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.     

Inactivation of Cryptosporidium parvum Oocysts in Fresh Apple Cider by UV Irradiation

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm². Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.     

Inactivation of Cryptosporidium parvum oocysts in fresh apple cider by UV irradiation

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm(2). Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.     

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°C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm² (=30 J/m²), the reduction reached the cell culture assay detection limit of ~3 log₁₀. At UV doses of 1.2 and 3 mJ/cm², the log₁₀ 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.     

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.     

Behavior of Mycoplasma hominis in a Human Diploid Cell Culture System

The behavior of Mycoplasma hominis in normal human embryonic lung fibroblast (HAIN-55) cell cultures was investigated. Multiplication patterns of cell-associated mycoplasmas and of extracellular mycoplasmas in the HAIN-55 cultures depended upon the size of the inoculum. This relationship did not vary with the number of days the cells had been cultured, nor with the number of HAIN-55 cell passages. The maximum mycoplasmal growth was obtained with inoculum sizes of 10⁵ to 10⁶ colony-forming units (CFU)/ml. The recovery of mycoplasmas decreased rapidly with inoculum size beyond 10⁷ CFU/ml, and growth of the HAIN-55 cells was inhibited. Growth of the cells was also inhibited by the addition of the cytoplasmic fraction of Mycoplasma hominis.     

Characterisation of small double stranded RNA molecule in Cryptosporidium hominis, Cryptosporidium felis and Cryptosporidium meleagridis

Coding regions of double stranded RNA molecules from 3 human faecal samples containing Cryptosporidium hominis, C. felis and C. meleagridis were characterised by sequencing and compared with that previously obtained for C. parvum. Sequences outside the coding regions were also obtained. Overall similarities of between 86% and 92% and between 86% and 93% were observed in the nucleotide and amino acid sequences respectively between these species. These larger sequences will allow further molecular tools for detection, identification and characterisation of Cryptosporidium spp.     

Inferences about the Global Population Structures of Cryptosporidium parvum and Cryptosporidium hominis

Cryptosporidium parvum and Cryptosporidium hominis are two related species of apicomplexan protozoa responsible for the majority of human cases of cryptosporidiosis. In spite of their considerable public health impact, little is known about the population structures of these species. In this study, a battery of C. parvum and C. hominis isolates from seven countries was genotyped using a nine-locus DNA subtyping scheme. To assess the existence of geographical partitions, the multilocus genotype data were mined using a cluster analysis based on the nearest-neighbor principle. Within each country, the population genetic structures were explored by combining diversity statistical tests, linkage disequilibrium, and eBURST analysis. For both parasite species, a quasi-complete phylogenetic segregation was observed among the countries. Cluster analysis accurately identified recently introduced isolates. Rather than conforming to a strict paradigm of either a clonal or a panmictic population structure, data are consistent with a flexible reproductive strategy characterized by the cooccurrence of both propagation patterns. The relative contribution of each pattern appears to vary between the regions, perhaps dependent on the prevailing ecological determinants of transmission.     

Substituted pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis thymidylate synthase inhibitors

Cryptosporidiosis, a gastrointestinal disease caused by a protozoan Cryptosporidium hominis is often fatal in immunocompromised individuals. There is little clinical data to show that the existing treatment by nitazoxanide and paromomycin is effective in immunocompromised individuals.1, 2 Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer and malaria. A novel series of classical antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines have been evaluated as Cryptosporidium hominis thymidylate synthase (ChTS) inhibitors. Crystal structure in complex with the most potent compound, a 2′-chlorophenyl with a sulfur bridge with a K_i of 8.83 ± 0.67 nM is discussed in terms of several Van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate. Of these interactions, two interactions with the non-conserved residues (A287 and S290) offer an opportunity to develop ChTS specific inhibitors. Compound 6 serves as a lead compound for analog design and its crystal structure provides clues for the design of ChTS specific inhibitors.     

Occurrence and Genetic Characteristics of Cryptosporidium hominis and Cryptosporidium andersoni in Horses from Southwestern China

A total of 333 fecal specimens from horses in southwestern China were genotyped based on analysis of the small subunit rRNA (SSU rRNA) gene. Cryptosporidium hominis and Cryptosporidium andersoni were identified in 2 and 4 stool specimens, respectively. The identification of C. hominis was confirmed by sequence analysis of the 70‐kDa heat shock protein (HSP70) and oocyst wall protein (COWP) genes. Subtyping analysis of the 60‐kDa glycoprotein (GP60) gene sequence of C. hominis revealed a new rare subtype Id, named IdA15; only three Id isolates have been reported in humans to date. Multilocus sequence typing (MLST) analysis indicated that the C. andersoni subtype was A6, A5, A2, and A1 at the four minisatellite loci (MS1, MS2, MS3, and MS16, respectively). This is the first report to identify the presence of C. andersoni and C. hominis in horses in southwestern China and the first to identify a rare zoonotic subtype Id of C. hominis in horses. These findings suggest that infected horses may act as potential reservoirs of Cryptosporidium to transmit infections to humans.     

UV Light Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk as Assessed by FASTPlaqueTB Phage Assay and Culture

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in Middlebrook 7H9 broth and whole and semiskim milk was investigated using a laboratory-scale UV machine that incorporated static mixers within UV-penetrable pipes. UV treatment proved to be less effective in killing M. avium subsp. paratuberculosis suspended in milk (0.5- to 1.0-log₁₀ reduction per 1,000 mJ/ml) than that suspended in Middlebrook 7H9 broth (2.5- to 3.3-log₁₀ reduction per 1,000 mJ/ml). The FASTPlaqueTB phage assay provided more rapid enumeration of surviving M. avium subsp. paratuberculosis (within 24 h) than culture on Herrold's egg yolk medium (6 to 8 weeks). Despite the fact that plaque counts were consistently 1 to 2 log₁₀ lower than colony counts throughout the study, UV inactivation rates for M. avium subsp. paratuberculosis derived using the phage assay and culture results were not significantly different (P = 0.077).     

