Development of an improved in vitro culture system for Cryptosporidium parvum

Abstract

The protozoan parasite Cryptosporidium parvum is a leading cause of pediatric diarrhea in developing countries, yet effective treatments for the disease are lacking due to the difficulty of studying Cryptosporidium in the laboratory. Specifically, current culturing methods rely on adenocarcinoma cell lines that do not support continual growth of C. parvum in vitro, hindering the development of reagents such as stage‐specific antibodies and genetic tools for this parasite. To better mimic the native intracellular niche of C. parvum, we have utilized primary intestinal epithelial cell (IEC) monolayers as an improved system for the study of C. parvum. We have found that human and mouse IECs support enhanced growth of C. parvum compared to adenocarcinoma cell lines. Importantly, parasite growth in mouse IECs, the first mouse cell line to support robust asexual C. parvum growth, enabled us to generate a unique panel of monoclonal antibodies against the intracellular life cycle stages of the parasite. We have also developed quantitative PCR assays to measure expression of genes associated with sexual‐stage development of the parasite independent of absolute parasite replication. With the development of these new tools and the enhanced culture system, we are now able to screen culture conditions mimicking the hypoxic, reducing environment of the small intestine to identify conditions that further improve C. parvum growth and/or complete development of the life cycle in vitro. Additionally, we are also screening a panel of gut microbial metabolites for their effects on C. parvum growth to gain insight into the complex community interactions that may contribute to C. parvum infection in vivo. In all, these new tools and improved culture system will facilitate the study of Cryptosporidium in vitro, allowing us to gain a better understanding of the parasite's biology as a means to more effective therapeutics for the disease in the future.Support or Funding InformationThis work is supported by the Bill and Melinda Gates Foundation.