The response of Cryptosporidium parvum to UV light

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Vol.21 No.2 February 2005

The response of Cryptosporidium parvum to UV light Paul A. Rochelle1, Steve J. Upton2, Beth A. Montelone2 and Keith Woods2 1 2

Metropolitan Water District of Southern California, Water Quality Laboratory, 700 Moreno Avenue, La Verne, CA 91750, USA Division of Biology, Ackert Hall, Kansas State University, Manhattan, KS 66506, USA

Ultraviolet (UV) light is being considered as a disinfectant by the water industry because it appears to be very effective for controlling potential waterborne pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms such as nucleotide excision repair and photolyase enzymes for repairing UV-induced DNA damage and regaining preirradiation levels of infectivity or population density. Genes encoding UV repair proteins exist in C. parvum, so the parasite should be able to regain infectivity following exposure to UV. Nevertheless, there is an increasing body of evidence that the organism is unable to reactivate following UV irradiation. This paper describes the effective inactivation of C. parvum by UV light, identifies nucleotide excision repair genes in the C. parvum and Cryptosporidium hominis genomes and discusses the inability of UV-exposed oocysts to regain infectivity.

Introduction Protozoan parasites of the Cryptosporidium genus (Phylum Apicomplexa) are very common in many animal species including mammals, marsupials, reptiles, birds and fish [1,2]. Some species are restricted to a narrow range of host animals, for example Cryptosporidium nasorum in fish and Cryptosporidium serpentis in reptiles, whereas others, such as Cryptosporidium parvum, occur in a wide range of mammals. They are obligately intracellular parasites that infect the epithelial cells lining the luminal surfaces of the digestive and respiratory tracts. They are excreted and exist in the environment as dormant resistant oocysts. Based on genetic, physiological and host animal differences, and the apparent lack of recombination between them, the two primary genotypes of C. parvum have been classified as separate species. The zoonotic genotype (genotype 2) retained the name C. parvum, whereas the anthroponotic genotype (genotype 1) was named Cryptosporidium hominis [3]. C. parvum and C. hominis oocysts (or organisms morphologically indistinguishable from C. parvum and C. hominis) have been reported in at least 152 species of mammals, including humans [2]. It has been reported that endemic levels of infection in developing regions of the world and the resulting persistent diarrhea in young children might lead to long-lasting impairment of growth and cognition [4]. Corresponding author: Rochelle, P.A. ([email protected]). Available online 7 December 2004

In addition, there is no Food and Drug Administrationapproved treatment for cryptosporidiosis. Although the disease is usually self-limiting in otherwise healthy patients, persistent infection can contribute to mortality in individuals with weakened immune systems. For many years, it was thought that C. parvum (including C. hominis) was the only species of Cryptosporidium that caused infection in humans. However, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium meleagridis, and Cryptosporidium muris have also been detected in immune-compromised individuals [5–10]. Nevertheless, most cases of human cryptosporidiosis are attributed to C. parvum and C. hominis. Cryptosporidium in drinking water Oocysts of various species are frequently detected in rivers and lakes and have also been detected in groundwater and treated drinking water [11,12]. A survey of 5838 untreated source waters throughout the USA reported an average occurrence of 6.8%, with a mean concentration of 0.07 oocysts per liter, although the detection method had considerable variability [13]. Correctly operating water treatment plants that utilize filtration usually remove oocysts from source water with high efficiency. However, oocysts have been detected in 3.8–40% of treated drinking water samples at concentrations up to 0.5 oocysts per liter [12]. Consequently, C. parvum presents a serious threat to public health and is a significant concern for the water industry. Twelve different genotypes of Cryptosporidium were detected in 93.1% of storm water samples using a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique targeting the small subunit rRNA gene [14]. None of the detected genotypes matched those typically found in human, farm animal or domestic animal samples. However, four were identical or closely related to Cryptosporidium baileyi and Cryptosporidium genotypes from opossums and snakes, indicating that wildlife was the primary source of oocyst contamination of surface water during storms. Using the same method, Cryptosporidium was also detected in 45.5% of untreated surface water samples and 24.5% of raw wastewater samples [10]. The predominant genotypes in surface water matched the profiles of C. parvum and C. hominis, whereas Cryptosporidium andersoni was most commonly detected in wastewater. There have been many outbreaks of cryptosporidiosis, associated with either drinking water or recreational use

