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

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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 Ose´e 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,1and Institut de Recherche pour le De´veloppement, UR34, Mahidol University at Salaya, Nakorn Prathom,4Thailand; Unite´ des

Rickettsies, CNRS UMR 6020, Faculte´ de Me´decine, Marseille, France2; Laboratory of Public Health Entomology, Harvard School of Public Health, Boston, Massachusetts3; and Bao Loc

General Hospital, Bao Loc, Lam Dong Province, Vietnam5

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 orderRickettsiales. ThreeAnaplasmaspp. were detected in ticks collected in

Thailand, including (i)Anaplasmasp. strain AnDa465, which was considered a genotype ofAnaplasma platys

(formerlyEhrlichia platys) and which was obtained fromDermacentor auratusticks collected from dogs; (ii)

Anaplasmasp. strain AnAj360, which was obtained fromAmblyomma javanenseticks collected on a pangolin;

and (iii)Anaplasmasp. strain AnHl446, which was closely related toAnaplasma bovisand 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 microplusticks collected from cattle from

Thailand; (ii) Ehrlichiasp. strain EHh324, which was closely related toEhrlichia chaffeensisand which was

detected in Haemaphysalis hystricis ticks collected from wild pigs in Vietnam; and (iii) Ehrlichiasp. strain

EHh317, which was closely related toEhrlichiasp. strain EBm52 and which was also detected inH. hystricis

ticks collected from wild pigs in Vietnam. TwoRickettsiaspp. were detected in Thailand, including (i)Rickettsia

sp. strain RDla420, which was detected inDermacentor auratusticks collected from a bear, and (ii)Rickettsia

sp. strain RDla440, which was identified from two pools ofDermacentorlarvae collected from a wild pig nest.

Finally, two bacteria namedEubacteriumsp. strain Hw124 andEubacteriumsp. strain Hw191 were identified

inHaemaphysalis wellingtoniticks 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 belong-ing to the orderRickettsiales. They are now recognized as im-portant 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, includ-ingRickettsia rickettsii(in the Americas),Rickettsia sibirica(in Asia),Rickettsia conoriiincluding 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), andRickettsia slovaca(in Europe) (16, 19). Further-more, Astrakhan fever rickettsia and Israeli tick typhus rick-ettsia, both of which are closely related toR. conorii, are known as agents of rickettsioses in Astrakhan and Israel, respectively (19). Finally, human infections due toRickettsia aeschlimannii

in Africa (18), Rickettsia helvetica and “Rickettsia mongoloti-monae” in Europe (4, 5), and “Rickettsia heilongjiangii” in Asia (30) have recently been reported.

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 ehr-lichioses have been reported since 1991. They include (i) hu-man monocytic ehrlichiosis due toEhrlichia chaffeensisin 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 granulo-cytic ehrlichia orEhrlichia 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 honeiand its strain, Thai tick typhusRickettsiastrain 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 threeEhrlichiaspecies has also

* Corresponding author. Mailing address: Unite´ des Rickettsies, CNRS UMR 6020, WHO Collaborative Center for Rickettsial Refer-ence and Research, Faculte´ de Me´decine, 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.

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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 ehrli-chioses 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, Kan-chanaburi 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 tick-borne pathogens in arthropods including ticks (25). Accord-ingly, 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 Interna-tional 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 Octo-ber 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 dis-carded 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 de-scribed 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 fromRickettsia montanensis) were included in each test. To amplify the main part of thegltAgene, 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 ofRickettsia bellii, which was the closest to that of Dal420, was de-signed.

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 Anaplas-mataceae, including the generaAnaplasma,Ehrlichia,Neorickettsia, and Wolba-chia(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 andAnaplasma phagocytophilumDNA 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 transillu-mination. A DNA molecular weight marker (marker VI; Boehringer Mannheim, Mannheim, Germany) was used to estimate the sizes of the products. 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).

