Mouse Models for Use in Cryptosporidium Infection Studies, Quantification of Parasite Burden Using Flow Cytometry, qPCR and Histopathology, and Confocal Imaging of Oocysts.

Abstract

Cryptosporidiosis threatens the lives of young children in developing countries and newborn calves around the world. No vaccine or therapy can prevent or cure this diarrhea-inducing enteric disease caused by Cryptosporidium spp. protozoan parasites. There is an essential need to discover new therapeutic drugs efficient in reducing parasite burden in infected individuals. Research therefore relies on reliable small animal models of cryptosporidiosis. Here, we present excellent mouse models which can efficiently mimic pathogenesis of human and bovine cryptosporidiosis. We also describe methods to purify Cryptosporidium parvum oocysts from stool and intestine of infected mice to facilitate oocyst quantification. Moreover, we present protocols using flow cytometry, quantitative polymerase chain reaction, and histopathology to accurately quantify parasite burden in stool or intestine samples. Also, we are presenting a high-resolution confocal microscopy protocol that constitutes a tool to image C. parvum sporozoites within oocysts. Polyclonal and monoclonal antibodies against Cryptosporidium p23 protein can be used to detect sporozoites within permeabilized or partially excysted oocysts. Additionally, our novel qPCR method, targeting the HSP70 genes, offers enhanced sensitivity for Cryptosporidium detection and quantification.