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1
Weir, C., G. Vesey, M. Slade, B. Ferrari, D. A. Veal, and K. Williams. "An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts." Clinical Diagnostic Laboratory Immunology 7, no. 5 (September 1, 2000): 745–50. http://dx.doi.org/10.1128/cdli.7.5.745-750.2000.
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ABSTRACT The detection of Cryptosporidium oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to Cryptosporidium oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of Cryptosporidiumoocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG1 monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies.
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Searcy, Kristin E., Aaron I. Packman, Edward R. Atwill, and Thomas Harter. "Association of Cryptosporidium parvum with Suspended Particles: Impact on Oocyst Sedimentation." Applied and Environmental Microbiology 71, no. 2 (February 2005): 1072–78. http://dx.doi.org/10.1128/aem.71.2.1072-1078.2005.
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ABSTRACT The association of Cryptosporidium parvum oocysts with suspended particles can alter the oocysts' effective physical properties and influence their transport in aquatic systems. To assess this behavior, C. parvum oocysts were mixed with various suspended sediments under a variety of water chemical conditions, and the resulting settling of the oocysts was observed. Direct microscopic observations showed that oocysts attached to suspended sediments. Settling column and batch experiments demonstrated that oocysts are removed from suspension at a much higher rate when associated with sediments. The rate of oocyst sedimentation depended primarily on the type of sediment with which the oocysts were mixed. Changes in background water conditions had a relatively small impact on the extent of oocyst-particle association and the resulting oocyst deposition. We believe that the ubiquitous association of C. parvum oocysts with suspended particles enhances the sedimentation of oocysts in natural waters and that this interaction should generally be considered when predicting the migration of pathogens in the environment.
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Gale, P. "Risk assessment model for a waterborne outbreak of cryptosporidiosis." Water Science and Technology 41, no. 7 (April 1, 2000): 1–7. http://dx.doi.org/10.2166/wst.2000.0108.
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Counts of Cryptosporidium oocysts in 100L volumes of treated water are simulated for conditions representative of a waterborne outbreak in a surface water-derived supply. Assuming oocysts act independently during infection, the risk of infection is directly related to the arithmetic mean oocyst density in the water supply, which is in turn related to the total number of oocysts which break through treatment. Spatial/temporal heterogeneity of oocyst concentrations in the treated water contributes to monitoring programmes based on “spot-sampling” underestimating the arithmetic mean oocyst density and hence the risk of infection. This could contribute to the reported lack of a clear association between oocyst concentrations measured in drinking water supplies and the risk of waterborne outbreak of cryptosporidiosis in the population. An increase in spatial heterogeneity of oocysts during treatment could also contribute to an overestimation of the net oocyst removal by treatment.
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Jenkins, Michael B., Barbara S. Eaglesham, Larry C. Anthony, Scott C. Kachlany, Dwight D. Bowman, and William C. Ghiorse. "Significance of Wall Structure, Macromolecular Composition, and Surface Polymers to the Survival and Transport of Cryptosporidium parvum Oocysts." Applied and Environmental Microbiology 76, no. 6 (January 22, 2010): 1926–34. http://dx.doi.org/10.1128/aem.02295-09.
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ABSTRACT The structure and composition of the oocyst wall are primary factors determining the survival and hydrologic transport of Cryptosporidium parvum oocysts outside the host. Microscopic and biochemical analyses of whole oocysts and purified oocyst walls were undertaken to better understand the inactivation kinetics and hydrologic transport of oocysts in terrestrial and aquatic environments. Results of microscopy showed an outer electron-dense layer, a translucent middle layer, two inner electron-dense layers, and a suture structure embedded in the inner electron-dense layers. Freeze-substitution showed an expanded glycocalyx layer external to the outer bilayer, and Alcian Blue staining confirmed its presence on some but not all oocysts. Biochemical analyses of purified oocyst walls revealed carbohydrate components, medium- and long-chain fatty acids, and aliphatic hydrocarbons. Purified walls contained 7.5% total protein (by the Lowry assay), with five major bands in SDS-PAGE gels. Staining of purified oocyst walls with magnesium anilinonaphthalene-8-sulfonic acid indicated the presence of hydrophobic proteins. These structural and biochemical analyses support a model of the oocyst wall that is variably impermeable and resistant to many environmental pressures. The strength and flexibility of oocyst walls appear to depend on an inner layer of glycoprotein. The temperature-dependent permeability of oocyst walls may be associated with waxy hydrocarbons in the electron-translucent layer. The complex chemistry of these layers may explain the known acid-fast staining properties of oocysts, as well as some of the survival characteristics of oocysts in terrestrial and aquatic environments. The outer glycocalyx surface layer provides immunogenicity and attachment possibilities, and its ephemeral nature may explain the variable surface properties noted in oocyst hydrologic transport studies.
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Tilley, Michael, Steve J. Upton, and Clarence E. Chrisp. "A comparative study on the biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum (Apicomplexa)." Canadian Journal of Microbiology 37, no. 12 (December 1, 1991): 949–52. http://dx.doi.org/10.1139/m91-163.
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Cryptosporidum sp. from guinea pigs and C. parvum were compared morphologically, electrophoretically, and for the ability to infect suckling mice. Oocysts from guinea pigs measured 5.4 × 4.6 (4.8–5.6 × 4.0–5.0) μm and had a shape index (length/width) of 1.17 (1.04–1.33). Oocysts of C. parvum were similar and measured 5.2 × 4.6 (4.8–5.6 × 4.2–4.8) μm with a shape index of 1.16 (1.04–1.33). All suckling mice inoculated with oocyts of C. parvum became infected, whereas most, but not all, mice fed oocysts of the guinea pig isolate also became infected. However, mice inoculated with oocysts from guinea pigs produced on average 100-fold fewer oocysts by day 7 postinoculation than did mice infected with C. parvum, and the resulting infections were sparse and patchy along the ileum. Electrophoretic profiles were similar, but 125I surface labeling of outer oocyst wall proteins revealed striking differences between the two isolates. Cryptosporidium parvum had a wide molecular size range of 125I-labeled bands, whereas C. sp. from guinea pigs had a banding pattern clustered between 39 and 66 kDa, with a smaller number of bands >100 kDa. Key words: Cryptosporidium parvum, coccidia, Apicomplexa, guinea pig, mouse.
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Kwa, B. H., M. Moyad, M. A. Pentella, and J. B. Rose. "A Nude Mouse Model as an in vivo Infectivity Assay for Cryptospomdiosis." Water Science and Technology 27, no. 3-4 (February 1, 1993): 65–68. http://dx.doi.org/10.2166/wst.1993.0323.
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Cryptosporidium parvum is an important patliogen of diarrlieal disease which has been implicated in several outbreaks associated with contamination of surface waters. In monitoring for C. parvum in drinking water sources, it is important to asce tain the viability, and more importantly, the infectivity of low numbers of recovered oocysts. Groups of 10 Balb/C nude (nu/nu) mice, 4-8 weeks old at time of inoculation, were infected with C. parvum oocysts from naturally infected calves and purified using Sheather's sucrose gradients. Oocysts were counted using the Merifluor IFA kit (Meridian). Each group of 10 mice were infected with 1,10,100 and 1000 oocysts respectively. Numbers of oocysts per inoculation were determined by limiting dilution, and parallel inocula were counted microscopically to ascertain the accuracy of the dilutions. Two uninfected nude mice were kept in each cage to serve as controls. Mouse stools were collected every 4 days, concentrated using the Fekal Kontrate Concentration Kit (Meridian) and oocysts were counted with a UV microscope using the Merifluor IFA Kit (Meridian). Oocyst counts were expressed in terms of number of oocyst/g feces. Mice inoculated with 1000 oocysts began to shed oocysts on day 32, mice inoculated with 100 oocysts began to shed on days 44-48, mice inoculated with 10 oocysts began to shed on days 56-60, and mice inoculated with 1 oocyst shed on days 68-88. All infected mice continued to shed oocysts intermittently and with variable oocyst counts until day 180 when the experiment was terminated. This study established that it is possible to infect nude mice with very low numbers, down to a single oocyst. We are currently in the process of correlating the nude mouse assay with other viability assays.
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Somiya, I., S. Fujii, N. Kishimoto, and R.-H. Kim. "Development of a mathematical model of Cryptosporidium inactivation by ozonation." Water Science and Technology 41, no. 7 (April 1, 2000): 173–80. http://dx.doi.org/10.2166/wst.2000.0130.
