Sequences obtained in this study.

Orientia tsutsugamushi (Ot), the causative agent of tsutsugamushi disease (TD), has been detected in Muridae, Soricidae, Cricetidae, Canidae, Artiodactyla, and birds, as well as in its trombiculid mite vectors. However, to date, scarce reports have documented Ot infection in bats. TD is an ancient zoonotic disease transmitted through the bite of infected trombiculid mites. The global disease burden of TD, particularly in impoverished regions, warrants renewed attention and a reevaluation of public health strategies. In this study, we analyzed bat samples for Ot using nested PCR (nPCR) and real-time quantitative PCR (qPCR) for both qualitative and quantitative detection. Genetic evolution and divergence time of the detected Ot sequences were assessed using bioinformatics tools, including BioAider, Clustal X2, MEGA-X, and BEAST. Ot was detected in 7.32% (44/601) of bat samples by qPCR. At least three genotypes, including Karp, Gilliam, and Kato, were identified in both insectivorous bats (Hipposideros larvatus and Hipposideros armiger) and frugivorous bats (Rousettus amplexicaudatus and Cynopterus sphinx). Ot DNA was detected in multiple tissues, including heart, kidney, spleen, lung, rectum, liver, and brain, with median copy numbers ranging from 28.60 to 1069.76 copies/μL. Notably, divergence analysis suggests that Ot isolated from bats emerged around 126 AD, later than its appearance in rodents, humans, and chiggers (approximately 4140 BC), indicating that Ot infection in bats may originate from other animals or vectors. Our findings recommend ongoing monitoring of Ot in bats and their ectoparasites, which will provide a basis for risk assessment and guide strategies for the prevention and control of scrub typhus.