Synergism in sequential inactivation of Cryptosporidium parvum with trypsin and UV irradiation

Cryptosporidium, a protozoan parasite, in wastewater presents a major public health concern for water safety. However, bactericidal efficiencies of conventional disinfection methods towards Cryptosporidium oocysts are still hampered owing to the presence of their thick outer wall. In this study, we present a novel UV inactivation process where the efficiency has been significantly enhanced by addition of a trypsin pretreatment stage. Notably, inactivation (log-reduction) of oocysts was noted to be 73.75-294.72% higher than that obtained by UV irradiation alone, under identical conditions. Experimental observations and supporting mechanistic analyses suggest that trypsin led to cleavage of the protein layers on the oocyst wall, facilitating penetration of UV radiation into the oocysts leading to degradation of their genomic DNA (gDNA). The dissociative effect of trypsin on the oocyst wall was indicated by the fact that 64.50% of oocysts displayed early apoptosis after trypsinization. Imaging by scanning electron microscopy indicated that this combined treatment led to substantial disruption of the oocyst coat, deforming their shape. This resulted in the release of cellular proteins and gDNA, their concentrations in bulk solution increasing by 1.22-8.60 times. As UV irradiation time was prolonged, gDNA was degraded into smaller fragments with lower molecular masses. Both laddering and diffuse smear patterns in gel analysis indicated significantly detrimental effects on gDNA and viability of oocysts. Overall, this study demonstrated enhancement of UV inactivation of Cryptosporidium oocysts by trypsin and explored the underlying mechanisms for the process.