Protoscolex evagination and pre-worm maintenance with bile are key processes for adult worm development of Echinococcus granulosus and Echinococcus multilocularis in vitro

Abstract

Abstract Background: In vitro cultivation of Echinococcus is essential for vaccine development to prevent transmission of echinococcosis to dogs. We optimized the conditions for Echinococcusprotoscolex evagination and adult worm development in vitro, including those of water, bile, bile salt, trypsin, and serum in the culture system. Results: Short stimulation (2 × 20 s) with water significantly increased the evagination of protoscoleces (pre-worms) of both E. granulosus and E. multilocularis. However, medium containing fetal calf serum (FCS) invaginated 92% of these evaginated protoscoleces. Preculture of the evaginated protoscoleces in no-serum RPMI1640 medium containing dog bile or bile salt for three days maintained 80.5% of the evaginated protoscoleces. Dog serum gel-base maintained 79.8% of the evaginated protoscoleces developing adult worms, which was higher than newborn bovine serum gel-base. The rapidly developing worms had 3–4 progloids after 56 days of culture. E. granulosus worms were longer and wider in size than E. multilocularis after five weeks of in vitro culture. Conclusions: Brief stimulation with water for proscolex evagination and pre-worm maintenance in no-serum medium are crucial for in vitro worm development of E. granulosus and E. multilocularis. Dog serum gel-base and bile salts are important for long-term tapeworm development. This study was supported by the National Natural Science Foundation of China (81830066, U1803282)