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ARTICLE IN PRESSG ModelTBDIS-337; No. of Pages 9

Ticks and Tick-borne Diseases xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases

j ourna l h o me page: w ww.elsev ier .com/ locate / t tbd is

riginal article

olecular detection of Rickettsia species in Amblyomma ticks collectedrom snakes in Thailand

halao Sumrandeea, Supanee Hirunkanokpunc, Kathryn Doornbosa,angvorn Kitthaweea, Visut Baimaia,b, Libor Grubhofferd,achareeporn Trinachartvanita, Arunee Ahantariga,b,∗

Biodiversity Research Cluster, Department of Biology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, ThailandCenter of Excellence for Vectors and Vector-Borne Diseases, Faculty of Science, Mahidol University at Salaya, Phutthamonthon 4 Road,akhon Pathom 73170, ThailandDepartment of Biology, Faculty of Science, Ramkhamhaeng University, Ramkhamhaeng Road, Bangkok 10240, ThailandInstitute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic & Faculty of Science, University of South Bohemia,ranisovská 31, CZ-37005 Ceské Budejovice, Czech Republic

r t i c l e i n f o

rticle history:eceived 26 October 2013eceived in revised form 4 April 2014ccepted 19 April 2014vailable online xxx

eywords:ickickettsia spp.mblyomma varanensemblyomma helvolum

a b s t r a c t

Some reptile ticks are potential vectors of pathogens such as spotted fever group (SFG) rickettsiae. Here,we report for the first time in detail the molecular evidence, DNA sequences and phylogenetic studies,for the presence of Rickettsia spp. in Amblyomma ticks (Amblyomma helvolum and Amblyomma varanense)from snakes in Thailand. A total of 24 tick samples was collected from 4 snake species and identified. Aphylogenetic analysis inferred from the partial sequences of the gltA gene indicated that the Rickettsiaspp. from 2 Amblyomma helvolum and 1 Amblyomma varanense belong to the same group as the SFGrickettsiae, which are closely related to Rickettsia raoultii strains. In contrast, there was 1 Rickettsia sp.from Amblyomma helvolum grouped into the same clade with other SFG rickettsiae (Rickettsia tamurae,Rickettsia monacensis, and a Rickettsia endosymbiont of Amblyomma dubitatum from Brazil). However,another Rickettsia sp. from Amblyomma varanense was closely related to Rickettsia bellii and Rickettsia sp.

nakehailand

strain RDa420 from Thailand. In addition, from phylogenetic results based on the 16S rRNA gene and aconcatenated tree of the 3 genes (gltA, ompA, and ompB), we found what may be a novel SFG rickettsiaspecies closely related to Rickettsia raoultii (from both Amblyomma varanense and Amblyomma helvolum).In conclusion, our findings are the first report on the presence of novel SFG rickettsiae in 2 snake tickspecies, Amblyomma varanense and Amblyomma helvolum in Thailand and in south-eastern Asia.

ntroduction

Ticks are haematophagous arthropods second only toosquitoes as vectors of multiple disease agents. There are

pproximately 130 species of Amblyomma ticks in the worldGugielmone et al., 2010) 10 of which species have been reportedn Thailand: Amblyomma cordiferum, Amblyomma cyprium, Ambly-mma geomydae, Amblyomma helvolum, Amblyomma supinoi,

Please cite this article in press as: Sumrandee, C., et al., Molecular desnakes in Thailand. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1

mblyomma testudinarium, Amblyomma varanense Amblyommarassipes, Amblyomma pattoni, and Amblyomma trimaculatumTanskul et al., 1983; Petney and Keirans, 1995, 1996; Doornbos

∗ Corresponding author at: Department of Biology, Faculty of Science, Mahidolniversity, Rama 6 Road, Bangkok 10400, Thailand. Tel.: +66 2 201 5380;

ax: +66 2 354 7161.E-mail address: arunee.aha@mahidol.ac.th (A. Ahantarig).

ttp://dx.doi.org/10.1016/j.ttbdis.2014.04.013877-959X/© 2014 Elsevier GmbH. All rights reserved.

