al., 2002). Addition-lly, in Europe A. phagocytophilum has been detected in a broaderrray of hosts, including wild boar (Sus scrofa), red fox (Vulpesulpes), brown bear (Ursus arctos), and hare (Lepus europaeus)Víchová et al., 2010; Hulínska et al., 2004; Stefancíková et al.,005). Among small mammals, wood mice (Apodemus sylvaticus),ellow-necked mice (Apodemus flavicollis), herb field mice (Apode-us microps), field voles (Microtus agrestis) and bank voles (Myodes

lareolus) have also been suggested as reservoir hosts for A. phago-ytophilum (Liz et al., 2000; Bown et al., 2006, 2008; Stefancíkovát al., 2008; Keesing et al., 2012; Víchová et al., 2014). Interestingly,enetic analyses on several molecular marker genes have shownhat A. phagocytophilum genotypes circulating in rodents and Ixodesicks in Europe differ from those circulating in the USA and AsiaBown et al., 2009; Zhan et al., 2010). Furthermore, in the UK,own et al. (2003) described separate enzootic cycle of A. phagocy-ophilum genotypes: rodent associated genotypes are transmittedy I. trianguliceps.

The genetic diversity of A. phagocytophilum strains has beentudied by analyzing several phylogenetically informative loci,ncluding the 16S rRNA gene (Massung et al., 1998), the heat-hock protein GroEL (Liz et al., 2002; Carpi et al., 2009), the majorurface proteins Msp4 (de la Fuente et al., 2005), the variable non-oding fragment DOV1 (Bown et al., 2009) and the ankA gene whichncodes for the ankyrin repeat-containing protein (Park et al.,004). The phylogenetic analyses of groEL (Petrovec et al., 2002;iz et al., 2002), ankA (Von Loewenich et al., 2003; Park et al., 2004;charf et al., 2011) and msp4 (de la Fuente et al., 2005) genes of. phagocytophilum strains from various vertebrate hosts and vec-or ticks suggested that intraspecific variability is linked to specificosts, vectors and geographic locations.

Rodents act as reservoirs of many tick-borne pathogens. Untilecently, it was thought that in Europe rodents are also reservoirosts of A. phagocytophilum strains that are vectored by I. ricinusicks (Liz et al., 2000; Beninati et al., 2006; Spitálska et al., 2008;

ˇtefancíková et al., 2008) and infect both humans and domesticnimals as in the USA (Telford et al., 1996; Massung et al., 2003).owever, recent studies show that this might not be the case for

urope, as strains where strains in rodents differ genetically fromhose circulating in I. ricinus ticks, domestic ruminants, wild boar,ogs, horses and humans (Bown et al., 2008; Majazki et al., 2013).

t was also suggested that I. trianguliceps might be the vector of

rne Diseases 5 (2014) 928–938 929

these rodent strains in UK (Bown et al., 2008, 2009). Furthermore,in Switzerland, Burri et al. (2014) did not detect A. phagocytophilumin I. ricinus ticks feeding on rodents even though A. phagocytophilumwas detected in questing I. ricinus from the same areas. It is stilldebated whether rodents play a role in maintaining A. phagocy-tophilum in continental Europe, and empirical evidence is lacking.In this study we aim to assess whether rodents contribute to theecology of A. phagocytophilum in Central Europe. More specifically,our goal was to assess and characterize the genetic diversity andecological associations of A. phagocytophilum genotypes circulat-ing in rodents, questing I. ricinus ticks and feeding I. ricinus and I.trianguliceps ticks in several sites in Slovakia (Central Europe).

Materials and methods

Study area

This study was conducted in four sampling sites in Eastern Slo-vakia (Cermel’, Hyl’ov, Botanical garden Kosice and Rozhanovce).Sites were selected to include areas with contrasting occurrenceof nidicolous I. trianguliceps ticks feeding on rodents. Specifically,two control sites were characterized by the presence of two ixo-did species, I. ricinus and I. trianguliceps- Cermel’ (208–600 masl.; 48◦45′46.67′′ N; 21◦8′8.17′′ E) and Hyl’ov (500–750 m asl.;48◦44′22.80′′ N; 21◦4′18.90′′ E); whereas two sites were char-acterized by the absence of I. trianguliceps ticks and presenceof I. ricinus ticks exclusively, Botanical garden Kosice (208 masl.; 48◦44′6.84′′ N; 21◦14′16.14′′ E) and Rozhanovce (215 m asl.;48◦4500′′ N; 21◦21′00′′ E). Study sites were located in sylvaticmixed forest (Hyl’ov and Cermel’), suburban deciduous forest(Botanical garden, Kosice) and game reserve (Rozhanovce).

Sample collection

Tick collections and trapping of rodents were performed in 2011and 2012 at the four investigated sites in Eastern Slovakia.

