Distinct Anaplasma Phagocytophilum Genotypes Associated with Ixodes

Home , Ixodes arboricola, Ixodes persulcatus, Ixodes ricinus, Ixodes trianguliceps

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 a,b c,d a

Lucia Blanarováˇ , Michal Stanko , Giovanna Carpi , Dana Miklisová ,

a a e a,b,∗

Bronislava Víchová , Ladislav Mosanskˇ y´ , Martin Bona , Markéta Derdáková

Institute of Parasitology SAS, Hlinkova 3, 040 01 Kosice,ˇ Slovakia

Institute of Zoology SAS, Dúbravská cesta 9, 845 06 Bratislava, Slovakia

Fondazione Edmund Mach, Trento, Italy

Yale School of Public Health, Department of Epidemiology of Microbial Diseases, 60 College Street, New Haven, USA

Department of Anatomy, Faculty of Medicine UPJS, Srobárováˇ 2, 041 80 Kosice,ˇ Slovakia

a r t i c l e i n f o a b s t r a c t

Article history: Rodents are important reservoir hosts of tick-borne pathogens. Anaplasma phagocytophilum is the

Received 27 April 2014

causative agent of granulocytic anaplasmosis of both medical and veterinary importance. In Europe,

Received in revised form 1 July 2014

this pathogen is primarily transmitted by the Ixodes ricinus tick among a wide range of vertebrate hosts.

Accepted 15 July 2014

However, to what degree A. phagocytophilum exhibits host speciﬁcity and vector association is poorly

Available online 13 August 2014

understood. 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 from

Keywords:

questing and feeding I. ricinus and Ixodes trianguliceps ticks, as well as from rodent’ tissue samples. Tick

Anaplasma phagocytophilum genotypes

collection and rodent trapping were performed during a 2-year study (2011–2012) in ecologically con-

Ixodes trianguliceps

trasting setting at four sites in Eastern Slovakia. Genetic variability of this pathogen was studied from

Ixodes ricinus

Rodents the collected samples by DNA ampliﬁcation and sequencing of four loci followed by Bayesian phyloge-

Genetic loci netic 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 prob-

ably transmitted solely by I. trianguliceps ticks, thus implying that rodent-associated A. phagocytophilum

strains may not pose a risk for humans.

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 granulocytic

anaplasmosis of both medical and veterinary importance is widely

∗

Corresponding author at: Institute of Parasitology SAS, Hlinkova 3, 040 01 Kosice,ˇ distributed in North America (USA), Europe and Asia. A. phago-

Slovakia. Tel.: +421 907977389. cytophilum is maintained in natural foci by a complex natural

E-mail addresses: [email protected] (L. Blanarová),ˇ [email protected]

transmission enzootic cycle which involves the vector ticks of the

(M. Stanko), [email protected] (G. Carpi), [email protected]

Ixodes ricinus complex (Telford et al., 1996; Richter et al., 1996;

(D. Miklisová), [email protected] (B. Víchová), [email protected]

Ogden et al., 1998; Levin and Fish, 2000; Cao et al., 2003; Eremeeva

(L. Mosanskˇ y),´ [email protected] (M. Bona),

[email protected], [email protected] (M. Derdáková). et al., 2006) and a wide range of vertebrate species as reservoir

http://dx.doi.org/10.1016/j.ttbdis.2014.07.012

L. Blanarováˇ et al. / Ticks and Tick-borne Diseases 5 (2014) 928–938 929

hosts (Petrovec et al., 2002; de la Fuente et al., 2005; Woldehiwet, these rodent strains in UK (Bown et al., 2008, 2009). Furthermore,

2006; Stuen, 2007; Carpi et al., 2009), whereas humans are gener- in Switzerland, Burri et al. (2014) did not detect A. phagocytophilum

ally incidental hosts. Nidicolous ticks such as Ixodes spinipalpis in in I. ricinus ticks feeding on rodents even though A. phagocytophilum

USA (Burkot et al., 2001; DeNatale et al., 2002) and Ixodes trian- was detected in questing I. ricinus from the same areas. It is still

guliceps in United Kingdom (UK) may also contribute to the natural debated whether rodents play a role in maintaining A. phagocy-

enzootic cycle of this bacterium (Bown et al., 2003, 2006, 2008, tophilum in continental Europe, and empirical evidence is lacking.

