A metagenomic survey of ticks reveals pathogenic rickettsia and francisella/coxiella-like endosymbionts in Anatolia

Abstract

Purpose: We investigated tick-borne bacteria in field-collected ticks via a metagenomic approach using high throughput sequencing. Methods & Materials: A total of 210 ticks, collected from various regions of Anatolia, were identified morphologically and processed in 39 pools. Homogenization, nucleic acid purification and library preparation in pools were performed using standard assays and protocols, followed by sequencing using the Illumina MiSeq system. Results: A near-complete Rickettsial genome of 1.485.148 nucleotides with 89,5% identity to Rickettsia felis strain URRWXCal2 was assembled from a pool of Haemaphysalis parva ticks. The identical pool further provided a 5690-nucleotide sequence of the Rickettsial conjugative plasmid. Varying lengths of 16S, OmpA, Sca1 and gltA sequences were obtained from 7 tick pools (17,9%) comprising Haemaphysalis parva, Rhipicephalus bursa, Rhipicephalus sanguineus sensu lato and Hyalomma marginatum ticks. The sequences were characterized as Rickettsia felis and Rickettsia hoogstraalii in 5 pools whereas strain identification remained obscure in two pools. 16S rRNA sequences of Coxiella-like endosymbionts were detected in 15 pools (38,4%) of Haemaphysalis parva, Rhipicephalus bursa, Rhipicephalus sanguineus s. l., Hyalomma marginatum, Hyalomma excavatum and Dermacentor marginatus ticks. These sequences formed three phylogenetically-distinct clusters with previously-characterized Coxiella-like endosymbionts from Rhipicephalus, Ixodes and Dermacentor species. Finally, Francisella-related 16S sequences were characterized in 9 pools (23%) of Rhipicephalus sanguineus s. l., Hyalomma marginatum, Dermacentor marginatus and Hyalomma aegyptium ticks. These sequences formed two distinct clusters and grouped with Francisella-like endosymbionts of Dermacentor ticks, sharing a common ancestor with Francisella percica. Conclusion: Rickettsia of the spotted fever - Rickettsia akari group were characterized in this preliminary tick screening study, with detailed sequence information obtained for various strains. Widespread infection of ticks with Francisella- and Coxiella-like symbionts were also identified. The impact of these endosymbiotic bacteria on the circulation and detection of pathogenic strains in ticks requires further investigation.