Challenges and Achievements in the In Vitro Culture of Balantioides coli: Insights into the Excystation Process

Balantioides coli is the only ciliate currently described as an intestinal parasite of humans, although it can also infect other animals, particularly pigs. Its in vitro cultivation remains challenging, and no axenic culture system is currently available. Cultures are initiated by adding small amounts of feces containing cysts or trophozoites to the culture medium. Implantation success is lower when starting from cysts, and the mechanisms and early events of excystation remain poorly understood. In this study, we describe the sequence of events involved in excystation and identify factors potentially important for culture establishment. Cysts were obtained from orangutan feces and genetically confirmed as B. coli. Only viable cysts, determined by trypan blue or methylene blue exclusion, were used. After artificial digestion with pepsin and trypsin, cysts were incubated at 28 °C for up to 72 h in DMEM supplemented with L-glutamine, yeast extract, fetal bovine serum, and starch granules. Excystation began with a fissure in the cyst wall, allowing for bacterial entry. This appeared to stimulate the trophozoites, the increased motility of which progressively weakened and ruptured the wall, allowing for their emergence. Wall rupture and bacterial entry were critical for activation., whereas starch type had no apparent influence. Excystation occurred within the first hours; otherwise, cysts degenerated.