2009). In this study we aim to assess whether rodents contribute to the

In the USA, small and medium sized mammals, ungulates ecology of A. phagocytophilum in Central Europe. More speciﬁcally,

(white-tailed deer) and birds can act as reservoirs (Belongia et al., our goal was to assess and characterize the genetic diversity and

1997; Magnarelli et al., 1999; Nicholson et al., 1999; Levin et al., ecological associations of A. phagocytophilum genotypes circulat-

2002; Massung et al., 2003; Keesing et al., 2012). Moreover, based ing in rodents, questing I. ricinus ticks and feeding I. ricinus and I.

on the 16S rRNA gene, speciﬁc pathogen–host associations of two trianguliceps ticks in several sites in Slovakia (Central Europe).

different A. phagocytophilum variants were described: The Ap-1

variant circulates in Ixodes scapularis ticks and free-living ungu-

Materials and methods

lates, whereas the Ap-ha variant is found in infected humans and

its ecology is linked to rodents as reservoir hosts (Levin et al.,

Study area

2002). In contrast to the USA, the role of vertebrate species as nat-

ural reservoir of human pathogenic strains of A. phagocytophilum

This study was conducted in four sampling sites in Eastern Slo-

in Europe and Asia is still poorly understood. In Europe, a higher

vakia (Cermel’,ˇ Hyl’ov,´ Botanical garden Kosiceˇ and Rozhanovce).

degree of genetic diversity of A. phagocytophilum strains from dif-

Sites were selected to include areas with contrasting occurrence

ferent hosts has been described compared to the USA (de la Fuente

of nidicolous I. trianguliceps ticks feeding on rodents. Speciﬁcally,

et al., 2005; Carpi et al., 2009; Bown et al., 2009; Derdáková et al.,

two control sites were characterized by the presence of two ixo-

2011; Rar and Golovljova, 2011), and wild and domestic ungu-

did species, I. ricinus and I. trianguliceps- Cermel’ˇ (208–600 m

lates have been suggested as reservoirs (Ogden et al., 1998, 2002; ◦ ◦

asl.; 48 45 46.67 N; 21 8 8.17 E) and Hyl’ov´ (500–750 m asl.;

Petrovec et al., 2002; Liz et al., 2002; Stuen et al., 2002). Addition- ◦ ◦

48 44 22.80 N; 21 4 18.90 E); whereas two sites were char-

ally, in Europe A. phagocytophilum has been detected in a broader

acterized by the absence of I. trianguliceps ticks and presence

array of hosts, including wild boar (Sus scrofa), red fox (Vulpes

of I. ricinus ticks exclusively, Botanical garden Kosiceˇ (208 m

vulpes), brown bear (Ursus arctos), and hare (Lepus europaeus) ◦ ◦

asl.; 48 44 6.84 N; 21 14 16.14 E) and Rozhanovce (215 m asl.;

(Víchová et al., 2010; Hulínska et al., 2004; Stefancíkováˇ et al., ◦ ◦

48 4500 N; 21 21 00 E). Study sites were located in sylvatic

2005). Among small mammals, wood mice (Apodemus sylvaticus),

mixed forest (Hyl’ov´ and Cermel’),ˇ suburban deciduous forest

yellow-necked mice (Apodemus ﬂavicollis), herb ﬁeld mice (Apode-

(Botanical garden, Kosice)ˇ and game reserve (Rozhanovce).

mus microps), ﬁeld voles (Microtus agrestis) and bank voles (Myodes

glareolus) have also been suggested as reservoir hosts for A. phago-

cytophilum (Liz et al., 2000; Bown et al., 2006, 2008; Stefanˇ cíkováˇ

Sample collection

et al., 2008; Keesing et al., 2012; Víchová et al., 2014). Interestingly,

genetic analyses on several molecular marker genes have shown

Tick collections and trapping of rodents were performed in 2011

that A. phagocytophilum genotypes circulating in rodents and Ixodes

and 2012 at the four investigated sites in Eastern Slovakia.

ticks in Europe differ from those circulating in the USA and Asia

Questing ticks were collected at each study site by a standard-

(Bown et al., 2009; Zhan et al., 2010). Furthermore, in the UK, 2

ized ﬂagging method (Falco and Fish, 1988) using a 1- m white

Bown et al. (2003) described separate enzootic cycle of A. phagocy-

corduroy cloth for 1 h to cover various types of forest/shrubland and

tophilum genotypes: rodent associated genotypes are transmitted

edge vegetation. Immediately after collection, ticks were stored and

by I. trianguliceps.

preserved in tubes with 70% ethanol until the DNA was extracted.

