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Clinical pathobiological studies on Sarcocystosis

肉孢子虫病的临床病理生物学研究

基本信息

批准号：
04454120
负责人：
ONO Kenichiro
金额：
$ 4.22万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

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 … More 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

通过酶学分析对4种肉孢子虫（阿里肉孢子虫、山羊肉孢子虫、希尔西卡尼斯肉孢子虫、羊肉孢子虫）进行临床病理学检查。 1．肉孢子虫属物种的体外包囊和冷冻保存：通过在室温下在次氯酸钠（NaOCl）溶液中预孵育孢子来进行标准预处理。然后，将孢子被在排胞液（RPMI 1640、10% FCS和15%牛胆汁）中于39°C孵育1小时。对预处理的孢子被进行额外的超声处理会增加胞囊排出率。 1-9 个月大的孢子被的脱囊率分别为 S.capracanis 77%、S.hircicanis 77%、S.arieticanis 72% 和 S.ovicanis 92%。子孢子也用含有10％FCS和7.5％DMSO的RPMI 1640进行冷冻保存。液氮保存后，证明子孢子对其中间宿主具有感染性。 2．体外排出的肉孢子虫子孢子的纯化：通过 DE 52 阴离子交换纤维素柱纯化排出的肉孢子虫子孢子。排出的子孢子通过柱后，S.capracanis 的平均回收率为 88%，S.hircicanis 为 71%，S.ovicanis 为 77%，S.arieticanis 为 74%。子孢子大部分为孢子状，虫悬液中无排泄碎片。3． 4个肉孢子虫子孢子的同工酶谱：通过冷冻和解冻，然后超声处理，将通过优化的排泄技术获得的4个肉孢子虫子孢子裂解。 16种与碳水化合物代谢相关的酶的酶筛选分析4个样品均检测到乳酸脱氢酶（LDH）、苹果酸脱氢酶（MDH）和葡萄糖-6-磷酸脱氢酶（G6PD）活性。在这 3 种酶的同工酶模式上，所有 4 个肉孢子虫属物种均显示出 1 个单条 LDH 活性带，其中 S.ovicanis 和 S.capracaniis 中的阳极迁移程度高于其余 2 个物种。在MDH活性中，在S.hircicanis中显示出1个单条带，而在S.arieticanis中在相同位置检测到更分散的条带，并且在S.ovicanis和S.capracanis中显示出多个条带。在G6PD活性方面，4个样品均检测到2条窄带，但迁移距离没有观察到差异。较少的