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A new mathematical model was developed to express the processes of Cryptosporidium inactivation by ozonation. In this model, five different stages were considered as state variables of Cryptosporidium oocysts for accurate expression of the inactivation. ATP, in vitro excystation and DAPI/PI permeability assays were used to describe the oocyst amounts of different stages. Some reaction constants were estimated by the structure components of oocysts or by the stoichiometry of reactions, while the others were by the oocysts population changes in ozonation. The calculated values of this model were well consistent with experimental inactivation data. Three-log inactivation of sporozoites required about 0.04 mgO3 per unit oocyst (mgC) from the simulation results. Before ozone reacts with sporozoites, more ozone was consumed to oxidize other parts of oocysts and DOC produced. The main path of inactivation of oocysts by ozonation was estimated to be P1 (intact oocysts)→P2 (oocysts with damaged outer oocyst wall)→P4 (oocysts without excystation function)→P5 (oocysts with inactivated sporozoites and no excystation function) from experimental and simulated results.
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Gale, P. "Simulating cryptosporidium exposures in drinking water during an outbreak." Water Science and Technology 38, no. 12 (December 1, 1998): 7–13. http://dx.doi.org/10.2166/wst.1998.0486.
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This paper predicts exposures to Cryptosporidium parvum oocysts through drinking water under conditions which are consistent with a waterborne outbreak. Sources of variation which contribute to the variation in oocyst exposures include the oocyst densities in the raw waters, the efficiency of oocyst removal by treatment and the daily consumption of unboiled tap water. Even under outbreak conditions the majority of consumers may not ingest any oocysts each day. Of those who are exposed, some ingest just one oocyst/d while others ingest higher doses, which in a small proportion approach the ID50 for C parvum. Ignoring this variation and using a single point average exposure predicts that a much larger proportion of the population is exposed each day but only ever to very low doses of oocysts. The impact of ignoring this variation on the predicted risks depends on the nature of the dose-response curve and, in particular, the assumptions made about the low dose extrapolation. The heterogeneity of oocyst densities in drinking water during an outbreak could contribute to the failure to detect oocysts in some waterborne outbreaks.
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DENG, MING QI, and DEAN O. CLIVER. "Inactivation of Cryptosporidium parvum Oocysts in Cider by Flash Pasteurization." Journal of Food Protection 64, no. 4 (April 1, 2001): 523–27. http://dx.doi.org/10.4315/0362-028x-64.4.523.
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Cryptosporidium parvum is a well-recognized pathogen of significant medical importance, and cider (apple juice) has been associated with foodborne cryptosporidiosis. This study investigated the effect of flash pasteurization on the viability of contaminant C. parvum oocysts. Cider inoculated with oocysts was heated at 70 or 71.7°C for 5, 10, or 20 s, and oocyst viability was measured by a semiquantitative in vitro infectivity assay. By infecting multiple wells of confluent Madin-Darby bovine kidney cells with serial dilutions of heat-treated oocysts and examining infected cells by indirect fluorescent antibody staining, the most probable number technique was applied to quantify log reduction of oocyst viability. Heating for 10 or 20 s at either temperature caused oocyst killing of at least 4.9 log (or 99.999%), whereas oocyst inactivation after pasteurization for 5 s at 70 and 71.7°C was 3.0 log (99.9%) and 4.8 log (99.998%), respectively. Our results suggested that current practices of flash pasteurization in the juice industry are sufficient in inactivating contaminant oocysts.
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Butkus, Michael A., J. Timothy Bays, and Michael P. Labare. "Influence of Surface Characteristics on the Stability of Cryptosporidium parvum Oocysts." Applied and Environmental Microbiology 69, no. 7 (July 2003): 3819–25. http://dx.doi.org/10.1128/aem.69.7.3819-3825.2003.
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ABSTRACT Microelectrophoresis is a common technique for probing the surface chemistry of the Cryptosporidium parvum oocyst. Results of previous studies of the electrophoretic mobility of C. parvum oocysts in which microelectrophoresis was used are incongruent. In this work we demonstrated that capillary electrophoresis may also be used to probe the surface characteristics of C. parvum oocysts, and we related the surface chemistry of C. parvum oocysts to their stability in water. Capillary electrophoresis results indicated that oocysts which were washed in a phosphate buffer solution had neutrally charged surfaces. Inactivation of oocysts with formalin did not influence their electrophoretic mobility, while oocyst populations that were washed in distilled water consisted of cells with both neutral and negative surface charges. These results indicate that washing oocysts in low-ionic-strength distilled water can impart a negative charge to a fraction of the oocysts in the sample. Rapid coagulation experiments indicated that oocysts did not aggregate in a 0.5 M NaCl solution; oocyst stability in the salt solution may have been the result of Lewis acid-base forces, steric stabilization, or some other factor. The presence of sucrose and Percoll could not be readily identified on the surface of C. parvum oocysts by attenuated total reflectance-Fourier transform infrared spectroscopy, suggesting that these purification reagents may not be responsible for the stability of the uncharged oocysts. These findings imply that precipitate enmeshment may be the optimal mechanism of coagulation for removal of oocysts in water treatment systems. The results of this work may help elucidate the causes of variation in oocyst surface characteristics, may ultimately lead to improved removal efficiencies in full-scale water treatment systems, and may improve fate and transport predictions for oocysts in natural systems.
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Sturbaum, Gregory D., Carrie Reed, Paul J. Hoover, B. Helen Jost, Marilyn M. Marshall, and Charles R. Sterling. "Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts." Applied and Environmental Microbiology 67, no. 6 (June 1, 2001): 2665–68. http://dx.doi.org/10.1128/aem.67.6.2665-2668.2001.
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ABSTRACT Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum,Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguishedC. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolatedC. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.
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Stadterman, K. L., A. M. Sninsky, J. L. Sykora, and W. Jakubowskii. "Removal and inactivation of cryptosporidium oocysts by activated sludge treatment and anaerobic digestion." Water Science and Technology 31, no. 5-6 (March 1, 1995): 97–104. http://dx.doi.org/10.2166/wst.1995.0572.
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To determine the fate of Cryptosporidium parvum oocysts during wastewater treatment, a model of an activated sludge treatment plant was designed with a flow of 17 ml/min and a detention time of 6 hours. Samples of raw sewage were seeded with oocysts and primary and secondary effluents were analyzed for C. parvum using an immunofluorescent technique. To compare removal efficiencies of oocysts by various wastewater treatment processes, raw sewage, activated sludge, trickling filter and biodisc effluents were seeded with oocysts and settled for 2 hr and for the respective detention times. Sludge produced by a wastewater treatment plant and anaerobically digested at 37° C in a laboratory digester was also seeded with C. parvum oocysts. Oocyst inactivation was measured by excystation and direct counts. Removal of oocysts in primary and secondary sedimentation averaged 83.4% and 90.7% respectively. The total oocyst removal in sewage treatment averaged 98.6%. In comparison with other treatment processes, activated sludge had the maximum oocyst removal efficiency at 92%. The anaerobic digestion process inactivated 90% of the oocysts within four hours of exposure. 99.9% of the oocysts were eliminated by anaerobic digestion after 24 hours. This demonstrates that the activated sludge process and anaerobic digestion can be effective for the removal and inactivation of C. parvum oocysts.
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Wolyniak, E. A., B. R. Hargreaves, and K. L. Jellison. "Seasonal Retention and Release of Cryptosporidium parvum Oocysts by Environmental Biofilms in the Laboratory." Applied and Environmental Microbiology 76, no. 4 (December 18, 2009): 1021–27. http://dx.doi.org/10.1128/aem.01804-09.
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ABSTRACT Cryptosporidium is a genus of waterborne protozoan parasites that causes significant gastrointestinal disease in humans. These parasites can accumulate in environmental biofilms and be subsequently released to contaminate water supplies. Natural microbial assemblages were collected each season from an eastern Pennsylvania stream and used to grow biofilms in laboratory microcosms in which influx, efflux, and biofilm retention were determined from daily oocyst counts. For each seasonal biofilm, oocysts attached to the biofilm quickly during oocyst dosing. Upon termination of oocyst dosing, the percentage of oocysts retained within the biofilm decreased to a new steady state within 5 days. Seasonal differences in biofilm retention of oocysts were observed. The spring biofilm retained the greatest percentage of oocysts, followed (in decreasing order) by the winter, summer, and fall biofilms. There was no statistically significant correlation between the percentage of oocysts attached to the biofilm and (i) any measured stream water quality parameter (including temperature, pH, conductivity, and dissolved organic carbon concentration) or (ii) experimental temperature. Seasonal differences in oocyst retention persisted when biofilms were tested with stream water from a different season. These data suggest that seasonal variation in the microbial community and resulting biofilm architecture may be more important to oocyst transport in this stream site than water quality. The biofilm attachment and detachment dynamics of C. parvum oocysts have implications for public health, and the drinking water industry should recognize that the potential exists for pathogen-free water to become contaminated during the distribution process as a result of biofilm dynamics.
