Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp., and Other Eubacteria in Ticks from the Thai-Myanmar Border and Vietnam

JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2003, p. 1600 1608 Vol. 41, No. 4 0095-1137/03/$08.00 0 DOI: 10.1128/JCM.41.4.1600 1608.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved. Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp., and Other Eubacteria in Ticks from the Thai-Myanmar Border and Vietnam Philippe Parola, 1,2,3 * Jean-Paul Cornet, 4 Yibayiri Osée Sanogo, 2 R. Scott Miller, 1 Huynh Van Thien, 5 Jean-Paul Gonzalez, 4 Didier Raoult, 2 Sam R. Telford III, 3 and Chansuda Wongsrichanalai 1 Department of Immunology and Medicine, Armed Forces Research Institute of Medical Sciences, Bangkok, 1 and Institut de Recherche pour le Développement, UR34, Mahidol University at Salaya, Nakorn Prathom, 4 Thailand; Unité des Rickettsies, CNRS UMR 6020, Faculté demédecine, Marseille, France 2 ; Laboratory of Public Health Entomology, Harvard School of Public Health, Boston, Massachusetts 3 ; and Bao Loc General Hospital, Bao Loc, Lam Dong Province, Vietnam 5 Received 2 May 2002/Returned for modification 6 November 2002/Accepted 23 January 2003 A total of 650 ticks, including 13 species from five genera, were collected from animals, from people, or by flagging of the vegetation at sites on the Thai-Myanmar border and in Vietnam. They were tested by PCR to detect DNA of bacteria of the order Rickettsiales. Three Anaplasma spp. were detected in ticks collected in Thailand, including (i) Anaplasma sp. strain AnDa465, which was considered a genotype of Anaplasma platys (formerly Ehrlichia platys) and which was obtained from Dermacentor auratus ticks collected from dogs; (ii) Anaplasma sp. strain AnAj360, which was obtained from Amblyomma javanense ticks collected on a pangolin; and (iii) Anaplasma sp. strain AnHl446, which was closely related to Anaplasma bovis and which was detected in Haemaphysalis lagrangei ticks collected from a bear. Three Ehrlichia spp. were identified, including (i) Ehrlichia sp. strain EBm52, which was obtained from Boophilus microplus ticks collected from cattle from Thailand; (ii) Ehrlichia sp. strain EHh324, which was closely related to Ehrlichia chaffeensis and which was detected in Haemaphysalis hystricis ticks collected from wild pigs in Vietnam; and (iii) Ehrlichia sp. strain EHh317, which was closely related to Ehrlichia sp. strain EBm52 and which was also detected in H. hystricis ticks collected from wild pigs in Vietnam. Two Rickettsia spp. were detected in Thailand, including (i) Rickettsia sp. strain RDla420, which was detected in Dermacentor auratus ticks collected from a bear, and (ii) Rickettsia sp. strain RDla440, which was identified from two pools of Dermacentor larvae collected from a wild pig nest. Finally, two bacteria named Eubacterium sp. strain Hw124 and Eubacterium sp. strain Hw191 were identified in Haemaphysalis wellingtoni ticks collected from chicken in Thailand; these strains could belong to a new group of bacteria. Spotted fever group (SFG) rickettsioses and ehrlichioses are caused by obligate intracellular gram-negative bacteria belonging to the order Rickettsiales. They are now recognized as important emerging vector-borne human infections worldwide (16, 19). These zoonoses are associated with arthropods, mainly ticks. Eight tick-borne rickettsioses with distinct species as agents have definitively been described throughout the world, including Rickettsia rickettsii (in the Americas), Rickettsia sibirica (in Asia), Rickettsia conorii including different strains (in Europe, Asia, and Africa), Rickettsia australis (in Australia), Rickettsia honei (in the Flinders Island, Australia), Rickettsia japonica (in Japan), Rickettsia africae (in sub-saharan Africa and the West Indies), and Rickettsia slovaca (in Europe) (16, 19). Furthermore, Astrakhan fever rickettsia and Israeli tick typhus rickettsia, both of which are closely related to R. conorii, are known as agents of rickettsioses in Astrakhan and Israel, respectively (19). Finally, human infections due to Rickettsia aeschlimannii in Africa (18), Rickettsia helvetica and Rickettsia mongolotimonae in Europe (4, 5), and Rickettsia heilongjiangii in Asia (30) have recently been reported. * Corresponding author. Mailing address: Unité des Rickettsies, CNRS UMR 6020, WHO Collaborative Center for Rickettsial Reference and Research, Faculté de Médecine, 27 Bd. Jean Moulin, 13385 Marseille Cedex 5, France. Phone: 33 4 91 32 43 75. Fax: 33 4 91 83 03 90. E-mail: phparola@yahoo.fr. Although ehrlichioses have been recognized as infectious diseases only in animals for a long time, they are now known to be important emerging zoonoses in people. Three human ehrlichioses have been reported since 1991. They include (i) human monocytic ehrlichiosis due to Ehrlichia chaffeensis in the United States, (ii) infections due to Ehrlichia ewingii in the United States, and (iii) human granulocytic ehrlichiosis due to Anaplasma phagocytophilum (formerly named human granulocytic ehrlichia or Ehrlichia phagocytophila), which occur both in the United States and in Europe (2, 3). In Asia, tick-borne SFG rickettsioses and ehrlichioses have been poorly studied. SFG rickettsioses have been reported from Thailand, for example, but to date the cases have been confirmed solely by general SFG serology (24). The etiologic agent(s) has never been specifically identified by isolation or molecular characterization. Two SFG rickettsiae have been identified in ticks in Thailand, including (i) Rickettsia honei and its strain, Thai tick typhus Rickettsia strain TT-118, and (ii) a new rickettsia of unknown pathogenicity, Rickettsia thailandii sp. nov. (10, 19). Their roles as agents of human diseases in Thailand are not known. Ehrlichioses of veterinary importance are known to occur in Thailand, mainly canine ehrlichiosis due to Ehrlichia canis, which is transmitted by the brown dog tick (Rhipicephalus sanguineus). Coinfection with three Ehrlichia species has also 1600

