FRANCISELLA-LIKE ENDOSYMBIONT IN A TICK IN THAILAND FRANCISELLA-LIKE ENDOSYMBIONT IN A TICK COLLECTED FROM A CHICKEN IN SOUTHERN THAILAND Pakavadee Rakthong1, Toon Ruang-Areerate2, Visut Baimai3,4, Wachareeporn Trinachartvanit3 and Arunee Ahantarig3,4 1Faculty of Science and Technology, Rajabhat Suratthani University, Mueang Surat Thani, Surat Thani; 2Epidemiology Section, Research Division, Armed Forces Research, Institute of Medical Sciences, Bangkok; 3Biodiversity Research Cluster, Department of Biology, Faculty of Science; 4Center of Excellence for Vectors and Vector-Borne Diseases, Faculty of Science, Mahidol University, Thailand Abstract. Francisella is a genus of bacterial pathogens potentially lethal to humans. We report here for the first time a novel Francisella-like endosymbiont discovered in a hard-tick (Rhipicephalus sanguineus s.l.) obtained from a chicken (Gallus domes- ticus) in Thailand. The phylogenetic results indicate the 16S rDNA sequences of this Francisella bacterium form a unique clade with the Francisella-like endosymbi- ont of the tick species, Amblyomma varanense and Amblyomma helvolum, that have previously been found on snakes in Thailand. This species of Francisella is in a different group from the otherFrancisella -like endosymbionts previously reported from other countries. No Francisella was detected in Haemaphysalis wellingtoni ticks obtained from chickens in this study. Keywords: ticks, Francisella-like endosymbiont, chicken, Thailand INTRODUCTION (Yamauchi, 2001) and Ixodes ricinus has been found on red grouse chicks (Kirby Ticks are important vectors of zoon- et al, 2004) . oses that affect human health worldwide Francisella is a genus of bacterial and can transmit viruses, bacteria and pathogens that can cause potentially protozoa. A variety of avian species fatal disease. Francisella tularensis causes have been reported to be hosts for ticks: tularemia, also known as rabbit fever and Amblyomma americanum and Ixodes brun- deer-fly fever (Francis, 1921). The disease neus have been found on wild turkeys can be transmitted by ticks, biting flies, (Meleagris gallopavo) (Scott et al, 2010), water, food and aerosols (Tärnvik, 2007). Ixodes turdus and Haemaphysalis flavahave Francisella novicida had been isolated from been found mainly on passerine birds a Thai patient from southern Thailand (Leelaporn et al, 2008). In Thailand, Fran- Correspondence: Dr Arunee Ahantarig, Depart- cisella-like endosymbionts (FLEs) have ment of Biology, Faculty of Science, Mahidol been reported from snake ticks (Sumran- University, Rama VI Road, Bangkok 10400, Thailand. dee et al, 2014). Tel: +66 (0) 2201 5380; Fax: +66 (0) 2354 7161 In southern Thailand, indigenous E-mail: [email protected] chickens live in close contact with people. Vol 47 No. 2 March 2016 245 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH The chickens walk around freely and for- 1992). For the detection of Francisella 16S age on the ground for their natural diet, rDNA in tick samples, each PCR reaction which consists of worms, insects, seeds, was carried out using primers and condi- and plants. Pathogen-carrying ticks on tions as described previously (Williams et chickens can cause health problems for al, 1992). The PCR fragment of the positive local people. Little is known about the sample was purified and sequenced. presence of Francisella spp in indigenous Phylogenetic analysis chickens in southern Thailand. Here, we Nucleotide sequences of Francisella report a novel Francisella-like endosymbi- 16S rDNA (1,054bp) were aligned by ont discovered in Rhipicephalus sanguineus the MegAlign tool using DNASTAR® s.l. obtained from a chicken (Gallus domes- Lasergene software. Neighbour-joining ticus) in Thailand for the first time. (NJ) was used to generate phylogenetic relationships using PAUP 4.0b10 soft- MATERIALS AND METHODS ware. Bootstrap values >50% were indi- cated above branches (1,000 replicates). Tick collection Francisella halioticida was selected as an Ticks were randomly collected (June outgroup. 