Abstract
The parasite Cryptosporidium parvum is a common waterborne pathogen that causes diarrhea in vertebrates, including humans. The oocysts shed in the feces of their hosts are resistant to a wide range of disinfectants, and they can thus survive in the environment for a long time. Ozone (O3) is a powerful disinfectant due to its high oxidative characteristics, and it is used to inactivate microorganisms in drinking water and wastewater. As an alternative to the gas dissolution system for producing ozone from oxygen, a simpler electrolytic ozone generation system has recently been developed. In this system, ozone molecules (ozonated water) are more easily, safely, and directly generated in water by electrolysis. In the present study, we evaluated for the first time the efficacy of the ozonated water produced by this system in inactivating Cryptosporidium parasites. The disinfectant capabilities of ozonated water produced by this system at different current intensity (which change the ozone concentrations) and short exposure times (15–60 s) were assessed using two in vitro viability assays, i.e., 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) staining and excystation assays. The DAPI/PI staining assay results showed that treatment with ozonated water for 1 min inactivated 80% – 90% of the oocysts when the CT value [= concentration of ozone (mg/l) × time (min)] was more than 0.01 mg·min/l. However, these results were not reflected in the excystation assay results. Scanning electron microscopy revealed that the oocysts were damaged by the ozone molecules, especially the oocyst walls, and it was found that the deformed oocysts (dead) and partially excysted oocysts (alive) could not be differentiated by the excystation assay. Thus, the excystation assay was found to be unsuitable for the evaluation of the inactivation efficacy of ozonated water produced by this system. Exposure to ozonated water produced with a low current intensity (0.3 A) for 15 and 120 s resulted in the inactivation of 96.8% (CT value: <0.003) and 99.5% (CT value: <0.020) of the oocysts, respectively. Thus, it was estimated that a CT value more than 0.020 was required to inactivate > 99% of the C. parvum oocysts. These results suggested that ozonated water produced by the electrolytic generation system might more effectively inactivate the parasites when compared to that produced by the previous ozone generation system (the gas dissolution system). Although further studies using additional approaches are needed to obtain more clear evidence, this system appears to have high potential for application in various situations and for other pathogenic organisms.