Bacteria in the order Rickettsiales include some of the most important zoonotic (re)emerging
pathogens for animals and humans. In 2012, a total of 1267 adult Haemaphysalis longicornis
ticks were collected from domestic animals (cattle and sheep) in Shenyang of Liaoning
Province, China. These ticks were grouped into 181 pools (each pool with 6-7 ticks).
Rickettsiales agents were identified in 93 (51.38%) tick pools using PCR targeting
rrs (16S rRNA). In addition to 16S rRNA gene sequences, gltA and groEL gene sequences
were also recovered from these positive samples. Phylogenetic analyses of these sequences
revealed the presence of five species of Rickettsiales bacteria in a single tick species
(H. longicornis) in nature in Shenyang, including Rickettsia japonica, Rickettsia
raoultii, Anaplasma centrale, Anaplasma bovis, and a potentially novel A. phagocytophilum
variant. Additionally, two Candidatus Ehrlichia spp. (Ehrlichia sp. Yonaguni138, Candidatus
Ehrlichia shimanensis) were also identified in these ticks, with the highest prevalence
of Ehrlichia sp. Yonaguni138 (73/181, 40.3%). Notably, these agents except the novel
A. phagocytophilum variant had close evolutionary relationships with those previously
identified in northeastern Asian countries including Korea, Japan, and Russia, indicating
a geographic clustering pattern. Our data also reinforce the need for vigilance in
recognition and prevention of rickettsiosis, ehrlichiosis, and anaplasmosis in humans
and animals in this region.