Questing ticks were collected at each study site by a standard-ized flagging method (Falco and Fish, 1988) using a 1- m2 whitecorduroy cloth for 1 h to cover various types of forest/shrubland andedge vegetation. Immediately after collection, ticks were stored andpreserved in tubes with 70% ethanol until the DNA was extracted.

Rodents were trapped alive using Swedish bridge metal trapsfollowing the protocol of Stanko (1994) and Stanko and Miklisova(1995). Rodent trapping were carried out using 100–150 traps/persite for two-night trapping. A total of 854 trapped individuals of 10species of small mammals (rodents and insectivores) were euth-anized according to the laws of the Slovak Republic under thelicenses of the Ministry of Environment of the Slovak RepublicNo. 297/108/06-3.1 and No. 6743/2008-2.1. Feeding ticks wereremoved from the rodents with sterile forceps, counted and iden-tified to life stage and species level using previously publishedtaxonomic keys (Filippova, 1977; Estrada-Pena et al., 2004) andpreserved in 70% ethanol until DNA was extracted. Moreover,spleen and ear biopsies were obtained from each rodent duringnecroscopy.

DNA extraction

A total of 1376 questing ticks and 740 rodent-fed ticks from854 rodents were used for DNA analyses. Genomic DNA was

isolated from individual ticks by alkaline-hydrolysis according toGuy and Stanek (1991). DNA from rodent tissues (407 spleensand 669 ears) was extracted using a commercial DNA extractionkit (NucleoSpin Blood kit, NucleoSpin Tissue kit, Machery Nagel,

930 L. Blanarová et al. / Ticks and Tick-borne Diseases 5 (2014) 928–938

Table 1Number of questing I. ricinus ticks (IR), feeding ticks (IR + IT), rodent biopsies (ear and spleen) that were detected as infected with A. phagocytophilum by PCR; number of totalquesting I. ricinus ticks, feeding ticks (IR + IT) and rodent biopsies used for molecular analysis at the study sites in years 2011 and 2012; infection prevalence (%).

Site model No. of positive questingIR ticks/no. of questingIR tick; prevalence-%

No. of positive feeding ticks(IR + IT)/no. of feedingticks; prevalence-%

No. of positive earbiopsies/no. of earbiopsies; prevalence-%

No. of positive spleenbiopsies/no. of spleenbiopsies; prevalence-%

F-test 0.695 0.002 0.001 0.008

Cermel’ 2/220 (0.9) 7/48 (14.6) 2/178 (1.1) 3/165 (1.8)95% CI 0.11–3.25 6.07–27.77 0.64–2.48 0.38–5.29Hyl’ov 2/266 (0.8) 3/150 (2.0) 9/87 (10.5) 6/77 (7.9)95% CI 0.09–2.69 0.41–5.81 4.89–18.94 4.26–23.03B. garden 2/176 (1.1) 0/375 0/46 0/2895% CI 0.13–4.05Rozhanovce 4/714 (0.6) 0/167 0/358 0/13795% CI 0.15–1.43

Total 10/1376 (0.7) 10/740 (1.4) 11/669 (1.6) 9/407 (2.2)

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95% CI 0.34–1.34 0.64–2.48

R – Ixodes ricinus, IT – Ixodes trianguliceps; F-test: p-value of Fisher’s exact test for c

ermany) according to the manufacturer’s protocol. Lysates weretored at −20 ◦C prior to use (Table 1).

olecular detection and characterization of A. phagocytophilum

Polymerase chain reaction (PCR) amplification of the tick mito-hondrial cytochrome b gene was performed for each sample as auality control for tick DNA (Black and Roehrdanz, 1998; Derdákovát al., 2003). Moreover in the rodent samples, 12S rRNA gene wassed to determine the quality control for the tissue DNA extractionHumair et al., 2007).

Samples were further screened for the presence of A. phago-ytophilum by real-time PCR using the primers ApMSP2f5′-ATGGAAGGTAGTGTTGGTTATGGTATT-3′), ApMSP2r (5′-TGGTCTTGAAGCGCTCGTA-3′) and the TaqManProbe ApMSP2p5′-TGGTGCCAGGGTTGAGCTTGAGATTG-3′) labeled with FAM,hich targeted a 77-bp long fragment of the msp2 gene (Courtney

t al., 2004). This assay was run on a CFX96 Real-Time PCR SystemBio-Rad, Hercules, CA, USA).