The genetic diversity of A. phagocytophilum strains has been

Rodents were trapped alive using Swedish bridge metal traps

studied by analyzing several phylogenetically informative loci,

following the protocol of Stanko (1994) and Stanko and Miklisova

including the 16S rRNA gene (Massung et al., 1998), the heat-

(1995). Rodent trapping were carried out using 100–150 traps/per

shock protein GroEL (Liz et al., 2002; Carpi et al., 2009), the major

site for two-night trapping. A total of 854 trapped individuals of 10

surface proteins Msp4 (de la Fuente et al., 2005), the variable non-

species of small mammals (rodents and insectivores) were euth-

coding fragment DOV1 (Bown et al., 2009) and the ankA gene which

anized according to the laws of the Slovak Republic under the

encodes for the ankyrin repeat-containing protein (Park et al.,

licenses of the Ministry of Environment of the Slovak Republic

2004). The phylogenetic analyses of groEL (Petrovec et al., 2002;

No. 297/108/06-3.1 and No. 6743/2008-2.1. Feeding ticks were

Liz et al., 2002), ankA (Von Loewenich et al., 2003; Park et al., 2004;

removed from the rodents with sterile forceps, counted and iden-

Scharf et al., 2011) and msp4 (de la Fuente et al., 2005) genes of

tiﬁed to life stage and species level using previously published

A. phagocytophilum strains from various vertebrate hosts and vec-

taxonomic keys (Filippova, 1977; Estrada-Penaˇ et al., 2004) and

tor ticks suggested that intraspeciﬁc variability is linked to speciﬁc

preserved in 70% ethanol until DNA was extracted. Moreover,

hosts, vectors and geographic locations.

spleen and ear biopsies were obtained from each rodent during

Rodents act as reservoirs of many tick-borne pathogens. Until necroscopy.

recently, it was thought that in Europe rodents are also reservoir

hosts of A. phagocytophilum strains that are vectored by I. ricinus

ticks (Liz et al., 2000; Beninati et al., 2006; Spitálskaˇ et al., 2008; DNA extraction

Stefanˇ cíkováˇ et al., 2008) and infect both humans and domestic

animals as in the USA (Telford et al., 1996; Massung et al., 2003). A total of 1376 questing ticks and 740 rodent-fed ticks from

However, recent studies show that this might not be the case for 854 rodents were used for DNA analyses. Genomic DNA was

Europe, as strains where strains in rodents differ genetically from isolated from individual ticks by alkaline-hydrolysis according to

those circulating in I. ricinus ticks, domestic ruminants, wild boar, Guy and Stanek (1991). DNA from rodent tissues (407 spleens

dogs, horses and humans (Bown et al., 2008; Majazki et al., 2013). and 669 ears) was extracted using a commercial DNA extraction

It was also suggested that I. trianguliceps might be the vector of kit (NucleoSpin Blood kit, NucleoSpin Tissue kit, Machery Nagel,

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

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 No. of positive feeding ticks No. of positive ear No. of positive spleen

IR ticks/no. of questing (IR + IT)/no. of feeding biopsies/no. of ear biopsies/no. of spleen

IR tick; prevalence-% ticks; prevalence-% biopsies; prevalence-% biopsies; prevalence-%

F-test 0.695 0.002 0.001 0.008

Cermel’ˇ 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 0.38–5.29

Hyl’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 4.26–23.03

B. garden 2/176 (1.1) 0/375 0/46 0/28

95% CI 0.13–4.05

Rozhanovce 4/714 (0.6) 0/167 0/358 0/137

95% CI 0.15–1.43

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 sequences were compared to GenBank entries by BlastN v.2.2.13

◦

−

stored at 20 C prior to use (Table 1). (Altschul et al., 1997). Obtained A. phagocytophilum sequences

were aligned with representative homologous sequences publicly

Molecular detection and characterization of A. phagocytophilum available in GenBank (December 2013, 180 groEL sequences, 270

msp4 sequences and April 2014, 21 DOV1 sequences) using the

Polymerase chain reaction (PCR) ampliﬁcation of the tick mito- MUSCLE program (Edgar, 2004) and adjusted manually to main-

chondrial cytochrome b gene was performed for each sample as a tain reading frame integrity in the protein coding genes using

quality control for tick DNA (Black and Roehrdanz, 1998; Derdáková the Se–Al v.20a11 alignment editing software (Rambaut, 1996).

et al., 2003). Moreover in the rodent samples, 12S rRNA gene was Unique haplotypes were identiﬁed using COLLAPSE 1.2 (David

used to determine the quality control for the tissue DNA extraction Posada; http://darwin.uvigo.es/software/collapse.html). jMODEL-

(Humair et al., 2007). TEST v.2.1.4 (Darriba et al., 2012) was employed to select the

Samples were further screened for the presence of A. phago- nucleotide substitution model most appropriate to the data

cytophilum by real-time PCR using the primers ApMSP2f set (groEL: HKY + I, msp4: GTR + I + G, DOV1: HKY). The selected

