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

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Original article

Distinct Anaplasma phagocytophilum genotypes associated with Ixodes trianguliceps ticks and rodents in Central Europe a ˇ Lucia Blanarová , Michal Stanko a,b , Giovanna Carpi c,d , Dana Miklisová a , Bronislava Víchová a , Ladislav Moˇsansky´ a , Martin Bona e , Markéta Derdáková a,b,∗ a

Institute of Parasitology SAS, Hlinkova 3, 040 01 Koˇsice, Slovakia Institute of Zoology SAS, Dúbravská cesta 9, 845 06 Bratislava, Slovakia c Fondazione Edmund Mach, Trento, Italy d Yale School of Public Health, Department of Epidemiology of Microbial Diseases, 60 College Street, New Haven, USA e Department of Anatomy, Faculty of Medicine UPJS, Sˇ robárová 2, 041 80 Koˇsice, Slovakia b

a r t i c l e

i n f o

Article history: Received 27 April 2014 Received in revised form 1 July 2014 Accepted 15 July 2014 Available online 13 August 2014 Keywords: Anaplasma phagocytophilum genotypes Ixodes trianguliceps Ixodes ricinus Rodents Genetic loci

a b s t r a c t Rodents are important reservoir hosts of tick-borne pathogens. Anaplasma phagocytophilum is the causative 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 poorly understood. To assess the extent of vector association of this pathogen and to clarify its ecology in Central Europe we have analyzed and compared the genetic variability of A. phagocytophilum strains from questing and feeding I. ricinus and Ixodes trianguliceps ticks, as well as from rodent’ tissue samples. Tick collection and rodent trapping were performed during a 2-year study (2011–2012) in ecologically contrasting setting at four sites in Eastern Slovakia. Genetic variability of this pathogen was studied from the collected samples by DNA amplification and sequencing of four loci followed by Bayesian phylogenetic analyses. A. phagocytophilum was detected in questing I. ricinus ticks (0.7%) from all studied sites and 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 were absent. Moreover, Bayesian phylogenetic analyses have shown the presence of two distinct clades, and tree 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 specific host and vector associations also in Central Europe similarly to A. phagocytophilum’ molecular ecology in United Kingdom. This study suggests that A. phagocytophilum genotypes associated with rodents are probably transmitted solely by I. trianguliceps ticks, thus implying that rodent-associated A. phagocytophilum strains may not pose a risk for humans. © 2014 Elsevier GmbH. All rights reserved.

Introduction

∗ Corresponding author at: Institute of Parasitology SAS, Hlinkova 3, 040 01 Koˇsice, Slovakia. Tel.: +421 907977389. ˇ E-mail addresses: [email protected] (L. Blanarová), [email protected] (M. Stanko), [email protected] (G. Carpi), [email protected] (D. Miklisová), [email protected] (B. Víchová), [email protected] ´ [email protected] (M. Bona), (L. Moˇsansky), [email protected], [email protected] (M. Derdáková). http://dx.doi.org/10.1016/j.ttbdis.2014.07.012 1877-959X/© 2014 Elsevier GmbH. All rights reserved.

Anaplasma phagocytophilum is a gram-negative, intracellular, tick-transmitted bacterium belonging to the Anaplasmataceae family (Dumler et al., 2001). This causative agent of granulocytic anaplasmosis of both medical and veterinary importance is widely distributed in North America (USA), Europe and Asia. A. phagocytophilum is maintained in natural foci by a complex natural transmission enzootic cycle which involves the vector ticks of the Ixodes ricinus complex (Telford et al., 1996; Richter et al., 1996; Ogden et al., 1998; Levin and Fish, 2000; Cao et al., 2003; Eremeeva et al., 2006) and a wide range of vertebrate species as reservoir