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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 dideoxy-nucleotide 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 familyAnaplasmataceaeused for phylogenetic studies are available in GenBank under the following accession numbers: AF303467 forAnaplasma platys(formerlyEhrlichia platys), AF286699 forA. platysdetected in Thailand, M73224 forA. phagocytophilum(formerlyEhrlichia phagocytophila), U03775 for Anaplasma bovis(formerlyEhrlichia bovis), AF283007 for Ana-plasma centrale, M60313 forAnaplasma marginale, AF318945 forAnaplasma ovis, AF069758 forEhrlichia ruminatium, M73222 forE. chaffeensis, U15527 for

Ehrlichia muris, M73227 forE. ewingii, M73221 forE. canis, AF311967 for

Ehrlichiasp. strain ERm58, AF311968 forEhrlichiasp. strain EHt224; AF414399 forEhrlichiasp. strain Tibet; AF179630 forWolbachia pipientis; U12457 for

Neorickettsia helminthoeca; M73225 forNeorickettsia sennetsu(formerlyEhrlichia sennetsu); M21290 forNeorickettsia risticii(formerlyE. sennetsu), U11021 for

R. rickettsii; and M21789 forRickettsia 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 forAnaplasmasp. strain AnDa465; AF497580 forAnaplasmasp. strain AnAj360; AF497579 for

Anaplasma sp. strain AnHl446; AF497581 for Ehrlichia sp. strain EBm52; AF497578 forEhrlichiasp. strain EHh324; AF497577 forEhrlichiasp. strain EHh317; AF497583 forEubacteriumsp. strain Hw124, and AF497582 for Eu-bacteriumsp. 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. conoriiSeven, U59730;R. rickettsii, U59729; Astrakhan fever rick-ettsia, U59728; Israeli tick typhus rickrick-ettsia, U59727; R. honei strain RB, AF018074; Thai tick typhus rickettsia, U59726;R. slovaca, U59725;R. japonica, U59724;Rickettsia rhipicephali, U59721;R. montanensisU74756;Rickettsia mas-siliae, U59719; Bar29, U59720;R. aeschlimannii, U59722;R. helvetica, U59723;

Rickettsiasp. strain IRS4, AF141906;Rickettsiasp. 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;Rickettsiasp. strain RpA4, AF120029;Rickettsiasp. strain DnS28, AF120027;Rickettsiasp. strain RDa420, AF497584; andRickettsia

sp. strain RDla440, AF497585. The nucleotide sequences of thegltAgenes found in this study have been deposited in GenBank under the following accession numbers: AF497584 forRickettsiasp. strain RDa420 and AF497585 forRickettsia

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 gen-era 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 familyAnaplasmataceae

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

frag-ments of the 16S rRNA gene were obtained from each of the 45 positive samples.

Three 16S rRNA sequences ofAnaplasmaspp. were identi-fied (Fig. 1). They included (i) AnDa465 (1,016 bp), which was obtained from 3 of 20 (15%; 95% CI, 0 to 30.6)Dermacentor auratusnymphs collected from dogs in Thailand and which was closely related (99.3% similarity) to the sequence ofA. platys

(formerly namedE. 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 withA. platysandA. bovis(formerlyE. 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 withA. bovis, 97.9% similarity withA. phago-cytophilum, and 96.5% similarity withA. platys.

Three 16S rRNA sequences ofEhrlichiaspp. were identified (Fig. 1). They included (i) EBm52 (1,380 bp), which was ob-tained from 24 of 109 (22%; 95% CI, 14.2 to 29.8%)Boophilus microplusticks collected from cattle from Thailand and which was shown to be closely related (99.9% similarity) to a se-quence identified inB. microplusticks collected from cattle in Tibet (GenBank accession number AF414399) and also to be closely related (99.6% similarity) to two ehrlichial DNA se-quences detected in cattle ticks from Africa (Erm58 and Eht224; GenBank accession numbers AF311967 and AF311968, re-spectively); (ii) EHh324 (902 bp), which was detected in 1 of 19 (5.3%; 95% CI, 0 to 15.3%)Haemaphysalis hystricisticks col-lected from wild pigs in Vietnam and which was closely related toE. chaffeensis(99.4% similarity); and (iii) EHh317 (902 bp), which was also detected in 1 of 19H. hystricisticks from Viet-namese 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 wellingtoninymphs 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 Acanthamoebasp.” (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 align-ments of 960 bp of the 16S rRNA genes, AnDa465 clustered withA. platyssequences. AnHl446 and AnAj360 were placed within theAnaplasmaclade as well. The bacteria from which these sequences originated were temporarily calledAnaplasma