(5 -ATGGAAGGTAGTGTTGGTTATGGTATT-3 ), ApMSP2r (5 - nucleotide substitution models (model selection using Akaike (AIC)

TTGGTCTTGAAGCGCTCGTA-3 ) and the TaqManProbe ApMSP2p and Bayesian (BIC) criteria) were used to infer Bayesian phylogeny

(5 -TGGTGCCAGGGTTGAGCTTGAGATTG-3 ) labeled with FAM, for three genes calculated by the computer program MrBayes v3.1.2

which targeted a 77-bp long fragment of the msp2 gene (Courtney (Ronquist and Huelsenbeck, 2003). Markov chains were run for

et al., 2004). This assay was run on a CFX96 Real-Time PCR System 2,000,000 generations, sampled every 10,000 generations, and the

(Bio-Rad, Hercules, CA, USA). ﬁrst 25% of each chain was discarded as burning and the remaining

To further characterize A. phagocytophilum- infected samples, trees were used to construct a 50% majority-rule consensus tree.

four molecular loci, 16S rRNA, msp4, groEL and DOV1 were ampli- Uncorrected pairwise genetic distances were estimated using PAUP

ﬁed and sequenced. Nested PCR was performed to amplify a 546-bp v.4.0 b10 (Swofford, 2003) and expressed as nucleotide diversity

fragment of the 16S rRNA gene as previously described (Massung ( ) (Nei, 1987).

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 Nucleotide sequence accession numbers

groEL gene (Liz et al., 2002). The 275-bp fragment of the DOV1 non-

coding region was ampliﬁed using seminested PCR as described Eighty-six new sequences of A. phagocytophilum were

previously (Bown et al., 2009). deposited in the GenBank database with accession numbers

␮

The PCR reactions were performed in a total volume of 25 l of KF383227–KF383265, KF420092–KF420117 and KF481928–

␮ × ␮ ␮

reaction mixture with 12.5 l 1 mix, 2.6 l 2.5 mM MgCl2, 2.3 l KF481948 (Table 6).

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) Statistical analysis

◦

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 The association between A. phagocytophilum infection (AP),

from A. phagocytophilum- positive questing I. ricinus ticks were used the response variable of interest, and the categorical variables

as positive controls and DNA-free molecular water was added as (design factors), locality (LOC), host species (HOST), tick species

template in negative controls. The PCR products were visualized by (TICK), larval stadium and coincident occurrence of I. trianguli-

electrophoresis on 2% agarose gels stained with GoldView Nucleic ceps and I. ricinus, was analyzed. As test did not conﬁrm

Acid Stain (Beijing SBS Genetech, Beijing, China). All positive PCR a relation between AF and larval stadium or coincident occur-

products were puriﬁed using a QIAquick PCR puriﬁcation kit (Qia- rence of both tick species, these factors were excluded from

gen, Hilden, Germany) and bidirectionally Sanger sequenced with the subsequent statistical analysis. The log-linear analysis of

the same primers as for the PCR ampliﬁcations. frequency tables with automatic selection of best model, that

ﬁnd the least complex model ﬁtting the data, was used to

Phylogenetic analysis assess relationships between factors. The software Statistica 9

(http://www.statsoft.com) was used for this analysis. Conﬁdence

The complementary strands of each sequenced product were intervals (CI) and differences in A. phagocytophilum infection preva-

manually assembled into consensus sequences. The consensus lences among sites tested by Fisher’s exact test were calculated

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

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 No. of positive feeding No. of positive feeding No. of positive feeding No. of positive female IT

larval IR ticks/no. of nymphal IR ticks/no. of larval IT ticks/no. of nymphal IT ticks/no. of ticks/no. feeding female IT

feeding larval IR ticks; feeding nymphal IR ticks; feeding larval IT ticks; feeding nymphal IT ticks; ticks; prevalence-%

prevalence-% prevalence-% prevalence-% prevalence-%

Cermel’ˇ 0/25 0/1 7/19 (36.8) 0/3 0

95% CI 16.28–61.65

Hyl’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 0.84–90.58

B. garden 0/348 0/27 0 0 0

Rozhanovce 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 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 (%)

Cermel’ˇ Hyl’ov´ Botanical garden Rozhanovce Total

Apodemus agrarius 0/20 1/8 (12.5) 0/54 0/214 296

Apodemus ﬂavicollis 1/54 (1.9) 1/55 (1.8) 0/26 0/104 239

Crocidura suaveolens 0 0 0/1 0/1 2

Microtus subterraneus 0/1 0/3 0/1 0/2 7

Microtus arvalis 0/1 0 0 0/10 11

Myodes glareolus 4/111 (3.6) 8/39 (20.5) 0 0/87 237

Sorex araneus 0/6 0/3 0/1 0/4 14

Sorex minutus 0/1 0 0 0/2 3

Neomys fodiens 0/2 0 0 0 2

Micromys minutus 0 0 0/1 0 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); in total 10/66, 15.2% were infected. None of the 674 rodent feeding

