Clinical pathobiological studies on Sarcocystosis

对4种肉孢子虫(肉孢子虫、山羊肉孢子虫、多毛肉孢子虫和卵巢肉孢子虫)进行了临床病理检查。肉孢子虫的体外培养和冷冻保存：标准的前处理方法是将孢子囊在次氯酸钠(NaOCl)溶液中于室温下预孵育。然后，孢子囊在39℃的培养液(RPMI1640，10%胎牛血清和15%牛胆汁)中孵育1小时。对处理过的孢子囊进行额外的超声波处理可增加排泄率。1~9月龄猪链球菌孢子囊率分别为77%、77%、72%和92%。子孢子在含有10%FCS和7.5%DMSO的RPMI1640中进行超低温保存，在液氮中保存后，证明子孢子对其中间宿主具有感染性。体外分离肉孢子虫子孢子的纯化：出胞肉孢子虫子孢子为Purifi…用DE 52阴离子交换纤维素柱分离。分离的子孢子通过柱层析后，平均回收率分别为88%、71%、77%和74%。子孢子多为致死性的，悬浮液中无激发物。4个肉孢子虫子孢子的同工酶图谱：用优化的提取工艺获得的4个肉孢子虫子孢子经冷冻、解冻、超声定位后进行裂解。与碳水化合物代谢相关的16种酶的酶筛选试验。测定乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)和葡萄糖-6-磷酸脱氢酶(G6PD)活性。在这3种酶的同工酶图谱上，4种肉孢子虫均表现出1条LDH活性的单条带，但在肉孢子虫和肉孢子虫中的迁移率高于其余2种肉孢子虫。在MDH活性上，多毛链霉菌只有1条单条酶带，而在同一部位有一条弥漫的条带，而华支链球菌和羊链球菌则有多条酶带。在G6PD活性上，4个样品均检测到2条窄带，但迁移距离无明显差异。较少

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