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NICHOLS, R. A. B., C. A. PATON, and H. V. SMITH. "Survival of Cryptosporidium parvum Oocysts after Prolonged Exposure to Still Natural Mineral Waters." Journal of Food Protection 67, no. 3 (March 1, 2004): 517–23. http://dx.doi.org/10.4315/0362-028x-67.3.517.
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The survival kinetics of purified Cryptosporidium parvum oocysts of both human and ovine origin, immersed in four still natural mineral waters (total dissolved salts ranging from 91 mg/liter to 430 mg/liter) and reverse osmosis water was assessed by inclusion or exclusion of the fluorogenic vital dyes 4′,6-diamidino-2-phenylindole and propidium iodide over a 12-week period. Semipermeable chambers were used to contain the oocysts while immersed in each mineral water type, permitting both intimate interactions between oocysts and matrices and straightforward sampling for viability assessments. The viability of both oocyst types, assessed at weekly intervals, remained unaltered after 12 weeks at 4°C, whereas a progressive decline in the viability of both oocyst isolates was observed when immersed in mineral waters at 20°C. At 20°C, approximately 30% of oocysts remained viable after 12 weeks incubation. Here, temperature was the major factor that adversely affected oocyst survival, although higher mineral content was also proportionally and significantly associated with this increased oocyst inactivation. The prolonged survival of oocysts at 4°C in our studies indicates that they could survive for prolonged periods of time in U.K. groundwaters (average temperature ~10°C) and thus represent a potential public health hazard if contamination of mineral water sources by viable oocysts were to occur.
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Wolyniak DiCesare, E. A., B. R. Hargreaves, and K. L. Jellison. "Biofilm Roughness Determines Cryptosporidium parvum Retention in Environmental Biofilms." Applied and Environmental Microbiology 78, no. 12 (April 6, 2012): 4187–93. http://dx.doi.org/10.1128/aem.08026-11.
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ABSTRACTThe genusCryptosporidiumis a group of waterborne protozoan parasites that have been implicated in significant outbreaks of gastrointestinal infections throughout the world. Biofilms trap these pathogens and can contaminate water supplies through subsequent release. Biofilm microbial assemblages were collected seasonally from three streams in eastern Pennsylvania and used to grow biofilms in laboratory microcosms. Daily oocyst counts in the influx and efflux flow allowed the calculation of daily oocyst retention in the biofilm. Following the removal of oocysts from the influx water, oocyst attachment to the biofilm declined to an equilibrium state within 5 days that was sustained for at least 25 days. Varying the oocyst loading rate for the system showed that biofilm retention could be saturated, suggesting that discrete binding sites determined the maximum number of oocysts retained. Oocyst retention varied seasonally but was consistent across all three sites; however, seasonal oocyst retention was not consistent across years at the same site. No correlation between oocyst attachment and any measured water quality parameter was found. However, oocyst retention was strongly correlated with biofilm surface roughness and roughness varied among seasons and across years. We hypothesize that biofilm roughness and oocyst retention are dependent on environmentally driven changes in the biofilm community rather than directly on water quality conditions. It is important to understand oocyst transport dynamics to reduce risks of human infection. Better understanding of factors controlling biofilm retention of oocysts should improve our understanding of oocyst transport at different scales.
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NICHOLS, ROSELY A. B., and HUW V. SMITH. "Optimization of DNA Extraction and Molecular Detection of Cryptosporidium Oocysts in Natural Mineral Water Sources." Journal of Food Protection 67, no. 3 (March 1, 2004): 524–32. http://dx.doi.org/10.4315/0362-028x-67.3.524.
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The numerous published methods for extracting DNA from Cryptosporidium oocysts for PCR identify the lack of an optimized standard method for clinical, environmental, and public health investigations of cryptosporidiosis. A method that maximizes DNA extraction reliably, particularly from small numbers of partially purified or purified oocysts present in mineral waters and environmental samples, is required. We describe a maximized method for liberating DNA from Cryptosporidium parvum oocysts by 15 cycles of freezing (liquid nitrogen) and thawing (65°C) in lysis buffer containing sodium dodecyl sulfate. The inhibitory effects of sodium dodecyl sulfate are abrogated by the addition of Tween 20 to the PCR reaction. We tested seven different C. parvum oocyst isolates, consistently detecting fewer than five oocysts following direct PCR amplification of a segment of the 18S rRNA gene. Older oocysts, which were more refractory to freeze-thawing, were disrupted effectively. A single oocyst in each of two mineral water concentrates was detected by both microscopy and PCR/Southern blotting. We recommend 15 cycles of freeze-thawing, with thawing at 65°C in lysis buffer, to maximize oocyst disruption and DNA extraction, particularly when isolate history and oocyst age are unknown. Both the DNA extraction method and the PCR described can be used for clinical, environmental, and public health investigations of cryptosporidiosis.
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LE GOFF, L., B. HUBERT, L. FAVENNEC, I. VILLENA, J. J. BALLET, A. AGOULON, N. ORANGE, and G. GARGALA. "Pilot-Scale Pulsed UV Light Irradiation of Experimentally Infected Raspberries Suppresses Cryptosporidium parvum Infectivity in Immunocompetent Suckling Mice." Journal of Food Protection 78, no. 12 (December 1, 2015): 2247–52. http://dx.doi.org/10.4315/0362-028x.jfp-15-062.
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Cryptosporidium spp., a significant cause of foodborne infection, have been shown to be resistant to most chemical food disinfectant agents and infective for weeks in irrigation waters and stored fresh vegetal produce. Pulsed UV light (PL) has the potential to inactivate Cryptosporidium spp. on surfaces of raw or minimally processed foods or both. The present study aimed to evaluate the efficacy of PL on viability and in vivo infectivity of Cryptosporidium parvum oocysts present on raspberries, a known source of transmission to humans of oocyst-forming apicomplexan pathogens. The skin of each of 20 raspberries was experimentally inoculated with five 10-μl spots of an oocyst suspension containing 6 × 107 oocysts per ml (Nouzilly isolate). Raspberries were irradiated by PL flashes (4 J/cm2 of total fluence). This dose did not affect colorimetric or organoleptic characteristics of fruits. After immunomagnetic separation from raspberries, oocysts were bleached and administered orally to neonatal suckling mice. Seven days after infection, mice were euthanized, and the number of oocysts in the entire small intestine was individually assessed by immunofluorescence flow cytometry. Three of 12 and 12 of 12 inoculated mice that received 10 and 100 oocysts isolated from nonirradiated raspberries, respectively, were found infected. Four of 12 and 2 of 12 inoculated mice that received 103 and 104 oocysts from irradiated raspberries, respectively, were found infected. Oocyst counts were lower in animals inoculated with 103 and 104 oocysts from irradiated raspberries (92 ± 144 and 38 ± 82, respectively) than in animals infected with 100 oocysts from nonirradiated raspberries (35,785 ± 66,221, P = 0.008). PL irradiation achieved oocyst reductions of 2 and 3 log for an inoculum of 103 and 104 oocysts, respectively. The present pilot-scale evaluation suggests that PL is an effective mode of decontamination for raspberries and prompts further applicability studies in industrial contexts.
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BUKHARI, Z., and H. V. SMITH. "SHORT PAPER Cryptosporidium parvum: oocyst excretion and viability patterns in experimentally infected lambs." Epidemiology and Infection 119, no. 1 (August 1997): 105–8. http://dx.doi.org/10.1017/s0950268897007590.
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Cryptosporidium parvum infections of domestic animals can have a considerable economic impact and as oocysts are voided in the faeces of infected hosts, environmental contamination with agricultural waste has also become a matter of concern. Since only viable oocysts are potentially infectious, the numbers of oocysts excreted during infection can have important implications for both veterinary and public health. During the course of infection in experimentally infected lambs, oocyst viability was assessed by a fluorogenic vital dyes assay and by a maximized in vitro excystation assay. The excreted oocyst populations contained a higher proportion of viable oocysts 5–11 days post infection (d.p.i.) than later in the infection. Oocyst viability declined consistently 11–15 d.p.i. and coincided with periods when peaks in serum and intestinal anti-Cryptosporidium antibodies have been reported to occur. Infected lambs excreted a mean of 4·8 (standard error [S.E.]±0·4)×109 oocysts per g of faeces, of which half were non-viable and therefore of no significance for disease transmission. This study demonstrates that the numbers of viable oocysts excreted by infected lambs is smaller than previously suspected.
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Guyot, K., M. F. Gireaudot-Liepmann, A. Cabon, I. Riveau-Ricard, M. Lange, J. M. Delattre, and E. Dei-Cas. "Influence of US EPA 1622 method successive steps on the viability of Cryptosporidium oocysts." Water Science and Technology 41, no. 7 (April 1, 2000): 189–96. http://dx.doi.org/10.2166/wst.2000.0132.