VOL. 41, 2003 TICK-ASSOCIATED BACTERIA IN THAILAND AND VIETNAM 1601 FIG. 1. Map showing the locations where ticks were collected in Thailand and Vietnam and the results of the molecular detection of rickettsial DNA (in the following order: bacteria/tick species/number of ticks tested/number of positive ticks/host). been reported in dogs (26). Although human ehrlichioses have been suspected in Thailand on the basis of serological data (7), no known human pathogens have been identified from patients or in ticks that bite humans. Tick-borne diseases including SFG rickettsioses and ehrlichioses have been suspected to occur among local Karen, Mon, Burmese, and Thai rural residents living in the central part of the Thai-Myanmar border region (Sangkhlaburi District, Kanchanaburi Province, Thailand) [R. S. Miller, P. McDaniel, S. Nedek, N. Thanoosingha, N. Buathong, S. Sriwichai, A. Weld, S. R. Telford III, and C. Wongsrichanalai, Program Abstr. 49th Annu. Meet. Am. Soc. Trop. Med. Hyg., Am. J. Trop. Med. Hyg. 62(Suppl. 3):469-470, 2000]. Recently, new molecular methods have enabled the development of useful, sensitive, and rapid tools for the detection and identification of tickborne pathogens in arthropods including ticks (25). Accordingly, in an effort to identify the possible etiologic agents for SFG rickettsioses and ehrlichioses affecting humans in these sites, we analyzed ticks collected from peridomestic or wild animals in the Sangkhlaburi District for evidence of rickettsial infections. Additional ticks collected in Vietnam were included in this work. (These results were presented in part at the 3rd International Conference on Emerging Infectious Diseases, 24 to 27 March 2002, Atlanta, Ga.) MATERIALS AND METHODS Tick sampling. From September 2001 to February 2002, ticks were collected in the central part of the Thai-Myanmar border region in the Sangkhlaburi District of Kanchanaburi Province, Thailand, in areas within a 10-km radius from Huay Malaï village (15 09 N latitude, 98 27 E longitude) (Fig. 1). Ticks were collected the first week of each month from domestic mammals from local Karen villages; from wild animals trapped or killed by hunters (independently of this research project); as well as by flagging of the vegetation in the villages, in rubber plantations, and in the nearby jungles. Ticks collected from animals in Vietnam (Bao Loc, Lam Dong Province [11 30 N latitude, 107 46 E longitude]) in October 2001 were also studied. The ticks were identified by the use of standard taxonomic keys by two of us (P.P. and J.-P.C.). PCR. Ticks were sterilized by immersion in iodinated alcohol for 10 min, rinsed with distilled water for 10 min, and dried on sterile filter paper in a laminar-flow hood. Each tick was cut in half lengthways (the blades were discarded after each tick was cut), and the DNA was extracted from one half as described previously (14). The remaining halves of the ticks were frozen at 80 C for subsequent studies. Rickettsial DNA was detected by PCR as described previously by using primers Rp877p and Rp1258r (Bioprobe Systems, Montrevilsous Bois, France), which amplify a 396-bp fragment of the citrate synthase gene (glta) of rickettsia (20). A negative control with distilled water instead of tick DNA template in the PCR master mixture and a positive control (DNA from Rickettsia montanensis) were included in each test. To amplify the main part of the glta gene, tick DNA samples that were found to be positive with the primers described above were amplified with primer pair CS1d and CS890r and primer pair Rp877n and CS1273r, as described previously (20). For strain Dal420, an additional primer pair (primer CS62.2F [CAA GTA TTG GGC AGG ATG] and primer CS539.3R [CAA GTA TTG GGC AGG ATG]), based on the sequence of Rickettsia bellii, which was the closest to that of Dal420, was designed. The DNA extracted from the ticks was also screened as described previously with primers EHR16SR and EHR16SD (Bioprobe Systems), which amplify a 345-bp fragment of the 16S rrna gene of bacteria within the family Anaplasmataceae, including the genera Anaplasma, Ehrlichia, Neorickettsia, and Wolbachia (17). To amplify the main part of the 16S rrna gene, tick DNA samples that were found to be positive with the primers described above were subjected to a second PCR with primers EHR16SR and EHR16SD coupled with universal primers fd1 and rp2, as described previously (8). Distilled water and Anaplasma phagocytophilum DNA were used in each test as negative and positive controls, respectively. After electrophoresis the amplification products were visualized on 1% agarose gels stained with ethidium bromide and examined by UV transillumination. A DNA molecular weight marker (marker VI; Boehringer Mannheim, Mannheim, Germany) was used to estimate the sizes of the products.

