1994 Fiscal Year Annual Research Report

DNAプローブによる住血微生物感染症の早期診断と鑑別法の開発

Research Project

Project/Area Number	04556043
Research Institution	HOKKAIDO UNIVERSITY
Principal Investigator	杉本千尋北海道大学, 獣医学部, 助教授 (90231373)
Co-Investigator(Kenkyū-buntansha)	岡部達二微生物化学研究所(株), 主任研究員高橋清志酪農学園大学, 酪農学部, 教授 (90048108) 小沼操北海道大学, 獣医学部, 教授 (70109510)
Keywords	DNAプローブ / PCR / 住血微生物 / 原虫 / ウシ
Research Abstract	前年度までにTheileria sergentiの高感度検出を可能とする主要表面抗原(p32)遺伝子を標的としたPCRを開発した。p32遺伝子には現在まで3種の対立遺伝子(allele)の存在が知られているが、PCRでの型別が可能か、また小型ピロプラズマ感染症の疫学研究に応用しうるかを今年度は主に検討した。すなわち池田型、千歳型、buffeli型のalleleに特異的な塩基配列からプライマーを設計し、PCRの反応条件を検討した結果、3つの型が特異的に増幅しうることが確認された。型の特異性は増幅産物を制限酵素で切断したパターンによって確認した。さらに、日本国内で分離された野外株および実験継代株計29株について本法で検査したところ、5株を除いて複数のp32 alleleが検出され、多数の株は複数のT.sergenti原虫が混在していることが明らかとなった。また、中国、韓国など東アジア諸国で分離されたT.sergenti株のDNAを解析したところ、いずれも池田型、千歳型、buffeli型の2-3型の混合であることが明らかにできた。また、本PCR法によって、持続感染牛体内で優勢に出現する原虫のalleleの変換が起こること、ダニ・牛間での病原体の伝播によっても同様な現象が起こることも確認できた。今回開発されたallele特異的PCR法は野外での小型ピロプラズマ感染症の分子疫学的調査に極めて有効な手法で、今後のワクチン開発と効果判定など防圧法の開発上重要な解析データを提供しうる手法である。本法はほぼ実用化の段階に達していると考えられ、今後野外調査での普及を図ってゆく。

Research Products

(13 results)

All Other

All Publications (13 results)

[Publications] TANAKA,M.: "Detection of Theileria sergenti infection in cattle by using polymerase chain reaction amplification of parasite specific DNA." Journal of Clinical Microbiology. 31. 2565-2569 (1993)
[Publications] ALI,S.: "Protein characterization of Babesia equi piroplasms isolated from infected horse erythrocytes." Parasitology Research. 79. 639-643 (1993)
[Publications] MATSUBA,T.: "Changes in the hybridization patterns of populations of Theileria sergenti during infection." Veterinary Parasitololgy. 47. 215-223 (1993)
[Publications] MATSUBA,T.: "Analysis of mixed parasite populations of Theileria sergenti using cDNA probes encoding a major piroplasm surface protein." Parasitology,. 107. 369-377 (1993)
[Publications] ZHONG,W.Z.: "Characterization of epitopes on a 32 kDa merozoite surface protein of Theileria sergenti." Parasite Immunology. 15. 113-119 (1993)
[Publications] KUBOTA,S.: "Molecular cloning and sequencing of hsp70cDNA of Theileria sergenti." Molecular and Biochemical Parasitology. 62. 317-318 (1993)
[Publications] ZHUANG,W.Z.: "Analyses of antigenic diversities of Theileria sergenti piroplasm surface proteins" Journal of Veterinary Medical Science. 56. 469-473 (1994)
[Publications] WATARAI,S.: "Alteration of ganglioside composition in erythrocytes associated with Theileria sergenti infection." Journal of Veterinary Medical Science. 56. 841-847 (1994)
[Publications] SUGIMOTO,C.: "Characterization of membrane proteions of Babesia equi piroplasm." Equine Infectious Diseases VII. (発表予定).
[Publications] ONOUE,S.: "Prenatal infection with Theileria sergenti in calves." Journal of Protozoology Research. (発表予定).
[Publications] WATARAI,S.: "Ganglioside as a possible receptor on the erythrocyte for Theileria sergenti." Journal of Veterinary Medical Science. (発表予定).
[Publications] ZHUANG,W.Z.: "Antigenic alteration in major piroplasm surface proteins of Theileria sergenti during infection." Veterinary Parasitology. (発表予定).
[Publications] S.KUBOTA: "A genetic analysis of mixed population in Theileria sergenti stocks and isolates using allele-specific polymerase chain reaction." Journal of Veterinary Medical Science. (発表予定).

1994 Fiscal Year Annual Research Report

DNAプローブによる住血微生物感染症の早期診断と鑑別法の開発

Principal Investigator

杉本 千尋 北海道大学, 獣医学部, 助教授 (90231373)

Research Products

[Publications] TANAKA,M.: "Detection of Theileria sergenti infection in cattle by using polymerase chain reaction amplification of parasite specific DNA." Journal of Clinical Microbiology. 31. 2565-2569 (1993)

[Publications] ALI,S.: "Protein characterization of Babesia equi piroplasms isolated from infected horse erythrocytes." Parasitology Research. 79. 639-643 (1993)

[Publications] MATSUBA,T.: "Changes in the hybridization patterns of populations of Theileria sergenti during infection." Veterinary Parasitololgy. 47. 215-223 (1993)

[Publications] MATSUBA,T.: "Analysis of mixed parasite populations of Theileria sergenti using cDNA probes encoding a major piroplasm surface protein." Parasitology,. 107. 369-377 (1993)

[Publications] ZHONG,W.Z.: "Characterization of epitopes on a 32 kDa merozoite surface protein of Theileria sergenti." Parasite Immunology. 15. 113-119 (1993)

[Publications] KUBOTA,S.: "Molecular cloning and sequencing of hsp70cDNA of Theileria sergenti." Molecular and Biochemical Parasitology. 62. 317-318 (1993)

[Publications] ZHUANG,W.Z.: "Analyses of antigenic diversities of Theileria sergenti piroplasm surface proteins" Journal of Veterinary Medical Science. 56. 469-473 (1994)

[Publications] WATARAI,S.: "Alteration of ganglioside composition in erythrocytes associated with Theileria sergenti infection." Journal of Veterinary Medical Science. 56. 841-847 (1994)

[Publications] SUGIMOTO,C.: "Characterization of membrane proteions of Babesia equi piroplasm." Equine Infectious Diseases VII. (発表予定).

[Publications] ONOUE,S.: "Prenatal infection with Theileria sergenti in calves." Journal of Protozoology Research. (発表予定).

[Publications] WATARAI,S.: "Ganglioside as a possible receptor on the erythrocyte for Theileria sergenti." Journal of Veterinary Medical Science. (発表予定).

[Publications] ZHUANG,W.Z.: "Antigenic alteration in major piroplasm surface proteins of Theileria sergenti during infection." Veterinary Parasitology. (発表予定).

[Publications] S.KUBOTA: "A genetic analysis of mixed population in Theileria sergenti stocks and isolates using allele-specific polymerase chain reaction." Journal of Veterinary Medical Science. (発表予定).

杉本千尋北海道大学, 獣医学部, 助教授 (90231373)