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Viable Cryptosporidium parvum oocysts were processed by the US EPA 1622 method to determine if the procedure that requires successive filtration, elutionand centrifugation alters their integrity and viability (determined by in vitro excystation). Oocyst seeded in tap water samples were also used to evaluate recovery efficiencies and impact of the whole procedure on oocyst viability. Filtration through Envirochek Gelman cartridge was found not to damage oocysts. The use of Laureth-12 buffer during the elution step was shown to lead to greater spontaneous oocysts excystation than other phosphate buffers containing between 80 and/or SDS (like the Gelman buffer). However, this drawback was widely balanced against the best efficiency of this buffer to elute oocysts captured by the cartridge filter and therefore against its high recovery efficiency. Thus, in water samples in which the oocyst concentration is expected to be low, it is more advantageous to employ the Laureth-12 buffer for the elution through it can influence viability. Centrifugation speeds (1,000–5,000 g) did not alter oocysts.
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Inomata, A., N. Kishida, T. Momoda, M. Akiba, S. Izumiyama, K. Yagita, and T. Endo. "Development and evaluation of a reverse transcription-loop-mediated isothermal amplification assay for rapid and high-sensitive detection of Cryptosporidium in water samples." Water Science and Technology 60, no. 8 (October 1, 2009): 2167–72. http://dx.doi.org/10.2166/wst.2009.599.
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We describe a novel assay for simple, rapid and high-sensitive detection of Cryptosporidium oocysts in water samples using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The detection limit of the developed RT-LAMP assay was as low as 6 × 10−3 oocysts/test tube, which theoretically enables us to detect a Cryptosporidium oocyst and perform duplicated tests even if water samples contain only one oocyst. The developed RT-LAMP assay could more sensitively detect Cryptosporidium oocysts in real water samples than the conventional assay based on microscopic observation.
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Nichols, Rosely A. B., Brian M. Campbell, and Huw V. Smith. "Molecular Fingerprinting of Cryptosporidium Oocysts Isolated during Water Monitoring." Applied and Environmental Microbiology 72, no. 8 (August 2006): 5428–35. http://dx.doi.org/10.1128/aem.02906-05.
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ABSTRACT 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.
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Kuczynska, Ewa, Daniel R. Shelton, and Yakov Pachepsky. "Effect of Bovine Manure on Cryptosporidium parvum Oocyst Attachment to Soil." Applied and Environmental Microbiology 71, no. 10 (October 2005): 6394–97. http://dx.doi.org/10.1128/aem.71.10.6394-6397.2005.
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ABSTRACT The objective of this work was to assess the effect of dilute bovine manure (1.0% and 0.1%) versus that of no manure on attachment and subsequent detachment of Cryptosporidium parvum oocysts to soil. Manure enhanced the attachment of oocysts to soil particles; the maximum attachment was observed with 0.1% manure. Oocyst attachment was partially reversible; maximum detachment was observed with dilute manure. These results indicate that oocyst attachment to soil is substantially affected by bovine manure in a complex manner and should have implications for how oocysts may be transported through or over soils.
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Chauret, Christian, Kerry Nolan, Ping Chen, Susan Springthorpe, and Syed Sattar. "Aging of Cryptosporidium parvum oocysts in river water and their susceptibility to disinfection by chlorine and monochloramine." Canadian Journal of Microbiology 44, no. 12 (December 1, 1998): 1154–60. http://dx.doi.org/10.1139/w98-113.
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Cryptosporidium parvum oocysts were aged in waters from both the St. Lawrence River and the Ottawa River. In situ survival experiments were carried out by incubating the oocysts in either dialysis cassettes or microtubes floated into an overflow tank. A significant portion of the oocysts survived in the test waters for several weeks. Oocyst survival in the St. Lawrence River was better in membrane-filtered (0.2-µm-pore diameter) water than in unfiltered water, suggesting that biological antagonism may play a role in the environmental fate of the parasite. Oocysts aged in river waters under in situ conditions and control oocysts kept refrigerated in synthetic water (100 ppm as CaCO3; pH 7.0) were subjected to the same disinfection protocol. Aged oocysts were at least as resistant as, if not more resistant than, the control oocysts to disinfection. This indicates that the oocysts surviving in the water environment may be just as difficult to inactivate by potable water disinfection as freshly shed oocysts. Therefore, water treatment should not be based on the assumption that environmental oocysts may be more easily inactivated than freshly shed oocysts. First-order kinetics die-off rates varied from one river to another (from 0.013 to 0.039 log10·day-1) and from one experiment to another with water from the same river collected at different times. Calculation of the die-off rates based on either in vitro excystation or in vitro excystation in combination with total counts (overall die-off rates) showed that the assessment of oocyst viability by microscopic methods must account for the total oocyst loss observed during long-term inactivation assays of river waters.Key words: Cryptosporidium, survival, disinfection, biological antagonism.
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KNIEL, K. E., and M. C. JENKINS. "Detection of Cryptosporidium parvum Oocysts on Fresh Vegetables and Herbs Using Antibodies Specific for a Cryptosporidium parvum Viral Antigen." Journal of Food Protection 68, no. 5 (May 1, 2005): 1093–96. http://dx.doi.org/10.4315/0362-028x-68.5.1093.
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The purpose of this study was to determine if the viral symbiont of Cryptosporidium parvum (CPV) sporozoites could be used as a target for sensitive detection of the parasite in food samples. Polyclonal sera specific to the recombinant viral capsid protein (rCPV40) was used in a dot blot hybridization assay to detect oocysts recovered from green onions and cilantro. Small batches of chopped green onions and cilantro leaves were artificially contaminated with three different concentrations of oocysts: 106, 102, and 101. rCPV40 was superior in detecting oocysts compared with other antibodies directed toward total oocyst protein and oocyst surface antigens. This study provides evidence that CPV is an excellent target for sensitive detection of C. parvum oocysts in foods.
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Pezzana, A., Ph Vilaginès, F. Bordet, D. Coquard, B. Sarrette, and R. Vilaginès. "Optimization of the Envirochek capsule method and immunomagnetic separation procedure for the detection of low levels of Cryptosporidium in large drinking water samples." Water Science and Technology 41, no. 7 (April 1, 2000): 111–17. http://dx.doi.org/10.2166/wst.2000.0122.
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The method for concentration of Cryptosporidium oocysts in large drinking water samples using the Envirocheck capsule has been optimized for the detection of low levels of oocysts. Elution from the filter by contact time and vortex agitation gave 68% oocyst recovery. Centrifugation (1,250 g; 30 min; 4°C) improved recovery to 94% without morphological damage of the oocysts. Increasing the ratio of magnetic beads to sample volume in the IMS procedure led to 69% efficiency. In these conditions, the overall recovery of the procedure was 49% as assessed with low oocysts spike doses in 100 litres tap water samples. The methodology described allows the detection of 0.1 oocyst per litre when 100 litres samples are processed.
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Jenkins, M. B., M. J. Walker, D. D. Bowman, L. C. Anthony, and W. C. Ghiorse. "Use of a Sentinel System for Field Measurements ofCryptosporidium parvum Oocyst Inactivation in Soil and Animal Waste." Applied and Environmental Microbiology 65, no. 5 (May 1, 1999): 1998–2005. http://dx.doi.org/10.1128/aem.65.5.1998-2005.1999.
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ABSTRACT A small-volume sentinel chamber was developed to assess the effects of environmental stresses on survival of sucrose-Percoll-purifiedCryptosporidium parvum oocysts in soil and animal wastes. Chambers were tested for their ability to equilibrate with external chemical and moisture conditions. Sentinel oocysts were then exposed to stresses of the external environment that affected their viability (potential infectivity), as indicated by results of a dye permeability assay. Preliminary laboratory experiments indicated that temperatures between 35 and 50°C and decreases in soil water potential (−0.003 to −3.20 MPa) increased oocyst inactivation rates. The effects of two common animal waste management practices on oocyst survival were investigated on three dairy farms in Delaware County, N.Y., within the New York City watershed: (i) piling wastes from dairy youngstock (including neonatal calves) and (ii) spreading wastes as a soil amendment on an agricultural field. Sentinel containers filled with air-dried and sieved (2-mm mesh) youngstock waste or field soil were wetted and inoculated with 2 million oocysts in an aqueous suspension and then placed in waste piles on two different farms and in soil within a cropped field on one farm. Controls consisted of purified oocysts in either phosphate-buffered saline or distilled water contained in sealed microcentrifuge tubes. Two microdata loggers recorded the ambient temperature at each field site. Sentinel experiments were conducted during the fall and winter (1996 to 1997) and winter (1998). Sentinel containers and controls were removed at 2- to 4-week intervals, and oocysts were extracted and tested by the dye permeability assay. The proportions of potentially infective oocysts exposed to the soil and waste pile material decreased more rapidly than their counterpart controls exposed to buffer or water, indicating that factors other than temperature affected oocyst inactivation in the waste piles and soil. The effect of soil freeze-thaw cycles was evident in the large proportion of empty sentinel oocysts. The potentially infective sentinel oocysts were reduced to <1% while the proportions in controls did not decrease below 50% potentially infective during the first field experiment. Microscopic observations of empty oocyst fragments indicated that abrasive effects of soil particles were a factor in oocyst inactivation. A similar pattern was observed in a second field experiment at the same site.
