Towards methods for detecting UV-induced damage in individual Cryptosporidium parvum and Cryptosporidium hominis oocysts by immunofluorescence microscopy.

Abstract

Water is an important transmission route for cryptosporidiosis, with at least 165 waterborne outbreaks of cryptosporidiosis documented. Cryptosporidium can be controlled through water treatment by physical removal and UV disinfection, and a method that can determine whether individual oocysts in a routine sample exposed to UV irradiation have been disinfected is of benefit as it offers increased confidence to water operators. The major effects of UV radiation on cell membranes are alterations of proteins, particularly protein cross-linking. UV-B radiation progressively inhibits protein synthesis. Specific free radical scavengers protect cells against killing and inhibition of protein synthesis by UV-B. UV light also crosslinks the complementary strands of DNA and causes the formation of single strand breaks and pyrimidine dimers. The major lesions induced are cyclobutyl pyrimidine dimers (CPDs; also known as thymine dimers, TD). UV-induced DNA lesions in living cells and in some microorganisms can be repaired by the enzyme-dependent nucleotide excision repair (NER), also named dark repair, and the light-dependent reaction known as photoreactivation (PHR). Dark repair and PHR enable UV-inactivated microorganisms to recover and may reduce the efficiency of UV inactivation. Cryptosporidium parvum oocysts are inactivated at 3-40 mJ/cm² using medium- and low-pressure UV light. Cryptosporidium parvum can undertake photoreactivation and dark repair at the genomic level and NER repair genes have been identified in C. parvum and C. hominis. However, UV inactivation of Cryptosporidium oocysts is irreversible, despite the presence of the UV repair genes. We investigated the following hypotheses for developing a method to demonstrate UV inactivation of Cryptosporidium oocysts: (i) UV disinfection induces the production of reactive oxygen species (ROS); (ii) UV disinfection induces apoptosis; and (iii) UV disinfection causes damage to DNA, which is detectable using fluorogenic DNA reporters. We developed assays for determining UV inactivation of Cryptosporidium oocysts which would remain compatible with, and integrated as much as possible with, existing UK and USA detection methodologies. Our development of suitable methods which can detect UV damage in individual organisms reliably and reproducibly was driven by a search for fluorogenic reporters which could enter and stain UV-killed and damaged Cryptosporidium oocysts. Antioxidants reduce ROS production and the antioxidant glutathione (GSH) plays a significant role in inhibiting the generation of mutagens by ionizing radiation. The presence of GSH, as a putative reporter of UV damage caused by the production of ROS, was investigated in intact, untreated C. parvum oocysts and sporozoites within intact UV-irradiated oocysts (40 mJ/cm²) using the fluorogenic vital dye monochlorobimane (MCB) to detect both GSH levels and activity in oocysts. MCB fluorescence localized GSH in purified, intact, recently excreted and aged C. parvum oocysts, at several nuclear and cytoplasmic sporozoite foci (n=2-6). We did not demonstrate the function of GSH as an endogenous free radical scavenger in UV-irradiated oocysts, and other free radical scavengers are more active than GSH in UV-treated C. parvum oocysts. MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts. The DNA intercalating dye YO-PRO1 (YP) has been used to determine apoptosis and was used to investigate the role of UV irradiation in inducing programmed cell death/apoptosis. YP detected DNA damage in UV-treated (40 mJ/cm²) C. parvum oocysts. YP was incorporated into sporozoite DNA of intact, irradiated oocysts (possibly apoptotic) which exhibited no apparent oocyst wall damage. However, control oocysts did not exclude YP entirely. YP is unlikely to provide a reliable estimate of the possible apoptotic changes that can occur in irradiated oocysts. An antibody raised against TDs (α-TD) was used to identify changes induced by UV light in C. parvum sporozoites and oocysts, and its nuclear location was validated by co-localization with DAPI. A freeze-thawing (five cycles) procedure improved α-TD antibody labelling within irradiated C. parvum oocysts. No α-TD localization was seen in non-irradiated oocysts. Both C. parvum and C. hominis oocysts exposed to different doses of UV light (range, 10-40 mJ/cm²) demonstrated TD lesions following irradiation. We conclude that an immunofluorescence assay using α-TD antibodies which, for C. parvum, has been validated against a neonatal mouse infectivity assay, is suitable for detecting thymine dimers in air-dried oocysts and air-dried sporozoites of C. parvum and C. hominis oocysts, and that the α-dsDNA antibody is a good candidate for a positive control for the assay.