Grant-in-Aid for Scientific Research (B)
|Allocation Type||Single-year Grants|
Applied veterinary science
|Research Institution||Azabu University|
KANEUCHI Choji Azabu University, School of Veterinary Medicine, Professor, 獣医学部, 教授 (20087597)
KIUCHI Akio Azabu University, School of Veterinary Medicine, Professor, 獣医学部, 教授 (60120953)
FUKUYAMA Masafumi Azabu University, College of Environmental Health, Professor, 環境保健学部, 教授 (40075932)
MATSUDA Motoo Azabu University, College of Environmental Health, Professor, 環境保健学部, 教授 (50139531)
CHUMA Takehisa Kagoshima University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (90201631)
ITOH Kikuji The University of Tokyo, Graduate School of Agricultural and Life sciences, Associate Professor, 大学院・農学生命科学研究科, 助教授 (50100045)
|Project Period (FY)
1997 – 1999
Completed(Fiscal Year 1999)
|Budget Amount *help
¥12,100,000 (Direct Cost : ¥12,100,000)
Fiscal Year 1999 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1998 : ¥3,900,000 (Direct Cost : ¥3,900,000)
Fiscal Year 1997 : ¥6,200,000 (Direct Cost : ¥6,200,000)
|Keywords||Campylobacter / Identification / Detection / Strain discrimination / PCR / Invasion into cells / Urease / Attachment to cells / ニワトリ / 血清型別 / コンセンサスプライマー / カンピロバクター / パルスフィールドゲル電気泳動 / 16SrDNA配列|
1. Recently much attention has been paid to urease-positive thermophilic Campylobacter (UPTC) isolated from natural environment. DNA analysis was carried out of approximately 60 strains of UPTC widely collected from Japan, North Ireland, England, France and the Netherlands. Most of the strains studied had different genotypes thus suggesting the diversity of genomes and possibility of strain discrimination. The genomes sizes were found to be 1.6 to 1.9Mb.
2. Multiplex PCR method was established for quick and simultaneous identification of C.jejuni, C.coli, and C.lari. Using this method as well as southern blot method, C.jejuni could directly be detected in the feces of chickens. Possibility of the vertical transmission of C.jejuni and C.coli from hen to chick was demonstrated to be quite low by the RFLP analysis of flagellin gene of C.jejuni strains isolated from chickens.
3. Attachment degrees to INT-407 cells were found to be similar for the strains of C.jejuni isolated from the disease
d and healthy chickens. However, the invasion activity into the cells differed from strain to strain with the clear tendency of the clinical strains showing higher activity of invasion than the strains from healthy chickens. Furthermore, the invasion activity was impeded by different anti-invasion agents. These suggested that the invasion mechanism is diverse in association with the diverse transmission modes and clinical symptoms by C.jejuni.
4. Identification of Campylobacter species and strain discrimination revealed the following details by using PCR-RFLP analysis based on the base sequence of 16S rRNA :
(1) C.upsaliensis was distinguished from C.jejuni, C.coli, and C.lari by digestion pattern with restriction enzyme. Dde I of the respective amplified RNA of the 4 species when consensus primer set _<45/>47 was used.
(2) C.coli strains did not yielded the PCR product with 6 different sets of primers suggesting that there are strain variations in the 5' and 3' terminal base sequences.
(3) C.lari strains amplified DNA segments only when consensus primer _<45/>47 was used. Less