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Sekikawa, Takahiro. "A new immunomagnetic bead separation–surfactant extraction treatment protocol for rapid and sensitive quantitative PCR detection of Cryptosporidium parvum DNA." Water Supply 17, no. 1 (July 27, 2016): 161–68. http://dx.doi.org/10.2166/ws.2016.125.
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The Cryptosporidium oocyst is encased in a robust wall that is extremely resistant to detrimental environmental factors such as chlorine used to disinfect potable water. Therefore, extracting oocyst DNA is not a trivial undertaking. Standard procedures used to extract DNA from oocysts, such as freeze–thaw (F/T) methods and DNA purification kits, are time-consuming and expensive and are difficult to implement in routine clinical practice. Therefore, we developed a surfactant extraction treatment (SET) that efficiently extracts DNA from the oocyst. Immunomagnetic separation (IMS) combined with quantitative real-time polymerase chain reaction (qPCR) detects pathogenic microorganisms with high sensitivity. The objective of the present study was to evaluate SET for its ability to simplify qPCR detection of 18S rDNA directly from immunomagnetic bead–oocyst conjugates. DNA extracted directly from the conjugates using SET did not affect DNA amplification in the qPCR assay. Further, the rate of DNA amplification using IMS–SET was greater than that using F/T combined with the DNA purification kit. The rate of recovery of oocysts from surface water samples spiked with oocysts did not differ significantly from previously published values. These data demonstrate that the new IMS–SET protocol using qPCR can simplify the recovery and detection of Cryptosporidium oocysts.
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Smith, H. V., B. M. Campbell, C. A. Paton, and R. A. B. Nichols. "Significance of Enhanced Morphological Detection of Cryptosporidium sp. Oocysts in Water Concentrates Determined by Using 4′,6′-Diamidino-2-Phenylindole and Immunofluorescence Microscopy." Applied and Environmental Microbiology 68, no. 10 (October 2002): 5198–201. http://dx.doi.org/10.1128/aem.68.10.5198-5201.2002.
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ABSTRACT Of 2,361 water concentrates analyzed for the presence of Cryptosporidium spp. oocysts between January 1992 and May 1998, 269 (11.4%) were positive, of which 235 (87.4%) were raw and 34 were final water concentrates. Of 740 oocysts enumerated in positive samples, 656 oocysts (88.7%) were detected in raw and 84 oocysts (11.3%) were detected in final water concentrates by using a commercially available fluorescein isothiocyanate-labeled anti-Cryptosporidium sp. monoclonal antibody and the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Of raw water positive samples, 66.8% had oocysts that contained nuclei, while 58.8% of final water samples had oocysts that contained nuclei. The most frequently identified oocysts had either no DAPI-positive nuclei and no internal morphology according to Nomarski differential interference-contrast microscopy (DIC) or four DAPI-positive nuclei together with internal contents according to DIC (39.5 and 32.8% of raw and 42.9 and 30.9% of final water positives, respectively). By use of the presence of DAPI-stained nuclei to support oocyst identification based upon oocyst wall fluorescence, 56.5% of oocysts were identified when at least one nucleus was present, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 32.8% in raw water concentrates. In final water concentrates, 51% of oocysts were identified using oocyst wall fluorescence and the presence of at least one nucleus, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 30.9%. By consolidating our identification criteria from the presence of at least one nucleus to the presence of four nuclei, we excluded approximately 20% of oocysts in either water type. Approximately 40% of oocysts detected in these United Kingdom samples were empty and could not be detected by alternative methods, including the PCR and fluorescence in situ hybridization.
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Delaunay, Agnès, Gilles Gargala, Xunde Li, Loic Favennec, and Jean Jacques Ballet. "Quantitative Flow Cytometric Evaluation of MaximalCryptosporidium parvum Oocyst Infectivity in a Neonate Mouse Model." Applied and Environmental Microbiology 66, no. 10 (October 1, 2000): 4315–17. http://dx.doi.org/10.1128/aem.66.10.4315-4317.2000.
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ABSTRACT The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.
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Slifko, Theresa R., Debra E. Huffman, and Joan B. Rose. "A Most-Probable-Number Assay for Enumeration of Infectious Cryptosporidium parvum Oocysts." Applied and Environmental Microbiology 65, no. 9 (September 1, 1999): 3936–41. http://dx.doi.org/10.1128/aem.65.9.3936-3941.1999.
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ABSTRACT Cryptosporidium is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20). A two-tailed Pvalue (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log10 inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.
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Machado, E. C. L., T. L. M. Stamford, L. C. Alves, R. G. Melo, and N. K. S. Shinohara. "Effectiveness of Cryptosporidium spp. oocysts detection and enumeration methods in water and milk samples." Arquivo Brasileiro de Medicina Veterinária e Zootecnia 58, no. 3 (June 2006): 432–39. http://dx.doi.org/10.1590/s0102-09352006000300023.
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Cryptosporidium spp. oocyst recovery in water and milk samples was evaluated. Samples were inoculated with a suspension of 1.2×10(7) Cryptosporidium spp. oocysts and submitted to centrifugal flotation, using different solutions (sucrose, NaCl, MgSO4, ZnSO4, AlSO4, NH4SO4 40% and NH4SO4 80%). Centrifugation of the samples was carried out in two stages for concentration using two methods that differed in the order in which the saturated solutions were used, namely only in the first stage of method I and only in the second stage of method II. Oocyst identification was performed using the Kinyoun and Koster histochemical staining techniques. Samples analyzed by method I showed different degree of oocyst recovery, namely 10.9% with NaCl and 42.5% with MgSO4 in water and milk samples, while those samples analyzed by method II showed 10.6% with NaCl and 5.3% with sucrose in water and milk, respectively. Histochemical staining methods have no influence on the degree of oocysts recovery. The efficiency of Cryptosporidium spp. oocysts recovery methods depends on the nature and composition of the sample and on the methodology used for oocyst concentration.
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Atwill, E. R., B. Hoar, M. das Graças Cabral Pereira, K. W. Tate, F. Rulofson, and G. Nader. "Improved Quantitative Estimates of Low Environmental Loading and Sporadic Periparturient Shedding of Cryptosporidium parvum in Adult Beef Cattle." Applied and Environmental Microbiology 69, no. 8 (August 2003): 4604–10. http://dx.doi.org/10.1128/aem.69.8.4604-4610.2003.
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ABSTRACT Our primary goal was to generate an accurate estimate of the daily environmental loading rate of Cryptosporidium parvum oocysts for adult beef cattle, using immunomagnetic separation coupled with direct immunofluorescence microscopy for a highly sensitive diagnostic assay. An additional goal was to measure the prevalence and intensity of fecal shedding of C. parvum oocysts in pre- and postparturient cows as an indicator of their potential to infect young calves. This diagnostic method could detect with a ≥90% probability oocyst concentrations as low as 3.2 oocysts g of feces−1, with a 54% probability of detecting just one oocyst g of feces−1. Using this diagnostic method, the overall apparent prevalence of adult beef cattle testing positive for C. parvum was 7.1% (17 of 240), with 8.3 and 5.8% of cattle shedding oocysts during the pre- and postcalving periods, respectively. The mean intensity of oocyst shedding for test-positive cattle was 3.38 oocysts g of feces−1. The estimated environmental loading rate of C. parvum ranged from 3,900 to 9,200 oocysts cow−1 day−1, which is substantially less than a previous estimate of 1.7 × 105 oocysts cow−1 day−1 (range of 7.7 × 104 to 2.3 × 105 oocysts cow−1 day−1) (B. Hoar, E. R. Atwill, and T. B. Farver, Quant. Microbiol. 2:21-36, 2000). Use of this highly sensitive assay functioned to detect a greater proportion of low-intensity shedders in our population of cattle, which reduced the estimated mean intensity of shedding and thereby reduced the associated environmental loading rate compared to those of previous studies.
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King, Brendon J., Alexandra R. Keegan, Paul T. Monis, and Christopher P. Saint. "Environmental Temperature Controls Cryptosporidium Oocyst Metabolic Rate and Associated Retention of Infectivity." Applied and Environmental Microbiology 71, no. 7 (July 2005): 3848–57. http://dx.doi.org/10.1128/aem.71.7.3848-3857.2005.