© 2014 Elsevier GmbH. All rights reserved.

et al., 2013; Sumrandee et al., 2014). The tick species A. helvolumis widely distributed throughout Thailand (Tanskul et al., 1983;Cornet et al., 2009; Doornbos et al., 2013). It has been collectedfrom various reptiles including Varanus bengalensis, Elaphe radiata,Python reticulatus, and Python sp. in Thailand (Tanskul et al., 1983).It has also been recorded on many other reptiles in south-easternAsia (Kohls, 1957; Keirans, 1985; Auffenberg, 1988; Kolonin,1995). Amblyomma varanense is a common tick of Asian reptiles.Its distribution extends from India to China, Indonesia (Kauffman,1972; Kolonin, 1995), and Thailand. The medical and veterinarysignificance of these tick species in the genus Amblyomma is poorlyunderstood.

Ticks can transmit a wide variety of pathogenic microor-

tection of Rickettsia species in Amblyomma ticks collected from016/j.ttbdis.2014.04.013

ganisms, such as viruses, bacteria, and protozoa, to vertebratehosts (Jongejan and Uilenberg, 2004). Among tick-borne diseases,spotted fever group (SFG) rickettsioses that are caused by obli-gate, intracellular Gram-negative bacteria of the genus Rickettsia

ARTICLE IN PRESSG ModelTTBDIS-337; No. of Pages 9

2 C. Sumrandee et al. / Ticks and Tick-borne Diseases xxx (2014) xxx–xxx

Table 1Oligonucleotide primers used for PCR amplification.

Primer name Gene targeted Species detected Nucleotide sequences (5′–3′) Size of amplifiedfragments (bp)

References

16S+116S−1

16S mitochondrial DNA Ticks CTGCTCAATGATTTTTTAAATTGCTGTGGCCGGTCTGAACTCAGATCAAGT

460 Black and Piesman (1994)

fD1 rP2 16S rDNA gene Eubacteria AGAGTTTGATCCTGGCTCAGACGGCTACCTTGTTAGGACTT

1484 Weisburg et al. (1989, 1991)Roux and Raoult (1995)

RpCS.877pRpCS.1258n

Citrate synthase (gltA) All Rickettsia GGGGGCCTGCTCACGGCGGATTGCAAAAAGTACAGTGAACA

381 Regnery et al. (1991)

Rr190.70p Outer membrane SFG Rickettsia ATGGCGAATATTTCTCCAAAATCGC

532 Regnery et al. (1991)

GAC GCGGTA

a(bAGhLr(hee(tmeaPmtr

SrRtastCm(Ippteasstpos

M

T

4

Rr190.602n protein (ompA) AGTGCAGCAT120-607

120-1497Outer membraneprotein (ompB)

SFG Rickettsia andtyphus group

AAT ATC GGTCCTATATCGC

re considered emerging diseases affecting humans worldwideRaoult and Roux, 1997). To date, at least 6 SFG rickettsiae haveeen classified as human pathogens and found in south-easternsia and Thailand (Kollars et al., 2001; Phongmany et al., 2006;aywee et al., 2007; Mediannikov and Tarasevich, 2008): Rickettsiaeilongjiangensis (Thailand, Laos), Rickettsia japonica (Thailand,aos), Rickettsia honei (Malaysia, Thailand, Laos), Rickettsia tamu-ae (Thailand, Laos), Rickettsia conorii, and Rickettsia helveticaThailand) (Parola et al., 2003a,b; Pickard et al., 2004). In Thailand, R.onei was first isolated from Rhipicephalus and Ixodes ticks (Kollarst al., 2001) and found to cause spotted fever in humans (Jiangt al., 2005). Rickettsia japonica was first reported in a human caseGaywee et al., 2007) and then isolated from Haemaphysalis shimogaicks (Takada et al., 2009). Many rickettsial species of undeter-

ined pathogenicity have been detected in ticks from Thailand,.g., Rickettsia sp. RDa420, Rickettsia sp. RDla440, Rickettsia sp. ATT,nd Rickettsia sp. HOT1 and HOT2 (Hirunkanokpun et al., 2003;arola et al., 2003a,b). The presence of these rickettsiae of undeter-ined pathogenicity suggests the potential medical importance of

icks, but the role of ticks as rickettsial pathogen vectors in Thailandemains largely unknown.

