Transactions of the Royal Society of Tropical Medicine and Hygiene

Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 10 15 Contents lists available at ScienceDirect Transactions of the Royal Society of Tropical Medicine and Hygiene journal homepage: http://www.elsevier.com/locate/trstmh Molecular evidence for Anaplasma phagocytophilum in Ixodes ricinus from Turkey Munir Aktas a,, Zati Vatansever b, Kursat Altay a, M. Fatih Aydin a, Nazir Dumanli a a Department of Parasitology, Faculty of Veterinary Medicine, Firat University, 23119 Elazig, Turkey b Department of Parasitology, Faculty of Veterinary Medicine, Kafkas University, Kars, Turkey article info abstract Article history: Received 17 February 2009 Received in revised form 21 July 2009 Accepted 21 July 2009 Available online 10 September 2009 Keywords: Ixodes Anaplasma phagocytophilum Nested PCR DNA sequence analysis 16S rrna Turkey This study investigated the presence of the pathogen Anaplasma phagocytophilum in ixodid ticks removed from humans living in three provinces (Giresun, Trabzon, Rize) in the east of the Black Sea Region of Turkey. A total of 1097 ixodid ticks were examined for the presence of A. phagocytophilum DNA. From the 95 pooled tick samples tested, species-specific fragments of A. phagocytophilum (11/95 samples, 11.6%) were amplified by nested PCR. Adult Ixodes ricinus (9/53 samples, 17.0%) and Ixodes spp. nymphs (2/9 samples, 22.2%) were infected with A. phagocytophilum. None of the remaining tick species gave a positive result for the presence of the pathogen. All nested PCR-positive samples were directly sequenced. The partial sequences (457 bp) of the amplicons obtained from the infected tick pools were 100% identical to one another and to previously isolated sequences from human patients. To obtain a longer 16S rrna gene sequence, one representative sample was reamplified with the universal primer set. The longer representative sequence (1306 bp) also shared 99.92% similarity (a single adenine deletion) with the recently reported complete sequence of A. phagocytophilum. 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. 1. Introduction Anaplasma and Ehrlichia species, in the Anaplasmataceae family, are known as important tick-borne pathogens of medical and veterinary importance. 1 Molecular studies suggest that the two genera are divided into various distinct genogroups. The genus Anaplasma comprises six species; A. phagocytophilum, A. centrale, A. marginale, A. bovis, A. ovis and A. platys. These pathogens are transmitted by ixodid ticks. 2 Anaplasma phagocytophilum causes a febrile disease in human and animals. 3 Ticks and reservoir hosts are crucial to the ecology of the disease. The agent is maintained in nature in a tick vertebrate cycle, especially small rodents Corresponding author. Tel.: +90 424 237 0000; fax: +90 424 238 8173. E-mail address: maktas@firat.edu.tr (M. Aktas). and deer. 4,5 The presence of A. phagocytophilum infection is closely related to the abundance of Ixodes spp. 5,6 The pathogen is transmitted to humans through the bites of infected ticks and through perinatal transmission. 7 The first report of a human case was a patient who died after contracting a severe febrile illness following a tick bite in Wisconsin, USA. 8 Acute cases caused by Ixodes bites have also been described in Europe. 9 Tick-borne pathogens such as Theileria and Babesia spp. have been documented in domestic ruminants and ticks in Turkey, 10 13 but there is little information about the presence of rickettsiae in the country. It has recently been reported that A. phagocytophilum has been detected in cattle and sheep; 14 however, the pathogen has not been reported in either humans or ticks in Turkey. This study investigates the presence of A. phagocytophilum in I. ricinus removed from humans in the Black Sea Region of Turkey. 0035-9203/$ see front matter 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2009.07.025

