SASAI Kazumi Osaka Pref.Univ., Col.of Agric., Res.associate, 農学部, 助手 (70211935)
FUKATA Tuneo Osaka Pref.Univ., Col.of Agric., Assistant prof., 農学部, 講師 (80081595)
馬場 栄一郎 大阪府立大学, 農学部, 助教授 (70081594)
|Budget Amount *help
¥7,200,000 (Direct Cost : ¥7,200,000)
Fiscal Year 1997 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1996 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1995 : ¥4,300,000 (Direct Cost : ¥4,300,000)
Invasive protozoa have been known to recognize specific sugar residues of host cell surface and use the apical complex at the penetration into cells. At the first step of this series of study, the effects of carbohydrates on the penetration of Eimeria tenella sporozoites into cultured cells were investigated. The penetration of sporozoites was sppressed when pretreated with peanut lectin that specifically recognizes D-galactos residues at a concentration of 50 ml.ml. At the second step, to producing chicken monoclonal antibody against apical complex of Eimeria to get more knowlede during the invasion. Many stable chicken hybridoma secreting a monoclonal antibody (mAb) that detect the apical complex of Eimeria acervulina sporozoites has been developed by fusing a thymidine kinase (TK) -deficient chicken myeloma with spleen cells from chickens immunized with sporozoite antigen. One of the mAbs, designated as 6D-12-G10, recognized the apical complex of a sporozoite of 20-21 kDa molecular
mass on western blots. Immunoelectron microscopic examination revealed that mAb 6D-12-G10 stained the conoid antigen. Furthermore, mAb 6D-12-G10 inhibited the invasion of sporozoites into CD8+ T cells in vitro. At the next step, the species-specificity and cross-reactivity of chicken developed against E.acervulina (EA) sporozoite were determined by confocal laser scanning microscopy and Western blotting analysis using the sporozoites of seven different avian Eimeria species such as EA,E.brunetti (EB), E.maxima (EM), E.mitis, E.necatrix, E.praecox and E.tenella. The five different mAbs, named 8E-1, HE-4,8D-2,5D-11 and 8C-3 were used in this study. In the immunofluorescent examination, these mAbs showed similar reactivity on the apical complex of the sporozoite. One of these mAbs, 8E-1 cross-reacted with all Eimeria species that were examined, HE-4 and 8D-2 mAbs reacted with EA and EB,5D-11 mAb reacted with EA and EM,8C-3 mAb reacted with only EA.
In Western blot analysis using sonicated aporozoites of the seven of the Eimeria species as antigen, all mAbs recognized multiple bands ; the predominant bands of mAbs had molecular weights of approximately 32,43 and 260 kilodaltons. The present results of specific sugar residues of host cell surface suggested that D-galactose residues on E.tenella sporozoite surfaces and the lectin-like receptors that recognize D-galactose of host cells are important factors for penetration.
Those monolconal antibodies might help more clear cut results of parasites invasion into the host cell. And also we might continue to study the relationship between the antigens which were recognized by mAbs and sugar residues. The availability of a technique to develop chicken mAbs should greatly enhance our ability to study the role of individual molecules involved in attachment, invasion, and motility. These molecules may prove to be important and novel targets for immunological and pharmacological therapy against coccidial infection. Less