title: Molecular characterization and specific detection of Anaplasma species (AP-sd) in sika deer and its first detection in wild brown bears and rodents in Hokkaido, Japan.
abstract: A previously undescribed Anaplasma species (herein referred to as AP-sd) has been detected in sika deer, cattle and ticks in Japan. Despite being highly similar to some strains of A. phagocytophilum, AP-sd has never been detected in humans. Its ambiguous epidemiology and the lack of tools for its specific detection make it difficult to understand and interpret the prevalence of this Anaplasma species. We developed a method for specific detection, and examined AP-sd prevalence in Hokkaido wildlife. Our study included 250 sika deer (Cervus nippon yesoensis), 13 brown bears (Ursus arctos yesoensis) and 252 rodents including 138 (Apodemus speciosus), 45 (Apodemus argenteus), 42 (Myodes rufocanus) and 27 (Myodes rutilus) were collected from Hokkaido island, northern Japan, collected during 2010 to 2015. A 770 bp and 382 bp segment of the 16S rRNA and gltA genes, respectively, were amplified by nested PCR. Results were confirmed by cloning and sequencing of the positive PCR products. A reverse line blot hybridization (RLB) based on the 16S rRNA gene was then developed for the specific detection of AP-sd. The prevalence of AP-sd by nested PCR in sika deer was 51% (128/250). We detected this Anaplasma sp. for the first time in wild brown bears and rodents with a prevalence of 15% (2/13) and 2.4% (6/252), respectively. The sequencing results of the 16S rRNA and gltA gene amplicons were divergent from the selected A. phagocytophilum sequences in GenBank. Using a newly designed AP-sd specific probe for RLB has enabled us to specifically detect this Anaplasma species. Besides sika deer and cattle, wild brown bears and rodents were identified as potential reservoir hosts for AP-sd. This study provided a high throughput molecular method that specifically detects AP-sd, and which can be used to investigate its ecology and its potential as a threat to humans in Japan.
authors.affiliation: Laboratory of Wildlife Biology and Medicine, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. Electronic address: m.abdallah@vetmed.hokudai.ac.jp.
authors.forename: Mohamed Abdallah Mohamed
authors.initials: MAM
authors.lastname: Moustafa
authors.affiliation: Laboratory of Wildlife Biology and Medicine, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. Electronic address: rith19@hotmail.com.
authors.forename: Kyunglee
authors.initials: K
authors.lastname: Lee
authors.affiliation: Laboratory of Wildlife Biology and Medicine, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. Electronic address: ky.t@ufl.edu.
authors.forename: Kyle
authors.initials: K
authors.lastname: Taylor
authors.affiliation: Unit of Risk Analysis and Management, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan. Electronic address: ryo.nakao@czc.hokudai.ac.jp.
authors.forename: Ryo
authors.initials: R
authors.lastname: Nakao
authors.affiliation: Laboratory of Wildlife Biology and Medicine, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. Electronic address: msashika@vetmed.hokudai.ac.jp.
authors.forename: Mariko
authors.initials: M
authors.lastname: Sashika
authors.affiliation: Laboratory of Wildlife Biology and Medicine, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. Electronic address: shimozuru@vetmed.hokudai.ac.jp.
authors.forename: Michito
authors.initials: M
authors.lastname: Shimozuru
authors.affiliation: Laboratory of Wildlife Biology and Medicine, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. Electronic address: tsubota@vetmed.hokudai.ac.jp.
authors.forename: Toshio
authors.initials: T
authors.lastname: Tsubota
mesh_terms: D000709:Anaplasma / Q000145:classification / Q000235:genetics*; D000712:Anaplasmosis / Q000382:microbiology*; D000818:Animals; D004269:DNA, Bacterial / Q000097:blood / Q000235:genetics; D003670:Deer / Q000382:microbiology*; D007564:Japan; D058889:Molecular Typing; D010802:Phylogeny; D012377:Rodentia / Q000382:microbiology*; D001503:Ursidae / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial
keywords: Anaplasma phagocytophilum; Anaplasma species (AP-sd); Reverse line blot hybridization