Abstract:

Objective To investigate the possibility of the emergence of praziquantel resistance in Schistosoma japonicum in Mainland China under drug pressure. Methods S. japonicum cercaria were released from the infected Oncomelania hupensis snails collected from the marshland in Hunan Province that was endemic for schistosomiasis japonica and raised in the laboratory of Jiangsu Institute of Parasitic Diseases, and mice were infected. O. hupensis snails were infected with miracidia hatched from the schistosome mature eggs that were isolated from the liver of the infected mice. The life cycles of a field isolate and a laboratory passage isolate of S. japonicum were established in laboratory via the cycle of mouse?snail. The mice were infected with 40 cercariae each, 35 days later post?infection, were grouped randomly into control and resistance?induced groups. All the mice in the control group were sacrificed on day 45 post?infection, and any adult S. japonicum worms in the hepatic and portomesenteric veins were recovered and counted, and the worm burdens were calculated. The mice in the resistance?induced group were administered orally with the sub?curative dose of praziquantel, and were sacrificed 22 days post?treatment. Any adult S. japonicum worms in the hepatic and portomesenteric veins were recovered and counted, and the worm burdens and reduction in the worms recovered which were obviously caused by the praziquantel treatment were calculated. The eggs in the liver of the mice in the resistance?induced group were isolated and hatched to yield miracidia, and then the snails were again infected with the newly hatched miracidia to complete the first?passage inducement. After raising in laboratory at 25 ℃ for 60-70 days post?infection, the infected snails were isolated and shed cercaria, and the mice were infected with the newly released cercaria to start a new pas? sage of resistance?inducement. The oral dose of praziquantel for the first?passage inducement was 100 mg/kg, and an additional 100 mg/kg was given every 2-3 passages. The mice were infected with cercariae of the parasite with 8?passge resistance?inducement and the isolate that was not induced, and 35 days post?infection, were administered with praziquantel at a single oral doses of 300 mg/kg and 600 mg/kg respectively. All the mice were sacrificed 14 days post?treatment, and any adult S. japonicum worms in the hepatic and portomesenteric veins were recovered and counted, and the reductions in the worm burdens were calculated to assess the sensitivity of praziquantel in the parasites after 8?passage resistance?inducement. Results Two isolates of Jiangsu laboratory passage of Jiangsu and field isolate of Hunan were established in the laboratory, and a total 8?passage resistance ?inducement was completed. For the laboratory passage isolate, the worm burden reduction was 22.3% post?treatment with 100 mg/kg praziquantel during the first?passage inducement, and 53.7% post?treatment with 300 mg/kg praziquantel during the 8?passage inducement, appearing that the worm burden reduction increased with the increasing dose of praziquantel. For the field?collected isolate, the worm burden reduction was 66.8% post?treatment with 100 mg/kg praziquantel during the first?passage induce? ment, and 20.6% post?treatment with 300 mg/kg praziquantel during the 8?passage inducement, indicating that the worm burden reduction markedly decreased with the increasing dose of praziquantel. The worm burden reductions were 71.5% and 97.4% respectively for the mice infected with the non?induced laboratory passage isolate, while administered with praziquantel at doses of 300 mg/kg and 600 mg/kg respectively. After 8?passage treatment with sub?curative praziquantel, the corresponding worm burden reductions decreased to 32.6% and 68.1%, respectively. For the field?collected isolate without inducement, the worm burden reductions in the mice were 70.8% and 97.5% respectively post?treatment with praziquantel at doses of 300 mg/kg and 600 mg/ kg respectively, and the corresponding worm burden reductions decreased to 45.7% and 61.9%, respectively after 8?passage treatment. Conclusions S. japonicum (strain of Mainland China) is able to develop resistance to praziquantel under continuous drug pressure. However, there are variations in the potential of the emergence of resistance due to various susceptibility of praziquantel among different isolates. The successful establishment of praziquantel?resistant strain of S. japonicum (Mainland China) will provide the basis for exploring the mechanism of praziquantel resistance in S. japonicum, and developing related techniques to detect and monitor praziquantel resistance.

Key words: Schistosoma japonicum, Praziquantel, Drug resistance, Resistance inducement

摘要：

目的 实验研究在吡喹酮药物压力下中国大陆日本血吸虫对吡喹酮产生抗药性的可能性。方法 取采自湖南省血吸虫病流行区湖滩现场和江苏省实验室传代的感染性钉螺实验室逸蚴， 获得日本血吸虫尾蚴感染小鼠， 从感染鼠肝脏分离成熟虫卵孵化毛蚴感染湖北钉螺， 建立现场采集株和实验室传代株日本血吸虫小鼠?钉螺实验室生活史循环。用定量尾蚴 （40条/鼠） 感染小鼠， 感染35 d后将小鼠随机分为对照组和抗性诱导组： 对照组小鼠感染后45 d解剖收集肠系膜静脉和肝门脉静活虫体， 计算虫负荷 （条/鼠）； 抗性诱导组小鼠采用灌胃法一次口服亚治疗剂量吡喹酮进行治疗， 服药22 d后解剖收集肠系膜静脉和肝门脉静存活虫体。计算虫负荷 （条/鼠） 和减虫率， 完成首轮诱导。取抗性诱导组小鼠肝脏， 分离虫卵， 实验室孵化出毛蚴重新感染钉螺， 感染后的钉螺经25 ℃生化培养箱内饲养60~70 d后， 分离感染性钉螺并逸蚴， 用成熟尾蚴感染小鼠， 开始新一轮循环诱导。首轮诱导吡喹酮口服剂量为100 mg/kg， 后每循环2~3轮增加100 mg/kg口服剂量。取完成8轮诱导后和未经诱导原代虫株的尾蚴感染小鼠， 感染35 d后分别采用300 mg/kg和600 mg/kg 吡喹酮一次性灌胃治疗感染小鼠， 服药后14 d解剖感染鼠， 收集活虫， 计算各虫株减虫率， 评价虫株经8轮诱导后对吡喹酮敏感性的变化。结果 在实验室内建立了江苏实验室传代株和湖南现场采集株2个虫株， 并对其实施了8轮诱导。江苏实验室传代株在小鼠体内经第1轮口服100 mg/kg吡喹酮治疗后减虫率为22.3%， 第8轮口服300 mg/kg吡喹酮治疗后减虫率为53.7%，减虫率随口服吡喹酮剂量增加而增加； 湖南现场采集株在小鼠体内经第1轮口服100 mg/kg 吡喹酮治疗后减虫率为 66.8%， 第8轮口服300 mg/kg 吡喹酮治疗后减虫率仅为20.6%， 减虫率随口服吡喹酮剂量增加而显著降低。未经吡喹酮诱导的江苏实验室传代株在小鼠体内经300 mg/kg和600 mg/kg 吡喹酮治疗后， 减虫率分别为71.5%和97.4%； 经8轮吡喹酮筛选治疗后该虫株减虫率降至32.6%和68.1%。未经吡喹酮诱导的湖南现场采集株在小鼠体内经300 mg/kg和600 mg/kg 吡喹酮治疗后， 减虫率分别为70.8%和97.5%； 经8轮吡喹酮筛选治疗后该虫株减虫率降至45.7%和61.9%。结论 中国大陆日本血吸虫在吡喹酮持续药物压力下可产生抗药性， 但不同虫株间对吡喹酮敏感性存在差异， 药物压力下产生抗性的潜能也存在差异。中国大陆日本血吸虫吡喹酮抗性株的建立， 可为研究日本血吸虫吡喹酮抗性机制及其检测和监测技术奠定基础。

关键词: 日本血吸虫；吡喹酮；抗药性；抗药性诱导