sp. strain AnDa465,Anaplasmasp. strain AnHl446, and Ana-plasmasp. strain AnAj360, respectively. EBm52, EHh324, and EHh317 were shown to belong to theEhrlichiaclade; and the bacteria from which these sequences originated were tempo-rarily called Ehrlichia sp. strain EBm52, Ehrlichia sp. strain EHh324, andEhrlichia sp. strain EHh317, respectively. Fur-thermore, this analysis suggested that strains Hw124 and Hw191 may originate from a new group of bacteria. The

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ganisms from which these sequences originated were tempo-rarily calledEubacteriumsp. strain Hw124 andEubacteriumsp. strain Hw191, respectively.

PCR detection of bacteria of the genusRickettsiaand

anal-yses of gltAgene sequences. DNA of Rickettsiaspp. was

de-tected 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. auratusticks collected from a bear. It was found to be different from all the known Rickettsia sp. se-quences deposited in GenBank. The most closely related rick-ettsia wasR. bellii(94.7% similarity); other rickettsiae were less than 91% similar. RDla440 (1,108 bp) was detected in two pools of 30Dermacentorlarvae collected from a wild pig nest. Its sequence appeared to be most closely related to the

se-quences ofRickettsiasp. strain DnS14 andRickettsiasp. 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 thegltAgenes of the rickettsiae, RDa420 formed a clade with

R. belliiand Dla440 clustered withRickettsiasp. strain DnS14,

Rickettsiasp. strain DnS28, andRickettsiasp. strain RpA4 (Fig. 3). The rickettsiae from which the gltA sequences originated were temporarily calledRickettsiasp. strain RDa420 and Rick-ettsiasp. strain Dla440, respectively.

DISCUSSION

Although the diverse species that comprise the 606 ticks that we collected in the Sangkhlaburi District had previously been

TABLE 1. Characteristics of ticks collecteda

Tick Origin No. of ticks Host(s)

Male Female Nymphs Larvae Total

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)

Dermacentorsp. 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)

aTicks were collected from September 2001 to March 2002 in the central part of the Thai-Myanmar border in Sangkhlaburi District (15°09N latitude and 98°27E

longitude), Kanchanaburi Province, Thailand, and in Bao Loc (11°30⬘N latitude and 107°46⬘E longitude), Lam Dong Province, Vietnam.

bNewly reported tick-host association. cNew tick-host association reported in Thailand.

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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

FIG. 2. Phylogenetic tree based on studies of 960 sites of the 16S RNA genes of bacteria of the generaAnaplasma,Ehrlichia,Neorickettsia, and

Wolbachiaand 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.

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forR. sanguineusticks, 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 impor-tance, becauseR. sanguineusis known as the main vector ofR. 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. wellingtoniin

Thailand (27). However, the ticks that mainly bit humans at our study site during the period of observation were Derma-centor 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 speci-mens 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-FIG. 3. Phylogenetic tree based on studies of 1,035 sites of the citrate synthase genes of bacteria of the genusRickettsia, drawn as described in the legend to Fig. 2. The bacteria detected in this work are highlighted.

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er ticks collected during our study period are also known to bite humans, includingDermacentor atrosignatus,Dermacentor steini, Dermacentor compactus, H. hystricis, and H. lagrangei

(11, 27). Thus, all these ticks hold the first component neces-sary to be involved in human diseases: an affinity to bite hu-mans.

