日本血吸虫虫卵杀灭剂的筛选及快速杀灭技术的研究
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摘要
血吸虫病是严重危害人类健康和影响社会经济发展的重大传染病之一。众多保虫宿主与大多数受感染者作为传染源在血吸虫传播流行中起着重要的作用。随着高效低毒治疗药品吡喹酮的问世并广泛应用,以及病原学和免疫学诊断技术的进步,我国血吸虫病防治策略也发生了重大改变,已由以控制钉螺为主综合性防治策略逐渐转变为以传染源控制为主的综合性防治策略。渔、船民是目前湖区流行区血吸虫病传播的重要传染源之一,控制渔船民粪便对水体的污染和杀灭粪便中血吸虫卵,是控制血吸虫病关键技术之一,但目前针对粪便中的虫卵尚缺乏相应的快速杀灭技术。本研究通过对40多种农药和杀虫剂杀灭日本血吸虫虫卵效果实验观察,筛选出对日本血吸虫虫卵具有高效杀灭作用的备选药物。通过对其杀灭日本血吸虫虫卵影响相关因素的研究,优化了杀卵条件,观察了其在现场应用中的杀卵效果,建立了一种快速杀灭血吸虫卵的技术方法。
本研究内容如下:
一化学杀卵剂的筛选
对市场购买6大类41种农药杀日本血吸虫卵的作用进行观察。用待筛选的化学药物配成1000mg/L、100mg/L、10mg/L和1ml/L溶液,将一定量日本血吸虫卵分别置于上述浓度溶液中浸泡24h,洗涤残余药溶液后,孵化、收集和计数毛蚴,计算虫卵孵化率。结果:药物浓度为1000mg/L时,所测37种样品虫卵孵化率均低于20%。药物浓度为100mg/L时, 34种样品孵化率低于20%,4种样品孵化率在20-40%之间,3种样品孵化率在40-60%之间。药物浓度为10mg/L时, 14种样品孵化率低于20%,16种样品孵化率在20-40%之间,8种样品孵化率在40-60%之间,3种样品孵化率在60-80%之间。药物浓度为1mg/L时,在6种样品孵化率低于20%,10种样品孵化率在20-40%之间,13种样品孵化率在40-60%之间,11种样品孵化率在60-80%之间,1种样品孵化率大于80%。在所有被筛选的样品中,以80%敌敌畏乳油对日本血吸虫虫卵的杀灭效果最好,其浓度为100mg/L,10mg/L,1mg/L,0.1mg/L时,虫卵孵化率分别为0%,0%,0%,1.37%。
二敌敌畏杀卵效果及快速灭卵技术的建立
1.药液浓度对杀血吸虫虫卵效果的影响
在温度25℃条件下,将定量日本血吸虫虫卵加入浓度为0.5mg/L、0.1mg/L、0.05mg/L、0.01mg/L 80%敌敌畏乳油溶液中浸泡24h,洗去残余药液后孵化、收集并计数毛蚴,计算虫卵孵化率。结果:浓度为0.1mg/L和0.5mg/L时虫卵孵化率均为0%,浓度为0.05mg/L和0.01mg/L的虫卵孵化率分别为8.40%和35.11%。
2.实验温度对杀血吸虫虫卵效果的影响
在4℃、15℃、25℃、35℃环境中,分别将定量日本血吸虫虫卵加入浓度为10mg/L、1mg/L、0.1mg/L、0.05mg/L 80%敌敌畏乳油溶液中浸泡24h,洗去药物残液后孵化,收集计数毛蚴,计算虫卵孵化率。结果:浓度为10mg/L、1mg/L和0.1mg/L时4种温度虫卵孵化率均为0%。浓度为0.05mg/L时,在4℃、15℃、25℃和35℃环境中的虫卵孵化率分别为8.33%、8.26%、8.06%和7.89%,其差异无统计学意义。
3.浸泡时间对杀血吸虫虫卵效果的影响
将定量日本血吸虫虫卵加入浓度为0.5mg/L、0.1mg/L、0.02mg/L和0.004mg/L 80%敌敌畏乳油溶液中,浸泡12h、8h、4h和2h后洗去药物残液孵化、收集并计数毛蚴,计算虫卵孵化率。结果:浸泡12h、8h、4h和2h后,0.5mg/L浓度组虫卵孵化率均为0%;0.1mg/L浓度组虫卵孵化率分别为0%、0%、1.33%和1.65%。0.02mg/L浓度组虫卵孵化率分别为14.10%、15.00%、17.33%和16.53%,0.004mg/L浓度组的虫卵孵化率分别为61.54%、63.75%、69.33%和73.55%。12h、8h、4h和2h LD50分别为0.00604mg/L、0.00635mg/L、0.00732mg/L、0.00782mg/L,其差异无统计学意义。结论:环境温度的变化和在一定范围内浸泡时间对杀日本血吸虫卵效果影响不明显,浓度为0.1mg/L 80%敌敌畏乳油2h可以快速杀灭日本血吸虫虫卵。
三敌敌畏现场杀灭血吸虫虫卵效果研究
分别在260目/25.4mm尼龙绢纱袋中和人粪便中加入定量虫卵,将尼龙绢纱袋和加入虫卵后的人粪置于粪便收集器中,再分别加入用人尿配制成的10mg/L、1mg/L、0.1 mg/L、0.05mg/L 80%敌敌畏溶液浸泡2h,取出尼龙绢袋和50g粪便用尼龙绢袋淘洗法洗去粪渣和残留药液并收集虫卵后,孵化、收集并计数毛蚴,计算虫卵孵化率。结果:尼龙绢袋内的虫卵在10mg/L、1mg/L、0.1mg/和0.05mg/L 80%敌敌畏乳油尿液中浸泡2h后孵化率分别为0%、0%、13.11%和60.66%。人粪中的虫卵在10mg/L、1mg/L、0.1mg/和0.05mg/L 80%敌敌畏尿液中浸泡2h后孵化率分别0、10.52%、52.63%和94.74%。1mg/L 80%敌敌畏对人粪尿中的虫卵具有良好的杀灭作用。
