| Schistosomiasis is a snail-borne neglected tropical diseases which is harmful tohuman health and draining socio-economic development. The disease is endemic in76countries or regions worldwide, with an estimated200million people infected and afurther779million individuals at risk of infection in the endemic areas. In China,schistosomiasis japonica remains a big public health problem and it is involved in oneof the four key infectious diseases that are given high priority. Currently, an estimated0.32million cases of schistosomiasis patients are detected, and about373596.18hm2ofOncomelania snail inhibitants were found in2010. Since1950the nationalschistosomiasis control program was initiated, snail control-based integrated controlstrategy has been involved in the National Schistosomiasis Control Program and playeda critical role in the control of the disease. Since1992the World Bank Loan Project forSchistosomiasis Control(WBLP) initiated in1992, as the only recommendedmolluscicide drug by the World Health Organization (WHO), niclosamide has beenwidely used for killing snails in areas of China where the parasite is endemic, which hascontributed significantly to the interruption and control of transmission of Schistosomajaponicum in China. It is reported that the molluscicidal efficacy of niclosamide isaffected by the field environment and climate conditions. In addition, the molluscicidaleffect of the same batch and dose of drug varies in different endemic foci. It is thereforeconsidered that niclosamide with the uniform dose may achieve various molluscicidalefficacies among different endemic foci in the country. On one hand, the expectedmolluscicidal effect is not achieved in some endemic areas, which leads to snailbreeding or spread and thus is not beneficial for the prevention and control of thedisease. On other hand, excessive drug are used in some endemic areas, leading towasting of resources and aggravation of environmental burden. However, the causes forthe inconsistence of molluscicidal efficacy remain unknown. The present study wasconducted to quantitatively assess the effects of environmental temperature and contacttime of snail with drug on sensitivity of Oncomelania snails to niclosamide, anddetermined the sensitivity of snails collected at different seasons and from various areasto niclosamide in laboratory, so as to establish the basic database of niclosamidesensitivity in snails in Chinese mainland. Such a study would provide theoretical evidence for selection of appropriate drug dose for snail control in different variousendemic foci. Based the current study, a standardized protocol of mollusciciding whichmeets the requirement of snail control in the field would be developed, so as to achievethe maximal molluscicidal efficacy and reduce the consumption of the drug. On onehand, the cost of snail control will be saved, and the resource collocation will beeffectively optimized, so as to facilitate the progress and development ofschistosomiasis control. On other hand, the environmental pollution and environmentalburden would be reduced to the maximal degree, so as to the sustained development ofmollusciciding.It is well known that molluscicidal effect is the key point in assessment ofmolluscicides. Assessment of molluscicidal efficacy either in field or in laboratorydepends on standardized method of assessment of molluscicidal effect. Theexperimental operation procedure should be complied with the international recognizedstandard, and the source of drugs, selection of snails, and the experimental conditionsall should be standardized to ensure the reliability and precision of the results and thecomparability among the results. Recently, the standardization method of screening andefficacy evaluation of molluscicides including niclosamide has been carried out in ourlaboratory. It is indicated use of10snails for immersion test of mollusciciding achievesreliable molluscicidal efficacy, high repeatibility, low mistake rate. The volume of themolluscicide solution is required to be more than3.3ml for each snail. In addition, thebreeding time of snails collected from the endemic field is required for less than11d.Otherwise, the sensitivity of snails to molluscicide would significantly reduce, andaffect the assessment of molluscicidal efficacy. The aforementioned results wouldprovide feasibility for establishment of standardization of efficacy assessment ofmolluscicides and screening of novel molluscicides. However, unlike the vectors likeBiomphalaria glabrata and mosquito which can reproduce and passage continuously,Oncomelania snails reproduce only one passage each year, and the period of life cycleis long. Therefore, there are no standardized strains of snails passaged in laboratory forrelated experiments. Currently, the snails used in experiment are collected from the field,and the snails used in experiments at different time are all used following collection.The experimental snails used in different laboratories are collected from differentregions and seasons, does the sensitivity of snails collected from different regions and seasons to niclosamide vary? Whether this variation affects the screening ofmolluscicides and efficacy assessment? All these issues remain unknown. The presentstudy was to quantitatively assess the effects of temperature and time on drug sensitivityin snails, and determine the sensitivity of snails collected from different regions andseasons to niclosamide, so as to explore the effect of snails collected from differentregions and seasons on efficacy evaluation and screening of novel drugs, and providetheoretical evidence for further improvement of standardization of efficacy assessmentand screening of novel drugs.Part Ⅰ Influence of temperature and time on sensitivity of snail toniclosamideIn this study, the snails were collected from the marshland of Zhenjiang City,Jiangsu Province, after being raised in laboratory for1-2days, the snails were randomlygrouped, and then immersed in50%wettable powder of niclosamide ethanolamine salt(WPN) at concentrations of1,0.1,0.01g/L and1mg/L under different roomtemperature with a humidity of60%in laboratory, the dead snails were counted fordifferent immersion time and the biometric data were analyzed by using the CLL modelto evaluate the molluscicidal efficacy of WPN and further explore the impact oftemperature and contact time of snail with drug on the sensitivity of snail to WPN.The results showed that the t values of temperature and time parameters were bothat