Molecular Surveillance of Spotted Fever Group Rickettsiae in Haemaphysalis longicornis Ticks from Two Ecologically Distinct Regions of China

Background: The Asian longhorned tick, Haemaphysalis longicornis, is a vector of significant public health importance, capable of transmitting numerous zoonotic pathogens. In China, comprehensive data on the geographic distribution and genetic diversity of these pathogens, particularly Spotted Fever Group Rickettsiae (SFGR), remain limited. This study aimed to investigate the prevalence and characteristics of key pathogens in questing H. longicornis ticks from two geographically and ecologically distinct regions of China. Methods: A total of 1,004 questing H. longicornis ticks were collected from Liaoning Province (Northeast China) and Anhui Province (East-Central China) and processed in 670 pools. Tick species was confirmed using mitochondrial cytochrome oxidase I (COI) gene sequencing. Nucleic acids from all tick pools were screened for Dabie bandavirus (formerly SFTSV) and bacteria using universal 16S rRNA gene primers. Bacterial-positive samples were subsequently subjected to multi-gene nested PCR assays for Rickettsia spp. and Coxiella-like endosymbionts (CLE). Phylogenetic analyses, including supertree construction from concatenated gene sequences, were performed to ensure robust species identification and explore genetic relationships. Results: No Dabie bandavirus was detected. SFGR were identified in 14 of the 670 tick pools, yielding an overall Minimum Infection Rate (MIR) of 1.4% (14/1,004). Notably, the detected SFGR species showed clear geographic separation: Candidatus Rickettsia jingxinensis was found exclusively in Liaoning (MIR 2.0%), while Rickettsia heilongjiangensis was detected only in Anhui (MIR 2.6%). Additionally, CLE were found in 20 tick pools (overall MIR 2.0%), with a significantly higher prevalence in Anhui (17.1%) compared to Liaoning (1.2%). Conclusions: Our surveillance reveals distinct geographic distributions of SFGR species within H. longicornis populations in China, confirming the circulation of Ca. R. jingxinensis in the northeast and R. heilongjiangensis in the east-central region. These findings provide crucial, multi-gene-supported epidemiological data that enhance our understanding of regional tick-borne disease risks, and underscore the importance of continued, geographically targeted surveillance.