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of water, worldwide [2]. The largest waterborne outbreak occurred in 1993 in Milwaukee, with estimates of the affected population ranging from 15 000 to 400 000 individuals [15,16] and up to 100 deaths attributed to the contamination. The primary disinfectants used throughout the water industry in the USA for inactivation of microorganisms in drinking water are chlorine and chloramines. However, standard chlorine-based disinfectants have little effect on C. parvum oocysts at concentrations typically applied in drinking water treatment plants. In fact, incubation in 10% bleach (0.5% sodium hypochlorite) is a routine pretreatment used to enhance excystation for cell culture [17]. Some estimates of the chlorine product of contact time and concentration (CT) for C. parvum oocysts are as high as 7200 mg min LK1 (mg LK1 multiplied by disinfectant contact time in minutes) for O99% inactivation [18] compared with a typical value of 0.04 mg min LK1 for Escherichia coli. Although chlorine dioxide is a more effective disinfectant than free chlorine, CT values of 75–1000 mg min LK1 were necessary to achieve 99% inactivation [19], values that are much higher than concentrations typically used in drinking water treatment. There have been over 40 reports describing the efficacy of ozone for inactivation of C. parvum oocysts in drinking water. In one study, 99% inactivation was achieved with a CT of 5.4 mg min LK1 at 14 8C [20]. However, the CT product increased by a factor of 4.5 for each 10 8C decrease in water temperature, limiting the utility of ozone in cold climates. Consequently, there is an incentive to develop alternative, more effective disinfection strategies such as UV light. UV inactivation of C. parvum UV light has been used for many years to disinfect water intended for pharmaceutical, medical and food preparation purposes and it is used routinely in some parts of Europe for disinfection of drinking water. The Metropolitan Water District of Southern California (MWD) operates a large-scale experimental UV treatment unit (11.6 million liters per day) and has conducted extensive evaluations of the efficacy of UV light for inactivating C. parvum oocysts using bench-scale equipment and an in vitro cell culture assay to measure infectivity and inactivation [21–24]. Although pulsed UV light has been evaluated, the majority of published studies have used low- and medium-pressure lamps. Low-pressure lamps emit monochromatic radiation with peak output at 254 nm, whereas medium-pressure UV lamps are typically polychromatic with emission peaks at 248, 266, 280 and 295 nm [25]. An infectivity assay using C. parvumspecific reverse transcriptase PCR (RT-PCR) to detect infections in HCT-8 cell monolayers demonstrated that polychromatic UV doses of 3 mJ cmK2 and 6 mJ cmK2 provided an average of 90% (1-log10) and 99% (2-log10) inactivation of the Iowa isolate of C. parvum, respectively [21]. This study [21] also demonstrated that there was no significant difference in the levels of inactivation achieved with medium-pressure or pulsed-UV lamps. A more robust version of this cell-culture assay with much greater experimental replication demonstrated 90% and 99% www.sciencedirect.com

