Chin J Schisto Control ›› 2011, Vol. 23 ›› Issue (6): 611-619.

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Studies on resistance of Schistosoma to praziquantel XIV Experimental comparison of susceptibility to praziquantel between PZQ?resistant isolates and PZQ?susceptible isolates of Schistosoma japonicum in stages of adult worms, miracidia and cercariae

Li Hong-jun, Liang You-sheng*, Dai Jian-rong, Wang Wei, Qu Guo-li, Li You-zi, Xing Yun-tian, Tao Yong-hui, Qian Ke, Jia Yue, Yang Zhen-kun, Wei Jian-ying   

  1. Jiangsu Institute of Parasitic Diseases|Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health|Jiangsu Provincial Key Laboratory on Molecular Biology of Parasites, Wuxi 214064, China
  • Online:2011-12-22 Published:2011-12-14
  • Contact: Liang You?sheng

血吸虫对吡喹酮抗药性的研究 XIV 日本血吸虫吡喹酮抗性株和敏感株成虫 毛蚴和尾蚴对吡喹酮敏感性比较

李洪军|梁幼生*|戴建荣|汪伟|曲国立|李幼子|邢云天|陶永辉|钱科|贾悦|杨振坤|魏剑英   

  1. 江苏省血吸虫病防治研究所、 卫生部寄生虫病预防与控制技术重点实验室、 江苏省寄生虫分子生物学重点实验室 (无锡 214064)
  • 通讯作者: 梁幼生
  • 作者简介:李洪军|男|硕士|助理研究员。研究方向:抗血吸虫药物与寄生虫分子生物学
  • 基金资助:

    国家科技支撑计划项目 (2009BAI78B06); 国家自然科学基金 (30471516); 江苏省自然科学基金 (BK2009076); 江苏省卫生厅项目(X200912); 江苏省预防医学研究课题 (Y201031)

Abstract:

Objective To investigate the changes of sensitivity to praziquantel (PZQ) about PZQ?resistant isolates of Schistosoma japonicum established in laboratory by means of the resistance?inducement method during the stages of adult worms, cercariae and miracidia, so as to provide the basis for establishing the sensitivity?detecting technique to praziquantel. Methods A Jiangsu laboratory?maintaining isolate and a Hunan field?collecting isolate of S. japonicum that were never treated with PZQ were as PZQ?susceptible isolates, and two PZQ?induced isolates that were established via drug?treated passage in laboratory were as PZQ?resistant isolates. Mice were infected with S. japonicum cercariae collected from above four isolates each. Thirty?five days after the infection, the mice were divided into 6 groups and administered orally with PZQ at dosages of 0, 37.5, 75, 150, 300 mg/kg and 600 mg/kg, respectively. All the mice were sacrificed two weeks after the treatment, and all the adult worms in the hepatic and portomesenteric veins were recovered and counted. The mean worm burden and reductions were calculated and input into Graphpad Prism 5.0 software, and the PZQ ED50 values of four isolates were calculated by the software. The cercariae of above four isolates were exposed to 10-5 , 5×10-6 , 10-6 , 5×10-7 , 10-7 mol/L PZQ solutions for 20, 40, 60, 80, 100 min and the changes of tail shedding were observed under a dissecting micro? scope, then the tail shedding rates of cercariae were calculated. The miracidia of above four isolates were exposed to 5× 10-6 , 10-6 , 5×10-7 , 10-7 mol/L PZQ solutions for 1, 3 and 5 min and the morphological changes were observed under a dissecting microscope, then the morphological change rates of miracidia were calculated. Results The PZQ ED50 values of PZQ?susceptible and PZQ?resistant isolates of Jiangsu were 147.7 mg/kg and 565.5 mg/kg, respectively, and the PZQ ED50 values of PZQ?susceptible and PZQ?resistant isolates of Hunan were 151.8 mg/kg and 467.2 mg/kg, respectively. When the cercariae were exposed to 10-5 mol/L PZQ solution over 20 min, the tail shedding rate of cercariae from PZQ?susceptible isolate of Jiangsu was 68.8%, and the tail shedding rate of cercariae from PZQ?resistant isolate of Jiangsu was 38.2% (P<0.01). When the cercariae were exposed to 10-7 mol/L PZQ solution over 100 min, the tail shedding rate of cercariae from PZQ?susceptible isolate of Jiangsu was 15.9%, and the tail shedding rate of cercariae from PZQ?resistant isolate of Ji? angsu was 6.7% (P<0.01). When the cercariae were exposed to 10-5 mol/L PZQ solution over 20 min, the tail shedding rates of cercariae from PZQ?susceptible isolate of Hunan was 59.4%, and the tail shedding rates of cercariae from PZQ?resistant isolate of Hunan was 54.6% (P<0.05). When the cercariae were exposed to 5×10-7 mol/L PZQ solution over 40 min, the tail shedding rates of cercariae from PZQ?susceptible isolate of Jiangsu was 34.3%, and the tail shedding rates of cercariae from PZQ?resistant isolate of Jiangsu was 18.4% (P<0.01). When the miracidia were exposed to 5×10-7 mol/L and 10-7 mol/L PZQ solutions for 1, 3 and 5 min respectively, the morphological change rates of miracidia from PZQ?susceptible isolates of Jiangsu and Hunan were significantly higher than those of PZQ?resistant isolates (P<0.01). Conclu? sions PZQ?resistant isolates of S. japonicum has been established in mice with sub?curative doses of PZQ by artificial selection in laboratory, and their sensitivities to PZQ are significantly lower than those of the isolates never treated with PZQ. The drug?resistance could exhibit in the stages of adult worms, cercariae and miracidia. The PZQ ED50 value of adult worms, the tail shedding rates of cercariae and the morphological change rates of miracidia as quantitative indicators can be used for monitoring the S. japonicum sensitivity to PZQ.