To further characterize A. phagocytophilum- infected samples,our molecular loci, 16S rRNA, msp4, groEL and DOV1 were ampli-ed and sequenced. Nested PCR was performed to amplify a 546-bp

ragment of the 16S rRNA gene as previously described (Massungt al., 1998). Nested PCRs were used to amplify a 498-bp fragment ofhe msp4 gene (de la Fuente et al., 2005) and a 1297-bp region of theroEL gene (Liz et al., 2002). The 275-bp fragment of the DOV1 non-oding region was amplified using seminested PCR as describedreviously (Bown et al., 2009).

The PCR reactions were performed in a total volume of 25 �l ofeaction mixture with 12.5 �l 1× mix, 2.6 �l 2.5 mM MgCl2, 2.3 �l20 nM of each primers and 0.3 �l 0.12 nM of probe using 5 �l asNA template. Kit Bioron SuperHot Master Mix (Bioron, Germany)as used. The cycling conditions were 95 ◦C for 120 s followed by

9 cycles (95 ◦C for 15 s; 60 ◦C for 60 s). In each PCR reaction, DNArom A. phagocytophilum- positive questing I. ricinus ticks were useds positive controls and DNA-free molecular water was added asemplate in negative controls. The PCR products were visualized bylectrophoresis on 2% agarose gels stained with GoldView Nucleiccid Stain (Beijing SBS Genetech, Beijing, China). All positive PCRroducts were purified using a QIAquick PCR purification kit (Qia-en, Hilden, Germany) and bidirectionally Sanger sequenced withhe same primers as for the PCR amplifications.

hylogenetic analysis

The complementary strands of each sequenced product wereanually assembled into consensus sequences. The consensus

1.09–3.88 1.11–4.56

ring prevalences.

sequences were compared to GenBank entries by BlastN v.2.2.13(Altschul et al., 1997). Obtained A. phagocytophilum sequenceswere aligned with representative homologous sequences publiclyavailable in GenBank (December 2013, 180 groEL sequences, 270msp4 sequences and April 2014, 21 DOV1 sequences) using theMUSCLE program (Edgar, 2004) and adjusted manually to main-tain reading frame integrity in the protein coding genes usingthe Se–Al v.20a11 alignment editing software (Rambaut, 1996).Unique haplotypes were identified using COLLAPSE 1.2 (DavidPosada; http://darwin.uvigo.es/software/collapse.html). jMODEL-TEST v.2.1.4 (Darriba et al., 2012) was employed to select thenucleotide substitution model most appropriate to the dataset (groEL: HKY + I, msp4: GTR + I + G, DOV1: HKY). The selectednucleotide substitution models (model selection using Akaike (AIC)and Bayesian (BIC) criteria) were used to infer Bayesian phylogenyfor three genes calculated by the computer program MrBayes v3.1.2(Ronquist and Huelsenbeck, 2003). Markov chains were run for2,000,000 generations, sampled every 10,000 generations, and thefirst 25% of each chain was discarded as burning and the remainingtrees were used to construct a 50% majority-rule consensus tree.Uncorrected pairwise genetic distances were estimated using PAUPv.4.0 b10 (Swofford, 2003) and expressed as nucleotide diversity(�) (Nei, 1987).

Nucleotide sequence accession numbers

Eighty-six new sequences of A. phagocytophilum weredeposited in the GenBank database with accession numbersKF383227–KF383265, KF420092–KF420117 and KF481928–KF481948 (Table 6).

Statistical analysis

The association between A. phagocytophilum infection (AP),the response variable of interest, and the categorical variables(design factors), locality (LOC), host species (HOST), tick species(TICK), larval stadium and coincident occurrence of I. trianguli-ceps and I. ricinus, was analyzed. As �2 test did not confirma relation between AF and larval stadium or coincident occur-rence of both tick species, these factors were excluded fromthe subsequent statistical analysis. The log-linear analysis offrequency tables with automatic selection of best model, thatfind the least complex model fitting the data, was used to

assess relationships between factors. The software Statistica 9(http://www.statsoft.com) was used for this analysis. Confidenceintervals (CI) and differences in A. phagocytophilum infection preva-lences among sites tested by Fisher’s exact test were calculated

L. Blanarová et al. / Ticks and Tick-borne Diseases 5 (2014) 928–938 931

Table 2Number of feeding ticks collected from rodents that were detected as infected with A. phagocytophilum by PCR; number of feeding ticks collected from rodents used formolecular analysis at each study site in years 2011 and 2012; infection prevalence (%).

