Ticks and Tick-borne Diseases 5 (2014) 928–938 Contents lists available at ScienceDirect Ticks and Tick-borne Diseases j ournal homepage: www.elsevier.com/locate/ttbdis Original article Distinct Anaplasma phagocytophilum genotypes associated with Ixodes trianguliceps ticks and rodents in Central Europe a a,b c,d a Lucia Blanarovᡠ, Michal Stanko , Giovanna Carpi , Dana Miklisová , a a e a,b,∗ Bronislava Víchová , Ladislav Mosanskˇ y´ , Martin Bona , Markéta Derdáková a Institute of Parasitology SAS, Hlinkova 3, 040 01 Kosice,ˇ Slovakia b 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, Srobárovᡠ2, 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. © 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 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 1877-959X/© 2014 Elsevier GmbH. All rights reserved. L. Blanarovᡠet 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.; ˇ (Víchová et al., 2010; Hulínska et al., 2004; Stefancíkovᡠet 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 cíkovᡠSample 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.