ISOLATION OF SINGLE OOCYSTS OF CRYPTOSPORIDIUM PARVUM FOR MOLECULAR ANALYSIS

Carrie Reed, Greg Sturbaum, Paul Hoover, Marilyn Marshall and Charles Sterling

Cryptosporidium parvum is a protozoal parasite that has garnered increased attention as a human pathogen. First recognized in AIDS and other immunocompromised patients, cryptosporidiosis is now recognized as an important cause of diarrheal illness in immunocompetent patients worldwide, especially in children. Two genotypes of C. parvum are currently recognized, Type I which infects only humans and Type II which infects both animals and humans. While numerous population studies have been done to define the population structure and epidemiology of the organism, none have ever been done at the level of a single oocyst. Without molecular analysis at this level, the true population structure and epidemiology cannot be completely understood. The goal of this study was to develop a technique to extract single oocysts for further molecular analysis. The oocysts were isolated and extracted from a purified sample of oocysts via micromanipulation and DNA was liberated using a freeze-thaw method. A set of nested primers were developed to amplify a 593 bp portion of the 18s rRNA gene, and conditions were optimized to amplify this region using the polymerase chain reaction (PCR). PCR products were analyzed using agarose gel elecrophoresis and staining with ethidium bromide (EtBr). Using this technique, it was demonstrated that the sensitivity of detection ranges from 100% detection of 10 oocysts, to 38% detection of single oocysts. For quick analysis without the delay or expense of sequencing, a restriction-fragment length polymorphism (RFLP) analysis was also developed. This analysis is able to distinguish Type I and Type II based on the banding pattern following digestion with the restriction enzyme VspI.