M. Aktas et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 10 15 11 Figure 1. Map of the Turkish provinces showing the locations surveyed in the current study. 2. Materials and methods 2.1. Study area and collection of tick samples The study was conducted from March to December 2007 in an area comprising three provinces: Giresun, Trabzon and Rize, in the east of the Black Sea Region of Turkey (Figure 1). The region covers two climatic zones: a typical temperate climate in the coastal region (humid bioclimatic zone), with frequent rainfall and mild temperatures, and a continental Mediterranean climate in the interior parts (semi-arid cold Mediterranean bioclimatic zone), with warmer summers and colder winters. At the request of Ministry of Health of Turkey, ticks recovered from humans in Giresun, Trabzon and Rize provinces were sent to our laboratory (Department of Parasitology, Veterinary Faculty, Firat University, Elazig, Turkey). Most ticks (adult and/or nymph) were removed from patients by medical staff. They were placed into 1.5 ml tubes filled with 70% ethanol or propanol and submitted to us along with documentation. Although samples included immature ticks, only adults were identified to the species level using standard taxonomic keys. 15 A total of 4783 ticks (3240 adults and 1543 nymphs) were submitted, of which 1097 (815 adults and 282 nymphs) originating from 60 correctly georeferenced sites were selected for DNA extraction. Ticks were washed in 70% ethanol, rinsed three times in sterile PBS, and dried on filter paper. They were separated by georeferenced site, species and nymphal stage into 95 pooled samples consisting of 85 adult (2 10 per pool) and 10 nymphal (10 30 per pool) pools and stored at 80 C until DNA extraction. 2.2. DNA extraction and PCR amplification The frozen tick pools were crushed, using sterile metal rods, in liquid nitrogen in 1.5 ml Eppendorf tubes. Total DNA was extracted using the AbsoGene DNA isolation kit according to the manufacturer s instructions (RTA Laboratories, Gebze, Turkey). A nested PCR assay was performed to identify samples infected with A. phagocytophilum, as described by Kawahara et al. 5 A pair of universal primers, EC12A (5 -TGATCCTGGCTCAGAACGAACG-3 ) and EC9 (5 -TACCTTGTTACGACTT-3 ), that amplify the 16S rrna gene of Anaplasma and Ehrlichia spp. was used for the first amplification. 5,8 Primers SSAP2f

12 M. Aktas et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 10 15 Table 1 Tick samples removed from humans, and infection rates of Anaplasma phagocytophilum in three provinces in the Black Sea region of Turkey Province Identified tick species No. of removed ticks No. of examined ticks No. of pools No. of positive pools Giresun Ixodes ricinus 812 198 14 7 Ixodes spp. nymph 280 64 2 Hyalomma marginatum marginatum 163 23 3 Haemaphysalis sulcata 43 28 7 Haemaphysalis spp. nymph 266 7 1 Dermacentor marginatus 42 24 7 Trabzon Ixodes ricinus 1058 257 23 2 Ixodes spp. nymph 705 148 5 1 Haemaphysalis sulcata 9 7 1 Dermacentor marginatus 6 3 1 Rhipicephalus bursa 55 23 3 Rize Ixodes ricinus 909 216 16 Ixodes hexaganus 14 2 1 Ixodes spp. nymph 292 63 2 1 Haemaphysalis sulcata 37 20 5 Haemaphysalis punctata 80 5 2 Dermacentor marginatus 12 9 2 Total 4783 1097 95 11 (11.6%) (5 -GCTGAATGTGGGGATAATTTAT-3 ) and SSAP2r (5 -ATGGCTGCTTCCTTTCGGTTA-3 ), specific for A. phagocytophilum, were used for nested amplification. 5 For the nested PCR amplification, 1 l of first-round PCR products was used as the template. The PCR was performed in a total reaction volume of 25 l containing 2.5 lof10 PCR buffer [100 mm Tris HCl (ph 9), 500 mm KCl, 1% Triton X-100], 250 M of each of the four deoxynucleotide triphosphates, 2 U Taq DNA polymerase (Promega, Madison, WI, USA) and 10 pmol of each primer. The template for PCR was 2.5 l of the DNA suspension. A touchdown programme was used to reduce nonspecific amplification. Distilled water and Anaplasma ovis DNA (previously isolated from naturally infected sheep, GenBank accession no. EU191232) were used in each test as negative and positive controls, respectively. The amplification products were visualized on 1% agarose gels stained with ethidium bromide and observed under UV illumination. 2.3. Sequencing and phylogenetic analyses All nested PCR-positive samples were directly sequenced. To obtain a longer 16S rrna gene sequence, DNA from one representative positive sample was reamplified with the universal primer set EC12A/EC9, which generated a 1446 bp product. The PCR products were purified from agarose gel using a commercial PCR purification kit (Wizard SV gel and PCR clean-up system; Promega, Madison, WI, USA). The nearly full-length 16S rrna gene sequence of A. phagocytophilum (1306 bp) obtained in this study was deposited in the EMBL/GenBank databases under accession no. FJ172530 Sequence homology searches were made by BLAST. 16 Multiple sequence alignments were performed using the Jalview program, 17 which is based on Clustal W. 18 A phylogenetic tree was created from the sequences of the 16S rrna genes of A. phagocytophilum isolates available from GenBank and the novel sequence described here, using the neighbour-joining method in MEGA version 3.1. 