First molecular evidence of Rickettsia massiliae and R. slovaca in Rhipicephalus microplus ticks from China.

Rhipicephalus microplus is a critical cattle ectoparasite and a vector of pathogens, including bacteria and protozoan parasites, that cause diseases such as bovine anaplasmosis and babesiosis. To better understand the pathogen profile of this tick in China, we collected 592 tick specimens from seven provinces. Using conventional or nested PCR, we detected a range of pathogens. Six Anaplasma species were identified (prevalence: 3 - 21%), with the highest detection rate in Hunan Province (24%). Spotted fever group rickettsiae, including R. raoultii (21%), Candidatus R. jingxinensis (12%), R. slovaca (8%), R. heilongjiangensis (3%), and R. massiliae (0.7%), were also prevalent. Low rates of Ehrlichia species (1 - 3%) were found. Among protozoans, 71 samples were positive for Theileria species (T. annulata, T. sergenti, T. buffeli, T. sinensis, and T. orientalis), while Babesia bigemina was only detected in Hunan (n = 5) and Henan (n = 1). No Borrelia species were detected. Bacterial co-infections were frequent, particularly between Anaplasma and Rickettsia species. This study reports the first detection of the emerging human pathogens R. slovaca and R. massiliae in R. microplus, revealing a distribution linked to geographic region and tick lineage. Our findings expand the knowledge of tick-borne pathogens in China and highlight their potential public health risks.