| Summary: | <i>Rhipicephalus microplus</i> ticks are vectors for multiple pathogens infecting animals and humans. Although the medical importance of <i>R. microplus</i> 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 <i>R. microplus</i> ticks from cattle (209 ticks) and goats (67 ticks) in three locations of Guizhou Province. The <i>Rickettsia</i>, <i>Anaplasma</i>, and <i>Ehrlichia</i> were detected by targeting the 16S rRNA gene and were further characterized by amplifying the key genes. One <i>Rickettsia</i> (<i>Ca.</i> Rickettsia jingxinensis), three <i>Ehrlichia</i> (<i>E. canis</i>, <i>E. minasensis</i>, <i>Ehrlichia</i> sp.), and four <i>Anaplasma</i> (<i>A. capra</i>, <i>A. ovis</i>, <i>A. marginale</i>, Ca. Anaplasma boleense) species were detected, and their <i>gltA</i> and <i>groEL</i> genes were recovered. <i>Candidatus</i> Rickettsia jingxinensis, a spotted fever group of <i>Rickettsia</i>, 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 <i>Rickettsia</i>. All the 16S, <i>gltA</i>, <i>groEL</i>, and <i>ompA</i> sequences of these strains are 100% identical to strains reported in Ngawa, Sichuan Province. <i>E. minasensis</i>, <i>A. marginale</i>, and <i>Candidatus</i> Anaplasma boleense are known to infect livestock such as cattle. The potential effects on local husbandry should be considered. Notably, <i>E. canis</i>, <i>A. ovis</i>, and <i>A. capra</i> 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 <i>E. minasensis</i> strains in this study may represent a variant or recombinant. Our results indicated the extensive diversity of Rickettsiales in <i>R. microplus</i> 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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