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ABSTRACT Cryptosporidium is a significant cause of water-borne enteric disease throughout the world and represents a challenge to the water industry and a threat to public health. In this study we report the use of a cell culture-TaqMan PCR assay to measure oocyst inactivation rates in reagent-grade and environmental waters over a range of temperatures. While oocysts incubated at 4°C and 15°C remained infective over the 12-week holding period, we observed a 4 log10 reduction in infectivity for both 20 and 25°C incubation treatments at 12 and 8 weeks, respectively, for all water types examined, a faster rate of inactivation for oocysts than previously reported. This temperature-dependent inactivation was further investigated using a simple and rapid ATP assay described herein. Time course experiments performed in reagent-grade water at incubation temperatures of 4, 15, 20, 25, 30, and 37°C identified a close relationship between oocyst infectivity and oocyst ATP content, demonstrating that temperature inactivation at higher temperatures is a function of increased oocyst metabolic activity. While water quality did not affect oocyst inactivation, biological antagonism appears to be a key factor affecting oocyst removal from environmental waters. Both the cell culture-TaqMan PCR assay and the ATP assay provide a sensitive and quantitative method for the determination of environmental oocyst inactivation, providing an alternative to the more costly and time-consuming mouse infection assay. The findings presented here relating temperature to oocyst inactivation provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in water.
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Bumstead, N., and B. J. Millard. "Variation in susceptibility of inbred lines of chickens to seven species ofEimeria." Parasitology 104, no. 3 (June 1992): 407–13. http://dx.doi.org/10.1017/s0031182000063654.
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The pattern of oocyst production of 8 inbred lines of chickens was compared for each of the 7 species ofEimeriawhich infect this host. Both the overall numbers and the pattern of oocyst production differed in the inbred lines, but there was no evidence of prolonged cycling of schizogenic developmental stages. Comparison of the numbers of oocysts produced by the different lines indicates that there may be common genetic factors affecting susceptibility to 6 of the 7 species. Surprisingly there appears to be an inverse relationship between susceptibility toE. tenellaand susceptibility to the other species: lines which produced most oocysts ofE. tenellaproduced least oocysts of the other species andvice-versa.
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Al-Adhami, B. H., R. A. B. Nichols, J. R. Kusel, J. O'Grady, and H. V. Smith. "Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy." Applied and Environmental Microbiology 73, no. 3 (September 29, 2006): 947–55. http://dx.doi.org/10.1128/aem.01251-06.
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ABSTRACT 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.
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Hirata, T., and A. Hashimoto. "Experimental assessment of the efficacy of microfiltration and ultrafiltration for Cryptosporidium removal." Water Science and Technology 38, no. 12 (December 1, 1998): 103–7. http://dx.doi.org/10.2166/wst.1998.0515.
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In order to evaluate the efficacy of microfiltration and ultrafiltration for Cryptosporidium oocyst removal, a bench-scale experiment was carried out using two 0.2m2 molecules, MF (nominal pore size 0.25μm) and UF (nominal cut-off MW 13,000 daltons) in cross-flow mode at an oocyst level of 106/L. Both of the membranes eliminated the oocysts from the influents with removal efficiency estimated to be >7 log10. As for the MF, an additional experiment was conducted at a much higher oocyst level up to 108 oocysts/L in both cross-flow and dead-end modes and which achieved >7 log10 removal, although some oocysts appeared in the filtrate in both modes. Based on these results, microfiltration and ultrafiltration are conclusively considered to be excellent processes for drinking water treatment as a single process that produces safe (an annual risk 10−4) water from highly polluted source waters.
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FRÖLICH, SONJA, and MICHAEL WALLACH. "F-actin distribution and function during sexual development in Eimeria maxima." Parasitology 142, no. 7 (March 24, 2015): 855–64. http://dx.doi.org/10.1017/s0031182015000207.
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SUMMARYTo determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling ‘beads on a string’. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.
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Upton, Steve J., and Chris T. McAllister. "The Coccidia (Apicomplexa: Eimeriidae) of Anura, with descriptions of four new species." Canadian Journal of Zoology 66, no. 8 (August 1, 1988): 1822–30. http://dx.doi.org/10.1139/z88-263.
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Four new species of Coccidia (Apicomplexa: Eimeriidae) are described from anurans from Texas and Arkansas, U.S.A. Oocysts of Eimeria flexuosa sp.n. were found in Pseudacris streckeri streckeri and are irregular in shape, long axis 17.0 (15.2–19.2) μm (mean, range), with a thin, flexible wall. Micropyle and oocyst residuum absent; polar granule present. Sporocysts ovoid, 10.3 × 7.3 (9.6–12.0 × 6.4–8.0) μm, with Stieda body and sporocyst residuum. Oocysts of Eimeria streckeri sp.n. were also found in P. s. streckeri and are spherical or subspherical, 18.8 × 18.7(16.8–21.5 × 16.8–20.8) μm, with a thin wall. Micropyle and polar granule absent; oocyst residuum present. Sporocysts ovoid, 11.1 × 7.7 (9.6–12.8 × 7.2–8.8) μm, with Stieda body and sporocyst residuum. Oocysts of Isospora delicatus sp.n. were found in P. s. streckeri and Pseudacris streckeri illinoensis and are spherical or subspherical, 15.8 × 15.7(12.8–16.8 × 12.8–16.8) μm, and have a thin wall. Micropyle, oocyst residuum, and polar granule absent. Sporocysts ovoid, 13.5 × 8.0(11.2–14.8 × 7.2–9.6) μm, with Stieda body and diffuse sporocyst residuum. Oocysts of Isospora fragosum sp.n. were recovered from Gastrophryne olivacea and are spherical, 18.5 (16.8–20.8) μm, and have a thin wall that ruptures easily. Micropyle, polar granule, and oocyst residuum absent. Sporocysts ovoid, 12.7 × 10.9 (11.2–14.4 × 9.6–12.0) μm, lacking Stieda and substieda bodies; large sporocyst residuum present.
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Kaucner, C., C. M. Davies, C. M. Ferguson, and N. J. Ashbolt. "Evidence for the existence of Cryptosporidium oocysts as single entities in surface runoff." Water Science and Technology 52, no. 8 (October 1, 2005): 199–204. http://dx.doi.org/10.2166/wst.2005.0264.
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There is uncertainty whether Cryptosporidium oocysts attach to particles or to each other under ambient water conditions. Particle size distributions of Cryptosporidium oocyst suspensions were determined over a range of ionic strengths and pHs to determine under those environmental conditions that may promote oocyst aggregation. Cryptosporidium oocysts were shown to only aggregate in high ionic strength solutions (>0.45 M) and remain largely as single entities at ionic strengths and pHs that were likely to be encountered in surface runoff. Similarly, in loam soil suspensions, rather than attaching to the soil particles the majority of oocysts also remained as single entities. Overall, oocysts are expected to remain largely unattached to either themselves or soil particles in overland runoff. This has implications for pathogen transport and modelling since oocysts that are freely suspended are more likely to be transported in runoff to surface waters than if attached to more dense soil/faecal particles.
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Sinclair, James L. "Enumeration of Cryptosporidium spp. in Water with U.S. EPA Method 1622." Journal of AOAC INTERNATIONAL 83, no. 5 (September 1, 2000): 1108–14. http://dx.doi.org/10.1093/jaoac/83.5.1108.
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Abstract The occurrence of Cryptosporidium parvum or other pathogenic Cryptosporidium species in water must be known in order to assess risk and determine the treatment needed to reduce Cryptosporidium oocysts to acceptable levels in finished drinking water. Because Cryptosporidium oocyst occurrence may be sparse, methods must concentrate a large volume of water and correctly identify oocysts in the concentrate. The U.S. Environmental Protection Agency Information Collection Rule (ICR) protozoan method gives low and variable recoveries of Cryptosporidium oocysts, making risk assessment difficult. Therefore, a method giving better oocyst recovery and more consistent results was needed. Method 1622 was developed with existing materials and procedures, and improvements were made in filtration, cleanup, and detection. Absolute porosity filters were used, with cleanup by immunomagnetic separation and detection by direct fluorescent antibody stain with 4′,6-diamidino-2-phenylindole (DAPI) staining for additional cell structures. Both the level and consistency of oocyst recovery were improved compared to recovery with the ICR method.
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Medema, G. J., M. Bahar, and F. M. Schets. "Survival of cryptosporidium parvum, escherichia coli, faecal enterococci and clostridium perfringens in river water: influence of temperature and autochthonous microorganisms." Water Science and Technology 35, no. 11-12 (June 1, 1997): 249–52. http://dx.doi.org/10.2166/wst.1997.0742.
