Cryptosporidium parvum isolate comparisons and improvements to in vitro cultivation
AuthorOrtega, Ynes Rosa, 1960-
AdvisorSterling, Charles R.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © 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.
AbstractCryptosporidium parvum infects numerous species of mammals, including man, and is a frequent cause of diarrhea. It can be life threatening to immunocompromised individuals. The mechanisms responsible for its pathogenesis are not completely understood. Differences between isolates originating from animal and human sources are not completely defined. Six isolates of human or bovine origin from different geographical areas were examined by various protein analysis (immunofluorescence assay, SDS-polyacrylamide gel electrophoresis, isoelectrofocusing) and nucleic acid (restriction fragment length polymorphism and random amplified polymorphic DNA) methods. Differences were observed between human and bovine isolates when examined by RFLP and by using series of primers in RAPDs but not in their protein profiles. The mechanisms of parasite invasion and replication are not well defined due to the lack of a defined in vitro cultivation system for C. parvum. An in vitro cultivation system for this parasite was developed using the Caco-2 cell line. Optimum infection was obtained using 1x 10⁵ purified sporozoites and maximal yield of intracellular parasites were obtained 45 hrs. PI. This system was used to evaluate the in vitro neutralization of Cryptosporidium infection using hyperimmune chicken egg yolk and hyperimmune bovine colostrum. Also, paromomycin, clarithromycin and 14-OH-clarithromycin were evaluated alone and in combinations using this in vitro system. Paromomycin was more effective, but when combined with clarithromycin and 14-OH-clarithromycin, a synergistic effect in promoting parasite death was observed. The merozoite is a pivotal life cycle stage in the asexual development of C. parvum. The mass production of purified merozoites from infected animals depends largely on the infection dynamics occurring within individual animals. Mass production of merozoites in vitro simplifies the process of purification and permits further studies aimed toward interrupting parasite invasion and proliferation. Various strategies for mass production were tested. The use of a collagen coated Dacron tube allowed the growth of Caco-2 cells on the inner surface area. After confluency, purified sporozoites were allowed to infect the cells. This system proved to be efficient because of its large inner surface area, small amount of media required, permeability, flexibility of the tubing and ease of handling the system.
Degree ProgramGraduate College
Microbiology and Immunology