Detection of UV-Induced Thymine Dimers in IndividualCryptosporidium parvumandCryptosporidium hominisOocysts by Immunofluorescence Microscopy

Abstract

To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ ⅐ cm ؊2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4,6-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Redlabeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ ⅐ cm ؊2 . With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ ⅐ cm ؊2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ ⅐ cm ؊2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.UV irradiation as a sterilization technology in the water and food industry is effective in killing contaminating organisms, such as viruses, bacteria, fungal spores, and parasites (8). The major DNA lesions caused by UV light are cyclobutyl pyrimidine dimers (CPD), which are responsible for UV-induced cytotoxicity and mutagenicity in living cells and microorganisms. UV-induced damage consists of chemical base modifications, whereby two adjacent pyrimidine residues (cytosine or thymine) form a dimer (thymine dimer [TD], cytosine dimer, or thymine-cytosine heterodimer) and 6-4 photoproducts. The formation of these lesions in genomic DNA inhibits normal replication and transcription of DNA and results in the inactivation of cells (12). However, UV-induced DNA lesions in living human epidermal cells (21) and in some unicellular microorganisms (16) can be repaired by one or more mechanisms. These mechanisms include the enzyme-dependent nucleotide excision repair, also named dark repair, and the light-dependent reaction known as photoreactivation. Dark repair and photoreactivation enable UV-inactivated microorganisms to recover, which can reduce the efficiency of UV inactivation (20).Cryptosporidium parvum oocysts have the ability to carry out photoreactivation and dark repair at the genomic level (13, 16). Also, nucleotide excision repair genes in C. parvum and Cryptosporidium hominis have been identified. However, UV inactivation of Cryptosporidium oocysts is irreversible, despite the presence of these UV repair genes (18). Rochelle et al. (19) demonstrated the accumulation of cyclobutane TDs in the genome of C. parvum oocysts exposed to increasing dosage...

UV light-induced DNA damage detection in the unicellular green alga Chlamydomonas reinhardtii

“…Moreover, monoclonal antibodies against cyclobutane thymine dimers are used in immunofluorescence microscopy for the detection of photorepair (Roza et al 1991;Al-Adhami et al 2007).…”

Stress-Induced Hsp70 Gene Expression and Inactivation of Cryptosporidium parvum Oocysts by Chlorine-Based Oxidants

“…As to other possible cellular defense mechanisms, researchers have investigated UV-induced DNA damage in C. parvum oocysts, but no evidence was found that DNA repair would be capable of restoring oocyst infectivity (2,20,21,23).…”

“…A batch of 3 ϫ 10 6 oocysts was subjected to MOS at a 25-mg/liter FAC concentration for 1 h. The oxidant was quenched, and the oocysts were briefly centrifuged and resuspended in distilled water. The oocyst suspension was incubated at 22°C, during which aliquots of 10 5 oocysts were taken at different time intervals (1,2,4,6,8,12,16, and 24 h), and a second heat shock was applied at the 24-hour time point to measure the extent of the oocysts' heat shock response during a 4-h interval, using the qRT-PCR amplification of the Hsp70 mRNA. The controls were (i) the same number of oocysts subjected initially to heat shock at 45°C for 20 min but not treated with chlorine oxidants and also (ii) oocysts completely killed by exposure to 65°C for 1 h in 10% ammonia.…”

Assessment of UV Pre-Treatment to Reduce Fouling of NF Membranes

“…This may be related to the well-established mechanism of UV disinfection, i.e. bactericidal and bacteriostatic effect of UV mainly through genetic damages induction (Gaid et al 2006, Al-Adhami et al 2007). This effect of UV irradiation can be potentialized by other pretreatments decreasing BDOC like ozonation/GAC adsorption since dissolved organic matters are necessary to repair cell damages (Alkan et al 2007, Camper 2004.…”