The human schistosomiases are major infectious diseases that harm human health and affect the socio-economic development. As the sources of infection, reservoir hosts and most of the infected patients play an important role in the transmission of schistosomiasis. With the development and wide application of praziquantel, a highly effective and well-tolerated drug for treatment of schistosomiasis, and the progress of parasitological and immunological techniques, the national control strategy of schistosomiasis has altered significantly, changing from control of snails to the comprehensive control strategy with an emphasis on control of infectious source. At present, The boat fishermen are one of the major infectious sources in lake areas, the control of contamination of feces of the boat fishmen to the water body and killing of the schistosome eggs in the feces is one of the key techniques for control of schistosomiasis. However, the rapid technique of killing schistosome eggs in the feces is not available so far. This study was to investigate the effect of killing schistosome by screening a variety of pesticide and insecticide, so as to obtain the candidated drugs which had high effect on killing eggs of Schistosoma japonicum. And the tenichque of killing eggs of S. japonicum quickly has also been established. The main contents are as follows:
1 Screening of chemical ovicides.
Forty-one kind of pesticides from 6 categories were screened for the egg-killing effect. The chemicals were formulated into solutions of 1000mg/L, 100mg/L, 10mg/L and 1mg/L, a certain eggs of S. japonicum were put into the solutions and immersed for 24 h. Following the clean of the remaining drug solutions, the eggs were hatched and the miracidia were collected and counted, and the hatching rates were calculated. The results showed, the hatching rates were less than 20% for 37 kind of chemicals after the immersion by the solution at the concentration of 1000mg/L. At the concentration of 100mg/L, the hatching rates were less than 20% for 34 kind of all tested chemicals, while 4 kind between 20% and 40%, and 3 kind between 40% and 60%. At the concentration of 10mg/L, the hatching rates were less than 20% for 14 kind in all tested chemicals, while that for other were 16 kind between 20% and 40%, 8 kind between 40% and 60% and 3 kind between 60% and 80%. At the concentration of 1mg/L, the hatching rates were less than 20% for 6 kind, while 10 kind between 20% and 40%, 13 kind between 40% and 60%, 11 kind between 60% and 80% and 1 kind more than 80%. Among all the screened chemicals, the dichlorvos emulsifiable had the best effect, with the haching rates of 0%, 0%, 0%, and 1.37% at the concentrations of 100mg/L, 10mg/L, 1mg/L and 0.1mg/L.
