Isolation of reptile-associated Borrelia spirochetes from Amblyomma kappa ticks infesting the endangered Ryukyu black-breasted leaf turtles, Geoemyda japonica, confiscated during illegal export in Japan

Illegal trade in live reptiles is increasing worldwide and can facilitate long-distance movement of ectoparasites and their associated microorganisms, posing emerging challenges for veterinary surveillance and wildlife health management. The Ryukyu black-breasted leaf turtle, Geoemyda japonica , is an endangered chelonian endemic to the Okinawa archipelago in the Central Ryukyus and is frequently targeted in the illegal pet trade. In this study, we investigated Borrelia spirochetes in ticks removed from G. japonica that were confiscated during an illegal export attempt and subsequently housed at an ex-situ conservation facility in Japan. A total of 77 ticks were collected from six turtles and were morphologically identified as Amblyomma kappa nymphs, with molecular confirmation based on mitochondrial 16S rRNA gene and COI gene sequences from four representative ticks. Ticks were surface-sterilized and inoculated into BSK-M medium for isolation of spirochetes. Motile Borrelia -like spirochetes were isolated from 24 of 77 ticks and were confirmed by PCR targeting flaB (nested PCR) and 16S rDNA. Phylogenetic analyses based on flaB and concatenated partial sequences of groEL and gyrB revealed that all isolates formed a well-supported clade within the reptile-associated (REP) Borrelia group, clustering with previously reported sequences from the A. geoemydae species complex infesting turtles in the Yaeyama Islands in the Southern Ryukyus and closely related to, but distinct from, Borrelia turcica . These findings suggest that REP Borrelia are likely circulating within tick–reptile communities associated with G. japonica in its native range in the Okinawa archipelago and provide regional baseline data on this tick-borne bacterial group. To our knowledge, this study provides the first isolation of REP Borrelia from A. kappa ticks parasitizing G. japonica and highlights illegal reptile trafficking as a potential route for transboundary dissemination of exotic ticks and their microorganisms, underscoring the need for systematic health screening and ectoparasite surveillance in confiscated wildlife within regional veterinary and wildlife health programs. ● REP Borrelia were isolated from Amblyomma kappa on Geoemyda japonica . ● Multilocus sequencing places isolates in the reptile-associated Borrelia clade. ● Isolates form a distinct sister lineage to Borrelia turcica within REP Borrelia based on multilocus analyses. ● Findings suggest REP Borrelia circulate in a local tick–turtle system in the Ryukyus. ● Confiscated turtles highlight illegal trade as a route for exotic ticks and bacteria.