Model site No. of positive feedinglarval IR ticks/no. offeeding larval IR ticks;prevalence-%

No. of positive feedingnymphal IR ticks/no. offeeding nymphal IR ticks;prevalence-%

No. of positive feedinglarval IT ticks/no. offeeding larval IT ticks;prevalence-%

No. of positive feedingnymphal IT ticks/no. offeeding nymphal IT ticks;prevalence-%

No. of positive female ITticks/no. feeding female ITticks; prevalence-%

Cermel’ 0/25 0/1 7/19 (36.8) 0/3 095% CI 16.28–61.65Hyl’ov 0/104 0/4 1/33 (3.0) 1/6 (16.7) 1/3 (33.3)95% CI 0.07–15.76 0.42–64.13 0.84–90.58B. garden 0/348 0/27 0 0 0Rozhanovce 0/153 0/12 0 0/2 0

Total 0/630 0/44 8/52 (15.4) 1/11 (9.1) 1/3 (33.3)

95% CI 6.88–28.09 0.22–41.28 0.84–90.58

IR – Ixodes ricinus; IT – Ixodes trianguliceps.

Table 3Number of rodents that were detected as infected with A. phagocytophilum by PCR and number of trapped rodents used for molecular analysis at study in years 2011 and2012; prevalence (%).

Species of rodents No. of positive rodents/no. of total trapped rodents/prevalence (%)

Cermel’ Hyl’ov Botanical garden Rozhanovce Total

Apodemus agrarius 0/20 1/8 (12.5) 0/54 0/214 296Apodemus flavicollis 1/54 (1.9) 1/55 (1.8) 0/26 0/104 239Crocidura suaveolens 0 0 0/1 0/1 2Microtus subterraneus 0/1 0/3 0/1 0/2 7Microtus arvalis 0/1 0 0 0/10 11Myodes glareolus 4/111 (3.6) 8/39 (20.5) 0 0/87 237Sorex araneus 0/6 0/3 0/1 0/4 14Sorex minutus 0/1 0 0 0/2 3Neomys fodiens 0/2 0 0 0 2Micromys minutus 0 0 0/1 0 1

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in both spleen and ear biopsies simultaneously. Two positive earbiopsies came from rodents trapped at Cermel’, nine positive earbiopsies were found at the Hyl’ov site where we also detected the

Table 4Number of ticks that were detected as infected with A. phagocytophilum byPCR/infestations of individual rodents that carried both I. ricinus and I. triangulicepsconcurrently.

Species of rodents I. ricinus I. trianguliceps

Larvae Nymphs Adult Larvae Nymphs Adult

A. agrarius 0/231 0/31 0 0/9 0/1 0A. flavicollis 0/274 0/8 0 0/14 0/3 1/1

Total 5/196 (2.6) 10/108 (9.3)

y Quantitative Parasitology on the Web (Reiczigel et al., 2005);http://www.univet.hu/qpweb/qp10/index.php).

esults

. phagocytophilum in questing I. ricinus ticks

In total, 2710 questing I. ricinus ticks from four sites (251 I. rici-us ticks from Cermel’, 689 from Hyl’ov, 986 from Botanical gardennd 784 from Rozhanovce) were collected. From these ticks, 1376220 I. ricinus ticks from Cermel’, 266 from Hyl’ov, 176 from Botani-al garden and 714 from Rozhanovce) were tested for the presencef A. phagocytophilum by real-time PCR targeting the msp2 geneesulting in an overall A. phagocytophilum infection prevalence of.7% (10/1376; CI 95%: 0.34–1.34). We found no significant dif-erences between the infection rates from different sites (F-value.695, p > 0.01) (Table 1).

. phagocytophilum in rodent feeding ticks and biopsies

A total of 1713 feeding ticks (29 I. ricinus ticks and 22 I. trian-uliceps ticks from Cermel’; 167 I. ricinus and 42 I. trianguliceps ticksrom Hyl’ov; 751 I. ricinus ticks from Botanical garden; 698 I. ricinusnd 4 I. trianguliceps ticks from Rozhanovce) were removed fromodents with sterile forceps. Seven-hundred and forty ticks (66 I. tri-nguliceps and 674 I. ricinus) were further tested. As some rodentsere infested with a very high number of I. ricinus ticks, we tested

p to 20 feeding and visibly engorged I. ricinus and I. triangulicepsicks per single rodent. A. phagocytophilum was detected in 10 outf 740 tested feeding ticks (1.4%). Out of the tested feeding ticksrom rodents, only I. trianguliceps carried A. phagocytophilum and

0/84 0/424 812

in total 10/66, 15.2% were infected. None of the 674 rodent feedingI. ricinus tested positive for A. phagocytophilum even if feeding on anA. phagocytophilum-infected rodent (Tables 1–3). Positive feeding I.trianguliceps ticks (8 larvae, 1 nymph and 1 female) were collectedfrom M. glareolus and A. flavicollis (Tables 3 and 4). We observedsignificant differences in A. phagocytophilum prevalences in rodentfeeding ticks between the study sites based on the occurrence of I.trianguliceps (F-value 0.002, p < 0.01) (Table 1).

