Sensitivity of Oncomelania hupensis to Niclosamide: A Nation-Wide Survey in China

Schistosomiasis japonica, transmitted by the intermediate host snail Oncomelania hupensis of the causative agent Schistosoma japonicum, remains a major public-health concern in China, and control of this snail is one of the major approaches used in attempts to interrupt the transmission of this neglected tropical disease. Niclosamide is currently the only commercial molluscicide available for the control of O. hupensis snails in China. The purpose of this study was to evaluate the current sensitivity of O. hupensis to niclosamide in China. O. hupensis snails derived from 17 sampling sites from eight schistosomiasis-endemic provinces of China were used for the molluscicidal tests. Active adult snails (10 for each drug concentration), were immersed in solutions of 1, 0.5, 0.25, 0.125, 0.063, 0.032, 0.016 and 0.008 mg/L of 50% wettable powder of niclosamide ethanolamine salt (WPN) for 24 and 48 h at 25°C, and then the snail mortality was estimated and LC50 values were calculated. All field-derived O. hupensis snails were dead following immersion in 0.5 and 1 mg/L WPN for 24 h, whereas no death was observed after immersion in 0.008 mg/L WPN for 24 h. Immersion in 0.5, 0.25, 0.125, 0.063, 0.032 and 0.016 mg/L WPN for 24 h resulted in 80%–100%, 63.33%–100%, 0%–85%, 0%–50%, 0%–15%, and 0%–5% snail mortalities, respectively. The 24 h WPN LC50 values for the O. hupensis snails derived from the 17 sampling sites in China ranged from 0.0743 to 0.2285 mg/L, and no significant difference was detected by the Kolmogorov-Smirnov test (p = 0.2). The results indicate that there is no regional variation in the current susceptibility to niclosamide in O. hupensis populations in China. It is suggested that the current sensitivity of niclosamide against O. hupensis remains high and has not changed after more than two decades of repeated, extensive application for snail control in the main endemic areas of China.

0.125, 0.063, 0.032 and 0.016 mg/L WPN for 24 h resulted in 80%-100%, 63.33%-100%, 0%-85%, 0%-50%, 0%-15%, and 0%-5% snail mortalities, respectively. The 24 h WPN LC 50 values for the O. hupensis snails derived from the 17 sampling sites in China ranged from 0.0743 to 0.2285 mg/L, and no significant difference was detected by the Kolmogorov-Smirnov test (p = 0.2). The results indicate that there is no regional variation in the current susceptibility to niclosamide in O. hupensis populations in China. It is suggested that the current sensitivity of niclosamide against O. hupensis remains high and has not changed after more than two decades of repeated, extensive application for snail control in the main endemic areas of China.

Introduction
Schistosomiasis, a snail-transmitted parasitic disease, is a major neglected tropical disease affecting more than 207 million people in the tropical and subtropical regions around the world [1]. Although great success has been achieved in its control in China, schistosomiasis japonica is still one of the four communicable diseases that have been given high priority by the central government [2][3][4]. It is estimated that over 0.7 million people are infected with the parasite in China, and the snail host Oncomelania hupensis is detected in habitats with an area of 3.73 billion m 2 [5][6][7]. It has been proved that the transmission of S. japonicum is governed by the geographical distribution of its snail host; therefore, control of the O. hupensis snails, as a major part of the National Schistosomiasis Control Program, is currently one of the major approaches used for schistosomiasis control and elimination in China [8][9][10].
In China, many approaches have been used for the control of O. hupensis; however, snail control with chemicals remains the most widely used method to kill the snail intermediate host till now [11][12][13]. Niclosamide is recommended by the WHO as the only molluscicide for snail control in the endemic foci [14]; however, the agent is difficult to dissolve in both water and organic solvents. Therefore, many novel niclosamide formulations have been developed attempting to improve its water solubility. Of these a 50% wettable powder of niclosamide ethanolamine salt (WPN) has been recommended as the only market-available molluscicide since the initiation of the World Bank Loan Project for Schistosomiasis Control in China, due to its high efficacy and easy use [15]. Currently, WPN is recommended by the Ministry of Health, P. R. China at a dose of 1 mg/L for snail control in the field [15]. Following extensive, long-term, repeated use for more than two decades, the possible emergence of resistance to niclosamide in the intermediate host snails has received much attention [16]. Therefore, a systematic survey of the molluscicidal activity of niclosamide against O. hupensis would be of great significance for understanding the current sensitivity of niclosamide in China, and the prevention and rapid management of niclosamide resistance in snail populations. Here, a nationwide determination was carried out in regions where O. hupensis snails are present in China to assess the current efficacy of O. hupensis to niclosamide.

Snails
Adult O. hupensis snails were collected by individual picking with forceps [17], from 17 sampling sites from eight schistosomiasis-endemic provinces of China (Table 1, Figure 1). After feeding in the laboratory for 24 h, active adult snails with 7-8 spirals were randomly divided into groups for the molluscicidal test.

Molluscicidal Test
One hundred milliliter flasks were filled with the solutions, then 10 active adult snails were added and the flasks covered with gauze to prevent their escape. Snails in flasks of dechlorinated water served as controls. After being immersed for 24 and 48 h at 25°C, the snails were washed with dechlorinated water and fed for a further 48 h. Those suspected of being dead were tested by the knocking method [18,19], and the snail mortality was estimated. All tests were performed in triplicate, and LC 50 values were calculated.

Statistical Analysis
All data were double entered into Microsoft Excel 2003 (Microsoft Corporation; Redmond, WA, USA) and all statistical analyses were performed using the statistical software SPSS version 16.0 (SPSS Inc.; Chicago, IL, USA). Differences of LC 50 values were tested for statistical significance with Kolmogorov-Smirnov test. A p value < 0.05 was considered statistically significant.

