Co-circulation of multiple species of Rickettsiales bacteria in one single species of hard ticks in Shenyang, China

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.