A total of 854 small mammals were captured (198 rodentsfrom Cermel’; 113 from Hyl’ov; 85 from Botanical garden and 458rodents from Rozhanovce). A. phagocytophilum was detected in 11of out of 669 tested ear biopsies (1.6%; CI 95%: 1.09–3.88) andin nine of 407 tested spleens (2.2%; CI 95%: 1.11–4.56) (Table 1).In five of 854 small mammals, A. phagocytophilum was detected

S. araneus 0/2 0 0 0 0 0M. glareolus 0/86 0/2 0 8/28 1/7 0/2M. subterraneus 0/36 0/4 0 0/1 0 0

Total 0/629 0/45 0 8/52 1/11 1/3

932 L. Blanarová et al. / Ticks and Tick-borne Diseases 5 (2014) 928–938

Table 5Comparison of the 16S rRNA gene of A. phagocytophilum obtained from ticks and rodent biopsies with selected GenBank sequences.

Sample Accession numbers16S rRNA

Host Origin Variant Nucleotide position

3 4 5 7 11 270 303

Paulauskas et al. (2012) JN181063 DRtick

Siluté, Lithuania 1 A A A A G A A

Paulauskas et al. (2012) JN181079 Engorged IR removed from raccoon dog Siluté, Lithuania 2 G A A A A A GPaulauskas et al. (2012) JN181081 Engorged IR removed from raccoon dog Siluté, Lithuania 3 G A A A G A GPaulauskas et al. (2012) JN181071 Engorged IR removed from chaffinch Jomfruland, Norway 4 A A A A A A G5BZNIRQ KF481932 Questing IR tick B. garden, Slovakia 4 A A A A A A G220166Bs

MGKF481928 Spleen biopsy from rodent Hyl’ov, Slovakia 4 A A A A A A G

228141LITMG KF481938 Engorged IT from rodent Hyl’ov, Slovakia 4 A A A A A A G

Paulauskas et al. (2012) JN181067 Questing IR tick Hitra, Norway 5 A A A A G A G227785LITMG KF481940 Engorged IT from rodent Cermel’, Slovakia 5 A A A A G A G2282171BeMG KF481945 Ear biopsy from rodent Hyl’ov, Slovakia 5 A A A A G A GPaulauskas et al. (2012) JN181068 Questing IR tick Jomfruland, Norway 6 A A G A A A GPaulauskas et al. (2012) JN181066 DR tick Kaisiadorys, Lithuania 7 A A A G G G A77HNIRQ KF481933 Questing IR tick Hyl’ov, Slovakia 8 A G A A G A G163HNIRQ KF481929 Cermel’, Slovakia 839FCIRQ KF481931 82MBZIRQ KF481930 Questing IR tick B. garden, Slovakia 9 A A A G A A G2204085BeMG KF481943 Ear biopsy from rodent Hyl’ov, Slovakia 9 A A A G A A GBown et al. (2003) AY082656 Rodent MGLA UK 10 A G A A A A G

D dy are

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S

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S

1

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R – Dermacentor reticulatus; IR – I. ricinus; IT – I. trianguliceps; samples of this stu

ighest number of infected I. trianguliceps ticks (Tables 1 and 2). A.hagocytophilum was detected in 12 biopsies from M. glareolus, inwo from A. flavicollis and one from A. agrarius. These three rodentpecies were also the most commonly trapped rodents during ourtudy. Positive samples from rodents originated only from the twoontrols sites, Cermel’ and Hyl’ov, where I. trianguliceps and I. ricinusicks co-occur. There were significant differences between the sitesn A. phagocytophilum positivity of ear (F-value 0.001, p < 0.01) andpleen (F-value 0.008, p < 0.01) biopsies based on the I. triangulicepsccurrence at the site (Tables 1–3).

tatistical analysis of A. phagocytophilum infection prevalence

Data from the two control sites, Cermel’ and Hyl’ov, and twoodents species, M. glareolus and A. flavicollis (where positive feed-ng ticks were found) were statistically tested. The log-linearnalysis showed that the least complex model that will fit the dataontains two-way associations (K = 2, max-Likelihood �2 = 63.6,

= 0.000) and does not contain any three-way associations (for = 3, max-Likelihood �2 = 1.95, p = 0.74). The best selected modelsrovided the following associations: AF-LOC, AF-TICK, LOC-HOSTnd HOST-TICK (max-Likelihood �2 = 5.49, df = 7, p = 0.601). Theseodels indicated that major factors associated with A. phagocy-

ophilum infection were the presence of I. trianguliceps ticks at theested locality.

equence analysis

6S rRNA gene sequencesSequencing of the 497 bp region of the 16S rRNA gene revealed

our different sequence types among 21 samples isolated fromuesting I. ricinus, feeding I. trianguliceps ticks and ear and spleeniopsies from Eastern Slovakia (Table 5). Sequencing confirmed aigh degree of similarity among 16S rRNA (99.0%). In rodent biop-ies and feeding I. trianguliceps ticks we detected two genotypesfour and five) corresponding to genotypes previously described

y Paulauskas et al. (2012). Moreover, we detected two unique 16SRNA genotypes in five samples, one from questing I. ricinus ticks,nd one from questing I. ricinus tick and ear biopsy. They weredentified as genotypes eight and nine. The analyzed sequences of

indicated in boldface.

genotype eight had nucleotide substitutions at position 4 and 303,and genotype nine had substitutions at 7, 11 and 303 (Table 5).