Results
All field-derived O. hupensis snails were dead following immersion in 1 mg/L WPN for 24 h, whereas no death was observed after immersion in 0.008 mg/L WPN for 24 h. Immersion with WPN at concentrations of 0.5, 0.25, 0.125, 0.063, 0.032 and 0.016 mg/L for 24 h resulted in 80%-100%, 63.33%-100%, 0%-85%, 0%-50%, 0%-15%, and 0%-5% mortalities of snails, respectively ( Table 2). The 24 h WPN LC 50 values for the O. hupensis snails derived from the 17 sampling sites in China ranged from 0.0743 to 0.2285 mg/L, and no significant difference was detected by the Kolmogorov-Smirnov test (p = 0.2).   The O. hupensis snails derived from 16 sampling sites (the number of snails sampled from Shimen county of Hunan province was not enough, due to death, to be used for assessing the mortality following immersion in WPN for 48 h) were employed to test the efficacy of WPN following immersion for 48 h. No snails survived the treatment with WPN at concentrations of 0.5 and 1 mg/L for 48 h, whereas all snails were alive by immersion in WPN at a concentration of 0.008 mg/L for 48 h. Immersion in WPN at concentrations of 0.25, 0.125, 0.063, 0.032 and 0.016 mg/L for 48 h resulted in 95%-100%, 10%-100%, 0%-55%, 0%-10%, and 0%-5% mortalities of snails, respectively (Table 3).

Discussion
In China, the description of schistosomiasis japonica dates back more than two millennia [2]. Since the control efforts initiated in the 1950s, snail control has been a key part of the National Schistosomiasis Control Program of China [8,12], and many approaches have been used for the control of the intermediate host snails, including environmental improvement, physical methods, building trees, molluscicide treatment and biological control [11]. Among all these strategies, molluscicide treatment is the most widely used method for snail control due to the wide application coverage, easy procedure and fast action [20]. A recent meta-analysis revealed that it is necessary to continuously apply molluscicidal treatments more than twice a year in the field to consolidate the schistosomiasis control achievements gained [21], and different molluscicidal treatment strategies should be utilized at different stages of the control programme to maximize cost-effectiveness [20].
Since 1950s, many chemicals have been tested for molluscicidal activity and several agents have been used for snail control in the schistosomiasis-endemic fields of China, including sodium pentachlorophenate, bromoacetamide, nicotinanilide, calcium cyanamide, niclosamide, META-Li, and so on [22][23][24][25]. In addition, some plants have been screened and tested for molluscicidal activity against O. hupensis in laboratories [26][27][28][29][30]. Since 1992 when the World Bank Loan Project for Schistosomiasis Control was initiated in China, WPN was introduced and has replaced other molluscicides to become the only molluscicide now used for snail control in the endemic areas of China [15]. Following the repeated, long-term, extensive use for more than two decades, there is a concern about the potential development of resistance to niclosamide in O. hupensis snails [16]. It is therefore of great importance to monitor the molluscicidal activity of niclosamide against the O. hupensis snails in regions where this is currently the only available chemical that is widely used in China.
WPN at a dose of 1 mg/L is recommended for snail control in the field [15]. Therefore, drug solutions at concentrations of 1, 0.5, 0.25, 0.125, 0.063, 0.032, 0.016 and 0.008 mg/L were designed for the molluscicidal tests in the laboratory. Our findings showed that all O. hupensis snails were dead following immersion in 1 mg/L WPN for 24 and 48 h or in 0.5 mg/L WPN for 48 h, and only 0%-20% survived the treatment with WPN at a concentration of 0.5 mg/L for 24 h. In addition, the 24 h WPN LC 50 values for the O. hupensis snails derived from 17 sampling sites in China ranged from 0.0743 to 0.2285 mg/L, with no significant differences were detected (p = 0.2), and the 48-h WPN LC 50 values for the O. hupensis snails derived from 16 sampling sites in China ranged from 0.049 to 0.1593 mg/L, with no significant differences detected (p = 0.197). The results from this study demonstrate that the molluscicidal activity of niclosamide against O. hupensis is still high in the main endemic areas of China after more than two decades of repeated, extensive use. This is important information for both the public health workers and health policy makers in the field of schistosomiasis control, considering that niclosamide, the currently only chemical of choice for snail control, plays an essential role in the current Chinese National Schistosomiasis Control Program of China.
It has been found that the environmental factors including temperature, vegetation, sunlight, soil and rainfall, the quality and concentration of the chemicals, as well as the technical skills may affect the molluscicidal actions in the endemic foci [11], whereas in the laboratory, the volume of drug solution [31], and the quantity, laboratorial breeding duration and the sampling time of snails used for molluscicidal test [32][33][34] are reported to affect the evaluation of the molluscicidal actions in the laboratory. Currently, a single WPN dose (1 mg/L) is employed for the field snail control in China [15], however, the variations in snail habitats, climate, temperature and rainfall may affect the concentration and duration of snail contact with niclosamide, thereby resulting in various molluscicidal efficacies. It is suggested that the dose of molluscicides should be appropriately adjusted based on the actual field settings, and temperature, climate and rainfall should be taken into account during the snail control in the field, so as to achieve the optimal molluscicidal efficacy.

Conclusions
The results indicate that there is no regional variation in the current susceptibility to niclosamide in O. hupensis populations, and the current activity of niclosamide against O. hupensis snails in China appears satisfactory; however, it does not mean that resistance cannot occur nor that in different geographical regions the response of O. hupensis snails will be the same. We, therefore, should not reduce our vigilance to the possible development of resistance to niclosamide in the intermediate host