The 16S rRNA sequences of theAnaplasmaspp. and Ehr-lichia 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 conclu-sion. 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 familyAnaplasmataceae, 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% diver-gence 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 identifiedAnaplasmasp. strain AnDa465 in

D. auratusticks from Thailand. According to the level of 16 sRNA gene sequence similarity (99.3%) and to our phyloge-netic analysis,Anaplasmasp. strain AnDa465 appeared to be closely related toA. 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 se-quences of strains of the same species as well. We have recently detectedA. platysisolates from a dog and a tick in the Dem-ocratic Republic of Congo, and although the 16S rRNA gene sequences of the isolates showed some differences from the

A. platyssequences deposited in GenBank, additional analyses based ongroESLandgltAgene phylogenies suggest that they are a strain ofA. 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 theA. platys

amplification products could have been feeding on bacteremic dogs.

Two moreAnaplasmaspp. were detected in this work. Ana-plasmasp. strain AnAj360 was detected fromA. javanenseticks from a pangolin. Two specimens of this animal were screened for ticks, but only ticks collected from one of them were pos-itive. The sequence of this Anaplasma strain presented less than 98% similarity with those of otherAnaplasmaspp., and it could represent a new species. On the other hand,Anaplasma

sp. strain AnHl446 was detected fromH. lagrangeiticks from a bear. It appeared to be closely related toA. bovisaccording to the level of 16S rRNA gene sequence similarity (99.6%) and phylogenetic analysis; in the phylogenetic tree, it is grouped

withA. boviswith 100 as a bootstrap value. Thus,Anaplasma

sp. strain AnHl446 could be a strain ofA. bovis, although as stated above, this cannot be definitely assumed.

Ehrlichiasp. strain EBm52 was obtained fromB. microplus

ticks collected from cattle. This tick species is well known as the vector ofA. marginale, an intraerythrocytic pathogen that causes bovine anaplasmosis (13). This agent is distributed worldwide, and it was recently detected in B. microplusticks collected from cattle in Myanmar (P. Parola, unpublished data). However,Ehrlichiasp. strain EBm52 was shown to be closely related to Ehrlichiasp. 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 genusEhrlichia, based on phylogenetic anal-yses of the 16S rRNA gene (29). Furthermore, bothEhrlichia

sp. strain Tibet andEhrlichiasp. strain EBm52 were shown to be closely related to two ehrlichiae which we detected in Af-rican ticks, includingRhipicephalus muhsamaeticks from Mali (Ehrlichia sp. strain Erm58) and Hyalomma truncatum ticks from Niger (Ehrlichia sp. strain Eht224) (15). The gene se-quences of all four of these ehrlichiae were detected in ticks removed from cattle. Given the high degree of genetic simi-larity, these organisms may represent strains of the same spe-cies, all of which are associated with cattle. Their zoonotic or veterinary potential remains to be described. However,B. mi-croplus rarely bites people (if ever), and the transmission of

Ehrlichiasp. strain EBm52 to humans byB. microplusis un-likely.

AlthoughE. chaffeensisandA. phagocytophilum seroreactivi-ties 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, twoEhrlichiaspp. were detected in ticks from Vietnam.Ehrlichiasp. strain EHh317 was detected inH. hystricisticks 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 becauseH. hystricisticks are known to feed on humans, human exposure to Ehrlichiasp. strain EHh317 might confound epidemiological surveys for evidence of infec-tion with known Ehrlichia spp. Serological cross-reactivity amongEhrlichiaspp. is well known, and it may be that human exposure to EHh317-like agents may give rise to a response detectable withE. chaffeensisantigen. This is also particularly applicable to Ehrlichiasp. strain EHh324: it was detected in another specimen ofH. hystricis ticks from Vietnam and is closely related toE. chaffeensis, the agent of cases of human monocytic ehrlichioses occurring in the United States. In-deed, the 16S rRNA gene sequence of Ehrlichiasp. strain EHh324 was shown to have 99.4% similarity with that ofE. chaffeensis, and the strain was shown to cluster with E. chaffeensis in our phylogenetic tree. Thus, Ehrlichia sp. strain EHh324 could be a strain ofE. chaffeensis. Although human monocytic ehrlichiosis has not yet been described in Asia, our work and that of others (1, 22) suggest the poten-tial for its existence, particularly where ticks such asH. hys-tricis (which feed on peridomestic animals as well as hu-mans) are common.