2 Ovicidal effect of dichlorvos emulsifiable and establishment of a rapid egg-killing technique
2.1 Impact of concentration of solutions on ovicidal effect of eggs of S. japonicum
Some eggs of S. japonicum were put into 80% dichlorvos emulsifiable solutions with concentrations of 0.5mg/L, 0.1mg/L, 0.05mg/L and 0.01mg/L for 24 h under the temperature of 25℃. Following the clean of remaining drug solutions, the eggs were hatched and the miracidia were collected and counted, and the hatching rates of the eggs were calculated. The results showed that both of the hatching rates of eggs were 0 at the concentrations of 0.1mg/L and 0.5mg/L, and 8.40% for 0.05mg/L and 35.11% for 0.01mg/L.
2.2 Impact of temperature on ovicidal effect against eggs of S. japonicum
Some eggs were put into the 80% dichlorvos emulsifiable at the concentrations of 10mg/L, 1mg/L, 0.1mg/L and 0.05mg/L for 24 h under the temperatures of 4℃, 15℃, 25℃and 35℃, Following the clean of remaining drug solutions, the eggs were hatched and the miracidia were collected and counted, and the hatching rates were calculated. The results showed that all the hatching rates were 0 with the solutions at concentrations of 10mg/L, 1mg/L, 0.1mg/L under the four temperatures. At the concentration of 0.05 mg/L, the hatching rates were 8.33%, 8.26%, 8.06% and 7.89%, respectively under the temperatures of 4, 15, 25 and 35℃. There was no significant statistical difference.
2.3 Impact of immersion time on ovicidal effect of the eggs of S. japonicum
Some eggs of S. japonicum were put into 80% dichlorvos emulsifiable with concentrations of 0.5 mg/L, 0.1 mg/L, 0.02 mg/L and 0.004 mg/L, after immersing for 12 h, 8 h, 4 h and 2 h, the remaining drug solutions were cleaned, the eggs were hachced and the miracidia were collected and counted, and the hatching rates caculated. The results showed that all hatching rates were 0 at the concentration of 0.5mg/L after treating for 12 h, 8 h, 4 h and 2 h, while the hatching rates were 0%, 0%, 1.33% and 1.65% at the concentration of 0.1mg/L, 14.10%, 15.00%, 17.33% and 16.53% at the concentration of 0.02mg/L, and 61.54%, 63.75%, 69.33% and 73.55% at the concentration of 0.004mg/L, respectively. The LD50 values were 0.00604mg/L, 0.00635mg/L, 0.00732mg/L and 0.00782 mg/L, respectively for 12 h, 8 h, 4 h and 2 h. There was no significant statistical difference. It is concluded that, the temperature had little impact on the ovicidal effect, and 0.1mg/L of 80% dichlorvos emulsifiable were able to killing the eggs of S. japonicum rapid for 2 h.