Some reptile ticks are potential vectors of pathogens includingFG rickettsiae and Coxiella burnetii. Amblyomma gervaisi has beeneported to be infected with Coxiella burnetii in India (Stephen andao, 1979). Amblyomma exornatum (formerly Aponomma exorna-um) collected from a captive-bred monitor lizard was infected byn SFG Rickettsia in the USA (Reeves et al., 2006). Amblyomma dis-imile ticks collected from iguanas in Colombia were also reportedo be infected with a novel SFG Rickettsia, Rickettsia sp. strainolombianensi (Miranda et al., 2012). Bothriocroton hydrosauri (for-erly Aponomma hydrosauri) is known to be a vector of R. honei

Flinders Island spotted fever) in Australia (Stenos et al., 2003).n Asia, only A. testudinarium was reported to be a vector of aathogenic Rickettsia, R. tamurae (Fournier et al., 2006). Of unknownathogenicity, the Rickettsia sp. strain ATT1 was detected from thisick species collected from vegetation in Thailand (Hirunkanokpunt al., 2003). A Rickettsia sp. closely related to Rickettsia raoultii inn A. helvolum has also been found (Doornbos et al., 2013). The tickpecies that predominantly feed on reptiles have been less welltudied in terms of medical importance. Here, we report for the firstime in detail the molecular evidence, details of DNA sequences andhylogenetic studies, for the presence of Rickettsia spp. in Ambly-mma ticks (A. helvolum and A. varanense) collected from 4 snakepecies in Thailand.

aterials and methods

Please cite this article in press as: Sumrandee, C., et al., Molecular desnakes in Thailand. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1

ick collection and identification

From May to July 2009, 24 adult ticks were removed from snake species from 3 different regions of Thailand (Table 2).

TCCCCCTGT CAA GGATT

890 Roux and Raoult (2000)

The snake hosts were immediately released at the collection sitesafter the ticks were removed. The valid taxonomic names of snakespecies followed David et al. (2004). Ticks were immediately storedin 70% alcohol and then transported to Mahidol University inBangkok for refrigeration at −20 ◦C. They were morphologicallyidentified using available standard taxonomic keys as described byRobinson (1926), Kohls (1957), Kauffman (1972), Hoogstraal andKim (1985), and Tanskul and Inlao (1989).

DNA extraction

The ticks were cleaned by sequential 5-min surface washes with70% ethanol, 10% sodium hypochlorite, and 3 washes with steriledistilled water. Then, the ticks were air-dried on sterile filter paperfor 3–5 min. A fine sterile scalpel blade was used to bilaterally bisecteach tick. The ticks were individually crushed using a sterile pes-tle, and the DNA was extracted using a DNA blood minikit (QiagenGmbH, Germany) following the manufacturer’s instructions.

Molecular analyses

The identification of ticks using morphological characteristicswas supplemented by PCR using a primer pair consisting of 16S+1and 16S−1 primers (Table 1) targeting the mitochondrial 16S rRNAgene of ticks (Black and Piesman, 1994).

The presence of Rickettsia in ticks was detected by PCR withprimers and standard PCR programmes (Table 1). Each PCR reactionwas carried out in a volume of 20 �l containing 11.2 �l of distilledwater, 2 �l of 10× buffer, 2 �l of 25 mM MgCl2, 0.5 �l of dNTPs(10 mM each), 1 �l of 10 �M forward and reverse primers, 1 unit ofTaq DNA polymerase, and 2 �l of DNA template. DNA amplificationswere carried out in a MJ MiniTM Personal Thermal Cycler modelPTC 1148 (BioRad, California, USA). For Rickettsia detection, multi-ple DNA targets were used to identify Rickettsia in tick samples,including the genes for citrate synthase (gltA), the outer mem-brane proteins A and B (ompA and ompB), and 16S rRNA (Table 1).Ticks that were found to be positive for the primers RpCS.877p andRpCS.1258n, which targeted the citrate synthase gene, as previ-ously described (Roux et al., 1997), were further assayed for thepresence of rickettsial ompA and ompB genes. A set of universalprimers (fD1and rP2) targeting the 16S rRNA gene of eubacteriawhich yielded an amplicon size of approximately 1484 bp in lengthwas performed.

The positive PCR products from tick samples were purified usingHigh Pure PCR Product Purification Kit (Roche) and cloned into thepGEM-T vector. DNA sequencing reactions were carried out usingthe M13 forward and reverse primers and performed with BigDye

tection of Rickettsia species in Amblyomma ticks collected from016/j.ttbdis.2014.04.013

Terminator v3.1 Cycle Sequencing Kits (Applied BioSystems, USA).The sequencing products were analysed using a Genetic Analyzer3730XL automated DNA sequencing system (Applied BioSystems)at Macrogen Inc., Seoul, South Korea.