Key words: Schistosoma japonicum, Praziquantel, Drug resistance, Adult worm, Miracidium, Cercaria, ED50

摘要:

目的 测定实验室诱导筛选获得的日本血吸虫吡喹酮抗性株成虫、 毛蚴和尾蚴对吡喹酮的敏感性变化, 为建立吡喹酮敏感性检测/监测技术提供依据。方法 以未暴露于吡喹酮的日本血吸虫江苏实验室传代株和现场采集并经实验室传代的湖南株作为吡喹酮敏感株, 以实验室传代并经吡喹酮抗性诱导的日本血吸虫江苏抗性诱导株及现场采集并经实验室吡喹酮抗性诱导的湖南抗性诱导株作为吡喹酮抗性株。分别收集各虫株尾蚴感染小鼠, 感染35 d后分为6组, 5个服药组分别一次性灌服剂量为37.5、 75、 150、 300 mg/kg和600 mg/kg 吡喹酮, 对照组不给药; 用药后14 d解剖小鼠, 收集小鼠体内成虫, 计算虫负荷和减虫率, 计算吡喹酮对各虫株的半数有效剂量 (ED50值)。取一定量各虫株尾蚴分别暴露于10-5 、 5×10-6 、 10-6 、 5×10-7 mol/L和10-7 mol/L 吡喹酮溶液中, 20、 40、 60、 80 min和100 min后, 在解剖镜下观察尾蚴的断尾变化, 计算各虫株尾蚴断尾率。取一定量各虫株毛蚴分别暴露于10-5 、 5×10-6 、 10-6 、 5×10-7 mol/L和10-7 mol/L 吡喹酮溶液中, 1、 3 min 和5 min后, 解剖镜下观察毛蚴的形态变化, 计算各虫株毛蚴变形率。结果 吡喹酮对江苏吡喹酮敏感株和抗性诱导株的 ED50值分别为147.7 mg/kg和565.5 mg/kg, 对湖南吡喹酮敏感株和抗性诱导株的ED50值分别为151.8 mg/kg和467.2 mg/kg。当暴露于10-5 mol/L 吡喹酮溶液中20 min, 江苏吡喹酮敏感株尾蚴断尾率为68.8%, 吡喹酮抗性诱导株尾蚴断尾率为 38.2%, 差异有统计学意义 (P<0.01); 当暴露于10-7 mol/L 吡喹酮溶液中100 min, 江苏吡喹酮敏感株尾蚴断尾率为15.9%,吡喹酮抗性诱导株尾蚴断尾率为6.7%, 差异有统计学意义 (P<0.01)。当暴露于10-5 mol/L 吡喹酮溶液中20 min, 湖南吡喹酮敏感株尾蚴断尾率为59.4%, 吡喹酮抗性诱导株尾蚴断尾率为54.6%, 差异有统计学意义 (P<0.05); 当暴露于5×10-7 mol/L 吡喹酮溶液中40 min, 湖南吡喹酮敏感株尾蚴断尾率为34.3%, 吡喹酮抗性诱导株尾蚴断尾率为18.4%, 差异有统计学意义 (P<0.01)。当暴露于5×10-7 mol/L和10-7 mol/L吡喹酮溶液1、 3 min和5 min后, 江苏吡喹酮敏感株和湖南吡喹酮敏感株毛蚴的变形率均明显高于吡喹酮抗性诱导株, 差异均有统计学意义 (P均<0.01)。结论 采用亚治疗剂量吡喹酮, 在小鼠体内经过诱导筛选所获得的中国大陆日本血吸虫吡喹酮抗性株对吡喹酮敏感性显著低于未经诱导筛选的原代虫株,其抗药性可表现在成虫、 毛蚴和尾蚴发育阶段。吡喹酮对成虫的ED50值、 尾蚴断尾率和毛蚴变形率作为量化指标, 可用于现场日本血吸虫对吡喹酮的敏感性检测/监测。

关键词: 日本血吸虫;吡喹酮;抗药性;成虫;毛蚴;尾蚴;半数有效剂量 (ED50 )

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