(http://www.univet.hu/qpweb/qp10/index.php). I. ricinus tested positive for A. phagocytophilum even if feeding on an

A. phagocytophilum-infected rodent (Tables 1–3). Positive feeding I.

Results 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

A. phagocytophilum in questing I. ricinus ticks

feeding ticks between the study sites based on the occurrence of I.

trianguliceps (F-value 0.002, p < 0.01) (Table 1).

In total, 2710 questing I. ricinus ticks from four sites (251 I. rici-

A total of 854 small mammals were captured (198 rodents

nus ticks from Cermel’,ˇ 689 from Hyl’ov,´ 986 from Botanical garden

from Cermel’;ˇ 113 from Hyl’ov;´ 85 from Botanical garden and 458

and 784 from Rozhanovce) were collected. From these ticks, 1376

rodents from Rozhanovce). A. phagocytophilum was detected in 11

(220 I. ricinus ticks from Cermel’,ˇ 266 from Hyl’ov,´ 176 from Botani-

of out of 669 tested ear biopsies (1.6%; CI 95%: 1.09–3.88) and

cal garden and 714 from Rozhanovce) were tested for the presence

in nine of 407 tested spleens (2.2%; CI 95%: 1.11–4.56) (Table 1).

of A. phagocytophilum by real-time PCR targeting the msp2 gene

In ﬁve of 854 small mammals, A. phagocytophilum was detected

resulting in an overall A. phagocytophilum infection prevalence of

in both spleen and ear biopsies simultaneously. Two positive ear

0.7% (10/1376; CI 95%: 0.34–1.34). We found no signiﬁcant dif-

biopsies came from rodents trapped at Cermel’,ˇ nine positive ear

ferences between the infection rates from different sites (F-value

biopsies were found at the Hyl’ov´ site where we also detected the

0.695, p > 0.01) (Table 1).

A. phagocytophilum in rodent feeding ticks and biopsies

Table 4

Number of ticks that were detected as infected with A. phagocytophilum by

A total of 1713 feeding ticks (29 I. ricinus ticks and 22 I. trian-

PCR/infestations of individual rodents that carried both I. ricinus and I. trianguliceps

guliceps ticks from Cermel’;ˇ 167 I. ricinus and 42 I. trianguliceps ticks concurrently.

from Hyl’ov;´ 751 I. ricinus ticks from Botanical garden; 698 I. ricinus

Species of rodents I. ricinus I. trianguliceps

and 4 I. trianguliceps ticks from Rozhanovce) were removed from

Larvae Nymphs Adult Larvae Nymphs Adult

rodents with sterile forceps. Seven-hundred and forty ticks (66 I. tri-

anguliceps and 674 I. ricinus) were further tested. As some rodents A. agrarius 0/231 0/31 0 0/9 0/1 0

A. ﬂavicollis 0/274 0/8 0 0/14 0/3 1/1

were infested with a very high number of I. ricinus ticks, we tested

S. araneus 0/2 0 0 0 0 0

up to 20 feeding and visibly engorged I. ricinus and I. trianguliceps

M. glareolus 0/86 0/2 0 8/28 1/7 0/2

ticks per single rodent. A. phagocytophilum was detected in 10 out

M. subterraneus 0/36 0/4 0 0/1 0 0

of 740 tested feeding ticks (1.4%). Out of the tested feeding ticks

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

from rodents, only I. trianguliceps carried A. phagocytophilum and

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

Table 5

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

Sample Accession numbers Host Origin Variant Nucleotide position

16S rRNA

3 4 5 7 11 270 303

Paulauskas et al. (2012) JN181063 DR Siluté,ˇ Lithuania 1 A A A A G A A

tick

Paulauskas et al. (2012) JN181079 Engorged IR removed from raccoon dog Siluté,ˇ Lithuania 2 G A A A A A G

Paulauskas et al. (2012) JN181081 Engorged IR removed from raccoon dog Siluté,ˇ Lithuania 3 G A A A G A G

Paulauskas et al. (2012) JN181071 Engorged IR removed from chafﬁnch Jomfruland, Norway 4 A A A A A A G

