Transcriptional control of the Cryptosporidium life cycle.

The parasite Cryptosporidium is a leading agent of diarrhoeal disease in young children, and a cause and consequence of chronic malnutrition1,2. There are no vaccines and only limited treatment options3. The parasite infects enterocytes, in which it engages in asexual and sexual replication4, both of which are essential to continued infection and transmission. However, their molecular mechanisms remain largely unclear5. Here we use single-cell RNA sequencing to reveal the gene expression programme of the entire Cryptosporidium parvum life cycle in culture and in infected animals. Diverging from the prevailing model6, we find support for only three intracellular stages: asexual type-I meronts, male gamonts and female gametes. We reveal a highly organized program for the assembly of components at each stage. Dissecting the underlying regulatory network, we identify the transcription factor Myb-M as the earliest determinant of male fate, in an organism that lacks genetic sex determination. Conditional expression of this factor overrides the developmental program and induces widespread maleness, while conditional deletion ablates male development. Both have a profound impact on the infection. A large set of stage-specific genes now provides the opportunity to understand, engineer and disrupt parasite sex and life cycle progression to advance the development of vaccines and treatments.

Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.

Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.

Quantification of viable protozoan parasites on leafy greens using molecular methods.

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.

Effect of Ginsenoside-Rh2 and Curcurbitacin-B on Cryptosporidium parvum in vitro.

Ginsenoside-Rh2 and cucurbitacin-B (CuB) are secondary metabolites of Ginseng (Panax ginseng) and Cucurbitaceae plants respectively. We assessed the anticryptosporidial activity of these two functional compounds in a cell culture model of cryptosporidiosis. The highest concentration of each compound that was not toxic to the host cells was used to assess the activity against C. parvum during infection/invasion and growth in HCT-8 cell monolayers. Monolayers were infected with pre-excysted C. parvum oocysts. Infected monolayers were incubated at 37 °C for 24 h and 48 h in the presence of different concentrations of each test compound. A growth resumption assay was performed by incubating infected monolayers in the presence of compounds for 24 h followed by a second 24-h incubation in the absence of compound. To screen for invasion inhibiting activity, freshly excysted C. parvum sporozoites were pre-treated with different concentrations of compounds prior to adding them to the cell monolayers. Paromomycin, a known inhibitor of C. parvum, and DMSO were used as positive and negative control, respectively. The level of infection was initially assessed using an immunofluorescent assay and quantified by real-time PCR. Both compounds were found to strongly inhibit C. parvum intracellular development in a dose-dependent manner. IC50 values of 25 µM for a 24 h development period and 5.52 µM after 48 h development were measured for Rh2, whereas for CuB an IC50 value of 0.169 µg/ml and 0.118 µg/ml were obtained for the same incubation periods. CuB also effectively inhibited resumption of growth, an activity that was not observed with Rh2. CuB was more effective at inhibiting excystation and/or host cell invasion, indicating that this compound also targets extracellular stages of the parasite.

Cryptosporidium parvum Subtilisin-Like Serine Protease (SUB1) Is Crucial for Parasite Egress from Host Cells.

Despite the severity and global burden of Cryptosporidium infection, treatments are less than optimal, and there is no effective vaccine. Egress from host cells is a key process for the completion of the life cycle of apicomplexan parasites. For Plasmodium species, subtilisin-like serine protease (SUB1) is a key mediator of egress. For Toxoplasma species, calcium-dependent protein kinases (CDPKs) are critical. In this study, we characterized Cryptosporidium SUB1 expression and evaluated its effect using an infection model. We found increased expression between 12 and 20 h after in vitro infection, prior to egress. We induced silencing of SUB1 (ΔSUB1) mRNA using SUB1 single-stranded antisense RNA coupled with human Argonaute 2. Silencing of SUB1 mRNA expression did not affect parasite viability, excystation, or invasion of target cells. However, knockdown led to a 95% decrease in the proportion of released merozoites in vitro (P < 0.0001). In contrast, silencing of CDPK5 had no effect on egress. Overall, our results indicate that SUB1 is a key mediator of Cryptosporidium egress and suggest that interruption of the life cycle at this stage may effectively inhibit the propagation of infection.

Cryptosporidium parvum disrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins.

Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrhoeal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrhoeal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJs) and adherens junctions (AJs) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilising in vitro, ex vivo, and in vivo models. CP infection (0.5 × 106  oocysts/well in Transwell inserts, 24 hr) increased paracellular permeability (FITC-dextran flux) in Caco-2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4, and E-cadherin. Claudin 3, zonula occludens-1 (ZO1) and α-catenin were also significantly decreased, whereas claudins 1 and 2 and ß-catenin were not altered. Substantial downregulation of occludin, claudin 4, and E-cadherin was also observed in response to CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP-infected mouse ileum and jejunum but were unaltered in Caco-2 cells. Further, bafilomycin-A, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco-2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection-induced diarrhoea.

Curcumin: A promising treatment for Cryptosporidium parvum infection in immunosuppressed BALB/c mice.

Members of the genus Cryptosporidium are frequent protozoan pathogens in humans and a wide range of animals. There is no consistently effective treatment against cryptosporidiosis, especially in immunodeficient patients. The present study was carried out to study the therapeutic effects of curcumin against cryptosporidiosis in immunosuppressed BALB/c mice. Mice were divided into five groups and immunosuppressed by dexamethasone. Three groups were inoculated with C. parvum oocysts, administered with curcumin, paromomycin, and without treatment. The reminders were regarded as controls. The oocysts in the fecal smear were counted daily. At days 0, 3, 7, and 11 post-treatment, the mice were sacrificed, and the efficacy of drugs was evaluated by comparing the histopathological alterations in jejunum and ileum, measuring the total antioxidant capacity, and malondialdehyde in the affected tissues. The infection was completely eliminated in the curcumin-treated group, and oocyst shedding stopped with no recurrence after drug withdrawal. On the contrary, paromomycin was unable to eliminate C. parvum infection completely, and oocyst shedding continued even 10 days after the drug withdrawal. Based on these findings, curcumin can be a trustworthy compound for the elimination of infection in immunosuppressed hosts. Further evaluation to find its accurate mechanism of action should be considered.

Health benefits of orally administered anti-IL-10 antibody in milk-fed dairy calves.

The primary objective of this randomized controlled trial was to determine whether anti-IL-10 egg yolk antibodies fed upon arrival to a calf ranch would lower the prevalence of Cryptosporidium parvum shedding in naturally challenged preweaned dairy calves. The secondary objectives included measuring the effect of anti-IL-10 antibodies on calf health, performance, and shedding of less common diarrheal pathogens. A total of 133 calves, enrolled at 24 to 72 h of age, received a daily dose of 0.96 g of egg yolk powder with anti-IL-10 antibodies (MAB, n = 71) or without anti-IL-10 antibodies (MEP, n = 62) split between 2 feedings for the first 11 d on feed at a calf ranch. Daily health evaluations were completed for 15 d after arrival and on d 56. Digital weights were collected at enrollment and d 56, and hipometer weights were collected at enrollment and d 7 and 56. Packed cell volume and serum total protein concentration were measured at enrollment and on d 7 and 14. Fecal pH was measured at enrollment and on d 5 and 14, and fecal pathogen (C. parvum, coronavirus, rotavirus, and Salmonella spp.) shedding was assessed at d 5 and 14. Continuous outcomes were compared between groups using a Student's t-test or Wilcoxon rank sum test. Fecal pathogen shedding at d 14, respiratory disease at d 56, and antibiotic usage were compared using relative risk (RR) and chi-squared test. Fecal pH (median and interquartile range) on d 14 was 6.65 (6.39-6.99) and 6.52 (5.97-6.81) for MAB and MEP, respectively. On d 56, the risk of respiratory disease was lower for MAB compared with MEP (RR = 0.40; confidence interval = 0.16-0.99). The risk for antibiotic treatment was lower for MAB- compared with MEP-treated calves (RR = 0.38; confidence interval = 0.17-0.88). The risk of shedding rotavirus was higher in MAB (RR = 1.38; confidence interval = 1.10-1.81) calves. After multivariable analyses, hipometer weights (least squares means ± standard error) were 1.7 ± 0.8 kg greater on d 56 in MAB compared with MEP; however, ADG was 0.04 ± 0.02 kg/d lower in MAB calves. Total health score, diarrhea days, average respiratory score, packed cell volume, and serum total protein were not affected by feeding anti-IL-10 egg antibodies. In summary, feeding anti-IL-10 antibodies was associated with increased fecal pH, reduced risk of respiratory disease later in the preweaning period, and decreased antibiotic usage despite higher rotavirus infection. These findings might be associated with improved mucosal immunity, enhanced host defenses, or reduced susceptibility and warrant further investigation.

5-Aminopyrazole-4-Carboxamide-Based Compounds Prevent the Growth of Cryptosporidium parvum.

Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) is a promising target for drug development against cryptosporidiosis. We report a series of low-nanomolar CpCDPK1 5-aminopyrazole-4-carboxamide (AC) scaffold inhibitors that also potently inhibit C. parvum growth in vitro Correlation between anti-CpCDPK1 and C. parvum growth inhibition, as previously reported for pyrazolopyrimidines, was not apparent. Nonetheless, lead AC compounds exhibited a substantial reduction of parasite burden in the neonatal mouse cryptosporidiosis model when dosed at 25 mg/kg.

In Vitro and In Vivo Activities of Sulfur-Containing Linear Bisphosphonates against Apicomplexan Parasites.

We tested a series of sulfur-containing linear bisphosphonates against Toxoplasma gondii, the etiologic agent of toxoplasmosis. The most potent compound (compound 22; 1-[(n-decylsulfonyl)ethyl]-1,1-bisphosphonic acid) is a sulfone-containing compound, which had a 50% effective concentration (EC50) of 0.11 ± 0.02 µM against intracellular tachyzoites. The compound showed low toxicity when tested in tissue culture with a selectivity index of >2,000. Compound 22 also showed high activity in vivo in a toxoplasmosis mouse model. The compound inhibited the Toxoplasma farnesyl diphosphate synthase (TgFPPS), but the concentration needed to inhibit 50% of the enzymatic activity (IC50) was higher than the concentration that inhibited 50% of growth. We tested compound 22 against two other apicomplexan parasites, Plasmodium falciparum (EC50 of 0.6 ± 0.01 µM), the agent of malaria, and Cryptosporidium parvum (EC50 of ∼65 µM), the agent of cryptosporidiosis. Our results suggest that compound 22 is an excellent novel compound that could lead to the development of potent agents against apicomplexan parasites.

Cryptosporidium parvum increases intestinal permeability through interaction with epithelial cells and IL-1ß and TNFα released by inflammatory monocytes.

Intestinal epithelial cells form a single layer separating the intestinal lumen containing nutriments and microbiota from the underlying sterile tissue and therefore play a key role in maintaining homeostasis. We investigated the factors contributing to the alteration of the epithelial barrier function during Cryptosporidium parvum infection. Infected polarized epithelial cell monolayers exhibit a drop in transepithelial resistance associated with a delocalization of E-cadherin and ß-catenin from their intercellular area of contact, the adherens junction complex. In neonatal mice infected by C. parvum, the increased permeability is correlated with parasite development and with an important recruitment of Ly6c+ inflammatory monocytes to the subepithelial space. TNFα and IL-1ß produced by inflammatory monocytes play a key role in the loss of barrier function. Our findings demonstrate for the first time that both the parasite and inflammatory monocytes contribute to the loss of intestinal barrier function during cryptosporidiosis.

Removals of cryptosporidium parvum oocysts and cryptosporidium-sized polystyrene microspheres from swimming pool water by diatomaceous earth filtration and perlite-sand filtration.

Removal of Cryptosporidium-sized microspheres and Cryptosporidium parvum oocysts from swimming pools was investigated using diatomaceous earth (DE) precoat filtration and perlite-sand filtration. In pilot-scale experiments, microsphere removals of up to 2 log were obtained with 0.7 kg·DE/m2 at a filtration rate of 5 m/h. A slightly higher microsphere removal (2.3 log) was obtained for these DE-precoated filters when the filtration rate was 3.6 m/h. Additionally, pilot-scale perlite-sand filters achieved greater than 2 log removal when at least 0.37 kg/m2 of perlite was used compared to 0.1-0.4 log removal without perlite both at a surface loading rate of 37 m/h. Full-scale testing achieved 2.7 log of microspheres and oocysts removal when 0.7 kg·DE/m2 was used at 3.6 m/h. Removals were significantly decreased by a 15-minute interruption of the flow (without any mechanical agitation) to the DE filter in pilot-scale studies, which was not observed in full-scale filters. Microsphere removals were 2.7 log by perlite-sand filtration in a full-scale swimming pool filter operated at 34 m/h with 0.5 kg/m2 of perlite. The results demonstrate that either a DE precoat filter or a perlite-sand filter can improve the efficiency of removal of microspheres and oocysts from swimming pools over a standard sand filter under the conditions studied.

Inhibition of Calcium-Dependent Protein Kinase 1 (CDPK1) In Vitro by Pyrazolopyrimidine Derivatives Does Not Correlate with Sensitivity of Cryptosporidium parvum Growth in Cell Culture.