1602 PAROLA ET AL. J. CLIN. MICROBIOL. Gene sequencing and phylogenetic analysis. The PCR products were purified with QIAquick PCR purification kits (QIAGEN GmbH, Hilden, Germany), and DNA sequencing was performed by use of the fluorescence-labeled dideoxynucleotide technology in an ABI 3100 automated DNA sequencer (Perkin- Elmer, Applied Biosystems Division). Sequences were assembled and edited with AutoAssembler software (version 1.4; Perkin-Elmer). Multiple-sequence alignments with the corresponding sequences were performed by use of the ClustalW program (28). Phylogenetic and molecular evolutionary analyses were conducted by using MEGA software (version 2.1) (12). Phylogenetic trees were inferred from the multiple-sequence alignments, after the removal of all gaps, by the neighbor-joining method (MEGA software, version 2.1). The distance matrix was calculated by use of Kimura-2 parameters. Five hundred bootstrap replicates were used to estimate the reliabilities of the nodes on the phylogenetic trees. Nucleotide sequence accession numbers. The nucleotide sequences of the 16S rrna genes of the bacteria of the family Anaplasmataceae used for phylogenetic studies are available in GenBank under the following accession numbers: AF303467 for Anaplasma platys (formerly Ehrlichia platys), AF286699 for A. platys detected in Thailand, M73224 for A. phagocytophilum (formerly Ehrlichia phagocytophila), U03775 for Anaplasma bovis (formerly Ehrlichia bovis), AF283007 for Anaplasma centrale, M60313 for Anaplasma marginale, AF318945 for Anaplasma ovis, AF069758 for Ehrlichia ruminatium, M73222 for E. chaffeensis, U15527 for Ehrlichia muris, M73227 for E. ewingii, M73221 for E. canis, AF311967 for Ehrlichia sp. strain ERm58, AF311968 for Ehrlichia sp. strain EHt224; AF414399 for Ehrlichia sp. strain Tibet; AF179630 for Wolbachia pipientis; U12457 for Neorickettsia helminthoeca; M73225 for Neorickettsia sennetsu (formerly Ehrlichia sennetsu); M21290 for Neorickettsia risticii (formerly E. sennetsu), U11021 for R. rickettsii; and M21789 for Rickettsia prowazekii. The nucleotide sequences of the 16S rrna genes of the bacteria found in this study have been deposited in GenBank under the following accession numbers: AF497576 for Anaplasma sp. strain AnDa465; AF497580 for Anaplasma sp. strain AnAj360; AF497579 for Anaplasma sp. strain AnHl446; AF497581 for Ehrlichia sp. strain EBm52; AF497578 for Ehrlichia sp. strain EHh324; AF497577 for Ehrlichia sp. strain EHh317; AF497583 for Eubacterium sp. strain Hw124, and AF497582 for Eubacterium sp. strain Hw191. The nucleotide sequences of the citrate synthase gene (glta) of the following rickettsiae used for comparison and phylogenetic studies are deposited in Gen- Bank under the indicated accession numbers: Rickettsia parkeri, U59732; R. sibirica, U59734; R. mongolotimonae, U59731; strain S, U59735; R. africae, U59733; R. conorii Seven, U59730; R. rickettsii, U59729; Astrakhan fever rickettsia, U59728; Israeli tick typhus rickettsia, U59727; R. honei strain RB, AF018074; Thai tick typhus rickettsia, U59726; R. slovaca, U59725; R. japonica, U59724; Rickettsia rhipicephali, U59721; R. montanensis U74756; Rickettsia massiliae, U59719; Bar29, U59720; R. aeschlimannii, U59722; R. helvetica, U59723; Rickettsia sp. strain IRS4, AF141906; Rickettsia sp. strain IRS3, AF140706; R. australis, U59718; Rickettsia akari, U59717; Rickettsia typhi, U59714; Rickettsia canadensis, 59713; AB bacterium, U59712; R. prowazekii, U59715; R. bellii, U59716; Rickettsia hulinii, AF172943; R. heilongjiangii, AF178034; Rickettsia sp. strain DnS14, AF120028; Rickettsia sp. strain RpA4, AF120029; Rickettsia sp. strain DnS28, AF120027; Rickettsia sp. strain RDa420, AF497584; and Rickettsia sp. strain RDla440, AF497585. The nucleotide sequences of the glta genes found in this study have been deposited in GenBank under the following accession numbers: AF497584 for Rickettsia sp. strain RDa420 and AF497585 for Rickettsia sp. strain RDla440. RESULTS Tick sampling. A total of 606 specimens, including nine species from four genera of ticks, were collected in the Thai- Myanmar border area of Sangkhlaburi District in Thailand. A total of 44 specimens, including seven species from three genera of ticks, were also collected in Vietnam. Details about the tick species and hosts are presented in Table 1. PCR detection of bacteria within the family Anaplasmataceae and analyses of the 16S rrna gene sequences. By use of broad-spectrum primers EHR16SR and EHR16SD, PCR products of 335 bp were detected for 45 of the 650 (6.9%; 95% confidence interval [CI], 4.9 to 8.9%) ticks studied. By use of primers EHR16SR and EHR16SD coupled with universal primers fd1 and rp2, respectively, sequences of longer fragments of the 16S rrna gene were obtained from each of the 45 positive samples. Three 16S rrna sequences of Anaplasma spp. were identified (Fig. 1). They included (i) AnDa465 (1,016 bp), which was obtained from 3 of 20 (15%; 95% CI, 0 to 30.6) Dermacentor auratus nymphs collected from dogs in Thailand and which was closely related (99.3% similarity) to the sequence of A. platys (formerly named E. platys); (ii) AnAj360 (955 bp), which was obtained from 16 of 54 (29.6%; 95% CI, 17.4 to 41.8%) adult Amblyomma javanense ticks collected from a pangolin and which showed 97.9% similarity with A. phagocytophilum and 97.8% similarity with A. platys and A. bovis (formerly E. bovis); and (iii) AnHl446 (1,014 bp), which was identified in 3 of 8 (37.5%; 95% CI, 4 to 71%) female Haemaphysalis lagrangei ticks collected from a bear from Thailand and which showed 99.6% similarity with A. bovis, 97.9% similarity with A. phagocytophilum, and 96.5% similarity with A. platys. Three 16S rrna sequences of Ehrlichia spp. were identified (Fig. 1). They included (i) EBm52 (1,380 bp), which was obtained from 24 of 109 (22%; 95% CI, 14.2 to 29.8%) Boophilus microplus ticks collected from cattle from Thailand and which was shown to be closely related (99.9% similarity) to a sequence identified in B. microplus ticks collected from cattle in Tibet (GenBank accession number AF414399) and also to be closely related (99.6% similarity) to two ehrlichial DNA sequences detected in cattle ticks from Africa (Erm58 and Eht224; GenBank accession numbers AF311967 and AF311968, respectively); (ii) EHh324 (902 bp), which was detected in 1 of 19 (5.3%; 95% CI, 0 to 15.3%) Haemaphysalis hystricis ticks collected from wild pigs in Vietnam and which was closely related to E. chaffeensis (99.4% similarity); and (iii) EHh317 (902 bp), which was also detected in 1 of 19 H. hystricis ticks from Vietnamese wild pigs (the tick was different from the tick positive for EHh324) but which appeared to be closely related to Ebm52 and the related sequences described above (99% similarity). Furthermore, two 16S rrna sequences designated Hw124 and Hw191 were identified in 2 of 55 (3.6%; 95% CI 0 to 8.5%) Haemaphysalis wellingtoni nymphs collected from chickens in Thailand (Fig. 1). Compared with the sequences available in GenBank, the sequences of both Hw124 and Hw191 appeared to differ from those of all known bacteria. The most closely related sequences available in GenBank had been deposited under the name endosymbiont of Acanthamoeba sp. (Gen- Bank accession number AF069962; 93% similarity when 790- bp sequences were compared) and the name Eubacterium ZI-8 (GenBank accession number AJ292457; 96% similarity when 536-bp sequences were compared). By a neighbor-joining analysis (Fig. 2) based on the alignments of 960 bp of the 16S rrna genes, AnDa465 clustered with A. platys sequences. AnHl446 and AnAj360 were placed within the Anaplasma clade as well. The bacteria from which these sequences originated were temporarily called Anaplasma sp. strain AnDa465, Anaplasma sp. strain AnHl446, and Anaplasma sp. strain AnAj360, respectively. EBm52, EHh324, and EHh317 were shown to belong to the Ehrlichia clade; and the bacteria from which these sequences originated were temporarily called Ehrlichia sp. strain EBm52, Ehrlichia sp. strain EHh324, and Ehrlichia sp. strain EHh317, respectively. Furthermore, this analysis suggested that strains Hw124 and Hw191 may originate from a new group of bacteria. The or-

VOL. 41, 2003 TICK-ASSOCIATED BACTERIA IN THAILAND AND VIETNAM 1603 Tick Origin TABLE 1. Characteristics of ticks collected a No. of ticks Male Female Nymphs Larvae Total Host(s) Boophilus microplus Thailand 12 57 6 75 Bos domesticus (domestic cattle) Thailand 3 31 34 Capra hircus (domestic goat) Haemaphysalis wellingtoni Thailand 3 2 39 9 53 Gallus gallus (red jungle fowl) Thailand 1 1 2 Canis familiaris (domestic dog) Thailand 1 1 Homo sapiens (human) b Thailand 12 5 7 7 31 Centropus sinensis (greater coucal) Haemaphysalis lagrangei Thailand 1 1 Flagging into the jungle 8 8 Helarctos malayanus (Malayan sun bear) b Haemaphysalis asiatica Thailand 1 1 Rattus sabanus (noisy rat) c 4 4 Menetes berdmorei (Indochinese ground squirrel) 1 1 Berylmys bowersi (Bowers giant rat) c Haemaphysalis hystricis Vietnam 15 4 19 Sus scrofa (wild pig) Haemaphysalis traguli Vietnam 1 5 6 Tragulus jananicus (mouse deer) Amblyomma javanense Thailand 37 3 8 6 54 Manis javanica (pangolin) Dermacentor sp. Thailand 200 200 Flagging the vegetation in rubber plantations Thailand 48 48 Wild pig nest Thailand 12 12 Homo sapiens (human) Dermacentor auratus Thailand 6 6 Helarctos malayanus (Malayan sun bear) c Thailand 20 20 Sus scrofa (wild pig) Thailand 24 24 Canis familiaris (domestic dog) b Thailand 18 18 Homo sapiens (human) Thailand 6 6 Rattus sabanus (noisy rat) c Thailand 2 2 Rattus surifer (yellow Rajah Rat) Thailand 1 1 Menetes berdmorei (Indochinese ground squirrel) Thailand 1 1 Berylmys bowersi (Bowers giant rat) Thailand 1 1 Rattus koratensis (Sladen s rat) Thailand 1 1 Rattus fulvescens (chestnut rat) Vietnam 3 1 4 Sus scrofa (wild pig) Dermacentor compactus Vietnam 1 1 Sus scrofa (wild pig) Dermacentor steini Thailand 1 1 2 Helarctos malayanus (Malayan sun bear) b Vietnam 1 1 2 Sus scrofa (wild pig) Dermacentor atrosignatus Thailand 1 1 Sus scrofa (wild pig) Vietnam 1 2 3 Sus scrofa (wild pig) Rhipicephalus sanguineus Vietnam 7 2 9 Canis familiaris (domestic dog) a Ticks were collected from September 2001 to March 2002 in the central part of the Thai-Myanmar border in Sangkhlaburi District (15 09 N latitude and 98 27 E longitude), Kanchanaburi Province, Thailand, and in Bao Loc (11 30 N latitude and 107 46 E longitude), Lam Dong Province, Vietnam. b Newly reported tick-host association. c New tick-host association reported in Thailand. ganisms from which these sequences originated were temporarily called Eubacterium sp. strain Hw124 and Eubacterium sp. strain Hw191, respectively. PCR detection of bacteria of the genus Rickettsia and analyses of glta gene sequences. DNA of Rickettsia spp. was detected in three samples (Fig. 1). Two glta sequences were identifed. RDa420 (1,086 bp) was detected from 1 of 8 (95% CI, 0 to 22.9%) D. auratus ticks collected from a bear. It was found to be different from all the known Rickettsia sp. sequences deposited in GenBank. The most closely related rickettsia was R. bellii (94.7% similarity); other rickettsiae were less than 91% similar. RDla440 (1,108 bp) was detected in two pools of 30 Dermacentor larvae collected from a wild pig nest. Its sequence appeared to be most closely related to the sequences of Rickettsia sp. strain DnS14 and Rickettsia sp. strain RpA4, with only 2 bp being different (99.7% similarity). In a neighbor-joining analysis based on the alignment of 1,035 bp of the glta genes of the rickettsiae, RDa420 formed a clade with R. bellii and Dla440 clustered with Rickettsia sp. strain DnS14, Rickettsia sp. strain DnS28, and Rickettsia sp. strain RpA4 (Fig. 3). The rickettsiae from which the glta sequences originated were temporarily called Rickettsia sp. strain RDa420 and Rickettsia sp. strain Dla440, respectively. DISCUSSION Although the diverse species that comprise the 606 ticks that we collected in the Sangkhlaburi District had previously been