5BZNIRQ KF481932 Questing IR tick B. garden, Slovakia 4 A A A A A A G

220166Bs KF481928 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 G

227785LITMG KF481940 Engorged IT from rodent Cermel’,ˇ Slovakia 5 A A A A G A G

2282171BeMG KF481945 Ear biopsy from rodent Hyl’ov,´ Slovakia 5 A A A A G A G

Paulauskas et al. (2012) JN181068 Questing IR tick Jomfruland, Norway 6 A A G A A A G

Paulauskas et al. (2012) JN181066 DR tick Kaisiadorys,ˇ Lithuania 7 A A A G G G A

77HNIRQ KF481933 Questing IR tick Hyl’ov,´ Slovakia 8 A G A A G A G

163HNIRQ KF481929 Cermel’,ˇ Slovakia 8

39FCIRQ KF481931 8

2MBZIRQ KF481930 Questing IR tick B. garden, Slovakia 9 A A A G A A G

2204085BeMG KF481943 Ear biopsy from rodent Hyl’ov,´ Slovakia 9 A A A G A A G

Bown et al. (2003) AY082656 Rodent MGLA UK 10 A G A A A A G

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. genotype eight had nucleotide substitutions at position 4 and 303,

phagocytophilum was detected in 12 biopsies from M. glareolus, in and genotype nine had substitutions at 7, 11 and 303 (Table 5).

two from A. ﬂavicollis and one from A. agrarius. These three rodent

species were also the most commonly trapped rodents during our

GroEL, msp4 and DOV1 phylogenetic analyses

study. Positive samples from rodents originated only from the two

controls sites, Cermel’ˇ and Hyl’ov,´ where I. trianguliceps and I. ricinus

In this study we generated 15, 26 and 24 sequences for groEL,

ticks co-occur. There were signiﬁcant differences between the sites

msp4 and DOV1, respectively (Table 6). The mean nucleotide diver-

in A. phagocytophilum positivity of ear (F-value 0.001, p < 0.01) and

sity () among the A. phagocytophilum sequences from this study

spleen (F-value 0.008, p < 0.01) biopsies based on the I. trianguliceps

was 0.021 (range 0.0–0.061), 0.04 (range 0.0–0.118) and 0.068

occurrence at the site (Tables 1–3).

(range 0.0–0.234) for groEL, msp4, and DOV1, respectively.

The phylogenetic relationships based on Bayesian analysis

Statistical analysis of A. phagocytophilum infection prevalence

between 15 A. phagocytophilum groEL sequences from this study

(8 haplotypes) and 180 (91 haplotypes) from GenBank, and the 26

Data from the two control sites, Cermel’ˇ and Hyl’ov,´ and two

msp4 sequences from this study (8 haplotypes) and 270 (80 hap-

rodents species, M. glareolus and A. ﬂavicollis (where positive feed-

lotypes) from GenBank are shown in Fig. 1A and B, respectively.

ing ticks were found) were statistically tested. The log-linear

The phylogenetic trees of the two loci displayed similar topology

analysis showed that the least complex model that will ﬁt the data

2 with strong support for two main clades. The ﬁrst clade (hereafter

contains two-way associations (K = 2, max-Likelihood = 63.6,

“clade 1) included haplotypes detected in questing I. ricinus ticks

p = 0.000) and does not contain any three-way associations (for

2 and various vertebrate hosts such as roe deer, red deer, birds, sheep,

K = 3, max-Likelihood = 1.95, p = 0.74). The best selected models

dog and humans from European countries and the USA. The sec-

provided the following associations: AF-LOC, AF-TICK, LOC-HOST

2 ond clade (hereafter “clade 2) was highly divergent from clade 1,

and HOST-TICK (max-Likelihood = 5.49, df = 7, p = 0.601). These

and included solely haplotypes from rodents, feeding I. trianguli-

models indicated that major factors associated with A. phagocy-

ceps from Slovakia and the UK and from questing Ixodes persulcatus

tophilum infection were the presence of I. trianguliceps ticks at the

from Russia.

tested locality.

The Bayesian phylogenetic analysis of 25 A. phagocytophilum

DOV1 sequences from this study (4 haplotype) and 21 (10 haplo-

Sequence analysis

types) from GenBank conﬁrmed the same tree topology as groEL

and msp4 trees showing high support for two clades- clade 1 includ-

16S rRNA gene sequences

ing A. phagocytophilum haplotypes from primary questing I. ricinus,

Sequencing of the 497 bp region of the 16S rRNA gene revealed

and clade 2 comprising solely A. phagocytophilum haplotypes from

four different sequence types among 21 samples isolated from

rodents and I. trianguliceps (Fig. 2).