3 Ovicidal effect of dichlorvos emulsifiable against the eggs of S. japonicum in field
Some eggs were put into nylon gauze bag with size of 260 mesh/25.4 mm and feces, respectively, then the nylon gauze bag and feces were moved to a excrement collector, containing the solutions of 80% dichlorvos emulsifiable in the concentration of 10mg/L,1mg/L,0.1 mg/L,0.05mg/L (prepared with urine at 29℃) for 2 h. Then fecal dregs and the chemical were washed off by elutriation, and eggs hatched. The miracidia were collected and counted, and the hatching rate calculated. The results showed that schistosome eggs maintained in nylon gauze bag completely lost the hatching ability when treated with 10mg/L and1mg/L of 80% dichlorvos emulsifiable for 2 h, while the hatching rates were 13.11% and 60.66% when treated with 0.1mg/ and 0.05mg/L solutions. Immersed in the feces, the hatching rates were 0%, 10.52%, 52.63% and 94.74% at the concentrations of 10mg/L, 1mg/L, 0.1mg/L and 0.05 mg/L, respectively for 2 h. It is indicated that 1mg/L of 80% dichlorvos emulsifiable has a good killing effect against schistosome eggs in human wastes.
本研究内容如下:
一化学杀卵剂的筛选
对市场购买6大类41种农药杀日本血吸虫卵的作用进行观察。用待筛选的化学药物配成1000mg/L、100mg/L、10mg/L和1ml/L溶液,将一定量日本血吸虫卵分别置于上述浓度溶液中浸泡24h,洗涤残余药溶液后,孵化、收集和计数毛蚴,计算虫卵孵化率。结果:药物浓度为1000mg/L时,所测37种样品虫卵孵化率均低于20%。药物浓度为100mg/L时, 34种样品孵化率低于20%,4种样品孵化率在20-40%之间,3种样品孵化率在40-60%之间。药物浓度为10mg/L时, 14种样品孵化率低于20%,16种样品孵化率在20-40%之间,8种样品孵化率在40-60%之间,3种样品孵化率在60-80%之间。药物浓度为1mg/L时,在6种样品孵化率低于20%,10种样品孵化率在20-40%之间,13种样品孵化率在40-60%之间,11种样品孵化率在60-80%之间,1种样品孵化率大于80%。在所有被筛选的样品中,以80%敌敌畏乳油对日本血吸虫虫卵的杀灭效果最好,其浓度为100mg/L,10mg/L,1mg/L,0.1mg/L时,虫卵孵化率分别为0%,0%,0%,1.37%。
二敌敌畏杀卵效果及快速灭卵技术的建立
1.药液浓度对杀血吸虫虫卵效果的影响
在温度25℃条件下,将定量日本血吸虫虫卵加入浓度为0.5mg/L、0.1mg/L、0.05mg/L、0.01mg/L 80%敌敌畏乳油溶液中浸泡24h,洗去残余药液后孵化、收集并计数毛蚴,计算虫卵孵化率。结果:浓度为0.1mg/L和0.5mg/L时虫卵孵化率均为0%,浓度为0.05mg/L和0.01mg/L的虫卵孵化率分别为8.40%和35.11%。
2.实验温度对杀血吸虫虫卵效果的影响
在4℃、15℃、25℃、35℃环境中,分别将定量日本血吸虫虫卵加入浓度为10mg/L、1mg/L、0.1mg/L、0.05mg/L 80%敌敌畏乳油溶液中浸泡24h,洗去药物残液后孵化,收集计数毛蚴,计算虫卵孵化率。结果:浓度为10mg/L、1mg/L和0.1mg/L时4种温度虫卵孵化率均为0%。浓度为0.05mg/L时,在4℃、15℃、25℃和35℃环境中的虫卵孵化率分别为8.33%、8.26%、8.06%和7.89%,其差异无统计学意义。
3.浸泡时间对杀血吸虫虫卵效果的影响
将定量日本血吸虫虫卵加入浓度为0.5mg/L、0.1mg/L、0.02mg/L和0.004mg/L 80%敌敌畏乳油溶液中,浸泡12h、8h、4h和2h后洗去药物残液孵化、收集并计数毛蚴,计算虫卵孵化率。结果:浸泡12h、8h、4h和2h后,0.5mg/L浓度组虫卵孵化率均为0%;0.1mg/L浓度组虫卵孵化率分别为0%、0%、1.33%和1.65%。0.02mg/L浓度组虫卵孵化率分别为14.10%、15.00%、17.33%和16.53%,0.004mg/L浓度组的虫卵孵化率分别为61.54%、63.75%、69.33%和73.55%。12h、8h、4h和2h LD50分别为0.00604mg/L、0.00635mg/L、0.00732mg/L、0.00782mg/L,其差异无统计学意义。结论:环境温度的变化和在一定范围内浸泡时间对杀日本血吸虫卵效果影响不明显,浓度为0.1mg/L 80%敌敌畏乳油2h可以快速杀灭日本血吸虫虫卵。
三敌敌畏现场杀灭血吸虫虫卵效果研究
分别在260目/25.4mm尼龙绢纱袋中和人粪便中加入定量虫卵,将尼龙绢纱袋和加入虫卵后的人粪置于粪便收集器中,再分别加入用人尿配制成的10mg/L、1mg/L、0.1 mg/L、0.05mg/L 80%敌敌畏溶液浸泡2h,取出尼龙绢袋和50g粪便用尼龙绢袋淘洗法洗去粪渣和残留药液并收集虫卵后,孵化、收集并计数毛蚴,计算虫卵孵化率。结果:尼龙绢袋内的虫卵在10mg/L、1mg/L、0.1mg/和0.05mg/L 80%敌敌畏乳油尿液中浸泡2h后孵化率分别为0%、0%、13.11%和60.66%。人粪中的虫卵在10mg/L、1mg/L、0.1mg/和0.05mg/L 80%敌敌畏尿液中浸泡2h后孵化率分别0、10.52%、52.63%和94.74%。1mg/L 80%敌敌畏对人粪尿中的虫卵具有良好的杀灭作用。