GroEL, msp4 and DOV1 phylogenetic analyses

In this study we generated 15, 26 and 24 sequences for groEL,msp4 and DOV1, respectively (Table 6). The mean nucleotide diver-sity (�) among the A. phagocytophilum sequences from this studywas 0.021 (range 0.0–0.061), 0.04 (range 0.0–0.118) and 0.068(range 0.0–0.234) for groEL, msp4, and DOV1, respectively.

The phylogenetic relationships based on Bayesian analysisbetween 15 A. phagocytophilum groEL sequences from this study(8 haplotypes) and 180 (91 haplotypes) from GenBank, and the 26msp4 sequences from this study (8 haplotypes) and 270 (80 hap-lotypes) from GenBank are shown in Fig. 1A and B, respectively.The phylogenetic trees of the two loci displayed similar topologywith strong support for two main clades. The first clade (hereafter“clade 1) included haplotypes detected in questing I. ricinus ticksand various vertebrate hosts such as roe deer, red deer, birds, sheep,dog and humans from European countries and the USA. The sec-ond clade (hereafter “clade 2) was highly divergent from clade 1,and included solely haplotypes from rodents, feeding I. trianguli-ceps from Slovakia and the UK and from questing Ixodes persulcatusfrom Russia.

The Bayesian phylogenetic analysis of 25 A. phagocytophilumDOV1 sequences from this study (4 haplotype) and 21 (10 haplo-types) from GenBank confirmed the same tree topology as groELand msp4 trees showing high support for two clades- clade 1 includ-ing A. phagocytophilum haplotypes from primary questing I. ricinus,and clade 2 comprising solely A. phagocytophilum haplotypes fromrodents and I. trianguliceps (Fig. 2).

Discussion

In the present study we investigated the infection preva-

lence and genetic diversity of A. phagocytophilum strains andtheir ecological associations with rodents, questing I. ricinus andfeeding Ixodid ticks (I. ricinus and I. trianguliceps) collected fromrodents. To shed light on the vector competence of I. ricinus and

L. Blanarová et al. / Ticks and Tick-borne Diseases 5 (2014) 928–938 933

Table 6Accession numbers (GenBank database) of A. phagocytophilum sequences for the 16S rRNA, msp4, groEL and DOV1 genes generated in this study.

Vector, host Sequences of 16S rRNA gene Sequences of msp4 gene Sequences of groEL gene Sequences of DOV1 gene

Questing I. ricinus nymph, Hyl’ov KF481929 KF420110 KF383241 KF383255Questing I. ricinus male, B. garden KF481930 KF420112 KF383237 KF383253Questing I. ricinus female, Cermel’ KF481931 KF420113 KF383239 KF383254Questing I. ricinus nymph, B. garden KF481932 KF420114 KF383238 a

Questing I. ricinus nymph, Hyl’ov KF481933 KF420115 KF383240 a

Questing I. ricinus tick, female, Rozhanovce a KF420116 a a

Questing I. ricinus tick, nymph, Rozhanovce a KF420117 a a

Questing I. ricinus tick, male, Cermel’ a KF420111 a KF383256

Feeding I. trianguliceps larva, M. glareolus KF481934 KF420104 KF383235 KF383264Feeding I. trianguliceps larva, M. glareolus KF481935 KF420105 KF383234 KF383262Feeding I. trianguliceps larva, M. glareolus KF481936 KF420106 a KF383260Feeding I. trianguliceps larva, M. glareolus KF481937 KF420107 KF383233 KF383259Feeding I. trianguliceps larva, M. glareolus KF481938 KF420109 KF383232 KF383257Feeding I. trianguliceps larva, M. glareolus KF481939 KF420108 a KF383258Feeding I. trianguliceps larva, M. glareolus KF481940 a a KF383261Feeding I. trianguliceps nymph, M. glareolus a KF420103 a a

Feeding I. trianguliceps female, A. flavicollis a a KF383236 KF383265Feeding I. trianguliceps larva, M. glareolus a a a KF383263

