Pathogen surveillance in cave-dwelling phlebotomine sand flies from Chiang Rai, northern Thailand, with the first detection of Hepatocystis.

Sand flies are important vectors of several pathogens of medical and veterinary significance, including Leishmania spp., Trypanosoma spp., Bartonella spp., and various arboviruses. Cave ecosystems provide suitable habitats for sand flies and often harbor large bat populations that serve as reservoirs for diverse microorganisms, potentially facilitating pathogen circulation within cave environments. The present study aimed to investigate the presence of sand fly-borne pathogens in cave-dwelling sand flies from three caves in Chiang Rai Province, northern Thailand. A total of 616 sand fly specimens were collected, morphologically identified, and pooled into 95 pools for nucleic acid extraction. Molecular screening was performed to detect RNA viruses (Phlebovirus, Orbivirus, and members of the family Rhabdoviridae), blood parasites (Leishmania spp., Trypanosoma spp., and haemosporidian parasites), and Bartonella spp. bacteria. Positive samples were subsequently confirmed by sequencing and phylogenetic analysis. One pool of Phlebotomus shadenae tested positive for Rhabdoviridae and clustered within the Vesiculovirus. PCR screening also detected Leishmania martiniquensis (2 pools), Trypanosoma spp. (5 pools), and Hepatocystis sp. (2 pools), while Bartonella DNA was not detected. Importantly, this study provides the first molecular detection of a species of Hepatocystis in cave-dwelling sand flies from Thailand. The findings expand current knowledge of pathogen diversity in cave ecosystems and provide further insight into the range of pathogens molecularly detected in sand flies.