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Rhipicephalus microplus , a vector that can transmit many pathogens to humans and domestic animals, is widely distributed in Yunnan province, China. However, few reports on the prevalence of tick-borne pathogens (TBPs) in  Rh. microplus  in Yunnan are available. The aim of this study was to detect TBPs in  Rh. microplus  in Yunnan and to analyze the phylogenetic characterization of TBPs detected in these ticks. The adult  Rh. microplus  ( n  = 516) feeding on cattle were collected. The pooled DNA samples of these ticks were evaluated using metagenomic next-generation sequencing (mNGS) and then TBPs in individual ticks were identified using genus- or group-specific nested polymerase chain reaction (PCR) combined with DNA sequencing assay. As a result,  Candidatus Rickettsia jingxinensis  (24.61%, 127/516),  Anaplasma marginale  (13.18%, 68/516),  Coxiella burnetii  (3.10%, 16/516), and  Coxiella- like endosymbiont (CLE) (8.33%, 43/516) were detected. The dual coinfection with  Ca. R. jingxinensis  and  A. marginale  and the triple coinfection with  Ca. R. jingxinensis ,  A. marginale , and CLE were most frequent and detected in 3.68% (19/516) and 3.10% (16/516) of these ticks, respectively. The results provide insight into the diversity of TBPs and their coinfections in  Rh. microplus  in Yunnan province of China, reporting for the first time that  C. burnetii  had been found in  Rh. microplus  in China. Multilocus variable number tandem repeat analysis with 6 loci (MLVA-6) discriminated the  C. burnetii  detected in  Rh. microplus  in Yunnan into MLVA genotype 1, which is closely related to previously described genotypes found primarily in tick and human samples from different regions of the globe, indicating a potential public health threat posed by  C. burnetii  in  Rh. microplus  in Yunnan.

Identification of Tick-Borne Pathogens and Genotyping of Coxiella burnetii in Rhipicephalus microplus in Yunnan Province, China