| Project/Area Number |
03660323
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Applied veterinary science
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| Research Institution | THE UNIVERSITY OF TOKUSHIMA |
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Principal Investigator |
OTA Fusao TOKUSHIMA UNIVERSITY, SCHOOL OF MEDICINE, PROFESSOR, 医学部, 教授 (90035478)
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| Co-Investigator(Kenkyū-buntansha) |
HIROTA Katsuhiko TOKUSHIMA UNIVERSITY, SCHOOL OF DENTISTRY, RESEATCH ASSOCIATE, 歯学部, 助手 (60199130)
ONO Tsuneko TOKUSHIMA UNIVERSITY, SCHOOL OF DENTISTRY, LECTURER, 歯学部, 講師 (40035514)
FUKUI Komei TOKUSHIMA UNIVERSITY, SCHOOL OF DENTISTRY, PROFESSOR, 歯学部, 教授 (40035407)
TAMATO Masayuki TOKUSHIMA UNIVERSITY, SCHOOL OF MEDICINE, RESEATCH ASSOCIATE, 医学部, 助手 (90210492)
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| Project Period (FY) |
1991 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1993: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1992: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1991: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Anti-phagocytic factor / Group E streptococci / St.porcinus / Monoclonal antibody / HPLC / Surface antigen / Antigenic determinant / Mannose(Mannosamine) / 捕食実験 / 貧食 / 補体 / EIA / 特異性 / 蛋白抗原 / 抗補食因子 / 多糖抗原 |
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| Research Abstract |
Group E streptococci(GAS) have been reported to cause many purulent infections such as lymphadenitis in pigs. A vaccine has yet to be invented to protect domestic animals to ensure a constant supply to the meat market throughout the world . Monoclonal antibodies(mAbs) have been reported to be useful for identifying surface antigens of streptococci. Ota et al. previously identified several antigenic polysaccharides in oral streptococci and analyzed their antigenic determinants immunochemically by the use of several specific mAbs. The purpose of this study is to identify a factor(s) in group E streptococci, which has previously been reported to protect the bacterial cells against phagocytosis in pigs, despite the faci that St. porcinus including group E streptococci are generally not regarded as being highly pathogenic.
Three mAbs were obtained after fusing myeloma cells to the spleen cells of mice immunised with serotype II, IV or V of GES.They were designated mAb S-3-9, II-t, V16 and IV
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21. mAb S3-9 reacted with serotype e strains of St. mutans and all group E streptococcal strains. mAb II-T reacted with only serothpes II and V of St. porcinus. mAb V16 reacted with most serotypes whereas mAb IV21 reacted only with serotype IV or GES, respectively. GES cells were found to react with mAb S3-9, and to recognize the same polysaccharide antigen with a determinant of methyglucopyranoside, indicating that group E streptococci carry the carbohydrate antigen on the cell surface, mAb II-T was found to recognise a polysaccharide antigen with a chemical structure similar to mannosamine. mAbs V16 and IV21 also seemed to recognise polysaccharide antigens on the cell surface of S.porcinus after SDS-PAGE analysis and hapten inhibition tests. A carbohydrate antigen reacting with mAb II-T was successfully purified from cultured cells of a serotype II GES strain after extraction in hot saline and then sequential ion-and gel chromotographies. Chemical analyzes using paper, gel chromatographies and high performance liquid chromatography revealed that the antigen consisted of more than 95% sugar(glucose, galactos, rhamnose and some aminosugars) and no substantial protein.
In order to determine if this polysaccharide antigen was anti-phagocytic, mouse peritoneal exudate cells were collected and incubated with radiolabelled cells of GES.When cultured GES cells were mixed with the exudate cells and centrifuged under different conditions, the sedimented cells did not show any significant increase in cpm as compared to the negative or positive control experiments. It was also found that fresh serum from homologous animals did noto increase phagocytosis of GES cells when added to the system, indicating that the bacterium may have a factor(s) that interferes with the enhancing action of complement. This is in contrast with the results previously obtained by us with group B streptococci. A more sensitive method to quantify precisely the ability of exudate cells(phagocytes) was attempted by use of a fluoroscan to detect fluorescent-labelled antibody. Unfortunately the results to further investigate into the anti-phagocytic nature of the purified polysaccharide antigen mentioned above were not fully successful. However, in consideration of some previous reports regarding the immune suppressive properties of mannosamine against killer cells, the mannose-like determinant on the surtace of group E streptococci may be a factor for resistance to phagocytosis in the host animal. Less
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