| Schistosomiasis japonica is a major infectious zoonosis, which damages seriously people's health and impairs the development of animal husbandry. The marshland and beaches are not only high-risk regions of schistosome infections but also nice natural pasture. The bovine always infects or re-infects with the parasite repeatedly due to the pasture in the marshland, and results in a major source of infection. A novel protective agent of niclosamide against bovine schistosomiasis, therefore, is developed in our lab, with a national invention patent number of 200610098192.x. The field experiment was carried out following the protocol that the drug was sprayed, with an interval of 15 or 30 d, and reductions of 90% in bovine infections were achieved. The preventive agent has advantages of easy use, low-cost, long-term prevention and effectiveness, and meets the requirements of the control of bovine schistosomiasis in the schistosomiasi-endemic areas. However, the mechanism of actions of the preventive agent remains unclear. To investigate the preventive mechanism can provide scientific evidence for further development or improvement of better preventive agents, and can expand new approaches for schistosomiasis control, so as to change the current preventive mode depending mainly on oral drugs or vaccine which is difficult to get a breakthrough. Such a study would be of great importance for control of schistosomiasis japonica in China.According to the process of S. japonicum cercariae from skin penestration to maturing in hosts, combined with the characteristics of diffusion, absorption, retention, and distribution of the preventive agent, the present study was designed to investigate the interactions between the procedures of schistosome invastion to adult worms and drug distrubtion, and make clear the preventive mechanism of the preventive agent of niclosamide against bovine schistosomiasis.PART I Study on the basic property of niclosamideShake-flask method was adopted to the solubility of niclosamide.The solubility of niclosamide was 0.033, 0.037, 0.078, 0.746, 0.464 and 2.518 mg/L at solutions with pH values of 2, 4, 5, 6.8, 7.4 and 8.0, respectively. The results showed that the solubility of niclosamide was low and was pH value-dependent.N-octyl alcohol and water system was selected to determine the oil-water partition coefficients of niclosamide. The logKapp was 3.93, 4.26, 4.65, 4.66, 5.20 and 5.35 at solutions with pH values of 2, 4, 5, 6.8, 7.4 and 8.0, respectively. The results showed that niclosamide has the characteristics of transdermal agent.Franz glass diffusion cells were uesd for the subsequent experiments. The mouse and bovine skins, after treatment, were placed between diffusion and receptor compartments, and then fixed. A total of 500μl of the preventive agent was coated on the skin surface evenly, and 18 ml of receiving solutions were added into the receptor compartments, and all bubbles were removed. The receptor cell was filled with pH 7.4 phosphate buffered saline containing 30% ethanol. The diffusion and receptor compartments were then maintained at (32±0.1)°C in a water bath, and stirred at 100 rpm. Samples(18ml) were collected at 1, 2, 4, 6, 8, 10 h and filtered using a micro-hole filtering film (mesh size of 0.45μm), and then determined by HPLC. The diffusion rates of mouse and bovine skins were 17.17μg/(cm2·h) and 0.0483μg/(cm2·h), respectively, and the delay time was 0.97 h in mouse skin and below zero in bovine skin. It is concluded that, niclosamide can penetrate mouse and bovine skins, and the diffusion rate of bovine skin is higher than that of mouse skin.PART II The in-vitro activity of protective agent of niclosamide against bovine schistosomiasisThe preventive agent was sprayed on the bovine skin, dried in the shade. Then the skin was mounted between diffusion and receptor compartments, resulting in the the epidermal surface facing the receptor compartment. The diffusion and receptor compartments were then maintained at (32±0.1)°C in a water bath, and stirred at 100 rpm. Samples(18ml) were collected at 6, 12, 24, 36, 48, 60 h and 72 h from the receptor compartment, following filtering in a micro-hole filtering film (mesh size of 0.45μm), the niclosamide was determined using HPLC. The in-vitro diffusion rate was 0.0657μg/(cm2·h).Niclosamide solutions with concentration of 0.500, 0.100, 0.050, 0.010, 0.005 mg/L were added to 48-well plate, each well containing 0.3 ml, and dechlorinated water was set as control. 