Detection of UV-induced thymine dimers in individual Cryptosporidium parvum and Cryptosporidium hominis oocysts by immunofluorescence microscopy

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 x 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 Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ x 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 x cm-2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ x 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.     

The response of Cryptosporidium parvum to UV light

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.     

Low-pressure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts.

Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25 degrees C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm(2) (=30 J/m(2)), the reduction reached the cell culture assay detection limit of approximately 3 log(10). At UV doses of 1.2 and 3 mJ/cm(2), the log(10) reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.     

Genotype analysis of Cryptosporidium spp. prevalent in a rural village in Hwasun-gun, Republic of Korea

Two species of Cryptosporidium are known to infect man; C. hominis which shows anthroponotic transmission between humans, and C. parvum which shows zoonotic transmission between animals or between animals and man. In this study, we focused on identifying genotypes of Cryptosporidium prevalent among inhabitants and domestic animals (cattle and goats), to elucidate transmittal routes in a known endemic area in Hwasun-gun, Jeollanam-do, Republic of Korea. The existence of Cryptosporidium oocysts was confirmed using a modified Ziehl-Neelsen stain. Human infections were found in 7 (25.9%) of 27 people examined. Cattle cryptosporidiosis cases constituted 7 (41.2%) of 17 examined, and goat cases 3 (42.9%) of 7 examined. Species characterizations were performed on the small subunit of the rRNA gene using both PCR-RFLP and sequence analysis. Most of the human isolates were mixtures of C. hominis and C. parvum genotypes and similar PCR-RFLP patterns were observed in cattle and goat isolates. However, sequence analyses identified only C. hominis in all isolates examined. The natural infection of cattle and goats with C. hominis is a new and unique finding in the present study. It is suggested that human cryptosporidiosis in the studied area is caused by mixtures of C. hominis and C. parvum oocysts originating from both inhabitants and domestic animals.     

Failure to propagate Cryptosporidium spp. in cell-free culture

The successful propagation of Cryptosporidium parvum in cell-free culture medium was recently reported. To investigate whether this phenomenon could be broadened to include other C. parvum isolates, as well as Cryptosporidium hominis, we attempted to propagate 3 isolates in cell-free medium under reported culture conditions. Cryptosporidium oocysts from C. parvum strains Moredun (MD) or IOWA or C. hominis strain TU502 were added to media containing coagulated newborn calf serum. The cultures were sampled at various times throughout a 45 (IOWA) or 78 (MD, TU502)-day period and were microscopically examined for various life stages of Cryptosporidium. Cell-free cultures harvested on days 45 and 68 postinoculation were tested for in vitro infectivity on Madrin-Darby bovine kidney cells. In vivo infectivity testing was performed using either infant or 2-wk-old immunosuppressed C57BL mice with cell-free cultures harvested on days 52 and 78. Fecal and gut samples collected from mice were examined by modified acid-fast staining. Data from wet mounts, electron microscopy, and in vitro and in vivo infectivity testing showed that the original oocysts did not complete their life cycle and produce new, viable, infectious oocysts in cell-free culture. Thus, we conclude that this is not a universal phenomenon or readily accomplished.     

Inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores by a mixed-oxidant disinfectant and by free chlorine.

Cryptosporidium parvum oocysts and Clostridium perfringens spores are very resistant to chlorine and other drinking-water disinfectants. Clostridium perfringens spores have been suggested as a surrogate indicator of disinfectant activity against Cryptosporidium parvum and other hardy pathogens in water. In this study, an alternative disinfectant system consisting of an electrochemically produced mixed-oxidant solution (MIOX; LATA Inc.) was evaluated for inactivation of both Cryptosporidium parvum oocysts and Clostridium perfringens spores. The disinfection efficacy of the mixed-oxidant solution was compared to that of free chlorine on the basis of equal weight per volume concentrations of total oxidants. Batch inactivation experiments were done on purified oocysts and spores in buffered, oxidant demand-free water at pH 7 an 25 degrees C by using a disinfectant dose of 5 mg/liter and contact times of up to 24 h. The mixed-oxidant solution was considerably more effective than free chlorine in activating both microorganisms. A 5-mg/liter dose of mixed oxidants produced a > 3-log10-unit (> 99.9%) inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores in 4 h. Free chlorine produce no measurable inactivation of Cryptosporidium parvum oocysts by 4 or 24 h, although Clostridium perfringens spores were inactivated by 1.4 log10 units after 4 h. The on-site generation of mixed oxidants may be a practical and cost-effective system of drinking water disinfection protecting against even the most resistant pathogens, including Cryptosporidium oocysts.     