Cryptosporidiosis is a serious diarrheal disease in immunocompromised patients and malnourished children, and treatment is complicated by a lack of adequate drugs. Recent studies suggest that the natural occurrence of a small gatekeeper residue in serine threonine calcium-dependent protein kinase 1 (CDPK1) of Cryptosporidium parvum might be exploited to target this enzyme and block parasite growth. Here were explored the potency with which a series of pyrazolopyrimidine analogs, which are selective for small gatekeeper kinases, inhibit C. parvum CDPK1 and block C. parvum growth in tissue culture in vitro. Although these compounds potently inhibited kinase activity in vitro, most had no effect on parasite growth. Moreover, among those that were active against parasite growth, there was a very poor correlation with their 50% inhibitory concentrations against the enzyme. Active compounds also had no effect on cell invasion, unlike the situation in Toxoplasma gondii, where these compounds block CDPK1, prevent microneme secretion, and disrupt cell invasion. These findings suggest that CPDK1 is not essential for C. parvum host cell invasion or growth and therefore that it is not the optimal target for therapeutic intervention. Nonetheless, several inhibitors with low micromolar 50% effective concentrations were identified, and these may affect other essential targets in C. parvum that are worthy of further exploration.

Silver Nanoparticles Decrease the Viability of Cryptosporidium parvum Oocysts.

Oocysts of the waterborne protozoan parasite Cryptosporidium parvum are highly resistant to chlorine disinfection. We show here that both silver nanoparticles (AgNPs) and silver ions significantly decrease oocyst viability, in a dose-dependent manner, between concentrations of 0.005 and 500 µg/ml, as assessed by an excystation assay and the shell/sporozoite ratio. For percent excystation, the results are statistically significant for 500 µg/ml of AgNPs, with reductions from 83% for the control to 33% with AgNPs. For Ag ions, the results were statistically significant at 500 and 5,000 µg/ml, but the percent excystation values were reduced only to 66 and 62%, respectively, from 86% for the control. The sporozoite/shell ratio was affected to a greater extent following AgNP exposure, presumably because sporozoites are destroyed by interaction with NPs. We also demonstrated via hyperspectral imaging that there is a dual mode of interaction, with Ag ions entering the oocyst and destroying the sporozoites while AgNPs interact with the cell wall and, at high concentrations, are able to fully break the oocyst wall.

The fine structure of sexual stage development and sporogony of Cryptosporidium parvum in cell-free culture.

The sexual stages and new oocysts development of Cryptosporidium parvum were investigated in a cell-free culture system using transmission electron microscopy (TEM). Sexual development was extremely rapid after inoculation of oocysts into the medium. The process began within 1/2-12 h and was completed with new oocyst formation 120 h post-inoculation. The macrogamonts were bounded by two membranes and had amylopectin granules and two distinct types of wall-forming bodies. The microgamonts had a large nucleus showing lobe projections and condensation of chromatin, giving rise to peripherally budding microgametes. The microgametes contained a large area of granular substance containing groups of microtubules surrounding the electron-dense nucleus. In some instances, the dividing microgamy was observed in cell-free cultures with no preceding merogonic process. Fertilization was observed with the bullet-shaped microgamete penetrating an immature macrogamont at 24 and 216 h. The new thin- and thick-walled oocysts had a large residuum with polysaccharide granules and sporogony noted inside these oocysts. Novel immature four-layer walled thick oocysts with irregular knob-like protrusions on the outer layer resembling the immature Eimeria oocysts were also observed. The present study confirms the gametogony and sporogony of C. parvum in cell-free culture and describes their ultra-structure for the first time.

Discovery of new variable number tandem repeat loci in multiple Cryptosporidium parvum genomes for the surveillance and investigation of outbreaks of cryptosporidiosis.

Cryptosporidium parvum is a protozoan parasite causing gastro-intestinal disease (cryptosporidiosis) in humans and animals. The ability to investigate sources of contamination and routes of transmission by characterization and comparison of isolates in a cost- and time-efficient manner will help surveillance and epidemiological investigations, but as yet there is no standardised multi-locus typing scheme. To systematically identify variable number tandem repeat (VNTR) loci, which have been shown to provide differentiation in moderately conserved species, we interrogated the reference C. parvum Iowa II genome and seven other C. parvum genomes using a tandem repeat finder software. We identified 28 loci that met criteria defined previously for robust typing schemes for inter-laboratory surveillance, that had potential for generating PCR amplicons analysable on most fragment sizing platforms: repeats ≥6 bp, occurring in tandem in a single repeat region, and providing a total amplicon size of <300 bp including 50 bp for the location of the forward and reverse primers. The qualifying loci will be further investigated in vitro for consideration as preferred loci in the development of a robust VNTR scheme.