TwoRickettsiaspp. have been detected in this work,

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ing Rickettsia sp. strain RDa420 and Rickettsia sp. strain RDla440, fromD. auratusticks and pools ofDermacentor lar-vae from Sangkhlaburi, Thailand, respectively. Rickettsia sp. strain RDla440 was found to be closely related toRickettsiasp. 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 genusDermacentorand from Rhipiceph-alus pumilio ticks (21, 23). On the other hand,Rickettsiasp. 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. akariandR. 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 be-long to the SFG of the genusRickettsia, 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 includingEubacteriumsp. strain Hw124 andEubacteriumsp. strain Hw191 were detected inH. well-ingtoni 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 Anaplasmataceaeand 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 ofAcanthamoebasp.” (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 ob-tained with primers EHR16SR and EHR16SD from ticks col-lected in the United States and Italy, and new sequences that clustered together in a clade includingEubacteriumsp. strain Hw124 andEubacteriumsp. strain Hw191 were obtained (Y. O. Sanogo, unpublished). Thus, we hypothesize that the microor-ganism 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 rigor-ous 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 Viet-nam, where little information on the subject exists. Bacteria closely related to animal or human pathogens as well as bac-teria 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

whetherR. sanguineus,Amblyomma testudinarium, andIxodes

spp. are also prevalent in Sangkhlaburi, Thailand. Indeed, two rickettsiae (including the pathogenR. honei) are known to be associated with Ixodes granulatusticks in another location in Thailand (10). A new rickettsia has also recently been detected inA. testudinariumticks from central Thailand (J.-P. Gonzalez, unpublished data). These findings should stimulate further in-vestigations of the epidemiology of SFG rickettsioses and ehr-lichioses 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 Sante´, Ministe`re Franc¸ais des Affaires Etrange`res (Programme Lavoisier), Fondation pour la Recherche Me´dicale, As-sistance Publique- Hoˆpitaux de Marseille, Institut de Recherche International Servier, Association des Professeurs de Pathologie In-fectieuses et Tropicales, and the European Society of Clinical Micro-biology 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.

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Figure

FIG. 1. Map showing the locations where ticks were collected in Thailand and Vietnam and the results of the molecular detection of rickettsialDNA (in the following order: bacteria/tick species/number of ticks tested/number of positive ticks/host).
FIG. 1. Map showing the locations where ticks were collected in Thailand and Vietnam and the results of the molecular detection of rickettsialDNA (in the following order: bacteria/tick species/number of ticks tested/number of positive ticks/host). p.2
TABLE 1. Characteristics of ticks collecteda

TABLE 1.

Characteristics of ticks collecteda p.4
FIG. 2. Phylogenetic tree based on studies of 960 sites of the 16S RNA genes of bacteria of the genera AnaplasmaWolbachiaobtained by the neighbor-joining method
FIG. 2. Phylogenetic tree based on studies of 960 sites of the 16S RNA genes of bacteria of the genera AnaplasmaWolbachiaobtained by the neighbor-joining method p.5
FIG. 3. Phylogenetic tree based on studies of 1,035 sites of the citrate synthase genes of bacteria of the genus Rickettsia, drawn as describedin the legend to Fig
FIG. 3. Phylogenetic tree based on studies of 1,035 sites of the citrate synthase genes of bacteria of the genus Rickettsia, drawn as describedin the legend to Fig p.6

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