Quantitative tracking of Cryptosporidium infection in cell culture with CFSE

Immunofluorescence-based assays have been developed to detect and quantitate Cryptosporidium parvum infection in cell culture. Here, we describe a method that tracks and quantifies the early phase of attachment and invasion of C. parvum sporozoites using a fluorescent dye. Newly excysted sporozoites were labeled with the amine-reactive fluorescein probe carboxyfluorescein diacetate succinimidyl esters (CFSE) using an optimized protocol. The initial invasion of cells by labeled parasites was detected with fluorescent or confocal microscopy. The infection of cells was quantified by flow cytometry. Comparative analysis of infection of cells with CFSE-labeled and unlabeled sporozoites showed that the infectivity of C. parvum was not affected by CFSE labeling. Quantitative analysis showed that C. parvum Iowa and MD isolates were considerably more invasive than Cryptosporidium hominis isolate TU502. Unlike immunofluorescent assays, CFSE labeling permitted the tracking of the initial invasion of C. parvum. Such an assay may be useful for studying the dynamics of host cell-parasite interaction and possibly for drug screening.     

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.     

Quantitative-PCR assessment of Cryptosporidium parvum cell culture infection

A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-QSD revealed that cell culture infection at 24 and 48 h postinoculation was approximately 20 and 60%, respectively, of the endpoint 72-h postinoculation infection. Evaluation of three different lots of C. parvum Iowa isolate oocysts revealed that the mean infection of 0.1 N HCl-treated oocysts was only 36% of the infection obtained with oocysts treated with acidified Hanks' balanced salt solution containing 1% trypsin. CC-QSD comparison of the C. parvum Iowa and TAMU isolates revealed significantly higher levels of infection for the TAMU isolate, which agrees with and supports previous human, animal, and cell culture studies. CC-QSD has the potential to aid in the optimization of Cryptosporidium cell culture methods and facilitate quantitative evaluation of cell culture infectivity experiments.     

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.     

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.     

Genotyping Cryptosporidium parvum with an hsp70 single-nucleotide polymorphism microarray

We investigated the application of an oligonucleotide microarray to (i) specifically detect Cryptosporidium spp., (ii) differentiate between closely related C. parvum isolates and Cryptosporidium species, and (iii) differentiate between principle genotypes known to infect humans. A microarray of 68 capture probes targeting seven single-nucleotide polymorphisms (SNPs) within a 190-bp region of the hsp70 gene of Cryptosporidium parvum was constructed. Labeled hsp70 targets were generated by PCR with biotin- or Cy3-labeled primers. Hybridization conditions were optimized for hybridization time, temperature, and salt concentration. Two genotype I C. parvum isolates (TU502 and UG502), two C. parvum genotype II isolates (Iowa and GCH1), and DNAs from 22 non-Cryptosporidium sp. organisms were used to test method specificity. Only DNAs from C. parvum isolates produced labeled amplicons that could be hybridized to and detected on the array. Hybridization patterns between genotypes were visually distinct, but identification of SNPs required statistical analysis of the signal intensity data. The results indicated that correct mismatch discrimination could be achieved for all seven SNPs for the UG502 isolate, five of seven SNPs for the TU502 isolate, and six of seven SNPs for both the Iowa and GCH1 isolates. Even without perfect mismatch discrimination, the microarray method unambiguously distinguished between genotype I and genotype II isolates and demonstrated the potential to differentiate between other isolates and species on a single microarray. This method may provide a powerful new tool for water utilities and public health officials for assessing point and nonpoint source contamination of water supplies.     

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.     

Genetic crosses in the apicomplexan parasite Cryptosporidium parvum define recombination parameters

Recombinant progeny lines of Cryptosporidium parvum were generated by coinfecting immunosuppressed mice with two genetically distinct isolates of C. parvum. Progeny lines were obtained from a cross of parental lines MD x TU114 through targeted propagation in mice of progeny oocysts originating from populations lacking one parental allele at one or more loci. For each infection lineage this process was repeated until only a single allele remained for each marker, indicating that the progeny line was clonal. To study genetic recombination, 16 progeny clones were genotyped at 40 loci located on each of the eight chromosomes. The inheritance of parental alleles was significantly skewed towards the more virulent parent isolate MD. A contiguous 476 kb segment of chromosome V displayed MD allele in all progeny recovered, while MD and TU114 alleles were detected at other loci throughout the genome. The absence of alleles from one parental isolate in this chromosomal region may indicate phenotypic selection for the MD allele during the generation of these lines. A range for the meiotic crossover frequency was determined on the basis of 40 markers and the number of meioses estimated to have taken place during the crossing experiment. C. parvum exhibits a high rate of recombination commensurate with other Apicomplexa.     

Immunohistochemistry based assay to determine the effects of treatments on Cryptosporidium parvum viability

Cell culture infectivity assays can provide an accurate means of detecting viable Cryptosporidium parvum oocysts from environmental samples or to test the effects of various treatments on oocyst infectivity. Cell culture assays can also be used to test candidate chemotherapeutic agents. The use of a human cell line provides a situation close to human infection. The present assay uses an anti-Cryptospordium primary antibody, combined with a biotinylated secondary antibody, and an immunoperoxidase detection system. Cryptosporidium parvum oocysts excysted in vitro when placed on monolayers of HCT-8 cells and developmental stages including schizonts and merozoites were visualized using light microscopy of the immunoperoxidase stained slides and by transmission electron microscopy of infected HCT-8 cell cultures. Because the immunoperoxidase system used gives a permanent preparation, the cell cultures can be retained and examined later. Dose titration of oocysts indicated that as few as 50 inoculated oocysts could be detected. The activity of paromomycin was evaluated in this system and 500 microg/ml produced a 97.8% reduction in infection.     