2014) from two indigenous chickens (Gal- lus domesticus) from one suburban house RESULTS in Surat Thani Province, southern Thai- land (9°07’51.2” N latitude, 99°21’44.0” E Five ticks (adults: three ticks from longitude). The ticks were removed from one chicken and another two ticks from neck and cockscomb of the chickens and another chicken) and two nymphs (from kept in 70% ethanol at 4°C before being a cat) were identified to the species or sent to the Faculty of Science of Mahidol genus levels (only adult tick can be iden- University in Bangkok, Thailand for tified to species, while larva and nymph species identification (Kohls, 1957). Ticks can be identified to the genus level by a were also collected from a cat body (Felis tick taxonomist): Rhipicephalus sanguineus catus) in the same household where the s.l. (two ticks, both female), Haemaphysa- ticks were collected from chickens. No lis wellingtoni (three ticks, all male) and ticks were found in chickens and cats Haemaphysalis spp (two nymphs). Both from the other nearby houses. R. sanguineus s.l. specimens and one H. DNA extraction and Francisella detection wellingtoni were obtained from one chick- The ticks were cleaned before DNA en. Another two H. wellingtoni ticks were extraction. DNA extraction was per- collected from a second chicken. Two Hae- formed individually for adult ticks while maphysalis nymphs were collected from a the nymphs (developmental stage) were cat from the same household, as well. All pooled, using the QIAamp DNA Ex- ticks were positive for the primers 16S + traction Kit for Tissue (Qiagen, Hilden, 1 and 16S - 1. Germany) following the manufacturer’s For Francisella detection, DNA sam- protocol. The quality of the extracted tick ples were checked for the presence of DNA was determined by amplification Francisella 16S rDNA sequences. Positive of the tick mitochondrial (mt) 16S rDNA results were obtained in one out of the gene using the primers 16S + 1 and 16S - five ticks collected from the two chickens; 1 as previously described (Williams et al, the positive result was obtained for a R. 246 Vol 47 No. 2 March 2016 FRANCISELLA-LIKE ENDOSYMBIONT IN A TICK IN THAILAND sanguineus s.l. female. The other ticks (H. wellingtoni) collected from the same chicken were negative for Francisella 16S rDNA. The Haemaphysalis nymphs col- lected from the cat (Felis catus) in the same house- hold were also negative for Francisella. The Francisella 16S rDNA sequences obtained from R. sanguineus s.l. (SRT 95) were submitted to Gen- Bank (accession number KP659194). This sequence showed the highest se- quence identity (1052/1056, 99.62%) with a Francisella endosymbiont, Amblyom- ma varanense clone APOH2 16S ribosomal RNA gene, partial sequence (acces- sion number KF268342), from a snake in Thailand. Using Blast search results, this Francisella 16S rDNA sequence was also 98.77% (1043/1056) identical to Dermacentor auratus clone SSPG3 16S ribosomal RNA gene, partial sequence (JQ764629) and 98.68% Fig 1–Neighbour-joining phylogenetic analysis of a Francisella 16S (1043/1057) identical to Or- rDNA sequence obtained from a Rhipicephalus sanguineus nithodoros moubata symbiote s.l. specimens obtained from a chicken. Francisella halioticida B gene for a 16S rRNA par- was selected as an outgroup. tial sequence (AB001522). The phylogenetic analysis results this group (Fig 1). This type of Francisella indicate the 16S rDNA sequences from forms a different clade than F. tularensis the Francisella infecting the R. sanguineus strains (pathogenic bacteria). s.l. taken from G. domesticus represent a unique clade within the Francisella DISCUSSION endosymbiont of A. varanense and the Francisella endosymbiont of A. helvolum FLE emerged from an infective an- and are evolutionarily closely related to cestral organism (Scoles, 2004). FLE have Vol 47 No. 2 March 2016 247 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH a worldwide distribution in both hard grant (SCJV1099000737) and Higher and soft ticks (Szigeti et al, 2014), namely, Education Research Promotion grants in the genera Ixodes, Amblyomma, Derma- (2557A15962002 and 2558A15962001). centor, and Ornithodoros (Scoles, 2004). Ivanov et al (2011) reported detecting FLE REFERENCES in R. sanguineus s.l., R. bursa, R. turanicus, Dermacentor marginatus, D. reticulatus, Francis E. 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