A comprehensive genetic and phylogenetic study of Trypanosoma spp. in bats and sand flies from shared habitats in Thailand.

BACKGROUND

Bats are known reservoirs for various pathogens, many of which can infect other animals through blood-feeding arthropods. Over 100 bat species have been identified as hosts for kinetoplastid protozoans, including ≥ 30 distinct Trypanosoma spp. However, bat trypanosomes remain relatively understudied owing to the nocturnal behavior of their hosts and legal restrictions on their capture for research. In Southeast Asia, particularly Thailand, only one study has investigated bat trypanosomes, leaving their distribution and transmission pathways largely unexplored.

METHODS

Between April 2021 and November 2023, bats were captured at ten locations across four provinces in Thailand. Blood samples were collected, examined microscopically, and screened for Trypanosoma DNA targeting the SSU rRNA and gGAPDH genes. Phlebotomine sand flies from bat sampling sites were collected and analyzed for Trypanosoma DNA and blood meal sources. Sequences were identified using BLASTn searches, while genetic relationships were assessed through pairwise genetic distance, phylogenetic reconstruction, and TCS haplotype network analyses. In addition, species delimitation was conducted to validate unidentified sequences at the species level.

RESULTS

Out of 368 bats, 40 (10.9%) tested positive for four Trypanosoma species (including two previously named: T. dionisii and T. noyesi). Out of 189 sand flies, a single one tested positive for an unnamed anuran trypanosome from a gravid female (Phlebotomus stantoni), and the study was unable to detect the blood source of this sand fly. In total, 37 pools (189 specimens) of female sand flies-comprising 159 unfed, 29 gravid, and 1 engorged specimen-were analyzed for vertebrate blood meals, but none tested positive. Multiple analyses (BLASTn, phylogenetics, haplotype networks, pairwise genetic distances, and species delimitation) also confirmed a Trypanosoma sp. in a gravid sand fly, along with T. dionisii and T. noyesi, plus two uncharacterized bat-associated species.

CONCLUSIONS

The Trypanosoma spp. detected in the present study aligns with prior reports of diverse trypanosomes in bat populations, reinforcing their role as key reservoirs. Notably, a single sand fly (Phlebotomus stantoni) tested positive for an unnamed anuran trypanosome, but its blood meal source could not be determined, leaving unresolved questions about potential transmission pathways.