Irreversible UV Inactivation of Cryptosporidium spp. Despite the Presence of UV Repair Genes¹

Abstract

Ultraviolet light is being considered as a disinfectant by the water industry because it appears to be very effective for inactivating pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms for repairing ultraviolet light-induced DNA damage, which may limit the utility of this disinfection technology. Inactivation of C. parvum was assessed by measuring infectivity in cells of the human ileocecal adenocarcinoma HCT-8 cell line, with an assay targeting a heat shock protein gene and using a reverse transcriptase polymerase chain reaction to detect infections. Oocysts of five different isolates displayed similar sensitivity to ultraviolet light. An average dosage of 7.6 mJ/cm2 resulted in 99.9% inactivation, providing the first evidence that multiple isolates of C. parvum are equally sensitive to ultraviolet disinfection. Irradiated oocysts were unable to regain pre-irradiation levels of infectivity, following exposure to a broad array of potential repair conditions, such as prolonged incubation, pre-infection excystation triggers, and post-ultraviolet holding periods. A combination of data-mining and sequencing was used to identify genes for all of the major components of a nucleotide excision repair complex in C. parvum and Cryptosporidium hominis. The average similarity between the two organisms for the various genes was 96.4% (range, 92-98%). Thus, while Cryptosporidum spp. may have the potential to repair ultraviolet light-induced damage, oocyst reactivation will not occur under the standard conditions used for storage and distribution of treated drinking water.

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

“…Previous studies have shown that C. parvum oocysts can be inactivated by low (Ͻ10-mJ ⅐ cm Ϫ2 ) doses of UV light using low-or medium-pressure UV lamps (7,16,18,22). We observed nuclear, anti-TD binding in oocysts irradiated at 40, 20, and 10 mJ ⅐ cm Ϫ2 but not in oocysts irradiated at Ͻ10 mJ ⅐ cm Ϫ2 .…”

“…TDs are common forms of CPD produced in cells when UV light is absorbed by the double bond in the thymine base in a DNA molecule, opening the bond and allowing it to react with the adjacent thymine base, forming a tight four-member ring (12). UV irradiation of C. parvum oocysts also produces CPD, and UV inactivation of C. parvum and C. hominis is irreversible, despite the presence of UV repair genes (10,16,18,19). An assay that can detect the presence of TD following UV inactivation in individual waterborne oocysts would prove beneficial to the water industry, public health professionals, and government regulators.…”

“…Oocysts were exposed to a low-pressure UV lamp with an output at 254 nm. The procedure was adapted from that described by Rochelle et al (18). Short-wave UV irradiation from a UVGL-58 Mineralight lamp was used.…”

“…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 dosages of UV light, using a chemiluminescent Western blot assay.…”

Potential molecular tools for assessing the public health risk associated with waterborne Cryptosporidium oocysts

“…Infectivity or viability of Cryptosporidium oocysts has been evaluated by several molecular, culture or infectivity assays such as: (i) evaluation of comparative infectivity in CD1 mice and in cell cultures using RT-PCR detection assay targeting hsp70 gene mRNA (Rochelle et al, 2002(Rochelle et al, , 2004, (ii) evaluation of infectivity in HC8 cells using qPCR targeting the 18S rRNA gene (Garvey et al, 2010), (iii) cell culture (CC)-PCR assay targeting hsp70 DNA (Aboytes et al, 2004), and (iv) CC-IFA assay on oocysts recovered by USEPA method 1623 (Quintero-Betancourt et al, 2003;Gennaccaro et al, 2003).…”