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hosts (Petrovec et al., 2002; de la Fuente et al., 2005; Woldehiwet, 2006; Stuen, 2007; Carpi et al., 2009), whereas humans are generally incidental hosts. Nidicolous ticks such as Ixodes spinipalpis in USA (Burkot et al., 2001; DeNatale et al., 2002) and Ixodes trianguliceps in United Kingdom (UK) may also contribute to the natural enzootic cycle of this bacterium (Bown et al., 2003, 2006, 2008, 2009). In the USA, small and medium sized mammals, ungulates (white-tailed deer) and birds can act as reservoirs (Belongia et al., 1997; Magnarelli et al., 1999; Nicholson et al., 1999; Levin et al., 2002; Massung et al., 2003; Keesing et al., 2012). Moreover, based on the 16S rRNA gene, specific pathogen–host associations of two different A. phagocytophilum variants were described: The Ap-1 variant circulates in Ixodes scapularis ticks and free-living ungulates, whereas the Ap-ha variant is found in infected humans and its ecology is linked to rodents as reservoir hosts (Levin et al., 2002). In contrast to the USA, the role of vertebrate species as natural reservoir of human pathogenic strains of A. phagocytophilum in Europe and Asia is still poorly understood. In Europe, a higher degree of genetic diversity of A. phagocytophilum strains from different hosts has been described compared to the USA (de la Fuente et al., 2005; Carpi et al., 2009; Bown et al., 2009; Derdáková et al., 2011; Rar and Golovljova, 2011), and wild and domestic ungulates have been suggested as reservoirs (Ogden et al., 1998, 2002; Petrovec et al., 2002; Liz et al., 2002; Stuen et al., 2002). Additionally, in Europe A. phagocytophilum has been detected in a broader array of hosts, including wild boar (Sus scrofa), red fox (Vulpes vulpes), brown bear (Ursus arctos), and hare (Lepus europaeus) ˇ cíková et al., (Víchová et al., 2010; Hulínska et al., 2004; Stefanˇ 2005). Among small mammals, wood mice (Apodemus sylvaticus), yellow-necked mice (Apodemus flavicollis), herb field mice (Apodemus microps), field voles (Microtus agrestis) and bank voles (Myodes glareolus) have also been suggested as reservoir hosts for A. phagoˇ cytophilum (Liz et al., 2000; Bown et al., 2006, 2008; Stefanˇ cíková et al., 2008; Keesing et al., 2012; Víchová et al., 2014). Interestingly, genetic analyses on several molecular marker genes have shown that A. phagocytophilum genotypes circulating in rodents and Ixodes ticks in Europe differ from those circulating in the USA and Asia (Bown et al., 2009; Zhan et al., 2010). Furthermore, in the UK, Bown et al. (2003) described separate enzootic cycle of A. phagocytophilum genotypes: rodent associated genotypes are transmitted by I. trianguliceps. The genetic diversity of A. phagocytophilum strains has been studied by analyzing several phylogenetically informative loci, including the 16S rRNA gene (Massung et al., 1998), the heatshock protein GroEL (Liz et al., 2002; Carpi et al., 2009), the major surface proteins Msp4 (de la Fuente et al., 2005), the variable noncoding fragment DOV1 (Bown et al., 2009) and the ankA gene which encodes for the ankyrin repeat-containing protein (Park et al., 2004). The phylogenetic analyses of groEL (Petrovec et al., 2002; Liz et al., 2002), ankA (Von Loewenich et al., 2003; Park et al., 2004; Scharf et al., 2011) and msp4 (de la Fuente et al., 2005) genes of A. phagocytophilum strains from various vertebrate hosts and vector ticks suggested that intraspecific variability is linked to specific hosts, vectors and geographic locations. Rodents act as reservoirs of many tick-borne pathogens. Until recently, it was thought that in Europe rodents are also reservoir hosts of A. phagocytophilum strains that are vectored by I. ricinus ˇ ticks (Liz et al., 2000; Beninati et al., 2006; Spitálska et al., 2008; ˇ Stefanˇ cíková et al., 2008) and infect both humans and domestic animals as in the USA (Telford et al., 1996; Massung et al., 2003). However, recent studies show that this might not be the case for Europe, as strains where strains in rodents differ genetically from those circulating in I. ricinus ticks, domestic ruminants, wild boar, dogs, horses and humans (Bown et al., 2008; Majazki et al., 2013). It was also suggested that I. trianguliceps might be the vector of

