MATSUKI Naoaki Fac.of Agr., Univ.of Tokyo Ass.Prof., 農学部, 助手 (40251417)
NAKAYAMA Hiroyuki Fac.of Agr., Univ.of Tokyo Aso.Prof., 農学部, 助教授 (40155891)
KONO Michiko Fac.of Agr., Univ.of Tokyo Ass.Prof., 農学部, 助手 (70092202)
HASEGAWA Atsuhiko Fac.of Agr., Univ.of Tokyo Prof., 農学部, 教授 (90011923)
SUZUKI Naoyoshi Proto.Res.Center, Obihiro Univ.Prof., 畜産学部, 教授 (10003071)
後飯塚 僚 東京大学, 農学部, 助手 (60205581)
|Budget Amount *help
¥6,600,000 (Direct Cost : ¥6,600,000)
Fiscal Year 1993 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1992 : ¥5,200,000 (Direct Cost : ¥5,200,000)
Clinical pathological examination was performed in 4 Sarcocystis species(Sarcocystis arieticanis, S.capracanis, S.hircicanis, and S.ovicanis) by the enzymatic analysis.
1. In vitro excystation and cryoreservation of Sarcocystis species : A standard pretreatment was performed by preincubation of sporocysts in sodium hypochlorite(NaOCl) solution at room temperature. Then, sporocysts were incubated for 1 hr at 39 C in excystaion fluid(RPMI 1640, 10% FCS and 15% bovine bile). Additional sonication of pretreated sporocysts increased excystation rates. Excystaion rates for 1-9 months old sporocysts were 77% for S.capracanis, 77 % for S.hircicanis, 72 % for S.arieticanis and 92 % for S.ovicanis. Sporozoites were also subjected to cryopreservation in RPMI 1640 containing 10% FCS and 7.5% DMSO.After storage in liquid nitrogen, sporozoites proved to be infective for their intermediate hosts.
2. Purification of in vitro excysted Sarcocystis sporozoites : Excysted Sarcocystis sporozoites were purifi
ed by DE 52 anion-exchange cellulose column. After passage of excysted sporozoites through a column, the mean recovery was 88% for S.capracanis, 71% for S.hircicanis, 77% for S.ovicanis, and 74% for S.arieticanis. Most of the sporozoites were mortile, and the parasite suspension was free of excystation debris.
3. Isoenzyme profiles of 4 Sarcocystis sporozoites : The 4 Sarcocystis sporozoites obtained by an optimized excystation techniques were lysed by freezing and thawing, followed by ultrasonocation. On enzyme screening assay for 16 enzymes related to carbohydrate metabolism. Lactae dehydrogenase(LDH), malate dehydrogenase(MDH), and glucose-6-phosphate dehydrogenase(G6PD) activities were detected in all 4 samples. On isoenzyme patterns of these 3 enzymes, all 4 Sarcocystis species showed 1 single band of LDH activity with a more anodical migration in S.ovicanis and S.capracaniis than in the remaining 2 species. In MDH activity, 1 single band was shown in S.hircicanis whereas a more diffuse band was detected at same position in S.arieticanis and multiple band was shown in S.ovicanis and S.capracanis. In G6PD activity, 2 narrow bands were detected in all 4 samples, however, no difference was observed on their migration distances. Less