Rhipicephalus microplus ticks are vectors for multiple pathogens infecting animals and humans. Although the medical importance of R. microplus has been well-recognized and studied in most areas of China, the occurrence of tick-borne Rickettsiales has seldom been investigated in Guizhou Province, Southwest China. In this study, we collected 276 R. microplus ticks from cattle (209 ticks) and goats (67 ticks) in three locations of Guizhou Province. The Rickettsia, Anaplasma, and Ehrlichia were detected by targeting the 16S rRNA gene and were further characterized by amplifying the key genes. One Rickettsia (Ca. Rickettsia jingxinensis), three Ehrlichia (E. canis, E. minasensis, Ehrlichia sp.), and four Anaplasma (A. capra, A. ovis, A. marginale, Ca. Anaplasma boleense) species were detected, and their gltA and groEL genes were recovered. Candidatus Rickettsia jingxinensis, a spotted fever group of Rickettsia, was detected in a high proportion of the tested ticks (88.89%, 100%, and 100% in ticks from the three locations, respectively), suggesting the possibility that animals may be exposed to this type of Rickettsia. All the 16S, gltA, groEL, and ompA sequences of these strains are 100% identical to strains reported in Ngawa, Sichuan Province. E. minasensis, A. marginale, and Candidatus Anaplasma boleense are known to infect livestock such as cattle. The potential effects on local husbandry should be considered. Notably, E. canis, A. ovis, and A. capra have been reported to infect humans. The relatively high positive rates in Qianxinan (20.99%, 9.88%, and 4.94%, respectively) may indicate the potential risk to local populations. Furthermore, the genetic analysis indicated that the E. minasensis strains in this study may represent a variant or recombinant. Our results indicated the extensive diversity of Rickettsiales in R. microplus ticks from Guizhou Province. The possible occurrence of rickettsiosis, ehrlichiosis, and anaplasmosis in humans and domestic animals in this area should be further considered and investigated.

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