The human schistosomiases are major infectious diseases that harm human health and affect the socio-economic development. As the sources of infection, reservoir hosts and most of the infected patients play an important role in the transmission of schistosomiasis. With the development and wide application of praziquantel, a highly effective and well-tolerated drug for treatment of schistosomiasis, and the progress of parasitological and immunological techniques, the national control strategy of schistosomiasis has altered significantly, changing from control of snails to the comprehensive control strategy with an emphasis on control of infectious source. At present, The boat fishermen are one of the major infectious sources in lake areas, the control of contamination of feces of the boat fishmen to the water body and killing of the schistosome eggs in the feces is one of the key techniques for control of schistosomiasis. However, the rapid technique of killing schistosome eggs in the feces is not available so far. This study was to investigate the effect of killing schistosome by screening a variety of pesticide and insecticide, so as to obtain the candidated drugs which had high effect on killing eggs of Schistosoma japonicum. And the tenichque of killing eggs of S. japonicum quickly has also been established. The main contents are as follows:
1 Screening of chemical ovicides.
Forty-one kind of pesticides from 6 categories were screened for the egg-killing effect. The chemicals were formulated into solutions of 1000mg/L, 100mg/L, 10mg/L and 1mg/L, a certain eggs of S. japonicum were put into the solutions and immersed for 24 h. Following the clean of the remaining drug solutions, the eggs were hatched and the miracidia were collected and counted, and the hatching rates were calculated. The results showed, the hatching rates were less than 20% for 37 kind of chemicals after the immersion by the solution at the concentration of 1000mg/L. At the concentration of 100mg/L, the hatching rates were less than 20% for 34 kind of all tested chemicals, while 4 kind between 20% and 40%, and 3 kind between 40% and 60%. At the concentration of 10mg/L, the hatching rates were less than 20% for 14 kind in all tested chemicals, while that for other were 16 kind between 20% and 40%, 8 kind between 40% and 60% and 3 kind between 60% and 80%. At the concentration of 1mg/L, the hatching rates were less than 20% for 6 kind, while 10 kind between 20% and 40%, 13 kind between 40% and 60%, 11 kind between 60% and 80% and 1 kind more than 80%. Among all the screened chemicals, the dichlorvos emulsifiable had the best effect, with the haching rates of 0%, 0%, 0%, and 1.37% at the concentrations of 100mg/L, 10mg/L, 1mg/L and 0.1mg/L.
