Molecular-based detection and indentification of Cryptosporidium Parvum
This thesis is an investigation of means to detect and identify ' Cryptosporidium parvum' through the utilization of molecular-based methods. Prior to the naming of 'C. hominis', a 'Cryptosporidium ' species that primarily infects humans, 'C. parvum' was recognized as having 2 main genotypes. One genotype primarily infected humans (genotype I) and one that primary infected animals (genotype II). Genotyping the SSU-rRNA gene of 9 geographically distinct isolates of 'C. parvum ' revealed that the one isolate (HLA) that had been acquired from a human patient infected with Cryptosporidium was distinct from the 8 other isolates at 5 nucleotide positions. The SSU-rRNA sequence of the 8 other isolates were identical. By infecting tissue culture with 'C. parvum' oocysts, the oocyst infectivity can be determined by detecting foci of infection through the use of fluorochrome-conjugated antibodies and epifluorescent microscopy. By exposing 'C. parvum' oocysts to various conditions, their survivability was examined. Oocysts that had been stored at cooler temperatures retained their ability to infect tissue culture for longer durations, greater than 14 weeks when stored at both 4°C and 10°C. Upon increasing the water temperature to 21--23°C, the duration that the oocysts remained infective decreased by 3-log units after 12 weeks. This decrease in infectivity was independent of whether the water was directly from a river source or had been filter-sterilized. Oocysts were also exposed to 9 laboratory disinfectants for durations of 4, 13, and 33 minutes. Seven of the 9 disinfectants were ineffective at decreasing oocyst infectivity. However, exposure to 6% hydrogen peroxide for 4 minutes reduced oocyst infectivity by 3-log units. An in-house ' Cryptosporidium' disinfectant (2.5% (v/v) ammonium hydroxide in windshield washer fluid) reduced oocyst infectivity by 2-log units after 13 minutes exposure. Two human semi-synthetic naive antibody libraries were screened to isolate antibodies that could bind to 'C. parvum' oocysts. Three recombinant antibodies (A11, B9, and mutB10) were obtained, all possessing the ability to bind to 'C. parvum' and block its infection in an HCT-8 tissue culture assay. The degree to which infectivity was neutralized was antibody dose dependant. Of the 3 scFv antibodies, mutB10 was the most effective at neutralizing 'in vitro' oocyst infectivity.