10-40 freshly released S. japonicum cercariae were placed in each well, and the survival of the cercariae was observed under a microscope at 1, 5, 10, 30 min and 60 min at different concentrations of niclosmiade, respectively. The cercariae swelling and inactivity under the stimulation of a dissecting needle indicated death. Our findings showed, no dead cercariae were found at a concentration of less than 0.010 mg/L within 60 min. When treated with 0.050 mg/L for 5 min, all cercariae were dead. While the concentration increased to 0.100 or 0.500 mg/L, all cercariae were dead within 1 min.A rectangular glass groove (size of 60 mm×60 mm×60 mm) was divided into 3 zones A, B and C, and filled with dechlorinated water. Freshly released cercariae were transferred and stilled for 1 h. 50μl of ethanol of niclosamide (1 mg/L) was added slowly into another side of the groove. The number of cercariae was counted in 3 zones of A, B and C at 0(immediately), 10, 20, 30, 60, 90, 120 min after adding the drug. The total number of cercariae was 58 and 59, respectively at 0 and 120 min in Zone A, 62 and 64 in Zone B, and 58 and 59 in Zone C. It is indicated that, cercariae do not move to the low-concentration of niclosamide, and no withdrawal was found in cercaria to niclosmide.The cylinder bucket (diameter=40 cm, height=50 cm) was filled with de-chlorinated water (25°C), and 1 mg/L of ethanol of niclosamide was sprayed on the surface of water after the water was still. Samples (each for 1 ml) were collected in the distances of 0, 10, 20, 30, 40, 50 cm from the water surface, at 0 (immediately), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 h, and the concentration of niclosamide was determined using HPLC. The concentration of niclosamide sampled from the water surface, dropped from 5.19 mg/L at 0 h to 1.06 mg/L at 23 h, and minute niclosamide (about 0.01 mg/L) was detected in the depth of 50 cm at 1 h. At 23 h, concentrations of sample from different sites all ranged from 0.91 mg/L to 1.06 mg/L. The findings showed that, the concentration of niclosamide, though sampled from different depthes, achieved similar levels within 23 h.The results from the above mentioned experiments showed that the preventive agent diffused to water after spraying on the bovine skin. However, the subsequent drug was not able to either damage, or lead to with drawal of the surrounding cercariae. It is concluded that, the preventive agent of niclosamide against bovine schistosomiasis could not achieve the preventive efficacy in the surrounding water body.PART III The activity of the preventive agent of niclosamide against bovine schistosomiasis in skinFive buffaloes, aged 2-3 years old, were purchased from Wucheng Town,Yongxiu County of Jiangxi Provience, each was sprayed with 500 ml of the preventive agent. 0 (immediately), 1, 2, 3, 5, 10, 15, 20, 25, 30 days later, a small piece of skin, was collected from the neck of each buffalo, then transferred in a bag, and immediately stored in an icebox. All skin samples were cut into pieces, homogenated with 50 ml of water and 10μl of acetic acid for 30 s, then the mixture was transferred to a 100 ml of bottle for ultrasonic effect for 30 min. Then the homogenate was cooled to room temperature and fixed to 100 ml, then centrifuged at 4000 rpm for 20 min. Clear fluid was filtered with a micro-pore filter membrane (mesh size 0.45μm), and then the concentration of niclosamide was determined with HPLC. In the buffalo skin taken at 0 (immediately), 1, 2, 3, 5, 10, 15, 20, 25, 30 days after spraying the agent, the average concentrations of niclosamide were 17.05, 14.72, 7.21, 4.90, 4.92, 3.63, 3.86, 3.63, 3.66μg/cm2, respectively, and the top concentration reached up to 19.25μg/cm2. Niclosamide had a long detention time in the buffalo skin and formed a protective barrier with high concentration of drug.Thirty mice were divided into 3 groups, namely the control group, the spraying group on abdoman and the spraying group on the back. After the preventive agent dried, all mice were infected with S. japonicum cercariae. Our findings showed, the total worm burdens were 30.7±2.5 in the control group, 31.5±2.7 in the spraying group on abdoman, and 0 in the spraying group on the back, respectively. It is indicated that spraying agent on the back can not prevent the infection with the parasite (no significant difference while comparing with the control group), but spraying on the abdoman can completely prevent the infections.The results of the experiments showed that niclosamide had a long detention time in the buffalo skin and formed a protective barrier with high concentration of drug. The drug concentration in the skin killed all cercariaes effectively in a short time, so as to interrupt the invasion of cercariae. It is speculated that, the preventive agent of niclosamide against bovine schistosomiasis exerts the preventive effect against S. japonicum infection through drug retention in the skin and the resultant barrier.PART IV The in vivo activity of the protective agent of niclosamide against bovine schistosomiasisCercariae were released from S. japonicum-infected snails. Supernatant water was placed into a 10 ml tube, then put in an ice bath for 10 min, and centrifuged at 3 000 rpm for 3 min.The water in the tube was poured slowly, and the cleaning mixture was added, the centrifuged at 3000 rpm for 3 min for 6-7 times. Then the cercariae were transferred into the medium, and the density was 100 per ml. 0.2 ml of the medium was placed into a 96-well plate, each well containing 10-30 cercariae. The plate was then placed in an incubator at 37°C containing 5% CO2. 24 h later, the cercariae losing tails were counted, and the medium was changed to those containing niclosamide with concentrations of 3, 0.3, 0.03, 0.003 mg/L, respectively. The death of schistosomulum was observed each day. Those schistosomula losing semi-transparent appearance or inactivity for more than 30 s indicated death. 24 h later, the percentage of cercaria developing to schistosomulum was 90%. 24 h post-treatment with niclosamide, the mortality of schistosomulum was 100% while treated with 3 mg/L of the drug. While treated with 0.300 mg/L of niclosamide for 24 h and 0.030 or 0.003 mg/L of niclosamide for 120 h, there were no significant differences in the mortality rate of schistosomulum between the treated and control groups(P<0.05). Five buffaloes, aged 2-3 years old, were purchased in Wucheng Town, Yongxiu Counity of Jiangxi Province. 5 ml blood was collected from the cubital veins of each buffalo at 0 (immediately), 1, 2 ,6, 12, 24, 48, 96, 240, 360, 480, 600, 720 h after spraying the agent. Serum was precipitated with centrifugation after adding 2 times of acetonitrile, and its super-stratum clear fluid was treated by using solid phase extraction, centrifuged at 4000 rpm for 5 min. Niclosamide on the column was eluted by 5% ammonia-methanol and the eluate was dried by nitrogen and redissolved with mobile phase, and the concentration of niclosamide was determined. The average concentration of niclosamide in the blood, taken at 0, 1, 2, 6, 8, 24, 48, 96, 240, 360, 480, 600, 720 h post-spraying, was 0.115, 0.157, 0.193, 0.285, 0.166, 0.129, 0.020, 0.016, 0.013, 0.014, 0.011, 0.008 mg/L, respectively. PKSolver 2.0 software was employed to carry out the data analysis.The time to peak parameter of niclosamide was 8 h, half-time 328 h, peak value 0.285 mg/L.The results of the experiments showed that niclosamide had a long detention time in the buffalo blood, but the concentration did not reach lethal concentration for schistosomulum. Therefore, there is little possibility that the agent performs preventive effect in buffalo blood.Our findings showed, the protective agent of niclosamide against bovine schistosomiasis was absorpted through buffalo skin and dissolved to water body, but cercariae were not killed in a low concentration of niclosamide and did not have the characteristics of withdrawl. The agent was unlikely to exert any effect in water body. Niclosamide had a long detention time in the buffalo blood, but the concentration did not reach lethal concentration for schistosomulum.The agent, therefore, was impossible to effect in buffalo blood. Niclosamide had a long detention time in the buffalo skin and formed a protective barrier with a high concentration of drug. The concentration in the skin could kill cercaria in a short time, so as to interrupt the invasion of cercariae. Therefore, the agent developed a protective barrier of drug to protect against the infections of S. japonicum.
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