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We collected 1,084 ticks by flagging vegetation in Tsukuba and Moriya (Ibaraki, Japan), where several cases of canine granulocytic anaplasmosis were reported. The DNA of the collected ticks was molecularly examined for infection with the family Anaplasmataceae. Twenty-six positive samples of Anaplasmataceae-specific PCR of partial 16S rRNA gene were subjected to semi-nested PCR, covering the divergent regions, the gene, and sequence analysis. Anaplasma phagocytophilum was detected in 3 pools of Haemaphysalis longicornis larvae and A. bovis from a H. flava male. Sequences of both amplicons had high homologies to those from dogs in our previous studies in Ibaraki. These results suggest that Haemaphysalis ticks are candidate vectors of A. phagocytophilum and A. bovis in Ibaraki, Japan.

We evaluated the prevalence of Anaplasma infection in 332 dogs from Ibaraki, Japan, using serological and molecular methods. An immunofluorescence antibody assay against Anaplasma phagocytophilum indicated that 7 of the 328 serum samples tested (2.1%) were positive for A. phagocytophilum. Screening by polymerase chain reaction (PCR) analysis demonstrated that 8 of the 331 peripheral blood samples tested (2.4%) were positive for Anaplasmataceae. Phylogenetic analysis of the partial 16S rRNA sequence of the PCR amplicons revealed that 6 sequences were most similar to the 16S rRNA sequence of a Wolbachia sp., and the remaining 2 to A. bovis. Further analysis by A. phagocytophilum-specific nested PCR demonstrated that 1 dog infected with A. bovis was also positive for A. phagocytophilum. This is the first study to report the dual infection of a dog in Japan with A. bovis and A. phagocytophilum.

Anaplasma phagocytophilum DNA was detected from a dog with canine granulocytic anaplasmosis (CGA) in Japan. Phylogenetic analysis of the DNA using 16S rRNA, gltA, and groEL sequences revealed that the strain was nearly identical to A. phagocytophilum detected from Apodemus agrarius (black-striped field mouse) in China and Korea. To our knowledge, this is the first report of molecular detection and phylogenetic analysis of A. phagocytophilum from a clinical case of CGA in Japan.