Genotyping of Cryptosporidium parvum with microsatellite markers

Recent outbreaks of cryptosporidiosis caused by Cryptosporidium parvum in the United States and other countries, as well as the emergence of cryptosporidiosis as a frequent cause of morbidity and mortality in immunodeficient individuals, have raised the interest of the research community in this parasite. The genus Cryptosporidium, phylum Apicomplexa, comprises an undefined number of species, of which only C. parvum is of public health concern. Cryptosporidiosis is contracted through the ingestion of oocysts, the stage of the parasite produced in large numbers by infected hosts. Because the oocysts are small, typically about 5 microm in diameter, and lack species-specific morphological features, there is a need for molecular markers to distinguish between human-infectious C. parvum and other species that do not (or only infrequently) cause disease in humans. Genetic characterization of Cryptosporidium oocysts using restriction fragment length or sequence polymorphism has revealed host-associated genotypes, that are often referred to as species. In addition, C. parvum was found to include two genotypes, designated type 1 and type 2. Type 1 is almost exclusively found in humans, whereas type 2 infects humans and various mammalian hosts. The frequent occurrence of Cryptosporidium oocysts in untreated surface water and the potential for contamination of drinking water have emphasized the need for molecular markers to track the source of oocysts within a watershed or water distribution system, and to discriminate between oocysts infectious to humans and nonpathogenic species. Genetic markers are also needed to study the taxonomy of Cryptosporidium. Several laboratories have identified microsatellites in the genome of C. parvum and have investigated the level of polymorphism at these loci. For instance, 10 alleles of marker 5B12 have been found to date among C. parvum isolates from various geographical and host origins. Multilocus haplotypes based on such markers are suitable for discriminating individual isolates of C. parvum. In an attempt to develop rapid and cost-effective methods for typing isolates of C. parvum, we have pursued two methods, a traditional polymerase chain reaction (PCR) method followed by gel electrophoresis, and real-time PCR using SYBR Green I melting curve analysis for allele identification.