IN PRESSG ModelT

ck-borne Diseases xxx (2014) xxx–xxx 3

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ARTICLETBDIS-337; No. of Pages 9

C. Sumrandee et al. / Ticks and Ti

olecular characterisation and phylogenetic analyses

Consensus sequences from the 3 amplicons of each geneere assembled by aligning and editing sequences usingLUSTALW programme (Thompson et al., 1994) and BIOEDITrogramme version 7.0.5.3 (Hall, 1999), respectively. The BLASTrogramme (http://www.ncbi.nlm.nih.gov) was used to deter-ine genetic relationships and the identities of sequences in

he GenBank database (Altschul et al., 1997). Multisequencelignments using CLUSTALW with excluded DNA gaps were con-tructed and used for phylogenetic tree analysis with MEGA5http://www.megasoftware.net/). For each gene analysed, aeighbour-joining distance method/algorithm (Saitou and Nei,987; Swofford et al., 1996) was conducted using a Kimura 2-arameter distance model. A bootstrap analysis used to estimatehe node reliability of the trees was conducted with 1000 replicatesFelsenstein, 1989) as implemented in MEGA5 (Tamura et al., 2011).

concatenated phylogenetic tree was constructed using availableequences of the gltA, ompA, and ompB genes by a neighbour-joiningethod for the total of 1636 bp.

esults

ick identification

A total of 24 ticks was collected from 4 snake species (Table 2).ollected ticks were semi- or fully engorged. Ticks collected from

king cobras were taxonomically identified as A. varanense (16emales and 3 males). Ticks collected from an Asiatic water snake3 female ticks), an Indochinese rat snake (1 female tick), and

Burmese python (1 female tick) were morphologically similarnd identified as belonging to the species A. helvolum (Table 2).n addition, a sequence analysis of amplified fragments of tick 16S

tDNA (451–454 bp) allowed us to identify the tick species as A.aranense and A. helvolum. The 16S mtDNA from 2 A. varanenserom king cobras and 3 A. helvolum ticks from 3 different snakepecies (see below) were amplified. The 454 bp of amplified DNArom A. varanense ticks were obtained. Sequence analysis revealedhat their sequences were identical and submitted to GenBanknder accession number KC170736. BLAST search exhibited thelosest matched to A. varanense (L34320) with 98.5% identity451/458 bp). Furthermore, the 451 bp of 16S mtDNA were ampli-ed from 3 A. helvolum. The 3 sequences were found to have

dentical gene sequences and were submitted to GenBank underccession numbers KC170738, KC170739, and KC170740 for the A.elvolum tick that was collected from Asiatic water snake, Burmeseython, and Indochinese rat snake, respectively. Sequence analysisf 16S mtDNA from 3 A. helvolum ticks showed the highest matcho Amblyomma latum with 90.2% identity (415/460 bp) and a

atch 88.3% and 88.0% to A. varanense (L34320) and A. variega-um (L34312), respectively. Although, there was no 16S mtDNAequence of A. helvolum available in GenBank, the morphologicaldentification of this tick was confirmed to be A. helvolum (Kohls,957).

ickettsia identification

We obtained all gene sequences for gltA, ompA, ompB, and 16SRNA from the same Rickettsia-infected A. varanense and A. helvolumicks that were collected from a king cobra and a Burmese python,espectively (Table 2). Although an A. helvolum tick collected fromn Indochinese rat snake was PCR-positive for both ompB and the

Please cite this article in press as: Sumrandee, C., et al., Molecular detection of Rickettsia species in Amblyomma ticks collected fromsnakes in Thailand. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1016/j.ttbdis.2014.04.013

6S rRNA genes, the amplified PCR product was so faint that weere unable to obtain DNA sequences from those genes.

