New host record of Amblyomma pakhtunensis on the Indian Pangolin (Manis crassicaudata) with detection of a distinct Borrelia lineage.

Pangolins are mammals of the order Pholidota, with most species listed as endangered or critically endangered. They are distributed in both Asia and Africa, with the Indian pangolin (Manis crassicaudata) found in South Asia, including Pakistan. The tick genus Amblyomma, in the family Ixodidae, is widely distributed in tropical and subtropical regions and infests a variety of terrestrial and semi terrestrial vertebrates. The bacterial genus Borrelia is Gram-negative and consists of three main groups, one of which is mainly linked to reptiles, some birds, and mammals. This study focuses on investigating the Indian pangolin as a potential host for Amblyomma pakhtunensis, a recently described tick species previously reported only on monitor lizards and with no known microbial associations. It also aims to determine whether ticks from pangolins contain Borrelia species and assesses their phylogenetic relationships. Ticks collected from a pangolin were morphologically identified as A. pakhtunensis. DNA from a subset of ticks was extracted to confirm their molecular identity and to detect Borrelia spp. in these ticks using conventional PCR. For the former, a partial fragment of tick 16S rRNA was targeted, while for the latter, partial fragments of the Borrelia 16S rRNA, flaB, and glpQ genes were targeted for amplification. A single live pangolin, conventionally identified as M. crassicaudata, was found. Twenty‑seven ticks were collected (16 males, 11 females), all morphologically identified as A. pakhtunensis. BLAST analysis of A. pakhtunensis 16S rDNA sequence showed 99.75% identity with the same tick species. BLAST analysis of Borrelia 16S rDNA and flaB sequences revealed highest identities of 99.25% and 95.91%, respectively, with Candidatus Borrelia javanense. The overall Borrelia infection rate was 50% (5/10), with infections detected in 4/5 female ticks and 1/5 male tick. In both 16S rDNA and flaB based phylogenetic analyses, the detected Borrelia species clustered with Candidatus B. javanense from China. In addition to expanding the vertebrate host diversity of A. pakhtunensis, this study also expands knowledge of the third group of Borrelia by extending their known vertebrate host range and genetic diversity. This study can enhance our understanding of the regional epidemiology of tick-borne diseases and may specifically aid pangolin conservation strategies by taking into account the control of ticks and their associated pathogens.