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inactivation with UV dosages of 2.8 mJ cm K2 and 5.7 mJ cmK2, respectively (Figure 1). These values are in close agreement with those in the Long Term 2 Enhanced Surface Water Treatment Rule [26], which provides the UV dose that water treatment systems must apply to receive inactivation credit. According to this proposed rule, UV doses of 2.5 and 5.8 mJ cmK2 enable inactivation credits of 1-log10 (90% inactivation) and 2-log10 (99% inactivation), respectively. Other studies using mouse assays to measure oocyst inactivation reported 99.99% inactivation at a medium-pressure UV dosage of 19 mJ cmK2 [27] and 99.0–99.9% inactivation at 10 to 25 mJ cm K2 [28]. An alternative cell culture-based method combined with epifluorescence microscopy to detect infections was used to demonstrate 99.9% inactivation at a dosage of 3 mJ cmK2 of low-pressure UV [29]. There is also general agreement that there is no difference in the levels of inactivation when comparing low- and medium-pressure UV lamps [25,28,30,31]. The majority of the UV disinfection studies conducted on C. parvum have utilized the Iowa isolate (a bovine isolate). However, we have also demonstrated that four other isolates of C. parvum have a similar sensitivity to UV light [24]. A UV dosage of 6 mJ cmK2 resulted in 99.4% inactivation of the Moredun isolate (originally from a deer), 99.5% for the KSU-1 isolate (bovine), 98.2% for the TAMU isolate (originally from a horse) and 99.8% for the Maine isolate (recovered from a human during an outbreak). Similar results were also obtained for the Moredun, TAMU, Maine and Glasgow isolates when using the CD-1 mouse assay to assess inactivation [32]. Although there is considerable scatter in the reported levels of inactivation, the overall agreement in UV inactivation data generated by various laboratories, using different assay methods and a variety of isolates, provides a strong measure of confidence in the conclusion that UV efficiently inactivates C. parvum. This is supported by the rapid accumulation of thymine dimers in the genome of oocysts exposed to increasing dosages of UV 4

Inactivation (Log10)

82

3

2

1

0 0

1

2

3

4 5 6 7 UV dosage (mJ cm–2)

8

9

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Figure 1. Summary of UV inactivation of Cryptosporidium parvum. Inactivation data were generated using an HCT-8 cell culture/RT–PCR assay (green) [21,24] and mouse assays (red) [24,27,28,30–32]. The regression line was plotted through the HCT-8 cell culture and RT–PCR data only and forced through the origin (log10 inactivationZ0.35!UV dose, R2Z0.68). The dashed lines indicate the UV dosage required to achieve 90% (1-log10), 99% (2-log10) and 99.9% (3-log10) inactivation.

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Figure 2. Detection of UV-induced thymine dimers using a chemiluminescent antibody assay. Yeast cells and C. parvum oocysts were enumerated using a hemacytometer and diluted so that 3!106 cells or oocysts were exposed to UV light in a 10 ml volume. Oocysts of the Iowa isolate of C. parvum (a) and S. cerevisiae cells (b) were exposed to the indicated dosages of low-pressure UV light. Following DNA extraction and transfer to a nylon membrane, UV-induced damage was detected with an antibody specific for thymine dimers. Reproduced, with permission, from Ref. [30].

light (Figure 2). There have been no published studies to measure the efficacy of UV disinfection for C. hominis because of difficulties in obtaining sufficient oocysts. Identification of UV repair genes in C. parvum The primary type of UV-induced DNA damage is the generation of photoproducts between adjacent bases. The major photoproducts are cyclobutane pyrimidine dimers (CPDs) between adjacent thymine residues and a pyrimidine–pyrimidone product (6–4 lesions [33]). Both types of DNA damage are highly toxic and mutagenic because they prevent both DNA replication and transcription. Thus, a cell with a UV-induced lesion cannot express the genetic information in the region of the genome affected, nor can it accurately copy its DNA and divide. Such a cell might die, or, if it survives, become altered and begin to accumulate genetic changes. Additional UV-induced damage includes: hydroxylation of cytosine and uracil, cytosine–thymine dimers, crosslinking between DNA and proteins, interstrand crosslinking of DNA, and chain breakage or denaturation of DNA [33]. Because many organisms are exposed to potentially high levels of UV irradiation on a daily basis, the ability to repair damaged DNA is essential for the organism’s survival and, consequently, many organisms, both prokaryotic and eukaryotic, are known to have DNA repair mechanisms that act to reduce or eliminate UV-induced damage. These repair mechanisms include nucleotide excision repair, light-activated photolyase enzymes, UV-specific endonucleases and pyrimidine glycosylase. In addition, some organisms also have the ability to delay cellular processes once DNA damage is detected. These cell-cycle delays are mediated by checkpoint genes and they increase a cell’s chances of surviving DNA damage by providing time for the repair machinery to operate before DNA replication [34]. Nucleotide excision repair (NER) is a versatile and ubiquitous repair process in which alterations of the DNA double helix are recognized by a multiprotein complex that then makes dual incisions in the strand of the DNA containing the damaged nucleotide(s), resulting in removal of a fragment of DNA 24–32 nucleotides in www.sciencedirect.com