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 biop- Discussion

sies and feeding I. trianguliceps ticks we detected two genotypes

(four and ﬁve) corresponding to genotypes previously described In the present study we investigated the infection preva-

by Paulauskas et al. (2012). Moreover, we detected two unique 16S lence and genetic diversity of A. phagocytophilum strains and

rRNA genotypes in ﬁve samples, one from questing I. ricinus ticks, their ecological associations with rodents, questing I. ricinus and

and one from questing I. ricinus tick and ear biopsy. They were feeding Ixodid ticks (I. ricinus and I. trianguliceps) collected from

identiﬁed as genotypes eight and nine. The analyzed sequences of rodents. To shed light on the vector competence of I. ricinus and

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

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´ KF481929 KF420110 KF383241 KF383255

Questing I. ricinus male, B. garden KF481930 KF420112 KF383237 KF383253

Questing I. ricinus female, Cermel’ˇ KF481931 KF420113 KF383239 KF383254

Questing I. ricinus nymph, B. garden KF481932 KF420114 KF383238

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

a a a

Questing I. ricinus tick, female, Rozhanovce KF420116

a aa

Questing I. ricinus tick, nymph, Rozhanovce KF420117

a a

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

Feeding I. trianguliceps larva, M. glareolus KF481934 KF420104 KF383235 KF383264

Feeding I. trianguliceps larva, M. glareolus KF481935 KF420105 KF383234 KF383262

Feeding I. trianguliceps larva, M. glareolus KF481936 KF420106 KF383260

Feeding I. trianguliceps larva, M. glareolus KF481937 KF420107 KF383233 KF383259

Feeding I. trianguliceps larva, M. glareolus KF481938 KF420109 KF383232 KF383257

Feeding I. trianguliceps larva, M. glareolus KF481939 KF420108 KF383258

a a

Feeding I. trianguliceps larva, M. glareolus KF481940 KF383261

a a a

Feeding I. trianguliceps nymph, M. glareolus KF420103

Feeding I. trianguliceps female, A. ﬂavicollis KF383236 KF383265

a a a

Feeding I. trianguliceps larva, M. glareolus KF383263

Spleen of M. glareolus KF481928 KF420092 KF383231 KF383252

Ear biopsy from A. ﬂavicollis KF481941 KF420093 KF383242

Ear biopsy from M. glareolus KF481942 KF420095 KF383229 KF383244

Ear biopsy from M. glareolus KF481943 KF420096 KF383227 KF383245

Ear biopsy from M. glareolus KF481944 KF420098 KF383230 KF383247

Ear biopsy from M. glareolus KF481945 KF420099 KF383248

Ear biopsy from M. glareolus KF481946 KF420100 KF383249

Ear biopsy from M. glareolus KF481947 KF420101 KF383250

Ear biopsy from A. agrarius KF481948 KF420102 KF383251

Ear biopsy from M. glareolus KF420094 KF383228 KF383243

a a

Ear biopsy from M. glareolus KF420097 KF383246

Did not sequenced.

I. trianguliceps in the transmission cycle of the A. phagocytophilum Ixodes arboricola, I. frontalis and I. ricinus. In the areas where I.

rodent-associated strains we conducted a comparative study in trianguliceps ticks were absent (control sites – Botanical garden,

two ecologically contrasting settings, two sites known for the Kosiceˇ and Rozhanovce), we did not detect A. phagocytophilum

occurrence of I. ricinus only (Botanical garden Kosice,ˇ Rozhanovce) in rodents. Moreover, none of the questing I. ricinus ticks carried

and two sites known for the occurrence of both tick species (Pet’ko the A. phagocytophilum rodent genotype as conﬁrmed by phylo-

et al., 1991). genetic analyses. Furthermore, none of the rodents were infected

The total prevalence of A. phagocytophilum infection in questing with the A. phagocytophilum genotypes that were present in I. rici-

I. ricinus ticks in our study was 0.7%. Previous ﬁndings from Slovakia nus ticks. Similarly in Switzerland, Burri et al. (2014) did not ﬁnd

showed the infection rate in questing I. ricinus varying from 1.1% up A. phagocytophilum-infected I. ricinus feeding on rodents and none

to 8% (Spitálskaˇ and Kocianová, 2002, 2003; Spitálskaˇ et al., 2008; of the feeding xenodiagnostic ticks became infected. Even though

Derdáková et al., 2011; Subramanian et al., 2012). In total, 15.2% the blood of the rodents in our study was not tested, the presence

of feeding I. trianguliceps ticks (all developmental stages including of A. phagocytophilum was detected in questing I. ricinus from the

larvae) were infected with A. phagocytophilum (Tables 1 and 2). To areas where rodents were captured. Altogether, these ﬁndings fur-

our knowledge this is the ﬁrst detection of A. phagocytophilum in ther support that A. phagocytophilum exhibits host speciﬁcity and

feeding I. trianguliceps in continental Europe. Our results strongly vector association.

