Molecular survey of Borrelia spp. in bats and soft tick infesting bats in Thailand

Bats are recognized reservoirs for numerous pathogens, including zoonotic agents. They harbor a variety of ectoparasites, such as ticks, which pose a potential risk for cross-species pathogen spillover. Borrelia , a genus of spirochete bacteria responsible for several vector-borne diseases, represents a significant public health concern due to its global distribution. However, its genetic diversity and circulation in Southeast Asia remain poorly understood. To address this knowledge gap, this study investigated the occurrence and genetic diversity of Borrelia spp. in Thai bats and the ticks associated with them based on PCR and sequencing, without bacterial isolation or clinical case data. A total of 1031 bats were sampled across nine provinces in Thailand between February 2018 and February 2023. Morphological and molecular analysis-based methods identified and confirmed 96 Reticulinasus sp. ticks infesting Craseonycteris thonglongyai and Eonycteris spelaea. Molecular screening of 48 pooled tick samples (from 96 individual ticks) detected four Borrelia -positive pools originating from three individual C. thonglongyai bats. PCR screening of 814 bats targeting the flaB gene revealed 13 positive individuals (1.6%; 95% CI: 0.9–2.7%), comprising Taphozous melanopogon , Hipposideros gentilis , and C. thonglongyai . Multilocus sequence analysis of the 16S rRNA, flaB, and glpQ genes revealed three distinct Borrelia lineages. Two clustered within the relapsing fever Borrelia group, highlighting the need for further investigation of their transmission potential and epidemiological significance in other animal hosts. The third lineage formed a highly divergent, bat-associated clade, suggesting possible cryptic speciation based on sequence divergence across the three loci; however, formal taxonomic classification will require broader sampling and additional genetic markers. This study presents the first molecular-level evidence of Borrelia in Thai bats and bat-infesting ticks. • First report of Borrelia spp. detected in bats and bat-associated ticks in Thailand. • Detected Borrelia lineages were phylogenetically related to human-infecting relapsing fever Borrelia spp. • The virulence and zoonotic potential of the Borrelia strains detected in Thailand remain unknown. • One Borrelia lineage was highly divergent, suggesting potential cryptic speciation.