significant levels, indicating significant temperature and time effect.Hosme-Lemeshow test revealed no heterogeneity in the established model. It issuggested that the model well fits with the biometric data of molluscicidal effect causedby immersion in WPN, demonstrating the changing trend of temperature-and contacttime with drug-dependent sensitivity of snails to niclosamide. The β values fortemperature effect when snails were immersed in different concentrations of WPN were2.5264,2.360,1.6953and4.5557revealed by the CLL model fitting. The maximumeffect values of time were:1g/L WPN immersion for20min,0.1g/L WPN immersionfor2h,0.01g/L WPN immersion for20h and1mg/L WPN immersion for48h,indicating the theoretical rapid death period of snails immersed in differentconcentrations of WPN. The lethal temperature of snails immersed in WPN was estimated by CLL model.The LT99were48.42,26.80,19.09,9.50and3.55℃, respectively when snails wereimmersed in1g/L WPN for1,3,5,10and20min, whereas being142.99,68.33,45.77,40.43,35.36and1.81℃while immersed in0.1g/L WPN for1,3,5,10,20,60and120min. When snails were immersed in0.01g/L WPN for less than4h, the temperaturerequired to be higher than100℃, while snails were immersed in WNP for8,12,16and20h, the temperatures were required to be81.96,58.76,27.61and1.20℃; when snailswere immersed in1mg/L WPN for less than8h, the temperature should be higher than100℃, while immersed for72h, the temperature was19.44℃. Obviously, the lethaltemperature was affected by the contact time with drug, in general, with the increasingcontact time, the lethal temperature was lower when snails were immersed in a certainconcentration of WPN. In addition, the lethal time of snails immersed in WPN atdifferent temperatures can be estimated by the CLL model. The mortality rate can reach99%when snails were immersed in1g/L WPN at0℃for1h, to get the same mortalityrate, snails were required to be immersed for20min at5℃,5min at20℃, and lessthan2min at30℃; when snails were immersed in0.1g/L WPN, the99%lethal timewas3h at0℃, less than1h at10℃, and30min at20℃; while snails were immersedin0.01g/L WPN, the99%lethal time was26h at0℃, and24h at5℃,14h at30℃.The molluscicide effect was very low while snails were immersed in1mg/L WPN atvery low temperature, while the99%lethal time was6d at15℃, and56,35,25h at20,25and30℃, respectively.These results indicate that the CLL model applies to the bioassay data of molluscicide efficacyanalysis, The study also show that interaction effect exist between the temperature and contact timeof snail with drug, which will affect the sensitivity of snails to drug, so in addition to the factor ofdrug concentration, we have to further consider the influence of temperature and contact time ofsnail with drug in the future field molluscicide，also the CLL model can be used to evaluate andpredicate the drug effect in the field.Part Ⅱ Investigation of the sensitivity of snails collected from differentseasons to niclosamideThe snails were collected from the marshland of Zhenjiang City, Jiangsu Province month by month from June2010to May2011. After being raised in laboratory for24h,the snails were randomly grouped, and then immersed in different concentrations ofWPN at (25±1)℃with a humidity of60%in laboratory.When the snails were immersed in WPN, the LC50value was0.143to0.209mg/L,and followed a normal distribution with a mean value of0.166mg/L, and95%confidence interval of0.152to0.180mg/L. When the snails were immersed in WPNfor48h, the LC50value ranged from0.112to0.170mg/L for48h, and followed anormal distribution with a mean value of0.146mg/L, and95%confidence interval of0.126to0.153mg/L. There were no significant differences in the mortality of snailscaused by WPN treatment observed at24and48hours by Logistic regression analysis(P values=0.374and0.267, respectively). And no significant differences were found inthe sensitivity of snails from different seasons to WPN treatment for24and48h.In summary, there are no changes in the sensitivity of snails collected fromdifferent months or seasons to WPN, with no fluctuations in LC50values. Such a findingindicates that it is feasible to adopt a uniform concentration in different seasonstheoretically, but the influence of temperature and contact time of snail with drug in thefuture field molluscicide should be further considered according to the study of partⅠ,and the snails collected from different months have few effects on screening ofmolluscicides and efficacy evaluation, and on the resistance monitoring of snails toniclosamide..Part Ⅲ Investigation of the sensitivity of snails collected fromdifferent areas to niclosamideSnails were collected from37sampling areas distributed in10provinces(municipalities) using random environmental sampling method accroding to differenttypes and categories of snail habitats. After being raised in laboratory for1-2days, thesnails were randomly grouped, and then immersed in different concentrations of WPNfor24and48h at (25±1)℃with a humidity of60%in laboratory.The mean value of LC50for snails immersed from37sampling for24h was0.157mg/L with a95%confidence interval of0.142-0.174mg/L, while the mean value ofLC50for snails immersed from36(except Shimen) sampling sites for48hours was 0.114mg/L with a95%confidence interval of0.102-0.126mg/L. There was not anyspecific regional or geographic clustering phenomenon in the sensitivity of snailsimmersed from all sampling sites for24and48h, and no significant difference wasdetected in sensitivity of snails to WPN among the sampling areas of10provinces; andno significant differences existed in snail sensitivity to WPN between different snailhabitat types. There were no significant differences between the present results and thedetermination conducted in2002, indicating that no resistance of WPN was detected insnails and no reduced sensitivity of snails to WPN was observed.In summary, no regional or geographic clustering phenomenon exists in thesensitivity of snails from different schistosomiasis-endemic areas to WPN in China..Compared with the determination in2002, there was no resistance to WPN observed inOncomelania hupensis. All these findings urge us to establish a database regarding thesensitivity of Oncomelania hupensis to WPN. In addition, the present study can be usedto guide the mollusciciding in field according to the field conditions. Also the snailscollected from different regions have few effects on screening of molluscicides andefficacy evaluation. |
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