support the previously proposed hypothesis by Bown et al. (2009) Our results were further supported by the obtained sequence

that A. phagocytophilum strains associated with rodents circulate data (Table 5, Figs. 1 and 2). The highly conserved 16S rRNA gene

in enzootic cycles separate from non-rodent associated strains and has been used for genotyping A. phagocytophilum strains in many

that they are transmitted by I. trianguliceps but not I. ricinus ticks. As studies. Four 16S genetic variants were detected in questing I. rici-

the presence of infectious agent in feeding ticks does not prove that nus, feeding I. trianguliceps ticks and in rodents in this study. Two

the tick is also a biological vector of that agent DNA, additional xen- (4 and 5) were detected in I. ricinus, I. trianguliceps and M. glare-

odiagnostic studies are further needed to validate this hypothesis. oulus and were identical to variants previously detected in feeding

On the other hand, our conclusions are supported by the ﬁndings and questing I. ricinus ticks in Lithuania (Paulauskas et al., 2012). In

in rodent necropsies, where M. glareolus, A. ﬂavicollis, and A. agrar- addition, we detected two new sequence types in questing I. ricinus

ius were infected with the same genotype of A. phagocytophilum ticks and a rodent ear biopsy (Table 5). Von Loewenich et al. (2003)

in those areas where I. trianguliceps were present. The highest A. described seven variants infecting I. ricinus ticks in Germany, and

phagocytophilum prevalence in rodents was found in vole species Katargina et al. (2012) described four variants infecting I. ricinus

as in the UK (Bown et al., 2003, 2006, 2008, 2009). Importantly, the ticks in Estonia, Belarus and Russia. In the USA, the Ap-variant 1

ﬁnding that none of the rodent-feeding I. ricinus ticks were found differs from a human strain (Ap-ha) and appears to be restricted to

to be infected with A. phagocytophilum although they were coin- ruminant species as reported by Massung et al. (1998). However, in

cidentally feeding on an infected rodent together with infected I. Europe, both the Ap-variant 1 and the Ap-ha 16S rRNA gene vari-

trianguliceps ticks, further supports the proposed hypothesis. Sim- ants were detected in sheep, cattle and cervids (Bown et al., 2009).

ilarly, Heylen et al. (2014) did not observe co-feeding transmission Nevertheless, this locus is not an adequate genetic marker to inves-

of B. burgdorferi s.l. on songbirds among the ornithophilic ticks tigate the possible ecological association between the strains and

934 L. Blanarováˇ 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 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.

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

Fig. 1. (Continued)

936 L. Blanarováˇ 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 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 phyloge- spleen biopsies of rodents (M. glareolus, A. ﬂavicollis and A. agrar-

netic analysis of groEL, msp4 and DOV1 gene sequences we have ius). At sites where I. trianguliceps was absent, we did not detect A.

shown similar topologies for all three genetic loci which support phagocytophilum in rodents. None of the feeding I. ricinus ticks from

two distinct clades. Clade 1 contained strains from questing I. rici- rodents were found infected with A. phagocytophilum albeit some of

nus ticks and feeding I. ricinus ticks from deer, sheep, humans and them were feeding on an infected rodent. Phylogenetic analysis of

dogs but also the strains from I. scapularis and rodents from US four genetic loci of A. phagocytophilum-infected samples revealed

and I. persulcatus. Clade 2 contained genotypes from rodents from that genotypes in questing I. ricinus were distinct from genotypes

Europe and Russia and feeding I. trianguliceps and I. persulcatus. found in rodents and rodent feeding I. trianguliceps. Our study from

In the msp4 tree, the single strain from roe-deer (JN005728) rep- Central Europe conﬁrms the previous ﬁndings from the UK that

resents a unique haplotype in Clade 2. Interestingly, the highest in Europe A. phagocytophilum variants associated with rodents are

diversity of A. phagocytophilum strains was detected in I. persulcatus transmitted by I. trianguliceps but not I. ricinus, and thus in Europe

which carried strains from both clades (Rar and Golovljova, 2011). rodents are not reservoir hosts of the human pathogenic genotypes

These results clearly show that the ecology of A. phagocytophilum of A. phagocytophilum in contrast to the epidemiological context in

differs between North America, Europe and Asia. 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.

L. Blanarováˇ et al. / Ticks and Tick-borne Diseases 5 (2014) 928–938 937

Acknowledgments genetic variability of Anaplasma species in small ruminants and ticks from Cen-

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