Development and application of a molecular viability assay for Cryptosporidium parvum based on heat shock protein 70 gene expression : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
The aquatic protozoan pathogen Cryptosporidium parvum causes serious problems in water treatment plants. Its small size allows it to evade most physical removal barriers, becoming a major cause of diarrhoeal disease. Cryptosporidiosis is self-limiting in immuno-competent people, but can be life-threatening to immuno-compromised people. Multiple physical and/or chemical barriers, e.g. UV light, are used to remove or inactivate oocysts. It is important to have a reliable method to estimate numbers of viable C. parvum in order to monitor water supplies and evaluate protection measures. This study aims to establish an RT-PCR assay for viable C. parvum based on expression of the heat shock protein 70 (hsp70) gene. For the assay, oocysts are heat shocked at 45°C to increase hsp70 mRNA (to increase assay sensitivity) and then lysed to release nucleic acids. Oligo (dT) 25
Dynabeads® are used to isolate mRNA and a DNaseI step removes genomic DNA. The RT-PCR step converts mRNA to cDNA and this product is analysed by electrophoresis on an agarose gel. Many studies have investigated UV as a means to inactivate oocysts, but none so far have used an RT-PCR viability assay. UV inactivation may take two forms: oocysts can either be metabolically inactive or they can remain metabolically active but are rendered non-infective. First, the cidal UV dose for C. parvum oocysts was determined and was found to be 1100 mJ/cm2; this value is much higher than previously reported. Next, photoreactivation of UV-killed oocysts was investigated and found not to occur in C. parvum, which is inline with other observations. But, contrary to previous studies, trials reported here showed that hsp70 mRNA is still detectable inside heat inactivated oocysts even after 70 hours at room temperature. Other experiments conducted include trials using β-tubulin gene as a viability marker and studying the effect of hsp70 mRNA stability on heat inactivation of oocysts. Using the β-tubulin gene as a genetic marker in the RT-PCR assay found a cidal dose of only 10 mJ/cm2 was required for C. parvum oocysts, which corroborates previous work and can be explained by β-tubulin mRNA being less stable than hsp70 mRNA. The RT-PCR oocyst viability assay developed here is both efficient and effective. Overall the results presented show the importance of a reliable viability testing method and also that the best choice of gene target depends on the method of oocyst inactivation. The high apparent stability of hsp70 mRNA within heat inactivated oocysts limits the utility of this gene target and this phenomenon warrants further investigation. Future applications of the general RT-PCR oocyst viability assay method include development into a high throughput system for routine determinations of viable oocyst numbers in contaminated water. Other gene loci can also be examined as alternative viability markers now that the complete C. parvum genome sequence is available.