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length. The minimum set of proteins or protein complexes required for damage recognition and the dual cleavage reaction is w13–15 in eukaryotes (Figure 3). Cleavage is followed by displacement of the damaged fragment and filling in of the gapped region by DNA polymerase. Members of the NER complex include proteins that are solely involved in NER (e.g. RAD4) and those that have additional functions including transcription processes. NER genes in C. parvum were identified by searching the complete genome sequence of C. parvum [35] using known sequences of DNA repair genes from a variety of organisms, including bacteria, Arabidopsis thaliana, Homo sapiens and Saccharomyces cerevisiae. Once open reading frames (ORFs) were identified based upon consensus sequences, primers were designed to flank the identified genes and used to amplify the genes from genomic preparations of the KSU-1 isolate of C. parvum, and the amplified fragments were sequenced. These sequences were then screened against the C. hominis genomic database [36]. All of the major genetic components of an NER complex were identified in C. parvum and C. hominis [24] (Table 1). Most of these genes were represented in their entirety in the database (indicated by the presence of complete ORFs bounded by initiation and stop codons) but CpRAD2 and TFB1 had only w50% coverage when originally identified in a preliminary version of the C. parvum genome database. The full sequences of these genes in the KSU-1 isolate are available in GenBank: CpRAD2, accession number AY307379; TFB1, AY327143 [24]. CpRAD3 and CpRAD10 were also identified by random sequencing of 200 clones of a genomic library of the KSU-1 isolate [37]. Genes putatively identified as RAD2, RAD4, RAD10, RAD14 and RAD25 have also been identified by other researchers [35,38,39]. The 13 NER genes identified in C. parvum were also present in the C. hominis genomic database, although most were spread over multiple sequenced fragments. The (b) Attract TFIIH complex (Rad3, Tfb1, Tfb2, Tfb3, Ssl1, Ssl2) Rad4

(c) Unwind DNA

Tfb2 Tfb3 Rad10 Rad3

Rad23 Tfb1

Ssl1

(d) Incises DNA downstream of damage

Rad2

Rad14

Rad1 Rad25 (e) Incises DNA upstream of damage

(a) Recognizes UV-damaged DNA TRENDS in Parasitology

Figure 3. Proteins involved in NER in S. cerevisiae (adapted from Ref. [47]). Damaged DNA is recognized and removed by the multiprotein NER complex in which Rad14 specifically binds to UV-induced DNA lesions and interacts with Rad23 and Rad4, which have a role in attracting the proteins that form the core transcription initiation factor (TFIIH, light blue). The Rad3 and Rad25 helicases unwind the DNA, and the damaged area is cut out by incisions on either side by Rad2 and the Rad1–Rad10 complex. Abbreviations: Rad, proteins involved in radiation sensitivity/tolerance; Ssl, proteins involved in suppression of stem-loop formation; TFIIH, one of five transcription initiation factors with specificity for RNA polymerase II; Tfb, transcription factor b subunit.

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Table 1. UV-repair genes identified in Cryptosporidium parvuma Gene

Function of proteinb

CpRAD1

Endonuclease, cleaves duplex or singlestranded DNA from unpaired 3 0 -end

CpRAD2

UV-inducible endonuclease, cleaves DNA junctions from unpaired 5 0 -end

Size (base pairs) 1893

Accession number and locationc AAEE01000008.1

Chromosome numberd 4

Similaritye (%) 47

[24]

3276

331 259–329 364 AY307379f;

4

46

[24,35]

7

66

[24]

6

66

[24,35]

6

65

[24,35]

4

49

[24,35]

7

54

[24]

8

64

[24,35]

7

56

[24]

3

50

[24]

6

49

[24]

2

52

[24]

1

47

[24]