1604 PAROLA ET AL. J. CLIN. MICROBIOL. FIG. 2. Phylogenetic tree based on studies of 960 sites of the 16S RNA genes of bacteria of the genera Anaplasma, Ehrlichia, Neorickettsia, and Wolbachia and drawn by using MEGA software (version 2.1) (12). The distance matrix was calculated by using Kimura-2 parameters. Trees were obtained by the neighbor-joining method. The numbers at the nodes are the proportions of 500 bootstrap resamplings that support the topology shown. The bacteria detected in this work are highlighted. found in Thailand, they have never been recorded in the part of the Thai-Myanmar border where they were collected (27). Moreover, some new tick-host associations are presented here (Table 1). We report the first record of D. auratus nymphs from dogs. The two infested dogs frequently followed their hunters-owners into the jungle. Interestingly, although about 30 dogs were screened each month in the villages during the period of the study, no brown dog tick (R. sanguineus) was found. However, climate is a key for the distribution of tick species and then for the epidemiology of tick-borne diseases (16). The climatic conditions (the end of the rainy season and the beginning of the cool, dry season), which are not favorable

VOL. 41, 2003 TICK-ASSOCIATED BACTERIA IN THAILAND AND VIETNAM 1605 FIG. 3. Phylogenetic tree based on studies of 1,035 sites of the citrate synthase genes of bacteria of the genus Rickettsia, drawn as described in the legend to Fig. 2. The bacteria detected in this work are highlighted. for R. sanguineus ticks, may explain the absence or the low rate of occurrence of this tick species in this rural border area during the period of the study. This is of epidemiologic importance, because R. sanguineus is known as the main vector of R. conorii, an agent of rickettsioses in humans (19). Thus, putative rickettsioses in humans at this site during this season should be associated with another tick that bites humans. We also report the first record of a human bite caused by H. wellingtoni in Thailand (27). However, the ticks that mainly bit humans at our study site during the period of observation were Dermacentor spp., particularly D. auratus. Most of the D. auratus nymphs (16 of 18) collected from people were removed from hunters coming back from the jungles. Two engorged specimens were removed from the ears of two children living in a village; their fathers had recently returned home from the jungles with ticks attached to their bodies. Some of the oth-

1606 PAROLA ET AL. J. CLIN. MICROBIOL. er ticks collected during our study period are also known to bite humans, including Dermacentor atrosignatus, Dermacentor steini, Dermacentor compactus, H. hystricis, and H. lagrangei (11, 27). Thus, all these ticks hold the first component necessary to be involved in human diseases: an affinity to bite humans. The 16S rrna sequences of the Anaplasma spp. and Ehrlichia spp. identified in this work were not identical to any sequence deposited in GenBank. Although a failure to match a sequence to one in GenBank is all too often taken to imply novelty, we prefer to remain conservative about such a conclusion. We provide designations to refer to the organisms that are represented by the DNA sequences that were found in ticks but do not suggest that these necessarily comprise novel entities. Within the family Anaplasmataceae, there is at present no consensus as to the degree of 16S rrna gene dissimilarity which should be evident to distinguish two bacterial species as opposed to that which should be evident to represent natural genetic variation. A recent study suggested that 0.5% divergence in the 16S RNA gene sequence of bacteria within this family could be considered a cutoff (29); however, it seems prudent to await phylogenetic studies based on sequences of other genes before such a recommendation is accepted. In this work, we identified Anaplasma sp. strain AnDa465 in D. auratus ticks from Thailand. According to the level of 16 srna gene sequence similarity (99.3%) and to our phylogenetic analysis, Anaplasma sp. strain AnDa465 appeared to be closely related to A. platys, the agent of asymptomatic to mild infectious cyclic thrombocytopenia in dogs. A similar sequence had also recently been detected in dogs from Thailand (26). As suggested in the latter work, we believe that the few differences noticed in the 16S rrna gene sequences compared to those available in GenBank might be due to sequencing or PCR error, but they may possibly be due to variations in the sequences of strains of the same species as well. We have recently detected A. platys isolates from a dog and a tick in the Democratic Republic of Congo, and although the 16S rrna gene sequences of the isolates showed some differences from the A. platys sequences deposited in GenBank, additional analyses based on groesl and glta gene phylogenies suggest that they are a strain of A. platys (Y. O. Sanogo, unpublished data). Our findings thus confirm the presence of A. platys in Thailand. This bacterium has recently been detected in Japan in the brown dog tick (R. sanguineus), which could serve as a vector (9). For the first time, D. auratus was implicated here as a potential vector of canine infectious cyclic thrombocytopenia, although it is possible that the ticks that yielded the A. platys amplification