Molecular evidence of Coxiella-like microorganism harbored by Haemaphysalis concinnae ticks in the Russian far east

Twenty female imago of Haemaphysalis concinnae ticks collected from wild vegetation in May of 2001 in the Obor region near Khabarovsk in the Russian Far East were studied. DNA was extracted with commercially available kits following manufacturer's instructions. We used broad range primers SFG3 and SFG6 proposed for amplification of 16S ribosomal RNA gene's portion of spotted fever group Rickettsia. Initial amplification resulted in positive results in all 20 ticks. Direct sequencing of five randomly chosen amplicons showed that obtained sequences belong to a new species. Closest homology was found in Coxiella burnetii 16S rDNA. Using the obtained sequence as a basis, we designed internal primers highly specific for that sequence. Then we confirmed our results by nested PCR amplification with newly designed primers Cox1 and Cox2-all 20 samples also were positive. Having combined these primers with universal eubacterial primers fD1 and rP2 we retrieved a 1,043 bp portion of the 16S rRNA gene. Consecutive homology search demonstrated that the closest sequence similarity belongs to new Coxiella sp. recently found in Rhipicephalus sanguineus ticks (95.2% of homology) and Coxiella burnetii (94% of homology). So, our results suggest that a novel Coxiella-like microorganism, provisionally called here "Cenerentola," is harbored by Haemaphysalis concinnae ticks in the Russian Far East.