CpRAD3

TFIIH component, 5 0 / 3 0 helicase activity

2523

CpRAD4

Interacts with Rad23 protein and TFIIH

1524

CpRAD10

Endonuclease, complexes with product of CpRAD1 gene

672

CpRAD14

Binds specifically to UV-damaged DNA

630

CpRAD23

Interacts with Rad4 protein and attracts TFIIH complex

1023

CpRAD25

TFIIH component, 3 0 / 5 0 helicase acitivity

2412

CpRFA1

Subunit of replication factor A, enhances recognition by Rad14 protein

2064

CpSSL1

Essential core TFIIH component

1128

CpTFB1

Essential core TFIIH component

594

CpTFB2

TFIIH component

573

CpTFB3

TFIIH component

771

AAEE01000009.1 104 416–107 702 AAEE01000001.1 193 207–190 670 AAEE01000002.1 275 128–273 601 AAEE01000002.1 519 936–519 262 AAEE01000009.1 164 219–163 585 AAEE01000001.1 1 071 758–1 072 783 AAEE01000003.1 343 227–345 639 AAEE01000001.1 1 054 082–1 056 148 AAEE01000004.1 699 850–698 716 AY327143f; AAEE01000002.1 14 631–14 023 AAEE01000005.1 319 588–320 163 AAEE01000006.1 737 521–736 740

Refs

a

Abbreviations: CpRAD, C. parvum UV repair genes (RADZradiation); CpRFA, C. parvum replication factor A; CpSSL1, C. parvum suppressor of stem-loop; CpTFB1, C. parvum transcription factor b subunit; TFIIH, transcription initiation factor with RNA polymerase II specificity. Function of the protein in S. cerevisiae [47]. c GenBank accession number of C. parvum whole genome fragments [35] and location (nucleotide positions) of identified genes within these fragments. d Chromosomal location of gene, identified by similarity search of gene sequences against whole C. parvum genome [35]. e Similarity of translated amino acid sequences between C. parvum genes and recognized nucleotide excision repair genes in other eukaryotes. f GenBank accession number for genes in KSU-1 isolate of C. parvum [24]. b

level of similarity between the two genomes for the various NER genes ranged from 92% to 98%, with an average similarity of 96.4%. At least three of the genes (CpRAD1, CpRAD2 and CpRAD3) were also detected by PCR-based amplification in five isolates of C. parvum (Iowa, TAMU, Maine, Moredun and Glasgow) [24]. The various NER genes are dispersed across all of the C. parvum chromosomes, with the exception of chromosome 5 (Table 1). Although the CpRAD1 and CpRAD2 genes from the KSU-1 isolate were successfully cloned and transformed into RAD1- and RAD2-knockout strains of yeast that were UV-sensitive, cross-species complementation was not successful as the genes were not expressed [30]. Consequently, the identification of NER genes in C. parvum is, at the moment, based solely on similarity between C. parvum genomic sequences and recognized NER genes in other organisms. There is currently no experimental evidence that the proteins encoded by these 13 genes are involved in repair of UV damage in C. parvum. Nevertheless, many of the genes displayed substantial similarity with NER gene families (e.g. CpRAD3, CpRAD4, CpRAD10 and CpRAD25). A genomic sequence from the Iowa isolate (GenBank accession AQ842386) [39] included an ORF that contained a central region www.sciencedirect.com

displaying significant similarity at the amino acid level with a domain that is conserved in eukaryotic transcription factor b1 (TFB1) proteins (62 kDa subunit of transcription initiation factor TFIIH). The entire ORF was amplified from the KSU-1 isolate of C. parvum and sequenced (594-base pair predicted gene product, AY327143) [24]. The central 88 amino acids of this predicted protein demonstrated 48% similarity with a conserved domain of TFB1 proteins but there was no significant similarity outside of this region (Figure 4). This BSD domain is named after the family of BTF2-like eukaryotic transcription factors, synapse-associated proteins and DOS2-like proteins in which it is found, and contains distinctive valine (V)– proline (P) and phenylalanine (F)– tryptophan (W) motifs at the carboxyterminus [40]. It occurs in a variety of species, ranging from primal protozoa to humans, and is essential for UV excision-repair [41]. A second gene encoding a protein containing the highly conserved V–P and F–W residues characteristic of the BSD domain is located on chromosome 5 (protein accession number EAK87535) [35] but appears to be unrelated at the sequence level to the putative TFB1 gene identified on chromosome 6 [24].