products could have been feeding on bacteremic dogs. Two more Anaplasma spp. were detected in this work. Anaplasma sp. strain AnAj360 was detected from A. javanense ticks from a pangolin. Two specimens of this animal were screened for ticks, but only ticks collected from one of them were positive. The sequence of this Anaplasma strain presented less than 98% similarity with those of other Anaplasma spp., and it could represent a new species. On the other hand, Anaplasma sp. strain AnHl446 was detected from H. lagrangei ticks from a bear. It appeared to be closely related to A. bovis according to the level of 16S rrna gene sequence similarity (99.6%) and phylogenetic analysis; in the phylogenetic tree, it is grouped with A. bovis with 100 as a bootstrap value. Thus, Anaplasma sp. strain AnHl446 could be a strain of A. bovis, although as stated above, this cannot be definitely assumed. Ehrlichia sp. strain EBm52 was obtained from B. microplus ticks collected from cattle. This tick species is well known as the vector of A. marginale, an intraerythrocytic pathogen that causes bovine anaplasmosis (13). This agent is distributed worldwide, and it was recently detected in B. microplus ticks collected from cattle in Myanmar (P. Parola, unpublished data). However, Ehrlichia sp. strain EBm52 was shown to be closely related to Ehrlichia sp. strain Tibet (99.9% sequence similarity) identified in B. microplus ticks collected in Tibet (29). The latter ehrlichia was recently presented as a new species within the genus Ehrlichia, based on phylogenetic analyses of the 16S rrna gene (29). Furthermore, both Ehrlichia sp. strain Tibet and Ehrlichia sp. strain EBm52 were shown to be closely related to two ehrlichiae which we detected in African ticks, including Rhipicephalus muhsamae ticks from Mali (Ehrlichia sp. strain Erm58) and Hyalomma truncatum ticks from Niger (Ehrlichia sp. strain Eht224) (15). The gene sequences of all four of these ehrlichiae were detected in ticks removed from cattle. Given the high degree of genetic similarity, these organisms may represent strains of the same species, all of which are associated with cattle. Their zoonotic or veterinary potential remains to be described. However, B. microplus rarely bites people (if ever), and the transmission of Ehrlichia sp. strain EBm52 to humans by B. microplus is unlikely. Although E. chaffeensis and A. phagocytophilum seroreactivities have previously been reported in humans in Thailand [7; Miller et al., Program Abstr. 49th Annu. Meet. Am. Soc. Trop. Med. Hyg., Am. J. Trop. Med. Hyg. 62(Suppl. 3):469-470, 2000], we failed to detect these known agents of human ehrlichioses in that country. However, two Ehrlichia spp. were detected in ticks from Vietnam. Ehrlichia sp. strain EHh317 was detected in H. hystricis ticks collected from wild pigs and clustered with Ehrlichia sp. strain EBm52 and Ehrlichia sp. strain Tibet. The zoonotic potentials of these entities remain undescribed, but because H. hystricis ticks are known to feed on humans, human exposure to Ehrlichia sp. strain EHh317 might confound epidemiological surveys for evidence of infection with known Ehrlichia spp. Serological cross-reactivity among Ehrlichia spp. is well known, and it may be that human exposure to EHh317-like agents may give rise to a response detectable with E. chaffeensis antigen. This is also particularly applicable to Ehrlichia sp. strain EHh324: it was detected in another specimen of H. hystricis ticks from Vietnam and is closely related to E. chaffeensis, the agent of cases of human monocytic ehrlichioses occurring in the United States. Indeed, the 16S rrna gene sequence of Ehrlichia sp. strain EHh324 was shown to have 99.4% similarity with that of E. chaffeensis, and the strain was shown to cluster with E. chaffeensis in our phylogenetic tree. Thus, Ehrlichia sp. strain EHh324 could be a strain of E. chaffeensis. Although human monocytic ehrlichiosis has not yet been described in Asia, our work and that of others (1, 22) suggest the potential for its existence, particularly where ticks such as H. hystricis (which feed on peridomestic animals as well as humans) are common. Two Rickettsia spp. have been detected in this work, includ-

VOL. 41, 2003 TICK-ASSOCIATED BACTERIA IN THAILAND AND VIETNAM 1607 ing Rickettsia sp. strain RDa420 and Rickettsia sp. strain RDla440, from D. auratus ticks and pools of Dermacentor larvae from Sangkhlaburi, Thailand, respectively. Rickettsia sp. strain RDla440 was found to be closely related to Rickettsia sp. strain DnS14 and Rickettsia sp. strain RpA4S14, which are within the SFG rickettsiae. These rickettsiae of unknown pathogenicity have only recently been detected in Russia from several species of the genus Dermacentor and from Rhipicephalus pumilio ticks (21, 23). On the other hand, Rickettsia sp. strain RDa420 was found to be very different from all the known SFG rickettsiae: it was not possible to amplify DNA by PCR with primers CS1d and CS890r, which are known to amplify most of the SFG rickettsiae with only some exceptions (for example, R. akari and R. australis). The strain most closely related to RDa420 was found to be R. bellii, a rickettsia of unknown pathogenicity which is no longer considered to belong to the SFG of the genus Rickettsia, although its taxonomic position is disputed (19). This is the first description of these rickettsiae, and therefore, their epidemiological importance has yet to be determined; but both were detected in the tick species that readily bite humans. Finally, two bacteria including Eubacterium sp. strain Hw124 and Eubacterium sp. strain Hw191 were detected in H. wellingtoni nymphs collected from chickens in Thailand. They may represent novel bacteria. Clearly, the specificity of our broad-spectrum primers for