929

these rodent strains in UK (Bown et al., 2008, 2009). Furthermore, in Switzerland, Burri et al. (2014) did not detect A. phagocytophilum in I. ricinus ticks feeding on rodents even though A. phagocytophilum was detected in questing I. ricinus from the same areas. It is still debated whether rodents play a role in maintaining A. phagocytophilum in continental Europe, and empirical evidence is lacking. In this study we aim to assess whether rodents contribute to the ecology of A. phagocytophilum in Central Europe. More specifically, our goal was to assess and characterize the genetic diversity and ecological associations of A. phagocytophilum genotypes circulating 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 Sloˇ ´ vakia (Cermel’, Hyl’ov, Botanical garden Koˇsice and Rozhanovce). Sites were selected to include areas with contrasting occurrence of nidicolous I. trianguliceps ticks feeding on rodents. Specifically, two control sites were characterized by the presence of two ixoˇ did species, I. ricinus and I. trianguliceps- Cermel’ (208–600 m ´ asl.; 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 characterized by the absence of I. trianguliceps ticks and presence of I. ricinus ticks exclusively, Botanical garden Koˇsice (208 m asl.; 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 sylvatic ˇ ´ and Cermel’), suburban deciduous forest mixed forest (Hyl’ov (Botanical garden, Koˇsice) and game reserve (Rozhanovce). Sample collection Tick collections and trapping of rodents were performed in 2011 and 2012 at the four investigated sites in Eastern Slovakia. Questing ticks were collected at each study site by a standardized flagging method (Falco and Fish, 1988) using a 1- m2 white corduroy cloth for 1 h to cover various types of forest/shrubland and edge vegetation. Immediately after collection, ticks were stored and preserved in tubes with 70% ethanol until the DNA was extracted. Rodents were trapped alive using Swedish bridge metal traps following the protocol of Stanko (1994) and Stanko and Miklisova (1995). Rodent trapping were carried out using 100–150 traps/per site for two-night trapping. A total of 854 trapped individuals of 10 species of small mammals (rodents and insectivores) were euthanized according to the laws of the Slovak Republic under the licenses of the Ministry of Environment of the Slovak Republic No. 297/108/06-3.1 and No. 6743/2008-2.1. Feeding ticks were removed from the rodents with sterile forceps, counted and identified to life stage and species level using previously published ˇ et al., 2004) and taxonomic keys (Filippova, 1977; Estrada-Pena preserved in 70% ethanol until DNA was extracted. Moreover, spleen and ear biopsies were obtained from each rodent during necroscopy. DNA extraction A total of 1376 questing ticks and 740 rodent-fed ticks from 854 rodents were used for DNA analyses. Genomic DNA was isolated from individual ticks by alkaline-hydrolysis according to Guy and Stanek (1991). DNA from rodent tissues (407 spleens and 669 ears) was extracted using a commercial DNA extraction kit (NucleoSpin Blood kit, NucleoSpin Tissue kit, Machery Nagel,

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Table 1 Number 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 total questing 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 questing IR ticks/no. of questing IR tick; prevalence-%

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

No. of positive ear biopsies/no. of ear biopsies; prevalence-%

No. of positive spleen biopsies/no. of spleen biopsies; prevalence-%

F-test

0.695

0.002

0.001

0.008

ˇ Cermel’ 95% CI ´ Hyl’ov 95% CI B. garden 95% CI Rozhanovce 95% CI

2/220 (0.9) 0.11–3.25 2/266 (0.8) 0.09–2.69 2/176 (1.1) 0.13–4.05 4/714 (0.6) 0.15–1.43

7/48 (14.6) 6.07–27.77 3/150 (2.0) 0.41–5.81 0/375

2/178 (1.1) 0.64–2.48 9/87 (10.5) 4.89–18.94 0/46

3/165 (1.8) 0.38–5.29 6/77 (7.9) 4.26–23.03 0/28

0/167

0/358

0/137

Total

10/1376 (0.7)

10/740 (1.4)

11/669 (1.6)

9/407 (2.2)

95% CI

0.34–1.34

0.64–2.48

1.09–3.88

1.11–4.56

IR – Ixodes ricinus, IT – Ixodes trianguliceps; F-test: p-value of Fisher’s exact test for comparing prevalences.

Germany) according to the manufacturer’s protocol. Lysates were stored at −20 ◦ C prior to use (Table 1). Molecular detection and characterization of A. phagocytophilum Polymerase chain reaction (PCR) amplification of the tick mitochondrial cytochrome b gene was performed for each sample as a quality control for tick DNA (Black and Roehrdanz, 1998; Derdáková et al., 2003). Moreover in the rodent samples, 12S rRNA gene was used to determine the quality control for the tissue DNA extraction (Humair et al., 2007). Samples were further screened for the presence of A. phagocytophilum by real-time PCR using the primers ApMSP2f (5 -ATGGAAGGTAGTGTTGGTTATGGTATT-3 ), ApMSP2r (5 TTGGTCTTGAAGCGCTCGTA-3 ) and the TaqManProbe ApMSP2p (5 -TGGTGCCAGGGTTGAGCTTGAGATTG-3 ) labeled with FAM, which targeted a 77-bp long fragment of the msp2 gene (Courtney et al., 2004). This assay was run on a CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). To further characterize A. phagocytophilum- infected samples, four molecular loci, 16S rRNA, msp4, groEL and DOV1 were amplified and sequenced. Nested PCR was performed to amplify a 546-bp fragment of the 16S rRNA gene as previously described (Massung et al., 1998). Nested PCRs were used to amplify a 498-bp fragment of the msp4 gene (de la Fuente et al., 2005) and a 1297-bp region of the groEL gene (Liz et al., 2002). The 275-bp fragment of the DOV1 noncoding region was amplified using seminested PCR as described previously (Bown et al., 2009). The PCR reactions were performed in a total volume of 25 ␮l of reaction mixture with 12.5 ␮l 1× mix, 2.6 ␮l 2.5 mM MgCl2 , 2.3 ␮l 920 nM of each primers and 0.3 ␮l 0.12 nM of probe using 5 ␮l as DNA template. Kit Bioron SuperHot Master Mix (Bioron, Germany) was used. The cycling conditions were 95 ◦ C for 120 s followed by 39 cycles (95 ◦ C for 15 s; 60 ◦ C for 60 s). In each PCR reaction, DNA from A. phagocytophilum- positive questing I. ricinus ticks were used as positive controls and DNA-free molecular water was added as template in negative controls. The PCR products were visualized by electrophoresis on 2% agarose gels stained with GoldView Nucleic Acid Stain (Beijing SBS Genetech, Beijing, China). All positive PCR products were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany) and bidirectionally Sanger sequenced with the same primers as for the PCR amplifications. Phylogenetic analysis The complementary strands of each sequenced product were manually assembled into consensus sequences. The consensus

