title: PCR detection of Babesia ovata from questing ticks in Japan.
abstract: Babesia ovata is a tick-transmitted hemoprotozoan parasite of cattle. In the present study, we analyzed tick DNA samples (n=1459) prepared from questing ticks collected from various cattle pastures in Hokkaido (Shibecha, Taiki, Otofuke, Memuro, and Shin-Hidaka districts) and Okinawa (Yonaguni Island) prefectures of Japan for B. ovata. When all the tick DNA samples were screened by a previously described B. ovata-specific apical membrane antigen-1 (AMA-1) gene-based polymerase chain reaction (PCR) assay, none of the DNA samples was positive. Therefore, we developed a PCR assay based on the protozoan beta-tubulin (β-tubulin) gene to detect B. ovata from ticks in Japan. In the specificity test, the PCR assay amplified the expected 444-bp target gene fragment from B. ovata DNA. No PCR products were amplified from DNA samples from other blood pathogens, bovine blood, or ticks. In addition, the PCR assay detected 100 fg of B. ovata-genomic DNA extracted from an in vitro culture of the parasites. Subsequently, when all the tick DNA samples were screened by this new PCR assay, 18 were positive for B. ovata. Positive samples were found only in the Yonaguni and Memuro areas. In Okinawa, where all the ticks were identified as Haemaphysalis longicornis, 9.7% of the samples were PCR-positive, while a single tick (Ixodes ovatus) from Memuro was infected with B. ovata. When the nucleotide sequences of the PCR amplicons were phylogenetically analyzed, they formed a separate clade containing a previously described β-tubulin gene sequence from B. ovata (Miyake strain), confirming that the PCR assay had detected only B. ovata from the tick DNA samples. This is the first report that describes the PCR detection of B. ovata in ticks. The findings warrant transmission experiments to evaluate I. ovatus as a potential vector of B. ovata.
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan.
authors.forename: Thillaiampalam
authors.initials: T
authors.lastname: Sivakumar
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan.
authors.forename: Muncharee
authors.initials: M
authors.lastname: Tattiyapong
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Okubo
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Suganuma
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Hayashida
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Igarashi
authors.affiliation: Okinawa Prefectural Institute of Animal Health, Naha, Okinawa 900-0024, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Zakimi
authors.affiliation: Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Matsumoto
authors.affiliation: Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Inokuma
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan. Electronic address: yokoyama@obihiro.ac.jp.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Yokoyama
mesh_terms: D000818:Animals; D001403:Babesia / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001404:Babesiosis / Q000453:epidemiology* / Q000469:parasitology; D001483:Base Sequence; D002417:Cattle; D002418:Cattle Diseases / Q000453:epidemiology / Q000469:parasitology*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D007564:Japan / Q000453:epidemiology; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000662:veterinary; D015800:Protozoan Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000662:veterinary; D013984:Tick Infestations / Q000453:epidemiology / Q000469:parasitology*; D013987:Ticks / Q000469:parasitology*; D014404:Tubulin / Q000235:genetics*
chemical_list: D016054:DNA, Protozoan; D015800:Protozoan Proteins; D014404:Tubulin
keywords: Babesia ovata; Beta-tubulin; Japan; PCR; Ticks