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Oocysts of Cryptosporidium parvum can survive for several months in surface water, one of the main factors determining their success in environmental transmission and thus their health hazard via water. Several factors in the environment, e.g. temperature, presence of predators and exo-enzymes will probably influence oocyst survival. The high persistence of oocysts may also limit the value of traditional faecal indicator bacteria. The aim of this study was to determine the rate at which C parvum oocysts, E coli, faecal enterococci and C perfringens spores die in surface water and the influence of temperature and the presence of autochthonous (micro)organisms on the die-off rate. Microcosms with autoclaved river water were inoculated with the organisms. Microcosms with untreated river water were inoculated with concentrated primary effluent containing the bacteria and with C parvum oocysts. Microcosms were incubated at 5°C or 15°C at 100rpm. Viability of oocysts was monitored by in vitro excystation and dye-exclusion; viability of the bacteria was determined on appropriate selective media. When pseudo first-order die-off kinetics were assumed, the die-off rate of oocysts at 5°C was 0.010 log10/d and at 15°C, 0.006–0.024 log10/d. These rates underestimate die-off since oocyst disintegration was not accounted for. Incubation in autoclaved or untreated water did influence the die-off rate of oocysts at 15°C but not at 5°C. The die-off rate of E coli and enterococci was faster in the non-sterile river water than in autoclaved water at both temperatures. At 15°C, E coli (and possibly E faecium) even multiplied in autoclaved water. In untreated river water, the die-off of E coli and enterococci was approximately 10x faster than die-off of oocysts but die-off rates of C perfringens were lower than those of oocysts. As for oocysts, die-off of the bacteria and spores was faster at 15°C than at 5°C. Oocysts are very persistent in river water: the time required for a 10x reduction in viability being 40–160d at 15°C and 100d at 5°C. Biological/biochemical activity influenced oocyst survival at 15°C and survival of both vegetative bacteria at 5 and 15°C. The rapid die-off of E coli and enterococci makes them less suitable as indicators of oocyst presence in water. As C perfringens survived longer in untreated river water than oocysts, it may prove useful as an indicator of the presence of C parvum.
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Kim, H. S., Y. Kobayashi, M. Akiba, and S. Kunikane. "Evaluation of Scenedesmus quadricauda as a surrogate of Cryptosporidium oocysts removal in direct filtration." Water Supply 2, no. 5-6 (December 1, 2002): 395–402. http://dx.doi.org/10.2166/ws.2002.0196.
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A comparative study on the evaluation of a Cryptosporidium removal surrogate was conducted using Scenedesmus quadricauda, green algae. Bench-scale direct filtration experiments were carried out at various initial concentrations: 500-5,000 oocysts/ml for C. parvum oocysts and 500-10,000 cells/ml for S. quadricauda. From the results, algal cell or Cryptosporidium oocyst counts of the filtrates (C) increased in proportion to their initial concentrations (C0). However, no significant differences in C/C0 profiles were observed over the examined range of the initial concentration, which implied that the removal efficiencies for S. quadricauda cells and C. parvum oocysts were not related to the initial concentrations. Examination of the deposition in the sand filter showed that a large part of S. quadricauda cells or C. parvum oocyst counts were captured in the upper layer of the sand filter, and the deposition rates were gradually reduced along the filter depth. Total cell or oocysts counts deposited in the sand filter increased commensurate with the initial concentration for both microorganisms. The ratio of the deposited cell or oocyst counts to the deposited amounts of flocs showed quite similar values between S. quadricauda and C. parvum, which meant that these two microorganisms were alike in their removal behavior. From these similar characteristics of removal, S. quadricauda was thought to be a reasonable and reliable surrogate of C. parvum oocysts removal in sand filtration.
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Kuczynska, Ewa, and Daniel R. Shelton. "Method for Detection and Enumeration ofCryptosporidium parvum Oocysts in Feces, Manures, and Soils." Applied and Environmental Microbiology 65, no. 7 (July 1, 1999): 2820–26. http://dx.doi.org/10.1128/aem.65.7.2820-2826.1999.
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ABSTRACT Eight concentration and purification methods were evaluated to determine percentages of recovery of Cryptosporidium parvumoocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 102, 103, 104, and 105oocysts g of bovine feces−1. The percentages of recovery ranged from 10.8% (25 oocysts g−1) to 17.0% (104 oocysts g−1) (r 2 = 0.996). A conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces−1. Percentages of recovery were determined for six different types of animal feces (cow, horse, pig, sheep, deer, and chicken feces) at a single oocyst concentration (104 oocysts g−1). The percentages of recovery were highest for bovine feces (17.0%) and lowest for chicken feces (3.2%). Percentages of recovery were determined for bovine manure after 3 to 7 days of storage. The percentages of recovery ranged from 1.9 to 3.5% depending on the oocyst concentration, the time of storage, and the dispersing solution. The percentages of oocyst recovery from soils were evaluated by using different flotation solutions (NaCl, cold sucrose, ZnSO4), different dispersing solutions (Triton X-100, Tween 80, Tris plus Tween 80), different dispersion techniques (magnetic stirring, sonication, blending), and different dispersion times (5, 15, and 30 min). Twenty-five-gram soil samples were used to reduce the spatial variability. The highest percentages of recovery were obtained when we used 50 mM Tris–0.5% Tween 80 as the dispersing solution, dispersion for 15 min by stirring, and saturated NaCl as the flotation solution. The percentages of oocyst recovery from freshly spiked sandy loam, silty clay loam, and clay loam soils were ca. 12 to 18, 8, and 6%, respectively. The theoretical detection limits were ca. 1 to 2 oocysts g of soil−1 depending on the soil type. The percentages of recovery without dispersant (distilled H2O or phosphate-buffered saline) were less than 0.1%, which indicated that oocysts adhere to soil particles. The percentages of recovery decreased with storage time, although the addition of dispersant (Tris-Tween 80) before storage appeared to partially prevent adhesion. These data indicate that the NaCl flotation method is suitable for routine detection and enumeration of oocysts from feces, manures, soils, or soil-manure mixtures.
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Lee, Lai Yoke, Say Leong Ong, Jiang Yong Hu, Wun Jern Ng, Yaoyu Feng, Xiaolan Tan, and Shih Wei Wong. "Use of Semiconductor Quantum Dots for Photostable Immunofluorescence Labeling of Cryptosporidium parvum." Applied and Environmental Microbiology 70, no. 10 (October 2004): 5732–36. http://dx.doi.org/10.1128/aem.70.10.5732-5736.2004.
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ABSTRACT Cryptosporidium parvum is a waterborne pathogen that poses potential risk to drinking water consumers. The detection of Cryptosporidium oocysts, its transmissive stage, is used in the latest U.S. Environmental Protection Agency method 1622, which utilizes organic fluorophores such as fluorescein isothiocyanate (FITC) to label the oocysts by conjugation with anti-Cryptosporidium sp. monoclonal antibody (MAb). However, FITC exhibits low resistance to photodegradation. This property will inevitably limit the detection accuracy after a short period of continuous illumination. In view of this, the use of inorganic fluorophores, such as quantum dot (QD), which has a high photobleaching threshold, in place of the organic fluorophores could potentially enhance oocyst detection. In this study, QD605-streptavidin together with biotinylated MAb was used for C. parvum oocyst detection. The C. parvum oocyst detection sensitivity increased when the QD605-streptavidin concentration was increased from 5 to 15 nM and eventually leveled off at a saturation concentration of 20 nM and above. The minimum QD605-streptavidin saturation concentration for detecting up to 4,495 ± 501 oocysts (mean ± standard deviation) was determined to be 20 nM. The difference in the enumeration between 20 nM QD605-streptavidin with biotinylated MAb and FITC-MAb was insignificant (P > 0.126) when various C. parvum oocyst concentrations were used. The QD605 was highly photostable while the FITC intensity decreased to 19.5% ± 5.6% of its initial intensity after 5 min of continuous illumination. The QD605-based technique was also shown to be sensitive for oocyst detection in reservoir water. This observation showed that the QD method developed in this study was able to provide a sensitive technique for detecting C. parvum oocysts with the advantage of having a high photobleaching threshold.
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KNIEL, KALMIA E., ADRIENNE E. H. SHEARER, JENNIFER L. CASCARINO, GARY C. WILKINS, and MARK C. JENKINS. "High Hydrostatic Pressure and UV Light Treatment of Produce Contaminated with Eimeria acervulina as a Cyclospora cayetanensis Surrogate." Journal of Food Protection 70, no. 12 (December 1, 2007): 2837–42. http://dx.doi.org/10.4315/0362-028x-70.12.2837.