Spleen of M. glareolus KF481928 KF420092 KF383231 KF383252

Ear biopsy from A. flavicollis KF481941 KF420093 a KF383242Ear biopsy from M. glareolus KF481942 KF420095 KF383229 KF383244Ear biopsy from M. glareolus KF481943 KF420096 KF383227 KF383245Ear biopsy from M. glareolus KF481944 KF420098 KF383230 KF383247Ear biopsy from M. glareolus KF481945 KF420099 a KF383248Ear biopsy from M. glareolus KF481946 KF420100 a KF383249Ear biopsy from M. glareolus KF481947 KF420101 a KF383250Ear biopsy from A. agrarius KF481948 KF420102 a KF383251Ear biopsy from M. glareolus a KF420094 KF383228 KF383243

a KF4 a

Irtoae

IstDolofstitttoOiiipafitctio

Ear biopsy from M. glareolus

a Did not sequenced.

. trianguliceps in the transmission cycle of the A. phagocytophilumodent-associated strains we conducted a comparative study inwo ecologically contrasting settings, two sites known for theccurrence of I. ricinus only (Botanical garden Kosice, Rozhanovce)nd two sites known for the occurrence of both tick species (Pet’kot al., 1991).

The total prevalence of A. phagocytophilum infection in questing. ricinus ticks in our study was 0.7%. Previous findings from Slovakiahowed the infection rate in questing I. ricinus varying from 1.1% upo 8% (Spitálska and Kocianová, 2002, 2003; Spitálska et al., 2008;erdáková et al., 2011; Subramanian et al., 2012). In total, 15.2%f feeding I. trianguliceps ticks (all developmental stages includingarvae) were infected with A. phagocytophilum (Tables 1 and 2). Tour knowledge this is the first detection of A. phagocytophilum ineeding I. trianguliceps in continental Europe. Our results stronglyupport the previously proposed hypothesis by Bown et al. (2009)hat A. phagocytophilum strains associated with rodents circulaten enzootic cycles separate from non-rodent associated strains andhat they are transmitted by I. trianguliceps but not I. ricinus ticks. Ashe presence of infectious agent in feeding ticks does not prove thathe tick is also a biological vector of that agent DNA, additional xen-diagnostic studies are further needed to validate this hypothesis.n the other hand, our conclusions are supported by the findings

n rodent necropsies, where M. glareolus, A. flavicollis, and A. agrar-us were infected with the same genotype of A. phagocytophilumn those areas where I. trianguliceps were present. The highest A.hagocytophilum prevalence in rodents was found in vole speciess in the UK (Bown et al., 2003, 2006, 2008, 2009). Importantly, thending that none of the rodent-feeding I. ricinus ticks were foundo be infected with A. phagocytophilum although they were coin-

identally feeding on an infected rodent together with infected I.rianguliceps ticks, further supports the proposed hypothesis. Sim-larly, Heylen et al. (2014) did not observe co-feeding transmissionf B. burgdorferi s.l. on songbirds among the ornithophilic ticks

20097 KF383246

Ixodes arboricola, I. frontalis and I. ricinus. In the areas where I.trianguliceps ticks were absent (control sites – Botanical garden,Kosice and Rozhanovce), we did not detect A. phagocytophilumin rodents. Moreover, none of the questing I. ricinus ticks carriedthe A. phagocytophilum rodent genotype as confirmed by phylo-genetic analyses. Furthermore, none of the rodents were infectedwith the A. phagocytophilum genotypes that were present in I. rici-nus ticks. Similarly in Switzerland, Burri et al. (2014) did not findA. phagocytophilum-infected I. ricinus feeding on rodents and noneof the feeding xenodiagnostic ticks became infected. Even thoughthe blood of the rodents in our study was not tested, the presenceof A. phagocytophilum was detected in questing I. ricinus from theareas where rodents were captured. Altogether, these findings fur-ther support that A. phagocytophilum exhibits host specificity andvector association.

Our results were further supported by the obtained sequencedata (Table 5, Figs. 1 and 2). The highly conserved 16S rRNA genehas been used for genotyping A. phagocytophilum strains in manystudies. Four 16S genetic variants were detected in questing I. rici-nus, feeding I. trianguliceps ticks and in rodents in this study. Two(4 and 5) were detected in I. ricinus, I. trianguliceps and M. glare-oulus and were identical to variants previously detected in feedingand questing I. ricinus ticks in Lithuania (Paulauskas et al., 2012). Inaddition, we detected two new sequence types in questing I. ricinusticks and a rodent ear biopsy (Table 5). Von Loewenich et al. (2003)described seven variants infecting I. ricinus ticks in Germany, andKatargina et al. (2012) described four variants infecting I. ricinusticks in Estonia, Belarus and Russia. In the USA, the Ap-variant 1differs from a human strain (Ap-ha) and appears to be restricted toruminant species as reported by Massung et al. (1998). However, in