sequences were compared to GenBank entries by BlastN v.2.2.13 (Altschul et al., 1997). Obtained A. phagocytophilum sequences were aligned with representative homologous sequences publicly available in GenBank (December 2013, 180 groEL sequences, 270 msp4 sequences and April 2014, 21 DOV1 sequences) using the MUSCLE program (Edgar, 2004) and adjusted manually to maintain reading frame integrity in the protein coding genes using the Se–Al v.20a11 alignment editing software (Rambaut, 1996). Unique haplotypes were identified using COLLAPSE 1.2 (David Posada; http://darwin.uvigo.es/software/collapse.html). jMODELTEST v.2.1.4 (Darriba et al., 2012) was employed to select the nucleotide substitution model most appropriate to the data set (groEL: HKY + I, msp4: GTR + I + G, DOV1: HKY). The selected nucleotide substitution models (model selection using Akaike (AIC) and Bayesian (BIC) criteria) were used to infer Bayesian phylogeny for three genes calculated by the computer program MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003). Markov chains were run for 2,000,000 generations, sampled every 10,000 generations, and the first 25% of each chain was discarded as burning and the remaining trees were used to construct a 50% majority-rule consensus tree. Uncorrected pairwise genetic distances were estimated using PAUP v.4.0 b10 (Swofford, 2003) and expressed as nucleotide diversity () (Nei, 1987). Nucleotide sequence accession numbers Eighty-six new sequences of A. phagocytophilum were deposited in the GenBank database with accession numbers KF383227–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. trianguliceps and I. ricinus, was analyzed. As 2 test did not confirm a relation between AF and larval stadium or coincident occurrence of both tick species, these factors were excluded from the subsequent statistical analysis. The log-linear analysis of frequency tables with automatic selection of best model, that find 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. Confidence intervals (CI) and differences in A. phagocytophilum infection prevalences among sites tested by Fisher’s exact test were calculated

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

No. of positive feeding larval IR ticks/no. of feeding larval IR ticks; prevalence-%

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

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

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

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

ˇ Cermel’ 95% CI ´ Hyl’ov 95% CI B. garden Rozhanovce

0/25

0/1

0/3

0

0/104

0/4

0/348 0/153

0/27 0/12

7/19 (36.8) 16.28–61.65 1/33 (3.0) 0.07–15.76 0 0

1/6 (16.7) 0.42–64.13 0 0/2

1/3 (33.3) 0.84–90.58 0 0

Total

0/630

0/44

8/52 (15.4)

1/11 (9.1)

1/3 (33.3)

6.88–28.09

0.22–41.28

0.84–90.58

95% CI IR – Ixodes ricinus; IT – Ixodes trianguliceps.

Table 3 Number 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 and 2012; prevalence (%). Species of rodents

No. of positive rodents/no. of total trapped rodents/prevalence (%) ˇ Cermel’

´ Hyl’ov

Botanical garden

Rozhanovce

Total

Apodemus agrarius Apodemus flavicollis Crocidura suaveolens Microtus subterraneus Microtus arvalis Myodes glareolus Sorex araneus Sorex minutus Neomys fodiens Micromys minutus

0/20 1/54 (1.9) 0 0/1 0/1 4/111 (3.6) 0/6 0/1 0/2 0

1/8 (12.5) 1/55 (1.8) 0 0/3 0 8/39 (20.5) 0/3 0 0 0

0/54 0/26 0/1 0/1 0 0 0/1 0 0 0/1

0/214 0/104 0/1 0/2 0/10 0/87 0/4 0/2 0 0

296 239 2 7 11 237 14 3 2 1

Total

5/196 (2.6)

10/108 (9.3)