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The prevalence, size, genome, and life cycle of Eimeria acervulina make this organism a good surrogate for Cyclospora cayetanensis, a protozoan that causes gastroenteritis in humans, including recent outbreaks in the United States and Canada associated with contaminated raspberries and basil. Laboratory studies of C. cayetanensis are difficult because of the lack of readily available oocysts and of infection models and assays. UV radiation and high-hydrostatic-pressure processing (HPP) are both safe technologies with potential for use on fresh produce. Raspberries and basil were inoculated with sporulated E. acervulina oocysts at high (106 oocysts) and low (104 oocysts) levels, and inoculated and control produce were treated with UV (up to 261 mW/cm2) or HPP (550 MPa at 40°C for 2 min). Oocysts recovered from produce were fed to 3-week-old broiler chickens, which were scored for weight gain, oocyst shedding, and lesions at 6 days postinoculation. Oocysts exhibited enhanced excystation on raspberries but not on basil. Birds fed oocysts from UV-treated raspberries had reduced infection rates, which varied with oocyst inoculum level and UV intensity. Birds fed oocysts from UV-treated raspberries (104 oocysts) were asymptomatic but shed oocysts, and birds fed oocysts from UV-treated basil (104 oocysts) were asymptomatic and did not shed oocysts. Birds fed oocysts from HPP-treated raspberries and basil were asymptomatic and did not shed oocysts. These results suggest that UV radiation and HPP may be used to reduce the risk for cyclosporiasis infection associated with produce. Both treatments yielded healthy animals; however, HPP was more effective, as indicated by results for produce with higher contamination levels.
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Casteel, M. J., M. D. Sobsey, and M. J. Arrowood. "Inactivation of Cryptosporidium parvum oocysts and other microbes in water and wastewater by electrochemically generated mixed oxidants." Water Science and Technology 41, no. 7 (April 1, 2000): 127–34. http://dx.doi.org/10.2166/wst.2000.0124.
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Alternative disinfectants of water and wastewater are needed because conventional chlorination is ineffective against C. parvum oocysts. Reliable indicators of disinfection efficacy against C. parvum also are needed. Mixedoxidants (MO) electrochemically generated from brine were evaluated in batch disinfection experiments for inactivation of C. parvum oocysts and Cl. perfringensspores in both oxidant demand-free (ODF) water and treated wastewater. Coliphage MS2 and Escherichia coli B were also tested under some conditions. C. parvum oocyst infectivity was quantified by cell culture assay, and the dyes DAPI (4′,6-diamidino-2-phenylindole) and propidium iodide (PI) were used to assess oocyst viability in wastewater experiments. In treated wastewater dosed with 10–13 mg/L MO, inactivation after 90 minutes was about 3 log10 for C. parvum and about 2.5 log10 for Cl. perfringens spores; MS2 and E. coli were rapidly inactivated by > 5 log10. In ODF water, a 4 mg/L dose of MO inactivated ∼3 log10 of C. parvum oocysts and ∼1.5 log10 of Cl. perfringens spores. Inactivation of C. parvum oocysts and Cl. perfringensspores was less extensive at a lower MO dose of 2 mg/L. The use of DAPI and PI to determine viability of oocysts treated with MO did not correlate with, and greatly overestimated, cell culture infectivity. At practical doses and contact times, MO disinfection of water and wastewater achieves appreciable inactivation of both C. parvum oocysts and Cl. perfringens spores. Cl. perfringens spores reliably indicated oocyst inactivation by MO, but E. coli and coliphage MS2 were inactivated much too rapidly to indicate C. parvum inactivation.
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Quilez, Joaquin, Caridad Sanchez-Acedo, Catalina Avendaño, Emilio del Cacho, and Fernando Lopez-Bernad. "Efficacy of Two Peroxygen-Based Disinfectants for Inactivation of Cryptosporidium parvum Oocysts." Applied and Environmental Microbiology 71, no. 5 (May 2005): 2479–83. http://dx.doi.org/10.1128/aem.71.5.2479-2483.2005.
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ABSTRACT Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4′,6′-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.
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Graczyk, Thaddeus K., Ronald Fayer, Michael R. Cranfield, and David Bruce Conn. "Recovery of Waterborne Cryptosporidium parvum Oocysts by Freshwater Benthic Clams (Corbicula fluminea)." Applied and Environmental Microbiology 64, no. 2 (February 1, 1998): 427–30. http://dx.doi.org/10.1128/aem.64.2.427-430.1998.
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ABSTRACT Asian freshwater clams, Corbicula fluminea, exposed for 24 h to 38 liters of water contaminated with infectiousCryptosporidium parvum oocysts (1.00 × 106 oocysts/liter; approximately 1.9 × 105 oocysts/clam) were examined (hemolymph, gills, gastrointestinal [GI] tract, and feces) on days 1, 2, 3, 7, and 14 postexposure (PE). No oocysts were detected in the water 24 h after the contamination event. The percentage of oocyst-containing clams varied from 20 to 100%, depending on the type of tissue examined and the technique used—acid-fast stain (AFS) or immunofluorescent antibody (IFA). The oocysts were found in clam tissues and feces on days 1 through 14 PE; the oocysts extracted from the tissues on day 7 PE were infectious for neonatal BALB/c mice. Overall, the highest number of positive samples was obtained when gills and GI tracts were processed with IFA (prevalence, 97.5%). A comparison of the relative oocyst numbers indicated that overall, 58.3% of the oocysts were found in clam tissues and 41.7% were found in feces when IFA was used; when AFS was used, the values were 51.9 and 48.1%, respectively. Clam-released oocysts were always surrounded by feces; no free oocysts or oocysts disassociated from fecal matter were observed. The results indicate that these benthic freshwater clams are capable of recovery and sedimentation of waterborne C. parvum oocysts. To optimize the detection of C. parvum oocysts in C. fluminea tissue, it is recommended that gill and GI tract samples be screened with IFA (such as that in the commercially available MERIFLUOR test kit).
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Chaidez, Cristobal, Marcela Soto, and Nohelia Castro-del Campo. "Effect of water suspended particles on the recovery of Cryptosporidium parvum from tomato surfaces." Journal of Water and Health 5, no. 4 (May 1, 2007): 625–31. http://dx.doi.org/10.2166/wh.2007.048.
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An increase in the number of outbreaks of foodborne disease associated with fresh produce consumption has been described. The objective of the present study was to evaluate the effect of water suspended particles during immersing/spraying disinfection processes and the recovery of Cryptosporidium parvum oocysts from tomato surfaces. Tomatoes (Lycopersicum esculetum Mill.) were immersed/sprayed with chlorinated water with low and high suspended particle content (10 and 1,000 mg/l) containing 100, 1,000 or 10,000 oocysts/l. Tomatoes were evaluated after a contact time of 120 seconds and 30 seconds for immersing and spraying procedures, respectively. The immersing procedure showed a high recovery of C. parvum oocysts from the tomato surface when the concentration was 10,000 oocysts/l and 10 mg/l suspended particles (295±94 [mean±standard deviation]). High particle content affected oocyst recovery and dissolved particles exerted a chlorine demand reducing the disinfectant residual. In the spraying procedure, the highest recovery was observed with 10,000 oocysts/l (225±72). Our understanding is that the association of C. parvum oocysts with suspended particles might promote the oocyst deposition in the wash water tanks and that this interaction should be considered when evaluating the quality of the water.
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Kong, Frederic E., Margaret A. Deighton, Nerida A. Thurbon, Stephen R. Smith, and Duncan A. Rouch. "Cryptosporidium parvum decay during air drying and stockpiling of mesophilic anaerobically digested sewage sludge in a simulation experiment and oocyst counts in sludge collected from operational treatment lagoons in Victoria, Australia." Journal of Water and Health 16, no. 3 (April 5, 2018): 435–48. http://dx.doi.org/10.2166/wh.2018.018.
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Abstract The inactivation of Cryptosporidium species oocysts during sewage sludge treatment is important to protect human health when the residual biosolids are applied to agricultural land. Quantifying the decay of Cryptosporidium species during sludge treatment for microbiological assurance purposes is difficult if low numbers are present in wastewater. The rate of decay of Cryptosporidium parvum oocysts during solar/air drying treatment and in sludge stockpiles in temperate environment conditions was simulated in laboratory inoculation experiments using sludge sampled from a mesophilic anaerobic digester. Oocyst numbers were also determined in settled lagoon sludge samples collected from three operational rural wastewater treatment plants (WWTPs). C. parvum oocysts were enumerated by immunomagnetic separation followed by staining with vital dyes and examination by confocal laser scanning microscopy. An air-drying/storage period equivalent to 11 weeks was required for a 1 log10 reduction of viable oocysts inoculated into digested sludge. Oocyst viability in air-dried and stored digested sludge decreased with time, but was independent of sludge desiccation and dry solids (DS) content. No oocysts were detected in sludge samples collected from the anaerobic digester, and the average concentration of oocysts found in settled lagoon sludge from the rural WWTP was 4.6 × 102 oocysts/g DS.