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Cryptosporidium parvum Homo sapiens Plasmodium yoelii Schizosaccharomyces pombe Caenorhabditis elegans Neurospora crassa Drasophila melanogaster Saccharomyces cerevisiae

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NSGGIDVTITADDLK-SILEEMPSIKTKIKEYVPHRLTEEEFWNRIIQS GCNGLRYNLTSDIIE-SIFRTYPAVKMKYAENVPHNMTEKEFWTRFFQS TIGGFMFDNKYVTWAsFLLKEITSLKKVRYNIVPKLISEDEFWLKYFST VDNQMKVSLTGQQIH-DMFEQHPLLRKVYDKHVP-PLAEGEFWSRFFLS CKEILKFTIQCEYLTrKISRSENYIQKKNLELVPHEMSEENFWKKFFQS ENGELKLNINHEQVQ-LIFQQHPLVKRIYNENVP-KLTESEFWSRFFLS GCNGLKYNLTSDVIH-CIFKTYPAVKRKHFENVPAKMSEAEFWTKFFQS SENKVNVNLSREKIL-NIFENYPIVKKAYTDNVPKNFKEPEFWARFFSS

88 227 168 180 275 273 227 290

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Figure 4. Amino acid alignment in a conserved region of the BSD domain, named after the family of BTF2-like eukaryotic transcription factors, synapse-associated proteins and DOS2-like proteins in which it is found [40]. The alignment was generated using the online Conserved Domain Database tool [48]. Highly conserved positions within the domain are shaded green and those with O65% conservation within the displayed sequences are shaded blue. Biochemically similar residues (based on amino acid volume, polarity and substitution rates) [49,50], are shaded orange, whereas additional Cryptosporidium parvum sites that are the same in at least three other organisms are shaded yellow. GenBank accession numbers for the amino acid sequences are: AAQ92758, P32780, EAA18674, O13745, AAB94149, Q9P5N7, Q960E8 and P32776.

Genes encoding other UV damage repair mechanisms such as photolyases (catalyse photoreactivation), CPD-specific DNA glycosylase (involved in base excision repair) and UV-specific endonuclease were not identified in the C. parvum genome. UV-specific endonucleases and CPD-specific glycosylases are not widely distributed in nature [42,43] and there have been no reports of either type of enzyme or photolyases in coccidial parasites. Evaluation of post-UV oocyst reactivation Earlier studies of UV disinfection of C. parvum using surrogate measures of viability such as in vitro excystation and uptake of vital dyes indicated that very high doses of UV (up to 8700 mJ cmK2) were necessary to achieve 99% inactivation [44]. It has also been reported that UV-exposed oocysts are able to repair thymine dimers under both light and dark incubation conditions, based on an endonuclease site-sensitive assay [45], and that oocysts exposed to 123 mJ cmK2 of UV light excyst with the same efficiency as nonexposed control oocysts [27]. Thus, UV-exposed oocysts are able to excyst and to perform some DNA repair functions. However, oocysts are readily inactivated by exposure to UV light, when assessed by both cell culture

and mouse assays (Figure 1). Several studies have exposed UV-treated oocysts to a variety of potential reactivation conditions following UV irradiation to assess the capacity of C. parvum to regain pre-exposure levels of infectivity (Box 1). All of these studies have demonstrated the failure of UV-treated oocysts to reactivate following exposure to these conditions [24,29,30,45,46]. Conclusion The most important effect of an organism’s exposure to UV irradiation is DNA damage. Such damage can be accurately repaired with no long-term consequences for the organism, result in silent or deleterious mutations or cause death of the organism. Reported biological effects of UV irradiation on protozoa include: lowering the lethal temperature and sensitization to heat; altering mobility and shape; delayed division and excystment of cysts; changes in contractile vacuole activity; reduced respiration and changes in resistance to hydrostatic pressure [33]. C. parvum and C. hominis contain most, if not all, of the genes necessary for NER, the most common repair mechanism following exposure to UV light. In addition,