ehrlichia-like bacteria (primers EHR16SR and EHR16SD) is not absolute, although they were designed to amplify a 345-bp fragment of the 16S rrna gene specific for the members of the family Anaplasmataceae and have successfully been used as epidemiologic tools in Africa and Japan (8, 15, 17). Our first hypothesis regarding these sequences was to consider them nonspecific amplification products as a result of a PCR or sequencing error. However, the sequences are related to those previously deposited in GenBank as an endosymbiont of Acanthamoeba sp. (6) and Eubacterium ZI-8 (the sequence available in GenBank is from an unpublished work), respectively. Furthermore, we have recently analyzed the sequences of PCR products obtained with primers EHR16SR and EHR16SD from ticks collected in the United States and Italy, and new sequences that clustered together in a clade including Eubacterium sp. strain Hw124 and Eubacterium sp. strain Hw191 were obtained (Y. O. Sanogo, unpublished). Thus, we hypothesize that the microorganism from which the sequences originated might represent a new group of bacteria associated with ticks. Further studies are needed, however, and in particular, isolation and polyphasic characterization of these bacteria would be required for rigorous testing of this hypothesis. In conclusion, PCR assays and sequence analysis of PCR products have enabled us to provide further information on the epidemiology of tick-associated bacteria in Thailand and Vietnam, where little information on the subject exists. Bacteria closely related to animal or human pathogens as well as bacteria of unknown pathogenicities have been detected in this work. However, DNA detection does not imply a transmission competence for the tick vectors concerned, because they could have been removed from bacteremic animals. Also, because of the limited study period and the seasonal variations in tick activity, other tick species could be prevalent during the other half of the year. It would be of particular interest to determine whether R. sanguineus, Amblyomma testudinarium, and Ixodes spp. are also prevalent in Sangkhlaburi, Thailand. Indeed, two rickettsiae (including the pathogen R. honei) are known to be associated with Ixodes granulatus ticks in another location in Thailand (10). A new rickettsia has also recently been detected in A. testudinarium ticks from central Thailand (J.-P. Gonzalez, unpublished data). These findings should stimulate further investigations of the epidemiology of SFG rickettsioses and ehrlichioses in that part of the world. ACKNOWLEDGMENTS We are grateful to Philip McDaniel for support and to the AFRIMS Fever Study Team for technical assistance. This work was supported by the U.S. Department of Defense Global Emerging Infections Surveillance Program (DoD-GEIS) and NIH grant AI 39002. This work is a part of the postdoctoral research project of Philippe Parola, who had been supported during different periods by Fondation Bayer Santé, Ministère Français des Affaires Etrangères (Programme Lavoisier), Fondation pour la Recherche Médicale, Assistance Publique- Hôpitaux de Marseille, Institut de Recherche International Servier, Association des Professeurs de Pathologie Infectieuses et Tropicales, and the European Society of Clinical Microbiology and Infectious Diseases. The views of the authors do not purport to reflect the position of the U.S. Army or the U.S. Department of Defense. The funding agencies take no responsibility for the data and the views expressed in this article. REFERENCES 1. Cao, W. C., Y. M. Gao, P. H. Zhang, X. T. Zhang, Q. H. Dai, J. S. Dumler, L. Q. Fang, and H. Yang. 2000. Identification of Ehrlichia chaffeensis by nested PCR in ticks from Southern China. J. Clin. Microbiol. 38:2778 2780. 2. Dumler, J. S., A. F. Barbet, C. P. Bekker, G. A. Dasch, G. H. Palmer, S. C. Ray, Y. Rikihisa, and F. R. Rurangirwa. 2001. 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. 51:2145 2165. 3. Dumler, J. S., and D. Walker. 2001. Human ehrlichioses. Lancet Infect. Dis. 2001:21 28. 4. Fournier, P. E., F. Grunnenberger, B. Jaulhac, G. Gastinger, and D. Raoult. 2000. Evidence of Rickettsia helvetica infection in humans, eastern France. Emerg. Infect. Dis. 6:389 392. 5. Fournier, P. E., H. Tissot-Dupont, H. Gallais, and D. R. Raoult. 2000. Rickettsia mongolotimonae: a rare pathogen in France. Emerg. Infect. Dis. 6:290 292. 6. Fritsche, T. R., M. Horn, S. Seyedirashti, R. K. Gautom, K. H. Schleifer, and M. Wagner. 1999. In situ detection of novel bacterial endosymbionts of Acanthamoeba spp. phylogenetically related to members of the order Rickettsiales. Appl. Environ. Microbiol. 65:206 212. 7. Heppner, D. G., C. Wongsrichanalai, D. S. Walsh, P. McDaniel, C. Eamsila, B. Hanson, and H. Paxton. 1997. Human ehrlichiosis in Thailand. Lancet 350:785 786. 8. Inokuma, H., P. Parola, D. Raoult, and P. Brouqui. 2001. Molecular survey of Ehrlichia infection in ticks from animals in Yamagushi Prefecture, Japan. Vet. Parasitol. 99:335 339. 9. Inokuma, H., D. Raoult, and P. Brouqui. 2000. Detection of Ehrlichia platys DNA in brown dog ticks (Rhipicephalus sanguineus) in Okinawa Island, Japan. J. Clin. Microbiol. 38:4219 4221. 10. Kollars, T. M., Jr., B. Tippayachai, and D. Bodhidatta. 2001. Short report: Thai tick typhus, Rickettsia honei, and a unique Rickettsia detected in Ixodes granulatus (Ixodidae: Acari) from Thailand. Am. J. Trop. Med. Hyg. 65:535 537. 11. Kolonin, G. V. 1995. Review of the Ixodid tick fauna (Acari: Ixodidae) of Vietnam. J. Med. Entomol. 32:276 282. 12. Kumar, S., K. Tamura, I. B. Jacobsen, and M. Nei. 2001. MEGA 2: molecular evolutionary genetics analysis software. Arizona State University, Tempe. 13. Kuttler, K. L. 1984. Anaplasma infections in wild and domestic ruminants: a review. J. Wildl. Dis. 20:12 20.