0/84

0/424

812

by Quantitative Parasitology on the Web (Reiczigel et al., 2005); (http://www.univet.hu/qpweb/qp10/index.php). Results A. phagocytophilum in questing I. ricinus ticks In total, 2710 questing I. ricinus ticks from four sites (251 I. riciˇ ´ 689 from Hyl’ov, 986 from Botanical garden nus ticks from Cermel’, and 784 from Rozhanovce) were collected. From these ticks, 1376 ˇ ´ (220 I. ricinus ticks from Cermel’, 266 from Hyl’ov, 176 from Botanical garden and 714 from Rozhanovce) were tested for the presence of A. phagocytophilum by real-time PCR targeting the msp2 gene resulting in an overall A. phagocytophilum infection prevalence of 0.7% (10/1376; CI 95%: 0.34–1.34). We found no significant differences between the infection rates from different sites (F-value 0.695, p > 0.01) (Table 1). A. phagocytophilum in rodent feeding ticks and biopsies A total of 1713 feeding ticks (29 I. ricinus ticks and 22 I. trianˇ guliceps ticks from Cermel’; 167 I. ricinus and 42 I. trianguliceps ticks ´ 751 I. ricinus ticks from Botanical garden; 698 I. ricinus from Hyl’ov; and 4 I. trianguliceps ticks from Rozhanovce) were removed from rodents with sterile forceps. Seven-hundred and forty ticks (66 I. trianguliceps and 674 I. ricinus) were further tested. As some rodents were infested with a very high number of I. ricinus ticks, we tested up to 20 feeding and visibly engorged I. ricinus and I. trianguliceps ticks per single rodent. A. phagocytophilum was detected in 10 out of 740 tested feeding ticks (1.4%). Out of the tested feeding ticks from rodents, only I. trianguliceps carried A. phagocytophilum and

in total 10/66, 15.2% were infected. None of the 674 rodent feeding I. ricinus tested positive for A. phagocytophilum even if feeding on an A. phagocytophilum-infected rodent (Tables 1–3). Positive feeding I. trianguliceps ticks (8 larvae, 1 nymph and 1 female) were collected from M. glareolus and A. flavicollis (Tables 3 and 4). We observed significant differences in A. phagocytophilum prevalences in rodent feeding 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 rodents ˇ ´ 113 from Hyl’ov; 85 from Botanical garden and 458 from Cermel’; rodents from Rozhanovce). A. phagocytophilum was detected in 11 of out of 669 tested ear biopsies (1.6%; CI 95%: 1.09–3.88) and in 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 in both spleen and ear biopsies simultaneously. Two positive ear ˇ nine positive ear biopsies came from rodents trapped at Cermel’, ´ biopsies were found at the Hyl’ov site where we also detected the

Table 4 Number of ticks that were detected as infected with A. phagocytophilum by PCR/infestations of individual rodents that carried both I. ricinus and I. trianguliceps concurrently. Species of rodents

I. ricinus

I. trianguliceps

Larvae

Nymphs

Adult

Larvae

Nymphs

Adult

A. agrarius A. flavicollis S. araneus M. glareolus M. subterraneus

0/231 0/274 0/2 0/86 0/36

0/31 0/8 0 0/2 0/4

0 0 0 0 0

0/9 0/14 0 8/28 0/1

0/1 0/3 0 1/7 0

0 1/1 0 0/2 0

Total

0/629

0/45

0

8/52

1/11

1/3

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

Accession numbers 16S rRNA

Host

Origin

3

4

5

7

11

270

303

Paulauskas et al. (2012)

JN181063

ˇ Siluté, Lithuania

1

A

A

A

A

G

A

A

Paulauskas et al. (2012) Paulauskas et al. (2012) Paulauskas et al. (2012) 5BZNIRQ 220166Bs MG 228141LITMG

JN181079 JN181081 JN181071 KF481932 KF481928

DR tick Engorged IR removed from raccoon dog Engorged IR removed from raccoon dog Engorged IR removed from chaffinch Questing IR tick Spleen biopsy from rodent

ˇ Siluté, Lithuania ˇ Siluté, Lithuania Jomfruland, Norway B. garden, Slovakia ´ Hyl’ov, Slovakia

2 3 4 4 4

G G A A A

A A A A A

A A A A A

A A A A A

A G A A A

A A A A A

G G G G G

KF481938

Engorged IT from rodent

´ Hyl’ov, Slovakia

4

A

A

A

A

A

A

G

Paulauskas et al. (2012) 227785LITMG 2282171BeMG Paulauskas et al. (2012) Paulauskas et al. (2012) 77HNIRQ 163HNIRQ 39FCIRQ 2MBZIRQ 2204085BeMG Bown et al. (2003)

JN181067 KF481940 KF481945 JN181068 JN181066 KF481933 KF481929 KF481931 KF481930 KF481943 AY082656

Questing IR tick Engorged IT from rodent Ear biopsy from rodent Questing IR tick DR tick Questing IR tick

