title: Quantitative PCR-based parasite burden estimation of Babesia gibsoni in the vector tick, Haemaphysalis longicornis (Acari: Ixodidae), fed on an experimentally infected dog.
abstract: Most causative agents of babesiosis, Babesia parasites, are transmitted transovarially in ixodid ticks. In this study, B. gibsoni, the causative agent of canine babesiosis which has transovarial transmission, was detected in tissues of the vector tick, Haemaphysalis longicornis using a modified quantitative PCR assay. Conventional PCR results showed that the newly designed primer set, which amplifies a 143-bp fragment of rhoptry-associated protein-1 (BgRAP-1) gene in B. gibsoni, was 100 times more sensitive than primers targeting P18 gene encoding 18 kDa protein of B. gibsoni, which was recently renamed as thrombospondin related adhesive protein (BgTRAP) gene, in an artificially generated sample solution containing metagenomic DNA (B. gibsoni DNA extracted from infected dog blood mixed with tick DNA). The TaqMan probe-based quantitative PCR (qPCR) for BgRAP-1 could also detect infected RBCs (iRBCs) at levels of 3.5 × 10(5) to 3.5 × 10(1)/μl, a range that is broader than that of a past SYBR Green-based qPCR method for P18/BgTRAP, which had a detection limit of 3.5 × 10(3) iRBCs/μl. Using this qPCR assay, we attempted to quantify the B. gibsoni burden in tick ovaries and embryonated eggs. Levels of infection were normalized to the copy number of tick's genomic DNA fragment of ribosomal DNA internal transcribed spacer region 2 (ITS2) for the standardization. According to this, low levels of parasite burden were quantified in ovaries and eggs. This detection system is sensitive and is recommended as a tool for elucidating the biological interactions between the vector tick H. longicornis and the parasite, B. gibsoni.
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hatta
authors.forename: Makoto
authors.initials: M
authors.lastname: Matsubayashi
authors.forename: Takeharu
authors.initials: T
authors.lastname: Miyoshi
authors.forename: Khyrul
authors.initials: K
authors.lastname: Islam
authors.forename: M Abdul
authors.initials: MA
authors.lastname: Alim
authors.lastname: Anisuzzaman
authors.forename: Kayoko
authors.initials: K
authors.lastname: Yamaji
authors.forename: Kozo
authors.initials: K
authors.lastname: Fujisaki
authors.forename: Naotoshi
authors.initials: N
authors.lastname: Tsuji
mesh_terms: D000818:Animals; D001403:Babesia / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004283:Dog Diseases / Q000469:parasitology*; D004285:Dogs; D005260:Female; D026863:Ixodidae / Q000469:parasitology*; D010053:Ovary / Q000469:parasitology; D010063:Ovum / Q000469:parasitology; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D013984:Tick Infestations / Q000662:veterinary*
chemical_list: D017931:DNA Primers; D015800:Protozoan Proteins; C097551:rhoptry-associated protein-1, Babesia