Complete development and multiplication of Cryptosporidium hominis in cell-free culture

The present study reports for the first time the completion of the life cycle of Cryptosporidium hominis in cell-free culture and multiplication of the parasite via qPCR. Individual life-cycle stages were characterised using Cryptosporidium-specific antibody staining (Sporo-Glo™) and fluorescent in situ hybridisation (FISH) staining on cultures inoculated with excysted oocysts and purified sporozoites. In both cultures, C. hominis successfully proliferated and completed its life cycle, however development in cultures inoculated with purified sporozoites lagged behind cultures inoculated with excysted oocysts. Some novel findings of the study include the visualisation of pairing and multiple associations between various developmental stages in a process similar to syzygy and the formation of Cryptosporidium stages (trophozoites and meronts) inside the oocysts without excystation. qPCR analysis revealed a 5–6-fold amplification of parasite DNA. Future studies are required to improve the amplification of the parasite. The present study confirms the suitability of this culturing model to support the growth and proliferation of C. hominis (which unlike C. parvum, cannot be readily cultured in small animal models) and will greatly assist in our understanding of the developmental biology of Cryptosporidium, its position within the Apicomplexa and its relationship to gregarine protozoa.