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.     

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.     

Efficacy of common laboratory disinfectants on the infectivity of Cryptosporidium parvum oocysts in cell culture

Nine liquid disinfectants were tested for their ability to reduce infectivity of Cryptosporidium parvum oocysts in cell culture. A 4-min exposure to 6% hydrogen peroxide and a 13-min exposure to ammonium hydroxide-amended windshield washer fluid reduced infectivity 1,000-fold. Other disinfectants tested (70% ethanol, 37% methanol, 6% sodium hypochlorite, 70% isopropanol, and three commercial disinfectants) did not reduce the infectivity after a 33-min exposure. The results indicate that hydrogen peroxide and windshield washer fluid or ammonium hydroxide disinfectant may be suitable laboratory disinfectants against C. parvum oocysts.     

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.     

Studies on the resistance/reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts exposed to medium-pressure ultraviolet radiation

The ex vivo and in vivo reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts after exposure to different doses of ultraviolet (UV) radiation was determined using animal infectivity. The infectivity of UV-treated parasites stored for 1-4 days (G. muris) or 1-17 days (C. parvum) at room temperature in the dark was similar to that of organisms administered immediately after UV treatment, indicating that the parasites did not reactivate ex vivo. In contrast, we observed in vivo reactivation of G. muris in three of seven independent animal infectivity experiments, when parasites were treated with relatively low doses of medium-pressure UV (<25 mJ/cm(2)). Our observations indicate that G. muris cysts and C. parvum oocysts exposed to medium-pressure UV doses of 60 mJ/cm(2) or higher did not exhibit resistance to and/or reactivation following treatment. This suggests that when appropriate doses of UV are used, significant and permanent inactivation of these parasites may be achieved.     

Die-off of Cryptosporidium parvum in soil and wastewater effluents

AIMS: To determine the effect of biotic and abiotic components of soil on the viability and infectivity of Cryptosporidium parvum, and evaluate the suitability of viability tests as a surrogate for oocyst infectivity under various environmental settings. METHODS AND RESULTS: The die-off of C. parvum in saturated and dry loamy soil was monitored over time by immunofluorescence assay (IFA) and PCR to estimate oocysts viability and by cell culture to estimate oocysts infectivity. Pseudomonas aeruginosa activity resulted in digestion of the outer layer of the oocysts, as demonstrated by loss of the ability to react in IFA. Whereas, P. aeruginosa activity did not affect the DNA amplification by PCR. A 1-log reduction in the oocysts infectivity was observed at 30 degrees C in distilled water and in saturated soil while oocysts viability was unchanged. Incubation for 10 days in dry loamy soil at 32 degrees C resulted in a 3-log(10) reduction in their infectivity while no change of oocysts viability was recorded. CONCLUSIONS: Under low temperature, C. parvum oocysts may retain their infectivity for a long time. Soil desiccation and high temperatures enhance the die-off rate of C. parvum. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous die-off studies of C. parvum used viability tests that do not necessarily reflect the oocyst infectivity. Under low temperatures, there was an agreement observed between viability and infectivity tests and oocysts retained their infectivity for a long time. Desiccation and high temperatures enhance the loss of infectivity of C. parvum. The presented die-off data have significant implications on the management of wastewater reuse in warm environments.     

Subtype analysis of Cryptosporidium isolates from children in Uganda

Cryptosporidium hominis and Cryptosporidium parvum isolates from children in Uganda were characterized by DNA sequence analysis of the GP60 gene. Eight alleles were identified, 4 C. hominis and 4 C. parvum, of which 3 represent new C. parvum families. The data show that it is highly likely that the route of transmission is anthroponotic.     

A technique for typing Cryptosporidium isolates.

Antigens extracted from Cryptosporidium oocysts, which had been purified from faeces or chick egg culture, were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels, and blotted onto nitrocellulose membranes. A Cryptosporidium genus-specific monoclonal antibody MAb-C1 bound to multiple bands using several detection techniques, and these corresponded to bands detected using immune rabbit antisera. Using a detection system with fluorescein isothiocyanate (FITC)-labelled MAb-C1 and alkaline phosphatase-labelled anti-FITC, bands were detected between 50 and 300 kDa. Blots were examined directly and by using a laser scanner. The system was shown to be specific for Cryptosporidium spp., giving no staining with a variety of other pathogens, and with negative samples. The oocyst antigen which bound MAb-C1 was stable, and banding patterns were not significantly affected by pretreatment of oocysts with proteinase K, trypsin, formalin, or sodium hypochlorite, methods commonly used during preparation and storage of C. parvum oocysts. However, banding was reduced with potassium dichromate. Of 76 samples containing Cryptosporidium oocysts, 53 showed one or more MAb-C1 staining bands. Cryptosporidium baileyi and C. parvum could be clearly differentiated by their banding patterns, indicating that the system will distinguish between species. Some isolates, including a single isolate of C. muris, produced weak bands which made interpretation difficult. The technique showed differences between isolates of C. parvum, with two different banding types found in human isolates, and other banding types seen in calf and lamb isolates. This method provides a useful way of characterising isolates which may be new species.