Hitra, Norway ˇ ermel’, Slovakia C ´ Hyl’ov, Slovakia Jomfruland, Norway Kaiˇsiadorys, Lithuania ´ Hyl’ov, Slovakia ˇ ermel’, Slovakia C

A A A A A A

A A A A A G

A A A G A A

A A A A G A

G G G A G G

A A A A G A

G G G G A G

Questing IR tick Ear biopsy from rodent Rodent MGLA

B. garden, Slovakia ´ Hyl’ov, Slovakia UK

5 5 5 6 7 8 8 8 9 9 10

A A A

A A G

A A A

G G A

A A A

A A A

G G G

Variant

Nucleotide position

DR – Dermacentor reticulatus; IR – I. ricinus; IT – I. trianguliceps; samples of this study are indicated in boldface.

highest number of infected I. trianguliceps ticks (Tables 1 and 2). A. phagocytophilum was detected in 12 biopsies from M. glareolus, in two from A. flavicollis and one from A. agrarius. These three rodent species were also the most commonly trapped rodents during our study. Positive samples from rodents originated only from the two ˇ ´ controls sites, Cermel’ and Hyl’ov, where I. trianguliceps and I. ricinus ticks co-occur. There were significant differences between the sites in A. phagocytophilum positivity of ear (F-value 0.001, p < 0.01) and spleen (F-value 0.008, p < 0.01) biopsies based on the I. trianguliceps occurrence at the site (Tables 1–3). Statistical analysis of A. phagocytophilum infection prevalence ˇ ´ Data from the two control sites, Cermel’ and Hyl’ov, and two rodents species, M. glareolus and A. flavicollis (where positive feeding ticks were found) were statistically tested. The log-linear analysis showed that the least complex model that will fit the data contains two-way associations (K = 2, max-Likelihood 2 = 63.6, p = 0.000) and does not contain any three-way associations (for K = 3, max-Likelihood 2 = 1.95, p = 0.74). The best selected models provided the following associations: AF-LOC, AF-TICK, LOC-HOST and HOST-TICK (max-Likelihood 2 = 5.49, df = 7, p = 0.601). These models indicated that major factors associated with A. phagocytophilum infection were the presence of I. trianguliceps ticks at the tested locality. Sequence analysis 16S rRNA gene sequences Sequencing of the 497 bp region of the 16S rRNA gene revealed four different sequence types among 21 samples isolated from questing I. ricinus, feeding I. trianguliceps ticks and ear and spleen biopsies from Eastern Slovakia (Table 5). Sequencing confirmed a high degree of similarity among 16S rRNA (99.0%). In rodent biopsies and feeding I. trianguliceps ticks we detected two genotypes (four and five) corresponding to genotypes previously described by Paulauskas et al. (2012). Moreover, we detected two unique 16S rRNA genotypes in five samples, one from questing I. ricinus ticks, and one from questing I. ricinus tick and ear biopsy. They were identified as genotypes eight and nine. The analyzed sequences of

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 diversity () among the A. phagocytophilum sequences from this study was 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 analysis between 15 A. phagocytophilum groEL sequences from this study (8 haplotypes) and 180 (91 haplotypes) from GenBank, and the 26 msp4 sequences from this study (8 haplotypes) and 270 (80 haplotypes) from GenBank are shown in Fig. 1A and B, respectively. The phylogenetic trees of the two loci displayed similar topology with strong support for two main clades. The first clade (hereafter “clade 1) included haplotypes detected in questing I. ricinus ticks and various vertebrate hosts such as roe deer, red deer, birds, sheep, dog and humans from European countries and the USA. The second clade (hereafter “clade 2) was highly divergent from clade 1, and included solely haplotypes from rodents, feeding I. trianguliceps from Slovakia and the UK and from questing Ixodes persulcatus from Russia. The Bayesian phylogenetic analysis of 25 A. phagocytophilum DOV1 sequences from this study (4 haplotype) and 21 (10 haplotypes) from GenBank confirmed the same tree topology as groEL and msp4 trees showing high support for two clades- clade 1 including A. phagocytophilum haplotypes from primary questing I. ricinus, and clade 2 comprising solely A. phagocytophilum haplotypes from rodents and I. trianguliceps (Fig. 2). Discussion In the present study we investigated the infection prevalence and genetic diversity of A. phagocytophilum strains and their ecological associations with rodents, questing I. ricinus and feeding Ixodid ticks (I. ricinus and I. trianguliceps) collected from rodents. To shed light on the vector competence of I. ricinus and

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Table 6 Accession 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 Questing I. ricinus male, B. garden ˇ Questing I. ricinus female, Cermel’