Molecular fingerprinting of Cryptosporidium oocysts isolated during water monitoring

We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.     

Comparison of resistance to γ-irradiation between Cryptosporidium parvum and Cryptosporidium muris using in vivo infection

In the genus Cryptosporidium, there are more than 14 species with different sizes and habitats, as well as different hosts. Among these, C. parvum and C. hominis are known to be human pathogens. As C. parvum can survive exposure to harsh environmental conditions, including various disinfectants or high doses of radiation, it is considered to be an important environmental pathogen that may be a threat to human health. However, the resistance of other Cryptosporidium species to various environmental conditions is unknown. In this study, resistance against γ-irradiation was compared between C. parvum and C. muris using in vivo infection in mice. The capability of C. muris to infect mice could be eliminated with 1,000 Gy of γ-irradiation, while C. parvum remained infective in mice after up to 1,000 Gy of γ-irradiation, although the peak number of oocysts per gram of feces decreased to 16% that of non-irradiated oocysts. The difference in radioresistance between these 2 Cryptosporidium species should be investigated by further studies.     

Comparison of assays for Cryptosporidium parvum oocysts viability after chemical disinfection

Abstract In vitro excystation, vital dyes (4', 6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)), and infeictivity in neonatal CD-1 mice were used to assess the viability of Cryptosporidium parvum oocysts after chemical disinfection. In vitro excystation and DAPI/PI staining provided similar estimates of viability in bench-scale experiments, but both of these methods significantly overestimated the viability when compared with infectivity (Pr ≤ 0.01). Infectivity was the most reliable measure of the viability of C. parvum oocysts following chemical disinfection.     

Cryptosporidium viatorum n. sp. (Apicomplexa: Cryptosporidiidae) among travellers returning to Great Britain from the Indian subcontinent, 2007-2011

A novel Cryptosporidium genotype was identified, among travellers with gastro-intestinal symptoms returning to Great Britain from the Indian subcontinent, for which we propose the name Cryptosporidium viatorum n. sp. The epidemiology of these cases was distinctly different from those with Cryptosporidium parvum and Cryptosporidium hominis. Of the 10 cases identified involving C. viatorum, most were in the first quarter of the year. One occurred in 2007, one in 2008, three in 2010 and five to end March 2011. The median age was 19 years but most were in the 20-29 years age group and seven were male. The symptoms included diarrhoea, abdominal pain, nausea, vomiting and fever. Compared with cases due to C. hominis and C. parvum, vomiting was reported less often, although the duration of gastro-intestinal symptoms was longer. The cases of C. viatorum were all travellers to the Indian subcontinent, whereas cases of C. hominis and C. parvum were more likely to have travelled elsewhere. Cryptosporidium viatorum isolates had indistinguishable sequences at each of the 70 kDa heat shock protein (HSP70), actin and ssrRNA loci which did not match any published previously and, although phylogenetically most similar to Cryptosporidium fayeri, they were distinct (<98% similarity) at the ssrRNA, HSP70 and actin genes. Morphologically, oocysts were typical of predominantly human-infecting species. Cryptosporidium viatorum n. sp. is proposed and work is warranted to investigate further the public health significance and occurrence elsewhere of this emerging parasite.     

Detection and discrimination of Cryptosporidium parvum and C. hominis in water samples by immunomagnetic separation-PCR

Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.     

UV Inactivation of Cryptosporidium hominis as Measured in Cell Culture

The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.     

Cryptosporidium Species and Genotypes in HIV-Positive Patients in Lima, Peru

ABSTRACT: Cryptosporidium parasites from a cross-sectional study conducted in two national hospitals in Lima, Peru were genetically characterized to deteimine the diversity of Cryptosporidium spp. in HIV-positive people. A total of 2,672 patients participated in this study and provided 13,937 specimens. Cryptosporidium oocysts were detected by microscopy in 354 (13.3%) of the patients. Analysis of 951 Cryptosporidium - positive specimens from 300 patients using a small subunit rRNA-based PCR-RFLP tool identified 6 genotypes; Cryptosporidium hominis was the species most frequently detected (67.5%), followed by C. meleagridis (12.6%) and C. parvum (11.3%). Cryptosporidium canis (4.0%), C. felis (3.3%), and Cryptosporidium pig genotype (0.5%) were also found. These findings indicate that C. hominis is the predominant species in Peruvian HIV-positive persons, and that zoonotic Cryptosporidium spp. account for about 30% of cryptosporidiosis in these patients.     