Ticks were first examined for Rickettsia spp. using a primerair (RpCS.877p and RpCS.1258n). Among the 19 A. varanense ticks Ta

ble

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ARTICLE IN PRESSG ModelTTBDIS-337; No. of Pages 9

4 C. Sumrandee et al. / Ticks and Tick-borne Diseases xxx (2014) xxx–xxx

Fig. 1. Phylogenetic analysis of rickettsiae based on partial sequences of the gltA gene (341 bp). The tree was constructed by MEGA 5 software using the neighbour-joiningm re ind

e(

g

s3t1aiwIsoAoA

ethod with 1000 bootstrap resampling. The sequences obtained from this study a

xamined, 7 (37%) were positive for rickettsiae, whereas 4 out of 580%) of the A. helvolum ticks were Rickettsia-positive.

ltA

Seven A. varanense ticks were positive for gltA, and all DNAamples were sequenced. A sequence homology analysis of the41-bp fragment of the gltA gene revealed that 4 sequences ofhe gltA gene from A. varanense ticks were identical and showed00% identity (341/341 bp) to Rickettsia sp. RDa420 (AF497584)nd were closely related to Rickettsia bellii (U59716) with 96.18%dentity (328/341 bp). The sequence of this gltA-positive species

as submitted to GenBank as Rickettsia sp. clone APOH4 (Table 2).n contrast, another set of 3 sequences from this type of tickpecies were also identical (341/341 bp). They matched to those

Please cite this article in press as: Sumrandee, C., et al., Molecular desnakes in Thailand. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1

f published sequences of more than 24 species of SFG rickettsiae.dditionally, most of these sequences matched to different strainsf R. raoultii and have also been submitted as Rickettsia sp. clonePOH8 (Table 2).

icated in bold type.

Four out of 5 (80%) A. helvolum ticks were Rickettsia-positive(gltA), and all of them were sequenced. Two sequences from A.helvolum ticks collected from an Asiatic water snake and a sequencefrom a Burmese python were identical to each other. A sequenceanalysis revealed that their sequences were identical to severalspecies of SFG rickettsiae in particular to different strains of R. raoul-tii. Furthermore, a sequence from an A. helvolum tick collected froman Indochinese rat was 99.41% (339/341 bp) identical to 3 unnamedRickettsia of unknown pathogenicity: a Rickettsia endosymbiont ofAmblyomma dubitatum (JN676158) isolated in Brazil; an unculturedRickettsia sp. isolate 10610RCRICK (EF662058); and a Rickettsiasp. ‘midichlorii’ (AF214567) detected in Ixodes scapularis fromthe USA. In addition, 99.12% identity (338/341 bp) was deter-mined for R. monacensis (JX040640), which was detected fromIxodes ricinus in Hungary, and for R. tamurae (AF394896), which

tection of Rickettsia species in Amblyomma ticks collected from016/j.ttbdis.2014.04.013

was isolated from A. testudinarium in Japan (Fournier et al.,2006). A gltA sequence analysis revealed that the Rickettsia sp.clone APOH8 was identical to AmhXP1 and AmhPV1 but not toAmhPK1.

ARTICLE IN PRESSG ModelTTBDIS-337; No. of Pages 9

C. Sumrandee et al. / Ticks and Tick-borne Diseases xxx (2014) xxx–xxx 5

F NA gej study

o

tfwAo9tair

lagss(vRT

o

m

ig. 2. Phylogenetic analysis of rickettsiae based on partial sequences of the 16S rRoining method with 1000 bootstrap resampling. The sequences obtained from this

mpA

Among the 7 A. varanense ticks that were PCR-positive for gltA, 3icks were positive for ompA [we could not amplify the ompA generom 4 ticks that were positive for gltA (those 4 ticks were infectedith a R. bellii-like sp.)]. The sequences of the 3 A. varanense and 4. helvolum ticks positive for ompA gene were identical. An analysisf fragments of the ompA gene revealed that their sequences had7.33–97.54% identity to 16 different strains of R. raoultii, includinghe R. raoultii isolates LYG32 (JQ792137), Khabarovsk (DQ365801),nd DMS (JN398480). In addition, they showed 96.92% and 95.32%dentity to R. amblyommii (EF689733) and R. massiliae (HM014444),espectively.

