Cryptosporidiosis is a major cause of diarrheal illness worldwide and characterized by several daily bowel movements, resulting in fluid loss and dehydration. Two species, Cryptosporidium hominis and C. parvum are the main causative agents in human infection. Complicating matters for disinfection, epidemiology, and treatment studies, C. parvum isolates infect multiple mammalian species while C. hominis solely infects humans. The purpose of this dissertation was to: (1) develop a C. parvum PCR based detection method and discuss its limitations; and (2) to extend current epidemiological and molecular data rationalizing the multiple C. parvum host specific infectivity patterns. To fulfill these two objectives, three separate experiments were designed and executed. The results from which are included in the appendix as peer reviewed published manuscripts and are the basis of this dissertation. The first manuscript outlines the use and validation of microscopic micromanipulation to isolate and deliver low numbers of C. parvum oocysts to a test vial of interest. In addition, a nested PCR primer set was developed targeting the 18S rRNA and tested for sensitivity using micromanipulation and specificity using isolated DNA from multiple different species. It was determined that micromanipulation is an accurate technique able to deliver low numbers of oocysts to a test vial of interest. The nested PCR protocol had LLOD, in replicates of 50 and laboratory grade water, of 100% with ten oocysts and 38% with a single oocyst. The second manuscript compared detection efficiencies of the EPA Method 1623 with the nested PCR protocol outlined in the first manuscript. Both methods had equal detection efficiencies giving positive detection at the five-oocyst level. In addition, non-specific PCR amplification results generated during the study revealed specificity issues that have implications effecting past, current and future molecular detection validation processes. The final manuscript describes nucleotide and deduced amino acid differences between the C. parvum and C. hominis attachment/invasion surface proteins Cpgp 40/15, p23, and GP900. This information has implications explaining host-specificity differences observed among Cryptosporidium spp.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/280454 |
| Date | January 2003 |
| Creators | Sturbaum, Gregory Dean |
| Contributors | Sterling, Charles R. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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