KF481929 KF481930 KF481931 KF481932 KF481933

KF420110 KF420112 KF420113 KF420114 KF420115 KF420116 KF420117 KF420111

KF383241 KF383237 KF383239 KF383238 KF383240

KF383255 KF383253 KF383254

a

a

Questing I. ricinus nymph, B. garden ´ Questing I. ricinus nymph, Hyl’ov Questing I. ricinus tick, female, Rozhanovce Questing I. ricinus tick, nymph, Rozhanovce ˇ Questing I. ricinus tick, male, Cermel’

a a a

a a

a

a

a

KF383256 KF383264 KF383262 KF383260 KF383259 KF383257 KF383258 KF383261

Feeding I. trianguliceps larva, M. glareolus Feeding I. trianguliceps larva, M. glareolus Feeding I. trianguliceps larva, M. glareolus Feeding I. trianguliceps larva, M. glareolus Feeding I. trianguliceps larva, M. glareolus Feeding I. trianguliceps larva, M. glareolus Feeding I. trianguliceps larva, M. glareolus Feeding I. trianguliceps nymph, M. glareolus Feeding I. trianguliceps female, A. flavicollis Feeding I. trianguliceps larva, M. glareolus

KF481934 KF481935 KF481936 KF481937 KF481938 KF481939 KF481940

KF420104 KF420105 KF420106 KF420107 KF420109 KF420108

KF383235 KF383234

a

a

a

KF420103

a

a

a

a

KF383236

a

a

a

KF383265 KF383263

Spleen of M. glareolus

KF481928

KF420092

KF383231

KF383252

Ear biopsy from A. flavicollis Ear biopsy from M. glareolus Ear biopsy from M. glareolus Ear biopsy from M. glareolus Ear biopsy from M. glareolus Ear biopsy from M. glareolus Ear biopsy from M. glareolus Ear biopsy from A. agrarius Ear biopsy from M. glareolus Ear biopsy from M. glareolus

KF481941 KF481942 KF481943 KF481944 KF481945 KF481946 KF481947 KF481948

KF420093 KF420095 KF420096 KF420098 KF420099 KF420100 KF420101 KF420102 KF420094 KF420097

a

KF383242 KF383244 KF383245 KF383247 KF383248 KF383249 KF383250 KF383251 KF383243 KF383246

a

a a

a

KF383233 KF383232 a

KF383229 KF383227 KF383230 a a a a

KF383228 a

Did not sequenced.

I. trianguliceps in the transmission cycle of the A. phagocytophilum rodent-associated strains we conducted a comparative study in two ecologically contrasting settings, two sites known for the occurrence of I. ricinus only (Botanical garden Koˇsice, Rozhanovce) and two sites known for the occurrence of both tick species (Pet’ko et al., 1991). The total prevalence of A. phagocytophilum infection in questing I. ricinus ticks in our study was 0.7%. Previous findings from Slovakia showed the infection rate in questing I. ricinus varying from 1.1% up ˇ ˇ to 8% (Spitálska and Kocianová, 2002, 2003; Spitálska et al., 2008; Derdáková et al., 2011; Subramanian et al., 2012). In total, 15.2% of feeding I. trianguliceps ticks (all developmental stages including larvae) were infected with A. phagocytophilum (Tables 1 and 2). To our knowledge this is the first detection of A. phagocytophilum in feeding I. trianguliceps in continental Europe. Our results strongly support the previously proposed hypothesis by Bown et al. (2009) that A. phagocytophilum strains associated with rodents circulate in enzootic cycles separate from non-rodent associated strains and that they are transmitted by I. trianguliceps but not I. ricinus ticks. As the presence of infectious agent in feeding ticks does not prove that the tick is also a biological vector of that agent DNA, additional xenodiagnostic studies are further needed to validate this hypothesis. On the other hand, our conclusions are supported by the findings in rodent necropsies, where M. glareolus, A. flavicollis, and A. agrarius were infected with the same genotype of A. phagocytophilum in those areas where I. trianguliceps were present. The highest A. phagocytophilum prevalence in rodents was found in vole species as in the UK (Bown et al., 2003, 2006, 2008, 2009). Importantly, the finding that none of the rodent-feeding I. ricinus ticks were found to be infected with A. phagocytophilum although they were coincidentally feeding on an infected rodent together with infected I. trianguliceps ticks, further supports the proposed hypothesis. Similarly, Heylen et al. (2014) did not observe co-feeding transmission of B. burgdorferi s.l. on songbirds among the ornithophilic ticks