A sequence of the ompA gene taken from an A. helvolum tick col-ected from an Indochinese rat snake (Rickettsia sp. clone AmPK1,ccession no. KC170761) had 98.57% identity with a homologousene fragment from R. massiliae strain AZT80 (CP003319), R. mas-iliae (HM014444), R. massiliae (HM050294), and R. rhipicephalitrain Do276 (EU109175), and 98.37% to R. aeschlimannii isolate WBJN944634). The ompA gene of Rickettsia sp. clone APOH8 (from an A.aranense) was identical to that of Rickettsia sp. clone AmhXP1 andickettsia sp. clone AmhPV1, but not to Rickettsia sp. clone AmPK1.his was the same trend as found for the gltA gene.

Please cite this article in press as: Sumrandee, C., et al., Molecular desnakes in Thailand. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1

mpB

An 841-bp sequence of the rickettsial ompB gene was deter-ined and analysed from 2 A. varanense and 2 A. helvolum ticks.

ne (1421 bp). The tree was constructed by MEGA 5 software using the neighbour- are indicated in bold type.

The ompB gene studied here was identical in all ticks examined(sequenced). An analysis of the ompB gene from these tick samplesrevealed more than 97% sequence identity to SFG rickettsiae, the R.massiliae strain AZT80 (DQ503428), R. rhipicephali (AF123719), andthe R. raoultii strain Khabarovsk (DQ365798).

16S rRNA

An analysis of 1421 bp of a 16S rRNA gene from 2 tick species[a sequence from an A. varanense tick collected from a king cobraand one from an A. helvolum tick from a Burmese python (Table 2)]demonstrated that they were more than 99% similar to the R. mas-siliae strain AZT80 (CP003319), the R. slovaca strain 13-B (L36224),the R. raoultii strain Khabarovsk (DQ365810), and the Rickettsiasp. strain TT-118 (L36220). The sequences of 16S rRNA genes ofrickettsiae from 2 tick species exhibited 99.44% (1413/1421 bp)similarity with only 8 bases difference. Also the Rickettsia sp.clone AmhPV1, accession no. KF368168, showed sequence sim-ilarity to the rickettsiae mentioned above and was more than99% identical to those of the 16S rRNA genes of JQ412124 andJQ412125.

Phylogenetic studies

tection of Rickettsia species in Amblyomma ticks collected from016/j.ttbdis.2014.04.013

The phylogenetic relationships between the rickettsiae thatwere detected in A. helvolum and A. varanense ticks were deter-mined by comparing their gltA, ompA, ompB, and 16S rRNAsequences using the neighbour-joining method. A phylogenetic

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Fig. 3. Phylogenetic analysis of rickettsiae based on concatenated sequences of genes using combined partial gltA, ompA, and ompB sequences (1636 bp). The tree wasc otstra

ai(ttgRbsR

fiw(gr

A(pttcaR

onstructed by MEGA 5 software using the neighbour-joining method with 1000 bo

nalysis inferred from the partial sequences of the gltA genendicated that the Rickettsia spp. APOH8 (JQ412116), AmhXP1JQ412118), and AmhPV1 (JQ412119) belong to the same group ashe members of SFG rickettsiae, which are closely related to R. raoul-ii strains (Fig. 1). Likewise, Rickettsia sp. AmhPK1 (JQ412120) wasrouped into the same clade with other SFG rickettsiae, R. tamurae,. monacensis (Merhej and Raoult, 2011), and a Rickettsia endosym-iont of A. dubitatum from Brazil (JN676158). However, Rickettsiap. APOH4 (JQ412117) was closely related to R. bellii (UU59716) andickettsia sp. strain RDa420.

A phylogenetic analysis of rickettsial 16S rRNA sequences ampli-ed from 2 tick species derived from 3 different host snake speciesas performed (Fig. 2). Based on this gene, Rickettsia sp. APOH8

JQ412124), AmhPV1 (JQ412125), and AmhXP1 (KF318168) wererouped together into a clade that was most closely related to R.aoultii.

Fig. 3 illustrates the phylogenetic tree of Rickettsia sp. APOH8,mhPV1, and AmhXP1 using concatenated sequences of the 3 genes

gltA, ompA, and ompB). The list of selected Rickettsia spp. used in thehylogenetic construction of Fig. 3 is displayed in Table 3. Similaro the gltA and 16S rRNA tree results, a concatenated tree revealed

Please cite this article in press as: Sumrandee, C., et al., Molecular desnakes in Thailand. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1

hat these 3 rickettsiae derived from 3 different host snake specieslustered together in a clade separate from other SFG rickettsiaend grouped together into a clade that was most closely related to. raoultii.

p resampling. The sequences obtained from this study are indicated in bold type.