Human enteropathogen load in activated sewage sludge and corresponding sewage sludge end products

This study demonstrated a significant reduction in the concentrations of Cryptosporidium parvum and Cryptosporidium hominis oocysts, Giardia lamblia cysts, and spores of human-virulent microsporidia in dewatered and biologically stabilized sewage sludge cake end products compared to those of the respective pathogens in the corresponding samples collected during the sludge activation process.     

Genetic characterization and transmission cycles of Cryptosporidium species isolated from humans in New Zealand

Little is known about the genetic characteristics, distribution, and transmission cycles of Cryptosporidium species that cause human disease in New Zealand. To address these questions, 423 fecal specimens containing Cryptosporidium oocysts and obtained from different regions were examined by the PCR-restriction fragment length polymorphism technique. Indeterminant results were resolved by DNA sequence analysis. Two regions supplied the majority of isolates: one rural and one urban. Overall, Cryptosporidium hominis accounted for 47% of the isolates, with the remaining 53% being the C. parvum bovine genotype. A difference, however, was observed between the Cryptosporidium species from rural and urban isolates, with C. hominis dominant in the urban region, whereas the C. parvum bovine genotype was prevalent in rural New Zealand. A shift in transmission cycles was detected between seasons, with an anthroponotic cycle in autumn and a zoonotic cycle in spring. A novel Cryptosporidium sp., which on DNA sequence analysis showed a close relationship with C. canis, was detected in two unrelated children from different regions, illustrating the genetic diversity within this genus.     

Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log(10) unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.     

Evidence supporting zoonotic transmission of Cryptosporidium in rural New South Wales

Cryptosporidium hominis, which has an anthroponotic transmission cycle and Cryptosporidium parvum, which is zoonotic, are the primary species of Cryptosporidium that infect humans. The present study identified the species/genotypes and subgenotypes of Cryptosporidium in 7 human and 15 cattle cases of sporadic cryptosporidiosis in rural western NSW during the period from November 2005 to January 2006. The species/genotype of isolates was determined by PCR sequence analysis of the 18S rRNA and C. parvum and C. hominis isolates were subgenotyped by sequence analysis of the GP60 gene. Fourteen of 15 cattle-derived isolates were identified as C. parvum and 1 as a C. bovis/C. parvum mixture. Of the human isolates, 4 were C. parvum and 3 were C. hominis. Two different subgenotypes were identified with the human C. hominis isolates and six different subgenotypes were identified within the C. parvum species from humans and cattle. All four of the C. parvum subtypes found in humans were also found in the cattle, indicating that zoonotic transmission may be an important contributor to sporadic human cases cryptosporidiosis in rural NSW.     

Protection of calves against cryptosporiosis by oral inoculation with gamma-irradiated Cryptosporidium parvum oocysts

The purpose of this study was to determine whether gamma-irradiated Cryptosporidium parvum oocysts could elicit protective immunity against cryptosporidiosis in dairy calves. Cryptosporidium parvum Iowa strain oocysts (1 x 10(6) per inoculation) were exposed to various levels of gamma irradiation (350-500 Gy) and inoculated into 1-day-old dairy calves. The calves were examined daily for clinical signs of cryptosporidiosis, and fecal samples were processed for the presence of C. parvum oocysts. At 21 days of age, the calves were challenged by oral inoculation with 1 x 10(5) C. parvum oocysts and examined daily for oocyst shedding and clinical cryptosporidiosis. Calves that were inoculated with C. parvum oocysts exposed to 350-375 Gy shed C. parvum oocysts in feces. Higher irradiation doses (450 or 500 Gy) prevented oocyst development, but the calves remained susceptible to C. parvum challenge infection. Cryptosporidium parvum oocysts exposed to 400 Gy were incapable of any measurable development but retained the capacity to elicit a protective response against C. parvum challenge. These findings indicate that it may be possible to protect calves against cryptosporidiosis by inoculation with C. parvum oocysts exposed to 400-Gy gamma irradiation.