Quantitation of Cryptosporidium hominis Oocyst Shedding in a Rodent Model of Human Cryptosporidiosis by Real Time Polymerase Chain Reaction

• Main Author: Wyman, Taylor Dawn
• Language: en
• Published: The University of Arizona. 2012
• Online Access: http://hdl.handle.net/10150/244822

A real time or quantitative polymerase chain reaction (rtPCR or qPCR) was developed to quantitate Cryptosporidium hominis oocysts shed in the feces of experimentally infected gerbils serving as a model for human cryptosporidiosis. Primers specific for the Cryptosporidium oocyst wall protein (COWP), a gene with 99% conservation between C. hominis and C. parvum, were used to detect DNA extracted from both C. hominis and C. parvum. The concentration of oocysts in the experimental fecal samples was calculated by sequence detection software, comparing these samples to standard curves generated from DNA extracted from purified C. parvum or C. hominis oocysts. The data show a trend of similar quantities when comparing the number of oocysts per ml found using qPCR to the number counted microscopically by immunofluorescence assay. With further optimization, this protocol should be useful for quantitating oocysts in experimental fecal samples from other animal models of cryptosporidiosis resulting from C. hominis and C. parvum infection.