The parasites Toxoplasma gondii and Cryptosporidium parvum can infect and cause disease in humans, each with specific pathogenesis etiologies. Currently, there are no in vitro platforms for studying the enteric stages of these parasites, in large part because robust methods to expand intestinal epithelial cells from animals that are definitive hosts for these parasites have been lacking. Recently, methods were developed to continuously grow intestinal crypt organoids from human and mouse tissue. We have adapted these methods to isolate and grow intestinal crypt organoids from various large farm and small companion animals that serve as definitive hosts for parasites. These cell lines have expanded over time and have been propagated to high passage number. We intend to characterize these lines for the presence of Lgr5+ crypt cells and other intestinal cell linages, and to develop a 2D growth platform for use in parasite infection and differentiation assays. Much work has been carried out to understand the biology of the intermediate stages of T. gondii, the tachyzoite and bradyzoite. These culturing methods have allowed researchers to develop an extensive molecular toolbox for Toxoplasma, resulting in an increased understanding of the infectious intermediate stages. As of yet, culturing methods for stages beyond the bradyzoite, the merozoite and sexual stages, have not been developed, hindering the ability to study a large portion of the parasites life cycle. We isolated merozoites for genome-wide expression profiling, showing the unique state of the merozoite stage in context with the rest of the life cycle and identifying merozoite specific genes. This information was vital in allowing us to make a transgenic T. gondii parasite, ME49-MSFp-DHFR-Ty (MSF), which expresses a Ty tagged drug marker only at the merozoite stage allowing for the selection of enteric stage growth in vitro and the determination of merozoite differentiation via expression of the Ty marker. We propose to use this transgenic parasite to infect cat intestinal epithelial cells derived from intestinal crypt organoids, and thus determine if the cell type itself is sufficient for enteric differentiation or if additional cell culture requirements will be needed. Culturing methods are also limited for Cryptosporidium parvum, and although there have been advances in propagating Cryptosporidium in IFN? -/- mice providing a platform to perform selection and transgenic experiments, an in vitro culture system that allows for the robust expansion of the parasite still does not exist. Using C. parvum sporozoites, we will perform merozoite differentiation assay in organoid derived cow intestinal epithelial cells, determining the growth and or expansion potential of parasites in this platform using IFA or qPCR.
The parasites Toxoplasma gondii and Cryptosporidium parvum are medically important parasites that, although each has specific life cycle aspects that determine their range and prevalence, share a commonality in that they must pass through the intestinal tract of a definitive host in order to differentiate into the sexual stages, or enteric stages. Continued research into the enteric stages of these parasites will increase our understanding of parasite development and mechanisms that determine the shedding of these organisms into the environment, from where humans can be exposed, resulting in parasite infection and the potential for disease complications.
|Shen, Bang; Powell, Robin H; Behnke, Michael S (2017) QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii. J Vis Exp :|
|Powell, Robin H; Behnke, Michael S (2017) WRN conditioned media is sufficient for in vitro propagation of intestinal organoids from large farm and small companion animals. Biol Open 6:698-705|