Molecular Analysis of Anaplasma phagocytophilum Isolated from Patients with Febrile Diseases of Unknown Etiology in China

Although anaplasmosis cases have been nationally identified in China, no human isolates of A. phagocytophilum have been obtained, which limits the analysis of any molecular and genetic contributions to patients' severe clinical manifestations and the study of the bacteria's pathogeneses in China. Given this situation, a joint project was conducted in 2009-2010. A total of 421 febrile cases of unknown etiology were collected and the patients' blood samples were collected for laboratory diagnoses including serologic diagnosis based on the four-fold rise in the anti- A. phagocytophilum IgG titer by indirect micro-immunofluorescence assay (IFA), positive PCR assay and confirmation of A. phagocytophilum DNA and positive culture of A. phagocytophilum and confirmed by amplification and sequencing of the 16S rRNA and ank A genes of the A. phagocytophilum isolates. A total of 570 ticks were collected from the patients' domestic animals (456) and from wild fields (114) for culturing and amplifying and sequencing the 16S rRNA gene of A. phagocytophilum. Phylogenetic analyses were performed on the 16S rRNA and ank A gene sequences of the isolates and the ticks tested in the study. A total of 46 (10.9%) confirmed and 16 (3.8%) probable cases were diagnosed and severe clinical features and higher mortality rates were observed in these Chinese patients. Five isolates were obtained and the 16S rRNA genes of the 5 isolates were conserved but variety for ank A genes. Two human isolates and 1 tick isolate from Shandong Peninsula, where all patients exhibited severe clinical manifestations, were grouped as one clan based on the phylogenetic analyses, while 2 other human isolates were clustered in a second clan. 43.5% of H. longicornis were infected with A. phagocytophilum.The present study is the first to obtain clinical isolates of A. phagocytophilum in China. The diversity of the ank A genes of Chinese isolates will help us to further discern the relationship between the variations in the ank A genes and the severity of the disease's clinical manifestations in China.