Assessment of polymorphic genetic markers for multi-locus typing of Cryptosporidium parvum and Cryptosporidium hominis

The use of high resolution molecular tools to study Cryptosporidium parvum and Cryptosporidium hominis intra-species variation is becoming common practice, but there is currently no consensus in the methods used. The most commonly applied tool is partial gp60 gene sequence analysis. However, multi-locus schemes are acknowledged to improve resolution over analysis of a single locus, which neglects potential re-assortment of genes during the sexual phase of the Cryptosporidium life-cycle. Multi-locus markers have been investigated in isolates from a variety of sampling frames, in varying combinations and using different assays and methods of analysis. To identify the most informative markers as candidates for the development of a standardised multi-locus fragment size-based typing (MLFT) scheme to integrate with epidemiological analyses, we examined the published literature. A total of 31 MLFT studies were found, employing 55 markers of which 45 were applied to both C. parvum and C. hominis. Of the studies, 11 had sufficient raw data, from three or more markers, and a sampling frame containing at least 50 samples, for meaningful in-depth analysis using assessment criteria based on the sampling frame, study size, number of markers investigated in each study, marker characteristics (>2 nucleotide repeats) and the combinations of markers generating all possible multi-locus genotypes. Markers investigated differed between C. hominis and C. parvum. When each scheme was analysed for the fewest markers required to identify 95% of all MLFTs, some redundancy was identified in all schemes; an average redundancy of 40% for C. hominis and 27% for C. parvum. Ranking markers, based on the most productive combinations, identified two different sets of potentially most informative candidate markers, one for each species. These will be subjected to technical evaluation including typability (percentage of samples generating a complete multi-locus type) and discriminatory power by direct fragment size analysis and analysed for correlation with epidemiological data in suitable sampling frames. The establishment of a group of users and agreed subtyping scheme for improved epidemiological and public health investigations of C. parvum and C. hominis will facilitate further developments and consideration of technological advances in a harmonised manner.     

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·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·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·cm⁻² of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ·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.     

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.     

Inactivation of Cryptosporidium parvum Oocysts by Ammonia

The survival of Cryptosporidium parvum oocysts in soil and water microhabitats may be affected by the environmental production and release of free ammonia. The objective of this study was to determine the effects of increasing free ammonia concentrations and times of exposure on oocyst viability. Wild-type oocysts were obtained from naturally infected calf feces by chemical (continuous-flow) centrifugation and sucrose gradients. Ammonia (NH₃) from a commercial solution was applied in concentrations ranging from 0.007 to 0.148 M. Exposure times ranged from 10 min to 24 h at a constant temperature of 24 ± 1°C. Viability of oocysts was determined with a dye permeability assay and an in vitro excystation assay (M. B. Jenkins, L. J. Anguish, D. D. Bowman, M. J. Walker, and W. C. Ghiorse, Appl. Environ. Microbiol. 63:3844–3850, 1997). Even the lowest concentration of ammonia decreased significantly the viability of oocysts after 24 h of exposure. Increasing concentrations of ammonia increased inactivation rates, which ranged from 0.014 to 0.066 h⁻¹. At the highest concentration of ammonia, a small fraction of viable oocysts still remained. Exposure to pH levels corresponding to those associated with the ammonia concentrations showed minimal effects of alkaline pH alone on oocyst viability. This study shows that environmentally relevant concentrations of free ammonia may significantly increase the inactivation of oocysts in ammonia-containing environments.     

Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species

A study was undertaken to compare the performance of five different molecular methods (available in four different laboratories) for the identification of Cryptosporidium parvum and Cryptosporidium hominis and the detection of genetic variation within each of these species. The same panel of oocyst DNA samples derived from faeces (n=54; coded blindly) was sent for analysis by: (i) DNA sequence analysis of a fragment of the HSP70 gene; (ii) DNA sequence analysis and the ssrRNA gene in laboratory 1; (iii) single-strand conformation polymorphism analysis of part of the ssrRNA; (iv) SSCP analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA region in laboratory 2; (v) 60 kDa glycoprotein (gp60) gene sequencing with prior species determination using PCR with restriction fragment length polymorphism analysis of the ssrRNA gene in laboratory 3; and (vi) multilocus genotyping at three microsatellite markers in laboratory 4. For detecting variation within C. parvum and C. hominis, SSCP analysis of ITS-2 was considered to have superior utility and determined 'subgenotypes' in samples containing DNA from both species. SSCP was also most cost effective in terms of time, cost and consumables. Sequence analysis of gp60 and microsatellite markers ML1, ML2 and 'gp15' provided good comparators for the SSCP of ITS-2. However, applicability of these methods to other Cryptosporidium species or genotypes and to environmental samples needs to be evaluated. This trial provided, for the first time, a direct comparison of multiple methods for the genetic characterisation of C. parvum and C. hominis samples. A protocol has been established for the international distribution of samples for the characterisation of Cryptosporidium. This can be applied in further evaluation of molecular methods by investigation of a larger number of unrelated samples to establish sensitivity, typability, reproducibility and discriminatory power based on internationally accepted methods for evaluation of microbial typing schemes.     

Determination of Pyrimidine Dimers in Escherichia coli and Cryptosporidium parvum during UV Light Inactivation, Photoreactivation, and Dark Repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.