Europe, both the Ap-variant 1 and the Ap-ha 16S rRNA gene vari-ants were detected in sheep, cattle and cervids (Bown et al., 2009).Nevertheless, this locus is not an adequate genetic marker to inves-tigate the possible ecological association between the strains and

934 L. Blanarová et al. / Ticks and Tick-borne Diseases 5 (2014) 928–938

Fig. 1. Midpoint rooted 50% majority rule consensus trees constructed using Bayesian analysis for (A) 99 groEL haplotypes (length 1119 bp), and (B) 88 msp4 haplotypes(length 300 bp). Posterior probabilities >0.50 are indicated at nodes. New sequences from this study are indicated by dots. Each A. phagocytophilum sequence at each tipcorresponds to a unique haplotype and only a representative Genbank accession number for that haplotype is indicated. Each A. phagocytophilum sequence is shown with itssource: questing tick (e.g., I. ricinus), feeding tick (e.g., I. ricinus-host), or rodent tissue (e.g., host name), international country code (ISO �-2) and Genbank accession numbersare in parentheses. Scale bars indicate nucleotide substitutions per site.

L. Blanarová et al. / Ticks and Tick-borne Diseases 5 (2014) 928–938 935

Fig. 1. (Continued)

936 L. Blanarová et al. / Ticks and Tick-borne Diseases 5 (2014) 928–938

Fig. 2. Midpoint rooted 50% majority rule consensus tree constructed using Bayesian analysis of 14 DOV1 haplotypes (length 214 bp); posterior probabilities >0.50 areindicated at nodes. New sequences from this study are indicated by dots. Each A. phagocytophilum sequence at each tip correspondes to a unique haplotype and only arepresentative Genbank accession number for that haplotype is indicated. Each A. phagocytophilum sequence is shown with its source: questing tick (e.g., I. ricinus), feedingt code (n

rnstndaEIrdwTd

C

a

ick (e.g., I. ricinus-host), or rodent tissue (e.g., host name), international country

ucleotide substitutions per site.

eservoir hosts. Herein, by further providing a Bayesian phyloge-etic analysis of groEL, msp4 and DOV1 gene sequences we havehown similar topologies for all three genetic loci which supportwo distinct clades. Clade 1 contained strains from questing I. rici-us ticks and feeding I. ricinus ticks from deer, sheep, humans andogs but also the strains from I. scapularis and rodents from USnd I. persulcatus. Clade 2 contained genotypes from rodents fromurope and Russia and feeding I. trianguliceps and I. persulcatus.n the msp4 tree, the single strain from roe-deer (JN005728) rep-esents a unique haplotype in Clade 2. Interestingly, the highestiversity of A. phagocytophilum strains was detected in I. persulcatushich carried strains from both clades (Rar and Golovljova, 2011).

hese results clearly show that the ecology of A. phagocytophilumiffers between North America, Europe and Asia.

onclusion

A. phagocytophilum was detected in questing I. ricinus ticks fromll studied sites, from rodent-feeding I. trianguliceps ticks, ear and

ISO �-2) and Genbank accession numbers are in parentheses. Scale bars indicate

spleen biopsies of rodents (M. glareolus, A. flavicollis and A. agrar-ius). At sites where I. trianguliceps was absent, we did not detect A.phagocytophilum in rodents. None of the feeding I. ricinus ticks fromrodents were found infected with A. phagocytophilum albeit some ofthem were feeding on an infected rodent. Phylogenetic analysis offour genetic loci of A. phagocytophilum-infected samples revealedthat genotypes in questing I. ricinus were distinct from genotypesfound in rodents and rodent feeding I. trianguliceps. Our study fromCentral Europe confirms the previous findings from the UK thatin Europe A. phagocytophilum variants associated with rodents aretransmitted by I. trianguliceps but not I. ricinus, and thus in Europerodents are not reservoir hosts of the human pathogenic genotypesof A. phagocytophilum in contrast to the epidemiological context inthe USA.

Conflict of interest

The authors declare no conflict of interest.

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cknowledgments

We thank Monika Onderová, Jana Fricová and Jasna Kraljikor help with tick collections, rodent trapping and examinationnd DNA isolation. Authors thank to L’ubomír Vidlicka for helpith the figure editing. The study was supported by the projectsEGA 2/0055/11, VEGA 1/0390/12 and EU grant FP7-261504DENext, and is catalogued by the EDENext Steering Committee asDENext223. The contents of this publication are the sole respon-ibility of the authors and do not necessarily reflect the views ofhe European Commission. Data are also partially the result of theroject implementation: Environmental protection against parasito-oonoses under the influence of global climate and social changes (codeTMS: 26220220116), supported by the Research & Developmentperational Programme funded by the ERDF (0.1).

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