Box 1. Potential post-UV reactivation conditions investigated* † Incubated for four hours at 25 8C in white light. Also incubated for four hours at 25 8C in darkness † Incubated for four hours at 37 8C in white light. Also incubated for 4 hours at 37 8C in darkness † Incubated for two hours in light at 20 8C, followed by 24 hours in darkness † Incubated for 1 hour at 20 8C in darkness, followed by 30 minutes at 37 8C in darkness, then 1 hour of exposure to white light and 18 hours at 4 8C † Incubated cell monolayers for up to 168 hours following inoculation † Incubated oocysts in 0.75% sodium taurocholate for 30 minutes at 37 8C † Incubated oocysts at 20 8C in darkness for two hours, followed by incubation in 0.75% sodium taurocholate for 15 minutes at 37 8C † Incubated at 4 8C in darkness for 18 hours, followed by incubation in 0.75% sodium taurocholate for 15 minutes at 37 8C † Incubated at 4 8C in darknes for 18 hours, followed by incubation for 15 minutes at 37 8C † Incubated in 0.75% sodium taurocholate for 15 minutes at 37 8C, followed by 1 hour at 37 8C in phosphate buffer † Incubated in 0.5% sodium hypochlorite for 10 min † Incubated in phosphate buffer at 37 8C for 30 minutes † Incubated for up to 72 hours at 5 8C under fluorescent grow lamps, *From [24,29,30,45,46]. www.sciencedirect.com

followed by incubation in 0.5% sodium hypochlorite for 10 minutes at 4 8C (Also incubated under grow lamps at 25 8C) † Incubated for up to 72 hours at 25 8C in darkness, followed by incubation in 0.5% sodium hypochlorite for 10 minutes at 4 8C; also incubated in darkness at 5 8C † Inoculated secondary cell monolayers with supernatant from cell monolayers inoculated with UV-exposed oocysts that have been incubated for 48 hours These repair conditions were selected for their potential either to stimulate oocysts into a metabolically active state following UV exposure, so that any repair mechanisms could function, or to provide holding periods to provide time for repair mechanisms to operate. Such conditions are typically used to demonstrate post-UV repair in a variety of organisms. These reactivation experiments used a variety of C. parvum isolates (Iowa, Maine, KSU-1, HNJ-1), low-pressure and medium-pressure UV lamps with dosages ranging from 0.9 mJ cmK2 to 9.5 mJ cmK2 and an array of infectivity assays: SCID, BALB/c and CD-1 mice, cell culture combined with RT–PCR and a cell cultureimmunofluorescence assay. Although there is variability within the published data, a general picture emerges of UV-exposed oocysts displaying the same levels of inactivation, regardless of whether or not they were exposed to potential reactivation conditions.

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oocysts can repair UV-induced thymine dimers [45]. However, there is currently no evidence that UV-exposed oocysts can repair sufficiently to regain pre-exposure levels of infectivity. There are several explanations for this lack of reactivation: (i) nucleotide excision repair is inefficient in Cryptosporidium spp. because of mutations in crucial NER genes or the absence of additional genes beyond the minimum set of 13 that have been identified; (ii) although DNA is repaired, proteins that are essential for infectivity might also be damaged by UV light; or (iii) oocysts might be able to regain infectivity but the conditions used to study post-UV reactivation are restrictive and some vital component that is necessary for reactivation has been missing from the assays conducted to date. Nevertheless, considering the range of reactivation conditions that have been investigated, it can be concluded that oocysts are irreversibly inactivated by UV irradiation. Therefore, oocysts in drinking water that are exposed to UV disinfection should not pose a threat to public health. Acknowledgements Much of the research discussed in this paper was funded by the Awwa Research Foundation (project funding agreement 2669). However, the comments and views detailed herein might not necessarily reflect the views of the Awwa Research Foundation, its officers, directors, affiliates or agents. We would like to thank the following staff members at MWD for their involvement in work related to these studies: Anagha Chitre, Connie Chou, Ricardo De Leon, Anne M. Johnson, Alexander Mofidi and Daffodil Robles. P.A.R. is also grateful to Marilyn M. Marshall (University of Arizona, Tucson) for propagating and purifying the C. parvum oocysts used for most of the infectivity and inactivation studies at MWD and for performing mouse infectivity assays.

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