19 3. Results 3.1. Tick species and distribution The distribution of ixodid tick species and the A. phagocytophilum infection rates of ticks from three provinces in the Black Sea Region of Turkey are shown in Table 1. A total of 4783 ticks (3240 adults, 1543 nymphs) were removed from humans in Giresun (n = 1606), Trabzon (n = 1833) and Rize (n = 1344). The ticks removed were identified as Ixodes ricinus, Ixodes hexagonus, Ixodes spp. nymph, Hyalomma marginatum, Haemaphysalis punctata, Hae. sulcata, Hae. spp. Table 2 Frequency of Anaplasma phagocytophilum infection identified by nested PCR in ixodid tick species removed from humans in the east Black Sea Region of Turkey Tick species No. removed ticks No. examined ticks No. pools No. positive pools Ixodes ricinus 2779 671 53 9 (17.0%) Ixodes hexagonus 14 2 1 0 Ixodes spp. nymph 1277 275 9 2 (22.2%) Haemaphysalis sulcata 89 55 13 0 Haemaphysalis punctata 80 5 2 0 Haemaphysalis spp. nymph 266 7 1 0 Dermacentor marginatus 60 36 10 0 Hyalomma marginatum marginatum 163 23 3 0 Rhipicephalus bursa 55 23 3 0 Total 4783 1097 95 11 (11.6%)

M. Aktas et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 10 15 13 nymph, Dermacentor marginatus and Rhipicephalus bursa. The dominant species was I. ricinus in all three provinces (Table 1). All Ixodes, Rhipicephalus and Haemaphysalis specimens were from the humid bioclimatic zone, whereas Hyalomma spp. were from the semi-arid bioclimatic zone (Figure 1). Dermacentor species showed sparse distribution. 3.2. Detection of 16S rrna gene of Anaplasma phagocytophilum and sequence analysis A total of 95 tick pools (35 from Giresun, 33 from Trabzon and 27 from Rize) were included in this study. Among the seven species examined, only I. ricinus and Ixodes spp. nymphs were positive for A. phagocytophilum. The remaining tick species did not contain A. phagocytophilum DNA. The proportion of positive pools among the sampled Ixodes spp. was higher in Giresun province (7/16), followed by Trabzon (3/28) and Rize provinces (1/19) (Table 1). All the infected ticks were from the humid bioclimatic zone (Figure 1). Most of the tested pools were I. ricinus adults (53 pools, 55.8%) followed by Hae. sulcata adults (13 pools, 13.7%), D. marginatus adults (10 pools, 10.5%) and Ixodes spp. nymphs (9 pools, 9.5%). The frequency of A. phagocytophilum infection in ixodid tick species is shown in Table 2. The overall infection rate of all tick pools was 11.6% (11/95). The pathogen was detected in 9 of 53 (17.0%) adult I. ricinus and 2 of 9 (22.2%) Ixodes spp. nymph pools. The partial sequences (457 bp) of the amplicons obtained from the infected tick pools were 100% identical to one another and to previously isolated sequences from human patients (GenBank accession nos. U02521 and AF093789). The longer representative sequence (1306 bp) also shared 99.92% similarity (with the exception of a single deletion of an adenine residue at position 106) with the recently reported complete sequence of A. phagocytophilum (GenBank accession no. AY055469) and the human isolates U02521 and AF093789. Sequence comparisons and phylogenetic analyses showed the agent detected in this study to be 98.77% identical to the sequences of A. phagocytophilum identified in wild deer (GenBank accession nos. AB196721 and AB196720). To investigate the genetic relationships of the A. phagocytophilum strain detected in I. ricinus in Turkey, we performed a phylogenetic analysis based on the 1306 nucleotide fragment of the 16S rrna gene sequence. The constructed phylogenetic tree demonstrated a close relationship between the A. phagocytophilum isolate identified in this study and recently reported sequences identified in humans (Figure 2). 4. Discussion Ticks act as vectors of diseases affecting both humans and animals. They are also responsible for direct damage and cause severe toxic conditions such as paralysis, irritation and allergy. Most ticks have a preference for feeding on a wide variety of wild animals, with some being host specific. However, a large number of tick species have adapted to feed on humans. 20 More than 20 ixodid tick species are reported to be frequently found on humans in infested areas. 2 This survey recovered I. ricinus, I. hexagonus, H. sul- Figure 2. Phylogenetic tree based on a 1306 bp fragment of the 16S rrna gene of the Anaplasma phagocytophilum isolate identified in this study, and known Anaplasma spp. present in the GenBank database, using MEGA software (version 3.1). The distance matrix was calculated by using Kimura-2 parameter method. The tree was created by the neighbour-joining method. The sequence described in this study is indicated in bold. Scale bar represents nucleotide substitutions per position.

