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Molecular Genotyping of Viable Cryptosporidium Oocysts
Citation:
Brescia, C., M. W. WARE, S. L. BARTELT-HUNT, A. EGOROV, AND E. VILLEGAS. Molecular Genotyping of Viable Cryptosporidium Oocysts. Presented at ASM Annual Meeting, Muncie, IN, March 28 - 29, 2008.
Impact/Purpose:
This task focuses on the development, evaluation, and standardization of innovative methods, technologies, and procedures to determine the parasite burden of source and drinking water. Information as to the presence of these organisms in water supplies may assist communities to make informed decisions concerning their public health and infrastructure. Objective: 1) Refine new, practical methods for the detection of CCL-related and emerging waterborne human protozoa. 2) Evaluate new technologies for their use in method development. 3) Evaluate the efficacy of disinfection procedures on protozoans in collaboration with NRMRL scientists. This work in this task supports CCL2 and 3 and is expected to be completed by 9/07.
Description:
Cryptosporidium is a chlorination-resistant protozoan parasite that causes a self-limiting diarrheal disease in the immunocompetent or severe chronic diarrhea in the immunocompromised. Two species, C. parvum and C. hominis, cause most cases of cryptosporidiosis in humans, while C. muris infects primarily rodents. The current US EPA standard methods for the detection of Cryptosporidium oocysts in water, Methods 1622 and 1623, are labor intensive and cannot determine the species or distinguish viable from nonviable oocysts. The goal of this project is to develop a rapid molecular method that specifically detects viable waterborne Cryptosporidium oocysts and that determines the species present in a sample. This alternative technique employs propidium monoazide (PMA) staining in conjunction with conventional PCR detection and genotyping. PMA is a DNA intercalating dye that only penetrates dead cells. Using traditional DNA extraction procedures, only the genomic DNA from live cells, which is not intercalated with PMA, is retained and detected in subsequent PCR analysis. To date, PMA has only been used to differentiate live/dead bacteria and fungi. It has not yet been applied to protozoan parasites. Preliminary results revealed that heat killed oocysts treated with PMA were not detected while live oocysts treated with PMA were detected using conventional PCR. This method was then paired with Cryptosporidium PCR-based genotyping methods to allow for species discrimination. When live C. parvum and dead C. muris oocysts were mixed at a 1:9 ratio, only live C. parvum oocysts were detected. These results indicate that PMA treatment is very specific at detecting DNA from live oocysts in the presence of a higher concentration of dead oocysts. We are currently investigating the application of this method in environmental water matrices.