Borrelia sp. phylogenetically different from Lyme disease- and relapsing fever-related Borrelia spp. in Amblyomma varanense from Python reticulatus

Species of the genus Borrelia are causative agents of Lyme disease and relapsing fever. Lyme disease is the most commonly reported vector-borne disease in the northern hemisphere. However, in some parts of the world Lyme borreliosis and relapsing fever may be caused by novel Borrelia genotypes. Herein, we report the presence of a Borrelia sp. in an Amblyomma varanense collected from Python reticulatus. Ticks were collected from snakes, identified to species level and examined by PCR for the presence of Borrelia spp. flaB and 16S rRNA genes. Phylogenetic trees were constructed using the neighbour-joining method. Three A. varanense ticks collected from P. reticulatus were positive for a unique Borrelia sp., which was phylogenetically divergent from both Lyme disease- and relapsing fever-associated Borrelia spp. The results of this study suggest for the first time that there is a Borrelia sp. in A. varanense tick in the snake P. reticulatus that might be novel.


Background
Tick infestation in snakes occurs worldwide and involves the following species: Amblyomma gervaisi in the northern region of western Ghats in India [1], Rhipicephalus sanguineus (sensu lato) in Malaysia [2], Amblyomma varanense and Amblyomma helvolum in Thailand [3,4] and Amblyomma hydrosauri in Australia [5]. Hirunkanokpun et al. [6] detected several bacterial species in the national parks of Thailand, but no Borrelia spp. were found. The aim of this study was to determine the presence of Borrelia spp. within Amblyomma spp. ticks collected from five snake species. In addition, phylogenetic analyses of Borrelia spp. are also presented.

Tick collection and identification
Tick collection from snakes was performed in February 2014 in Lopburi Province, Thailand (14°48′1.61″N, 100°38′10.75″E). We observed snake scales to identify partial protrusions of tick bodies outside of the scales. Ticks were collected from the skin beneath the scales using forceps. Ticks were identified according to their morphology using standard taxonomic keys [7][8][9][10][11].

Phylogenetic analysis
Phylogenetic trees were constructed using the neighbourjoining method (PAUP 4.0b1) [12]. DNA gaps or missing data were excluded from the analyses. Confidence values for individual branches of the resulting tree were determined by bootstrap analysis with 1000 replicates.

Results and discussion
Ticks were collected from the following five snake species: Python reticulatus, Ophiophagus hannah, Ptyas korros, Naja kaouthia and Elaphe radiata. Four Amblyomma varanense ticks (three males and one female) were collected from one P. reticulatus. One A. varanense tick (one female) and two Amblyomma pattoni (males) were collected from O. hannah. Two A. pattoni (males) were collected from N. kaouthia. In addition, two A. pattoni males were collected from E. radiata. Finally, one A. varanense tick (male) was collected from E. radiata. Tick species reported in this study have hypostomal dentition 3/3. The male of A. pattoni has coxa I with an inconspicuous internal spur, which is sometimes fused with the more prominent external spur, and cervical pits are commashaped. The male of A. varanense has coxa I with the external spur noticeably longer than the internal and the female has coxa I with the internal spur smaller than the external spur, but always separated from the latter (Fig. 1).
A total of 12 ticks was collected from snakes and examined by PCR for the presence of the Borrelia spp. genes. Of these, three ticks, all identified as A. varanense isolated from P. reticulatus, were positive for Borrelia spp. No Borrelia spp. were detected in A. pattoni or in A. varanense collected from other snake species.
Borrelia sp. DNA sequences were compared with sequences in the NCBI GenBank database by nucleotide BLAST. The 16S rRNA gene sequence of this Borrelia sp. is 100 % identical (1449/1449 bp) to Borrelia sp. BF16 (GenBank: AB473538) and was submitted to GenBank and assigned as KU497718 (Borrelia sp. in Phylogenetic trees were constructed using the neighbour-joining method (PAUP 4.0b1) [12]. Individual branch confidence values were determined by bootstrap analysis with 1000 pseudoreplicates (Figs. 2 and 3). The phylogenetic trees for both genes of Borrelia spp. inferred from the complete 16S rRNA gene sequences (Fig. 2) and partial sequences of the flaB genes ( Fig. 3) indicated that Borrelia sp. from the present study is related to Borrelia sp. (GenBank: AB473538) from reptiles and to B. turcica IST7 (GenBank: KF422815) flagellin gene, respectively, but belongs in a different group from Borrelia burgdorferi. The phylogenetic relationships among relapsing fever-associated Borrelia spp. and Lyme diseaseassociated Borrelia spp. were reported previously using rrs and 16S rDNA [13,14]. The flaB and 16S rRNA gene sequences of Borrelia sp. of A. varanense from P. reticulatus isolated in this study, formed a separate branching root from both Lyme disease-associated Borrelia species and relapsing fever-associated Borrelia species.

Conclusion
Our findings suggest that Borrelia sp. in A. varanense from P. reticulatus might be novel and phylogenetically divergent from both Lyme disease-and relapsing feverassociated Borrelia species.