14 M. Aktas et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 10 15 cata, H. punctata, D. marginatus, H. marginatum and R. bursa from humans, and showed I. ricinus was the most prevalent tick on humans in the eastern Black Sea Region of Turkey. Human granulocytic anaplasmosis is an emerging infectious disease caused by A. phagocytophilum, which is typically transmitted to humans by ixodid ticks. The transmission cycle of the agent is not fully understood. Among non-human vertebrates it is believed to be maintained in a tick vertebrate cycle with Ixodes spp. 4 I. ricinus has been found to be the primary vector of A. phagocytophilum in Europe. 21 The importance of this tick for the pathogen is its widespread distribution and feeding habits. It is the most common tick that bites humans. 22 However, A. phagocytophilum has also been associated with other ixodid species. 23,24 In Turkey, the pathogen has not previously been described in vector ticks, or in humans. Here we present the first evidence of A. phagocytophilum in Ixodes ticks removed from humans in Turkey. In the present study, the prevalence of A. phagocytophilum infection in ixodid ticks removed from humans was 11.6%. The prevalence of the infection also varied among the life stages of the tick. In this study, 17.0% of adult I. ricinus and 22.2% of Ixodes spp. nymphs were infected with A. phagocytophilum. Previous reports have reported both nymphs and adults infected with A. phagocytophilum, with a higher infection rate in nymphs. 25 In a recent study, however, a much higher infection rate was observed in adult ticks (34%) than in nymphs (2%). 26 In the present study, the prevalence of A. phagocytophilum was higher in Giresun province than in the other two provinces. This result indicates that Giresun may be a focal point for the infection, depending on the abundance of mammalian reservoir hosts. Anaplasma phagocytophilum has been reported in cattle and sheep in the Black Sea Region of Turkey, 14 but the pathogen has not been described hitherto in ticks or humans. This study presents evidence for A. phagocytophilum in Ixodes ticks removed from humans. It is possible that people exposed to Ixodes bites may become infected with A. phagocytophilum. However, no clinical cases of human granulocytic anaplasmosis have been reported in Turkey. We speculate that the lack of reports of clinical cases may be because of low virulence of the pathogen strains, or lack of awareness among clinicians of the existence of the disease together with the limited number of diagnostic tools available. In conclusion, the results of present study provide molecular evidence for the presence of A. phagocytophilum in Ixodes ticks in Turkey, and suggest that human granulocytic anaplasmosis should be included in the differential diagnosis when dealing with a febrile patient with history of possible tick bite. Authors contributions: MA conceived and designed the study; ZV constructed the map and edited the manuscript; MFA conducted the molecular studies; KA participated in the phylogenetic analysis; ND coordinated the collection of ticks. All authors helped to draft the article or revised it critically for intellectual content, and read and approved the final version. MA is guarantor of the paper. Funding: This work was supported financially by a grant (106 O 416) from the Scientific and Technical Research Council of Turkey (TUBITAK). Conflicts of interest: None declared. Ethical approval: Not required. References 1. Dumler JS, Barbet AF, Bekker CP, Dasch GA, Palmer GH, Ray SC, et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and HGE agent as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol 2001;51: 2145 65. 2. Estrada-Pena A, Jongejan F. Ticks feeding on humans: a review of records on human-biting Ixodoidea with special reference to pathogen transmission. Exp Appl Acarol 1999;23:685 715. 3. Kocan KM, de la Fuente J, Blouin EF, Garcia-Garcia JC. 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M. Aktas et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 10 15 15 19. Kumar S, Tamura K, Nei M. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 2004;5:150 63. 20. Jongejan F, Uilenberg G. The global importance of ticks. Parasitology 2004;129(Suppl):S3 14. 21. Strle F. Human granulocytic ehrlichiosis in Europe. Int J Med Microbiol 2004;293(Suppl 37):27 35. 22. Gray JS. Biology of Ixodes species ticks in relation to tick-borne zoonoses. Wien Klin Wochenschr 2002;114:473 8. 23. Alberti A, Addis MF, Sparagano O, Zobba R, Chessa B, Cubeddu T, et al. Anaplasma phagocytophilum, Sardinia, Italy. Emerg Infect Dis 2005;11:1322 4. 24. Ogden NH, Bown K, Horrocks BK, Woldehiwet Z, Bennett M. Granulocytic Ehrlichia infection in ixodid ticks and mammals in woodlands and uplands of the U.K. Med Vet Entomol 1998;12:423 9. 25. Radzijevskaja J, Paulauskas A, Rosef O. Prevalence of Anaplasma phagocytophilum and Babesia divergens in Ixodes ricinus ticks from Lithuania and Norway. Int J Med Microbiol 2008;298:218 21. 26. Christova I, Schouls L, van De Pol I, Park J, Panayotov S, Lefterova V, et al. High prevalence of granulocytic Ehrlichiae and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Bulgaria. J Clin Microbiol 2001;39:4172 4.