Quantitative PCR-Based Parasite Burden Estimation of Babesia gibsoni in the Vector Tick, Haemaphysalis longicornis (Acari : Ixodidae), Fed on an Experimentally Infected Dog

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  • HATTA Takeshi
    Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan
  • MATSUBAYASHI Makoto
    Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan
  • MIYOSHI Takeharu
    Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan
  • ISLAM Md. Khyrul
    Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan
  • ALIM M. Abdul
    Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan
  • Anisuzzaman
    Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan
  • YAMAJI Kayoko
    Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan
  • FUJISAKI Kozo
    Laboratory of Emerging Infectious Diseases, School of Frontier Veterinary Medicine, Kagoshima University, Korimoto, Kagoshima 890–0065, Japan
  • TSUJI Naotoshi
    Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan

書誌事項

タイトル別名
  • Quantitative PCR-Based Parasite Burden Estimation of <i>Babesia gibsoni</i> in the Vector Tick, <i>Haemaphysalis longicornis </i>(Acari: Ixodidae), Fed on an Experimentally Infected Dog

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抄録

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 × 105 to 3.5 × 101/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 × 103 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.

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