Discussion

In this study, the infection rate of A. varanense ticks with Rick-ettsia sp., closely related to Rickettsia sp. strain RDa420 and R.bellii, was 21% (4/19 ticks). A phylogenetic analysis revealed thatRickettsia sp. strain RDa420 was clustered with R. bellii, a Rick-ettsia of unknown pathogenicity to vertebrate hosts (Parola et al.,2003a,b) (Fig. 1). Rickettsia bellii has been detected in many tickgenera including Amblyomma, Dermacentor, Haemaphysalis, Ixodes,Ornithodoros, and Rhipicephalus (Beati et al., 1995; Labruna et al.,2007; Fournier and Raoult, 2009; Tomassone et al., 2010; Merhejand Raoult, 2011). Very different infection rates with R. bellii havebeen reported in many tick species: The infection rates in Ambly-omma sp., Dermacentor sp., and Ixodes loricatus, for example, were17.4%, 2.2%, and 60.9%, respectively (Socolovschi et al., 2009), andthey were as high as 100% in Amblyomma rotundum (Labruna et al.,2004). We report here for the first time the infection with a Rick-ettsia sp. that belongs to an ancestral group of rickettsiae (Gillespieet al., 2007) in A. varanense ticks from Thailand.

The introduction of exotic ticks via the international reptiletrade has been of concern due to the potential establishment

tection of Rickettsia species in Amblyomma ticks collected from016/j.ttbdis.2014.04.013

of exotic ticks and their associated pathogens in new geograph-ical areas (Burridge et al., 2000a,b; Burridge, 2001; Burridgeand Simmons, 2003; Kenny et al., 2004; Pietzsch et al., 2006).Imported exotic ticks with pathogens such as Rickettsia, Ehrlichia,

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Table 3GenBank accession numbers of the gene sequences from validly published Rickettsia species used in the present study.

Rickettsia sp. Strain GenBank accession nos. for PCR target genes

gltA ompA ompB

R. sibirica sibirica 246 U59734 U43807 AF123722R. sibirica mongolitimonae HA-91 U59731 U43796 AF123715R. parkeri maculatum 20 U59732 U43802 AF123717R. africae ESF-5 U59733 U43790 AF123706R. conorii caspia A-167 U59728 U43791 AF123708R. conorii israelensis ISTT CDC1 U59727 U43797 AF123712R. conorii indica Seven U59730 U43794 AF123726R. conorii conorii Malish 7 U59730 U43806 AF123721R. slovaca 13-B U59725 U43808 AF123723R. honei TT-118 U59726 U43809 AF123724R. honei RB AF018074 AF018075 AF123711R. rickettsii R (Bitterroot) U59729 U43804 X16353R. japonica YM U59724 U43795 AF123713R. heilongjiangensis 054 AF178034 AF179362 AY260451R. montanensis M/5-6 U74756 U43801 AF123716R. raoultii Khabarovsk DQ365804 DQ365801 DQ365798R. raoultii Marne DQ365803 DQ365799 DQ365797R. raoultii Elanda EU036985 EU036986 EU036984R. aeschlimannii MC16 U59722 U43800 AF123705R. masslliae Mtu1 U59719 U43799 AF123714R. massiliae AZT80 CP003319 CP003319 CP003319R. rhipicephali 3-7-6 U59721 U43803 AF123719R. tamurae AT-1 AF394896 DQ103259 DQ113910R. australis Phillips 32 U59718 AF149108 AF123709R. monacensis IrR/Munich DQ100163 DQ100169 EF380356R. felis URRWXCal2 AF210692 AF210694 AF210695R. bellii 369L42-1 U59716 NA NAR. canadensis 2678 U59713 NA NAR. akari MK (Kaplan) U59717 NA AF123707R. prowazekii Breinl M17149 NA AF123718R. typhi Wilmington U59714 NA L04661Rickettsia sp. RDa420 – AF497584 NA NA

Ng enate

aKtiDpfli

akawboic

S2emSsinihne

A, not available.ltA (338 bp), ompA (467 bp), ompB (831 bp) = a total of 1636 bp to analyse in concat

nd haemoparasites have been reported (Burridge et al., 2000a,b;enny et al., 2004; Reeves et al., 2006). Various species of exotic

icks from Asia have been recorded on imported exotic reptilesn many countries especially in Europe and the USA (Keirans andurden, 2001; Pietzsch et al., 2006; Nowak, 2010). For exam-le, Varanus salvator brought from Indonesia to Poland have beenound to be parasitised by A. varanense (Nowak, 2010). Pythons andizards from Sumatra, Indonesia, and New Guinea were found to benfested with A. varanense as well (Keirans and Durden, 2001).