2 Ovicidal effect of dichlorvos emulsifiable and establishment of a rapid egg-killing technique
2.1 Impact of concentration of solutions on ovicidal effect of eggs of S. japonicum
Some eggs of S. japonicum were put into 80% dichlorvos emulsifiable solutions with concentrations of 0.5mg/L, 0.1mg/L, 0.05mg/L and 0.01mg/L for 24 h under the temperature of 25℃. Following the clean of remaining drug solutions, the eggs were hatched and the miracidia were collected and counted, and the hatching rates of the eggs were calculated. The results showed that both of the hatching rates of eggs were 0 at the concentrations of 0.1mg/L and 0.5mg/L, and 8.40% for 0.05mg/L and 35.11% for 0.01mg/L.
2.2 Impact of temperature on ovicidal effect against eggs of S. japonicum
Some eggs were put into the 80% dichlorvos emulsifiable at the concentrations of 10mg/L, 1mg/L, 0.1mg/L and 0.05mg/L for 24 h under the temperatures of 4℃, 15℃, 25℃and 35℃, Following the clean of remaining drug solutions, the eggs were hatched and the miracidia were collected and counted, and the hatching rates were calculated. The results showed that all the hatching rates were 0 with the solutions at concentrations of 10mg/L, 1mg/L, 0.1mg/L under the four temperatures. At the concentration of 0.05 mg/L, the hatching rates were 8.33%, 8.26%, 8.06% and 7.89%, respectively under the temperatures of 4, 15, 25 and 35℃. There was no significant statistical difference.
2.3 Impact of immersion time on ovicidal effect of the eggs of S. japonicum
Some eggs of S. japonicum were put into 80% dichlorvos emulsifiable with concentrations of 0.5 mg/L, 0.1 mg/L, 0.02 mg/L and 0.004 mg/L, after immersing for 12 h, 8 h, 4 h and 2 h, the remaining drug solutions were cleaned, the eggs were hachced and the miracidia were collected and counted, and the hatching rates caculated. The results showed that all hatching rates were 0 at the concentration of 0.5mg/L after treating for 12 h, 8 h, 4 h and 2 h, while the hatching rates were 0%, 0%, 1.33% and 1.65% at the concentration of 0.1mg/L, 14.10%, 15.00%, 17.33% and 16.53% at the concentration of 0.02mg/L, and 61.54%, 63.75%, 69.33% and 73.55% at the concentration of 0.004mg/L, respectively. The LD50 values were 0.00604mg/L, 0.00635mg/L, 0.00732mg/L and 0.00782 mg/L, respectively for 12 h, 8 h, 4 h and 2 h. There was no significant statistical difference. It is concluded that, the temperature had little impact on the ovicidal effect, and 0.1mg/L of 80% dichlorvos emulsifiable were able to killing the eggs of S. japonicum rapid for 2 h.
3 Ovicidal effect of dichlorvos emulsifiable against the eggs of S. japonicum in field
Some eggs were put into nylon gauze bag with size of 260 mesh/25.4 mm and feces, respectively, then the nylon gauze bag and feces were moved to a excrement collector, containing the solutions of 80% dichlorvos emulsifiable in the concentration of 10mg/L,1mg/L,0.1 mg/L,0.05mg/L (prepared with urine at 29℃) for 2 h. Then fecal dregs and the chemical were washed off by elutriation, and eggs hatched. The miracidia were collected and counted, and the hatching rate calculated. The results showed that schistosome eggs maintained in nylon gauze bag completely lost the hatching ability when treated with 10mg/L and1mg/L of 80% dichlorvos emulsifiable for 2 h, while the hatching rates were 13.11% and 60.66% when treated with 0.1mg/ and 0.05mg/L solutions. Immersed in the feces, the hatching rates were 0%, 10.52%, 52.63% and 94.74% at the concentrations of 10mg/L, 1mg/L, 0.1mg/L and 0.05 mg/L, respectively for 2 h. It is indicated that 1mg/L of 80% dichlorvos emulsifiable has a good killing effect against schistosome eggs in human wastes.
引文
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