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Cryptosporidium capture and detection of ultraviolet radiation induced DNA damage

University of British Columbia

Majority of the waterborne outbreaks in USA and Canada are associated with protozoa. Cryptosporidium, an obligate intra-cellular human pathogen, is responsible for more than 50% of these outbreaks and has become one of the major public health concerns as they can survive typical chemical disinfection treatments. Early detection of this parasite in the water and determination of ultraviolet treatment efficacy can play a role in reducing this disease burden. An antibody based capture surface was developed to detect Cryptosporidium oocysts in treated water. The surface was able to capture three different species of Cryptosporidium: C. parvum; C. muris; C. hominis but not E. coli indicating that the capture surface is Cryptosporidium genus specific rather than species specific and the chances of capturing microorganisms other than Cryptosporidium from water are low. IgG3 was selected as better candidate for the capture surface development due to its higher capture efficiency (~84%-90%) compared to that of IgG1 (~54%-74%). Though the oocysts were successfully released in intact form from the capture surface at pH 1.0, it was not possible to reuse the surface because the capture performance decreased after pH treatment. An indirect ELISA protocol was optimized to detect UV induced photoproducts (CPDs) in the DNA of UV treated Cryptosporidium oocysts using cuvettes in a spectrophotometer. Power soil kit was selected as the preferred DNA extraction kit because of its high recovery from low concentration of Cryptosporidium in water with high concentrations of other solids. The optimized ELISA protocol was applied on the samples spiked with different doses (0, 3, 6, 10 and 40 mJ/cm²) of UV irradiated Cryptosporidium oocysts. The signal generated from DNA-antibody reaction resulted in an exponential rise to maximum curve which showed that the absorbance (indication of DNA damage) increased with the increase in UV dose. Adaptation of these techniques for Cryptosporidium detection & UV treatment validation is expected to improve the standards for water quality monitoring, providing the communities with assurance that their water is safe to consume.

Text

2017-03-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/61058

Biochemistry and Molecular Biology

University of British Columbia

UBCO

http://creativecommons.org/licenses/by-nc-nd/4.0/