For reptiles imported from Thailand to the USA, A. helvolumnd A. varanense ticks were recorded on imported snakes anding cobras, respectively (Keirans and Durden, 2001; Burridgend Simmons, 2003). The importation of a king cobra infestedith A. varanense and A. cordiferum from Malaysia to Taiwan has

een reported (Norval et al., 2009). In our study, novel strainsf SFG rickettsiae in snake ticks were detected. Therefore, thenternational trade of snakes should be undertaken with greatare.

To our knowledge, this is the first report of the presence ofFG rickettsiae (rickettsia species closely related to R. raoultii) in

snake tick species, A. varanense and A. helvolum in Asia. Stenost al. (2003) were the first to report Bothriocroton hydrosauri (for-erly Aponomma hydrosauri), a reptile tick, as the reservoir of a

FG Rickettsia, R. honei, in Australia. In addition, Amblyomma dis-imile ticks collected from iguanas in Colombia were reported to benfected with a novel SFG rickettsia, Rickettsia sp. strain Colombia-ensi (Miranda et al., 2012). The natural cycle of rickettsial infection

Please cite this article in press as: Sumrandee, C., et al., Molecular desnakes in Thailand. Ticks Tick-borne Dis. (2014), http://dx.doi.org/10.1

n ticks and snakes of this study remains unknown. Although A.elvolum and A. varanense are common reptile ticks, they haveever been reported to feed on humans (Tanskul et al., 1983; Cornett al., 2009). It is unlikely that they are responsible for rickettsial

d NJ tree.

transmission to humans. Many tick species other than reptile ticks(A. helvolum and A. varanense) that have been recorded from snakesin south-eastern Asia may be involved in natural transmissioncycles of Rickettsia strains and in the transmission to other ver-tebrate hosts. Dermacentor auratus and Dermacentor compactus, forexample, were found to be parasites of pythons (Hoogstraal andWassef, 1984, 1985), but they were also collected from variousmammal hosts including wild boar, deer, dog, and rat (Tanskul et al.,1983; Hoogstraal and Wassef, 1984; Parola et al., 2003a,b). In addi-tion, a Rhipicephalus sanguineus tick was collected from a Burmesepython in Malaysia (Mariana et al., 2011).

D. auratus ticks that feed on the same infective vertebrate hosts(e.g., pythons) as A. varanense or A. helvolum may acquire thispathogen and transmit it to humans later on as humans havefrequently been reported to be an accidental host of D. auratus(Tanskul et al., 1983; Estrada-Pena and Jongejan, 1999; Ajithkumaret al., 2012). The infection with an SFG rickettsia, Rickettsia sp.strain RDla440 has been reported in Dermacentor larvae in Thailand(Parola et al., 2003a,b). Various mammals, such as otter, cattle,palm civet, mongoose, wild boar, and dog, have been recorded as A.varanense hosts (Kauffman, 1972). Therefore, these mammals maybe the hosts of this tick species in Thailand rather than snakes orreptiles and may serve as a reservoir of rickettsia. The systematicsurvey and collection of tick samples from various animal hosts isnecessary to better understand the epidemiology and transmissioncycle of this probable pathogen. Furthermore, systematic samplingsare necessary to elucidate the natural cycle of SFG rickettsiae infec-

tection of Rickettsia species in Amblyomma ticks collected from016/j.ttbdis.2014.04.013

tions in other tick species and their vertebrate hosts. Although thepathogenicity of novel rickettsial strains has not been determined,their pathogenicity should not be neglected in Thai patients sus-pected of having tick-borne rickettsiosis.

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cknowledgements

This work was supported by a research grant from the Faculty ofcience, Mahidol University, Thailand (SCM55-004). This work waslso supported by a grant from Strategic Scholarships Fellowshipsrontier Research Networks, Office of Higher Education Commis-ion, Ministry of Education and a Mahidol University Research grantSCJV1099000737).

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