Electron microscopic observation of the early stages of Cryptosporidium parvum asexual multiplication and development in in vitro axenic culture

• Early and mature C. parvum trophozoites were noticed.
• Dividing and mature meronts were observed within the first 24 h of cultivation.
• The observation of new budding merozoite and the formation of new banana-, crescent- and rod-shape morozoites were seen.
• The asexual reproduction of C. parvum was fit to the pattern of both eimerian and neogregarines.

The stages of Cryptosporidium parvum asexual exogenous development were investigated at high ultra-structural resolution in cell-free culture using transmission electron microscopy (TEM). Early C. parvum trophozoites were ovoid in shape, 1.07 × 1.47 μm2 in size, and contained a large nucleus and adjacent Golgi complex. Dividing and mature meronts containing four to eight developing merozoites, 2.34 × 2.7 μm2 in size, were observed within the first 24 h of cultivation. An obvious peculiarity was found within the merozoite pellicle, as it was composed of the outer plasma membrane with underlying middle and inner membrane complexes. Further novel findings were vacuolization of the meront's residuum and extension of its outer pellicle, as parasitophorous vacuole-like membranes were also evident. The asexual reproduction of C. parvum was consistent with the developmental pattern of both eimerian coccidia and Arthrogregarinida (formerly Neogregarinida). The unique cell-free development of C. parvum described here, along with the establishment of meronts and merozoite formation, is the first such evidence obtained from in vitro cell-free culture at the ultrastructural level.