Ixodes arboricola, I. frontalis and I. ricinus. In the areas where I. trianguliceps ticks were absent (control sites – Botanical garden, Koˇsice and Rozhanovce), we did not detect A. phagocytophilum in rodents. Moreover, none of the questing I. ricinus ticks carried the A. phagocytophilum rodent genotype as confirmed by phylogenetic analyses. Furthermore, none of the rodents were infected with the A. phagocytophilum genotypes that were present in I. ricinus ticks. Similarly in Switzerland, Burri et al. (2014) did not find A. phagocytophilum-infected I. ricinus feeding on rodents and none of the feeding xenodiagnostic ticks became infected. Even though the blood of the rodents in our study was not tested, the presence of A. phagocytophilum was detected in questing I. ricinus from the areas where rodents were captured. Altogether, these findings further support that A. phagocytophilum exhibits host specificity and vector association. Our results were further supported by the obtained sequence data (Table 5, Figs. 1 and 2). The highly conserved 16S rRNA gene has been used for genotyping A. phagocytophilum strains in many studies. Four 16S genetic variants were detected in questing I. ricinus, feeding I. trianguliceps ticks and in rodents in this study. Two (4 and 5) were detected in I. ricinus, I. trianguliceps and M. glareoulus and were identical to variants previously detected in feeding and questing I. ricinus ticks in Lithuania (Paulauskas et al., 2012). In addition, we detected two new sequence types in questing I. ricinus ticks and a rodent ear biopsy (Table 5). Von Loewenich et al. (2003) described seven variants infecting I. ricinus ticks in Germany, and Katargina et al. (2012) described four variants infecting I. ricinus ticks in Estonia, Belarus and Russia. In the USA, the Ap-variant 1 differs from a human strain (Ap-ha) and appears to be restricted to ruminant species as reported by Massung et al. (1998). However, in Europe, both the Ap-variant 1 and the Ap-ha 16S rRNA gene variants were detected in sheep, cattle and cervids (Bown et al., 2009). Nevertheless, this locus is not an adequate genetic marker to investigate the possible ecological association between the strains and

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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 tip corresponds to a unique haplotype and only a representative Genbank accession number for that haplotype is indicated. Each A. phagocytophilum sequence is shown with its source: 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 numbers are in parentheses. Scale bars indicate nucleotide substitutions per site.

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Fig. 1. (Continued)

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Fig. 2. Midpoint rooted 50% majority rule consensus tree constructed using Bayesian analysis of 14 DOV1 haplotypes (length 214 bp); posterior probabilities >0.50 are indicated 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 a representative Genbank accession number for that haplotype is indicated. Each A. phagocytophilum sequence is shown with its source: 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 numbers are in parentheses. Scale bars indicate nucleotide substitutions per site.

reservoir hosts. Herein, by further providing a Bayesian phylogenetic analysis of groEL, msp4 and DOV1 gene sequences we have shown similar topologies for all three genetic loci which support two distinct clades. Clade 1 contained strains from questing I. ricinus ticks and feeding I. ricinus ticks from deer, sheep, humans and dogs but also the strains from I. scapularis and rodents from US and I. persulcatus. Clade 2 contained genotypes from rodents from Europe and Russia and feeding I. trianguliceps and I. persulcatus. In the msp4 tree, the single strain from roe-deer (JN005728) represents a unique haplotype in Clade 2. Interestingly, the highest diversity of A. phagocytophilum strains was detected in I. persulcatus which carried strains from both clades (Rar and Golovljova, 2011). These results clearly show that the ecology of A. phagocytophilum differs between North America, Europe and Asia.

spleen biopsies of rodents (M. glareolus, A. flavicollis and A. agrarius). At sites where I. trianguliceps was absent, we did not detect A. phagocytophilum in rodents. None of the feeding I. ricinus ticks from rodents were found infected with A. phagocytophilum albeit some of them were feeding on an infected rodent. Phylogenetic analysis of four genetic loci of A. phagocytophilum-infected samples revealed that genotypes in questing I. ricinus were distinct from genotypes found in rodents and rodent feeding I. trianguliceps. Our study from Central Europe confirms the previous findings from the UK that in Europe A. phagocytophilum variants associated with rodents are transmitted by I. trianguliceps but not I. ricinus, and thus in Europe rodents are not reservoir hosts of the human pathogenic genotypes of A. phagocytophilum in contrast to the epidemiological context in the USA.

Conclusion Conflict of interest A. phagocytophilum was detected in questing I. ricinus ticks from all studied sites, from rodent-feeding I. trianguliceps ticks, ear and

The authors declare no conflict of interest.

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Acknowledgments We thank Monika Onderová, Jana Friˇcová and Jasna Kraljik for help with tick collections, rodent trapping and examination and DNA isolation. Authors thank to L’ubomír Vidliˇcka for help with the figure editing. The study was supported by the projects VEGA 2/0055/11, VEGA 1/0390/12 and EU grant FP7-261504 EDENext, and is catalogued by the EDENext Steering Committee as EDENext223. The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. Data are also partially the result of the project implementation: Environmental protection against parasitozoonoses under the influence of global climate and social changes (code ITMS: 26220220116), supported by the Research & Development Operational Programme funded by the ERDF (0.1).

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