1608 PAROLA ET AL. J. CLIN. MICROBIOL. 14. Parola, P., L. Beati, M. Cambon, and D. Raoult. 1998. First isolation of Rickettsia helvetica from Ixodes ricinus ticks in France. Eur. J. Clin. Microbiol. Infect. Dis. 17:95 100. 15. Parola, P., H. Inokuma, J. L. Camicas, P. Brouqui, and D. Raoult. 2001. Detection and identification of spotted fever group rickettsiae and ehrlichiae in African ticks. Emerg. Infect. Dis. 7:1014 1017. 16. Parola, P., and D. Raoult. 2001. Ticks and tick-borne bacterial human diseases, an emerging infectious threat. Clin. Infect. Dis. 32:897 928. (Erratum, 33:749.) 17. Parola, P., V. Roux, J. L. Camicas, I. Baradji, P. Brouqui, and D. Raoult. 2000. Detection of ehrlichiae in African ticks by PCR. Trans. R. Soc. Trop. Med. Hyg. 94:707 708. 18. Raoult, D., P. E. Fournier, P. Abboud, and F. Caron. 2002. First documented human Rickettsia aeschlimannii infection. Emerg. Infect. Dis. 8:748 749. 19. Raoult, D., and V. Roux. 1997. Rickettsioses as paradigms of new or emerging infectious diseases. Clin. Microbiol. Rev. 10:694 719. 20. Roux, V., E. Rydkina, M. Eremeeva, and D. Raoult. 1997. Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae. Int. J. Syst. Bacteriol. 47:252 261. 21. Rydkina, E., V. Roux, N. Rudakov, M. Gafarova, I. Tarasevich, and D. Raoult. 1999. New rickettsiae in ticks collected in territories of the former Soviet Union. Emerg. Infect. Dis. 5:811 814. 22. Shibata, S., M. Kawahara, Y. Rikihisa, H. Fujita, Y. Watanabe, C. Suto, and T. Ito. 2000. New Ehrlichia species closely related to Ehrlichia chaffeensis isolated from Ixodes ovatus ticks in Japan. J. Clin. Microbiol. 38:1331 1338. 23. Shpynov, S., P. Parola, N. Rudakov, I. Samoilenko, M. Tankibaev, I. Tarasevich, and D. Raoult. 2001. Detection and identification of spotted fever group rickettsiae in Dermacentor ticks from Russia and Central Kazakhstan. Eur. J. Clin. Microbiol. Infect. Dis. 20:903 905. 24. Sirisanthana, T., V. Pinyopornpanit, V. Sirisanthana, D. Strickman, D. J. Kelly, and G. A. Dasch. 1994. First cases of spotted fever group rickettsiosis in Thailand. Am. J. Trop. Med. Hyg. 50:682 686. 25. Sparagano, O. A., M. T. Allsopp, R. A. Mank, S. G. Rijpkema, J. V. Figueroa, and F. Jongejan. 1999. Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): a review. Exp. Appl. Acarol. 23:929 960. 26. Suksawat, J., C. Pitulle, C. Arraga-Alvarado, K. Madrigal, S. I. Hancock, and E. B. Breitschwerdt. 2001. Coinfection with three Ehrlichia species in dogs from Thailand and Venezuela with emphasis on consideration of 16S ribosomal DNA secondary structure. J. Clin. Microbiol. 39:90 93. 27. Tanskul, P., H. E. Stark, and I. Inlao. 1983. A checklist of ticks of Thailand (Acari: Metastigmata: Ixodoidea). J. Med. Entomol. 20:330 341. 28. Thompson, J. C., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting position, specific gap penalties, and weight matrix choice. Nucleic Acids Res. 22:4673 4680. 29. Wen, B., R. Jian, Y. Zhang, and R. Chen. 2002. Simultaneous detection of Anaplasma marginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis by sequence analyses of 16S ribosomal DNA in Boophilus microplus ticks from Tibet. J. Clin. Microbiol. 40:3286 3290. 30. Zhang, J. Z., M. Y. Fan, Y. M. Wu, P. E. Fournier, V. Roux, and D. Raoult. 2000. Genetic classification of Rickettsia heilongjiangii and Rickettsia hulinii, two Chinese spotted fever group rickettsiae. J. Clin. Microbiol. 38: 3498 3501. Downloaded from http://jcm.asm.org/ on August 31, 2018 by guest