Quantitative analysis of Cryptosporidium growth in in vitro culture--the impact of parasite density on the success of infection.

Cryptosporidium is an important waterborne pathogen for which no treatment or vaccination is available. This study set out to quantify DNA replication of Cryptosporidium parvum in vitro. Cryptosporidium DNA could be detected at up to 60 % of input level in both host-cell-free and host cell containing cultures 6 days after infection with living sporozoites, but was lost within 2 days in cultures inoculated with UV-inactivated sporozoites. Total DNA increased between days 2 and 6, evidence of successful DNA replication in both cell-free and host-cell-containing cultures. Overall however, only a small fraction (up to 5 %) of parasite DNA could be found associated with host cells or bound to plastic of the cell-free cultures, and the majority of parasite DNA was present in the cell culture medium, separable by simple decantation. After 2 days, in host-cell-containing cultures, the parasite DNA could be concentrated by slow centrifugation, suggesting that it was associated with intact parasite cells, but at 6 days, the majority could not be centrifuged and is therefore thought to have represented copies associated with dead and degraded parasites. In cell-free cultures and in larger plates, the majority of DNA was in this form. Performance of the parasite was best in small culture plates, and least in the largest plate sizes. We interpret these results as suggesting that Cryptosporidium sporozoites first bind to the host cell monolayer or to the plasticware, but then by 2 days, there has been a substantial release of parasites back into the medium. Host-cell-free cultures also supported modest replication and may have represented DNA synthesis in cells beginning merogony. The role of the host cells is unclear, as so much of the parasite DNA is released into the medium. Host cells may provide a feeder role, conditioning the medium for Cryptosporidium development.

Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum.

BACKGROUND: Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis-associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent yearsIaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome. RESULTS: Altogether, 8.59-9.05 Mb of Cryptosporidium sequences in 45-767 assembled contigs were obtained from the four specimens, representing 94.36-99.47% coverage of the expected genome. These genomes had complete synteny in gene organization and 96.86-97.0% and 99.72-99.83% nucleotide sequence similarities to the published genomes of C. parvum and C. hominis, respectively. Several major insertions and deletions were seen between C. hominis and C. parvum genomes, involving mostly members of multicopy gene families near telomeres. The four C. hominis genomes were highly similar to each other and divergent from the reference IaA25R3 genome in some highly polymorphic regions. Major sequence differences among the four specimens sequenced in this study were in the 5' and 3' ends of chromosome 6 and the gp60 region, largely the result of genetic recombination. CONCLUSIONS: The sequence similarity among specimens of the two dominant outbreak subtypes and genetic recombination in chromosome 6, especially around the putative virulence determinant gp60 region, suggest that genetic recombination plays a potential role in the emergence of hyper-transmissible C. hominis subtypes. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of MEDLE family secreted proteins and insulinase-like proteases indicate that telomeric gene duplications could potentially contribute to host expansion in C. parvum.

Effect of cyanuric acid on the inactivation of Cryptosporidium parvum under hyperchlorination conditions.

Cyanuric acid (CYA) is a chlorine stabilizer used in swimming pools to limit UV degradation of chlorine, thus reducing chlorine use and cost. However, CYA has been shown to decrease the efficacy of chlorine disinfection. In the event of a diarrheal incident, CDC recommends implementing 3-log10 inactivation conditions for Cryptosporidium (CT value = 15 300 mg·min/L) to remediate pools. Currently, CYA's impact on Cryptosporidium inactivation is not fully determined. We investigated the impact of multiple concentrations of CYA on C. parvum inactivation (at 20 and 40 mg/L free chlorine; average pH 7.6; 25 °C). At 20 mg/L free chlorine, average estimated 3-log10 CT values were 17 800 and 31 500 mg·min/L with 8 and 16 mg/L CYA, respectively, and the average estimated 1-log10 CT value was 76 500 mg·min/L with 48 mg/L CYA. At 40 mg/L free chlorine, 3-log10 CT values were lower than those at 20 mg/L, but still higher than those of free chlorine-only controls. In the presence of ∼100 mg/L CYA, average 0.8- and 1.4-log10 reductions were achieved by 72 h at 20 and 40 mg/L free chlorine, respectively. This study demonstrates CYA significantly delays chlorine inactivation of Cryptosporidium oocysts, emphasizing the need for additional pool remediation options following fecal incidents.

Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages.