Abstract:

Objective To evaluate the effects of clorsulon and ornidazole against Echinococcus granulosus protoscoleces and E. multilocularis metacestodes in vitro. Methods E. granulosus protoscoleces were isolated from livers of sheep infected with E. granulosus, and E. multilocularis metacestodes were isolated from the peritoneal cavity of infected Meriones unguiculatus. They were cultured in Dulbecco’s modified eagle medium (DMEM) seeded with human hepatoma cells for 2 months, then vesicles of 1-5 mm in diameter were collected. Four groups were set in the experiment: the clorsulon treatment group (experimental group), ornidazole group (experimental group), albendazole group (positive control), and 0.2% DMSO group (vehicle control group). The experiment was performed in duplicate, and repeated twice. All drugs were used at a final concentration of 40 μmol/L. Each well contained about 100 E. granulosus protoscoleces, or 25-35 E. multilocularis vesicles. After being treated for 24, 48, 72, 96, 120, 144 and 168 h, the protoscoleces were stained by trypan blue and observed by microscopy. The viability of protoscoleces was calculated and analyzed with analysis of chi-square test. The E. multilocularis vesicles were treated for 36 and 120 h, followed by morphological observation by microscopy and measurement of alkaline phosphatase activity in the culture supernatant. The results were analyzed with ANOVA. Result After treatment with clorsulon, ornidazole and albendazole, the protoscoleces showed a darkened color, decreased calcium precipitates, hook shedding, and scolex evagination and elongation, while 0.2% DMSO did not show such effects. After treatment with clorsulon, ornidazole or albendazole for 24, 48, 72, 96, 120, 144 and 168 h, the viability of protoscoleces was 79%, 70%, 56%, 42%, 33%, 16%, 15%; 86%, 67%, 63%, 48%, 32%, 28%, 21%; and 85%, 71%, 45%, 36%, 21%, 15%, 8%, respectively. In the 0.2% DMSO group, 100% of protoscoleces survived. The viability after clorsulon, ornidazole and albendazole treatment was significantly lower than that in the 0.2% DMSO group (χ² = 147.83, 130.58, 170.37; P < 0.05). Compared to the intact vesicles after 0.2% DMSO treatment, clorsulon, ornidazole and albendazole all caused collapse and shrinkage of E. multilocularis vesicles. At 36 h of treatment with clorsulon, ornidazole, albendazole and 0.2% DMSO treatment, the A₄₀₅ values of alkaline phosphatase were 0.196 ± 0.030, 0.186 ± 0.004, 0.244 ± 0.049 and 0.131 ± 0.020, respectively, and at 120 h, they were 0.431 ± 0.006, 0.271 ± 0.004, 0.423 ± 0.007 and 0.116 ± 0.004, respectively. There were significant differences in the alkaline phosphatase activity in the three drug treatment groups versus the 0.2% DMSO group (t = 0.006, 0.004, 0.007; P < 0.05). Conclusion Both clorsulon and ornidazole have strong effects against E. granulosus protoscoleces and E. multilocularis metacestodes in vitro, and are potential drugs for echinococcosis.

Key words: Echinnococcus granulosus, E. multilocularis, Clorsulon, Ornidazole