雷丸及吡喹酮对猬迭宫绦虫裂头蚴感染性及超微结构的影响

Effects of Omphalia lapidescens and Praziquantel on the Infectivity and Ultrastructure of Spirometra erinacei Plerocercoids

目的了解雷丸及吡喹酮对猬迭宫绦虫裂头蚴感染性及超微结构的影响。方法从黑斑蛙体内检获裂头蚴,用20、40和80 mg/ml雷丸粉末混悬培养液以及20、80和320μg/ml吡喹酮培养液分别体外培养裂头蚴4、12和24 h,单纯培养液培养4、12和24 h为感染对照组。168只昆明小鼠均分为21组（每组8只）,每组小鼠经口感染相应条件培养后的裂头蚴各5条/鼠。感染后1周剖杀各组小鼠,计数每鼠裂头蚴检出数,并计算每组小鼠的减虫率。扫描电镜和透射电镜分别观察不同培养处理后的裂头蚴超微结构变化。结果小鼠感染经40和80 mg/ml雷丸粉末混悬液分别培养4、12和24 h的裂头蚴1周后,每组小鼠裂头蚴平均检出数分别为1.6、1.0和0.3条,0.3、0和0条,均显著低于对照组（4.1、3.5和3.3条）（P〈0.05）,且两组间差异有统计学意义（P〈0.05）;减虫率分别为60.0%、71.4%和90.1%,92.7%、100%和100%,且两组间差异有统计学意义（P〈0.05）。小鼠感染经320μg/ml吡喹酮溶液分别培养4、12和24 h的裂头蚴1周后,每组小鼠裂头蚴平均检出数分别为1.9、1.3和0.4条,与对照组间差异均有统计学意义（P〈0.05）;减虫率分别为53.7%、62.9%和87.9%,显著高于20μg/ml吡喹酮处理组的14.6%、2.9%和6.1%以及80μg/ml吡喹酮处理组的24.4%、17.1%和24.2%（P〈0.05）。扫描电镜和透射电镜观察可见,经40 mg/ml雷丸粉末混悬液培养4 h以及320μg/ml吡喹酮溶液培养4和12 h后,裂头蚴虫体出现轻度挛缩;经40 mg/ml雷丸粉末混悬液培养12和24 h,虫体微毛凝集、融合或脱落,质膜破裂,石灰小体释出,皮质组织受损;经80 mg/ml雷丸粉末混悬液培养4-12 h,虫体损害逐渐加重,至24 h,裂头蚴组织结构严重受损,无法辨清;经320μg/ml吡喹酮溶液培养24 h,虫体前端挛缩明显,质膜水肿、泡状隆起,石灰小体形态改变,糖原颗粒耗损,分泌颗粒增加及焰细胞核质浓缩。经20 mg/ml雷丸粉末混悬液、20和80μg/ml吡喹酮溶液培养4、12和24 h的裂头蚴虫体与对照组相比,超微结构无明显改变。结论随着吡喹酮和雷丸体外培养浓度的增加以及时间的延长,裂头蚴对小鼠的感染性逐渐降低,并引起虫体广泛的组织结构损伤;雷丸的作用较吡喹酮明显。

Objective To study the effects of Omphalia lapidescens and praziquantel on the infectivity and ultrastructure of Spironetra erinacei plerocercoids. Methods The plerocercoids were taken from frogs（Rana nigromaculata）.A total of 168 mice were divided into 21 groups（8 mice per group）, each of them was orally infected with 5 plerocercoids. The mice in group 1-9 were inoculated with plerocercoids cultured in media respectively containing different con-centrations of O. lapidescens suspension（20, 40 or 80 mg/ml） for 4, 12 or 24 h, respectively. The mice in group 10-18 were inoculated with plerocercoids cultured in media respectively containing different concentrations of praziquantel（20, 80 or 320 μg/ml） for 4, 12 or 24 h, respectively. The mice in group 19-21 were inoculated with plerocercoids cultured in normal culture fluid for 4, 12 or 24 h, respectively, and served as controls. One week after infection, the mice were sacrificed to collect the plerocercoids. Worm reduction rate was calculated. The ultrastructure changes of ple-rocercoids were observed under transmission electron microscope（ TEM） and scanning electron microscope（SEM）, respectively. Results The average number of plerocercoids detected from mice infected by pleroceroids treated with 40,80 mg/ml O. lapidescens suspension for 4, 12 or 24 h were 1.6, 1.0, and 0.3; 0.3, 0, and 0, respectively, and significantly lower than that of the infected controls（4.1, 3.5 and 3.3）（P〈0.05）; the worm reduction rates were 60.0%,71.4%, and 90.1%; 92.7%, 100%, and 100%, respectively. The average number of pleroceroids detected from mice infected with pleroceroids treated with 320 μg/ml praziquantel for 4, 12, or 24 h were 1.9, 1.3, and 0.4, and significantly lower than that of the infected controls（P〈0.05）; the worm reduction rates were 53.7%, 62.9%, and 87.9%, and lower than that of 20 μg/ml praziquantel group（14.6%, 2.9%, and 6.1%） and 80 μg/ml praziquantel group（24.4%,17.1%, and 24.2%）（P〈0.05）. The ultrastructure of plerocercoids cultured in 20 mg/ml O. lapidescens suspension, 20 or80 μg/ml praziquantel for 4, 12 or 24 h had no significant difference compared with control groups. The plerocercoids cultured in 40 mg/ml O. lapidescens for 4 h or 320 μg/ml praziquantel for 4 or 12 h, showed mild contracture. The pleroceroids cultured in 40 mg/ml O. lapidescens for 12-24 h showed： agglutinate, fusion, fracture or abscission of microtriches, breakdown of plasma membrane, excretion of calcareous corpuscles, and tegument tissue damages. After cultured in80 mg/ml of O. lapidescens for 24 h, the tissues of plerocercoid were damaged seriously. After cultured in 320 μg/ml praziquantel for 24 h, the plerocercoids showed： obvious contracture in the anterior end of plerocercoid, edema and bulge of plasma membrane, morphological changes of calcareous corpuscles, increase of secretory granules, glycogen depletion, and chromatin compaction in flame cells. Conclusion The infectivity of Spironetra erinacei plerocercoids decreases along with the time of culture and the increase of drug concentration. Omphalia lapidescens and praziquantel can cause extensive tissue damage to the plerocercoids in vitro, and the effect of O. lapidescens on the infectivity and ultrastructure of plerocercoid is more considerable than that of praziquantel.