Spatial distributions of Cryptosporidium oocysts in porous media: evidence for dual mode deposition

Spatial distributions of Cryptosporidium parvum oocysts in columns packed with uniform glass-bead collectors were measured over a broad range of physicochemical conditions. Oocyst deposition behavior is shown to deviate from predictions based on classical colloid filtration theory (CFT) in the presence of repulsive (unfavorable) colloidal interactions. Specifically, CFT tends to predict greater removal of oocysts (less transport) than that observed in controlled laboratory experiments. Comparison of oocyst retention with results obtained using polystyrene latex particles of similar size suggests that mechanisms controlling particle deposition are the same in both systems. At a given ionic strength, the deposition of Cryptosporidium oocysts is generally greater than that of the microspheres; however, this discrepancy is partly attributable to large differences in oocyst and microsphere zeta potentials. A dual deposition mode (DDM) model is applied which considers the combined influence of "fast" and "slow" oocyst deposition due to the concurrent existence of favorable and unfavorable oocyst-collector interactions. Model simulations of retained oocyst profiles and suspended oocyst concentration at the column effluent are consistent with experimental data. Because classic CFT does not account for the effect of dual mode deposition (i.e., simultaneous "fast" and "slow" oocyst deposition), these observations have important implications for predictions of oocyst transport in subsurface environments, where repulsive electrostatic interactions predominate. Supporting elution experiments further suggest that specific surface interactions between oocyst wall macromolecules and the glass bead collectors could retard or even completely inhibit oocyst release upon perturbation in solution chemistry.     

Transport of Cryptosporidium oocysts in porous media: role of straining and physicochemical filtration

The transport and filtration behavior of Cryptosporidium parvum oocysts in columns packed with quartz sand was systematically examined under repulsive electrostatic conditions. An increase in solution ionic strength resulted in greater oocyst deposition rates despite theoretical predictions of a significant electrostatic energy barrier to deposition. Relatively high deposition rates obtained with both oocysts and polystyrene latex particles of comparable size at low ionic strength (1 mM) suggest that a physical mechanism may play a key role in oocyst removal. Supporting experiments conducted with latex particles of varying sizes, under very low ionic strength conditions where physicochemical filtration is negligible, clearly indicated that physical straining is an important capture mechanism. The results of this study indicate that irregularity of sand grain shape (verified by SEM imaging) contributes considerably to the straining potential of the porous medium. Hence, both straining and physicochemical filtration are expected to control the removal of C. parvum oocysts in settings typical of riverbank filtration, soil infiltration, and slow sand filtration. Because classic colloid filtration theory does not account for removal by straining, these observations have important implications with respect to predictions of oocyst transport.     

Deposition of Cryptosporidium parvum Oocysts in Porous Media: A Synthesis of Attachment Efficiencies Measured under Varying Environmental Conditions

An extensive set of column experiments was performed with freshly harvested Cryptosporidium parvum oocysts to evaluate the effects of solution chemistry, surface coatings, interactions with other suspended particles, and pore fluid velocity on the fate and transport of this widely occurring waterborne pathogen in sandy porous media. We synthesized our data set with a comprehensive literature survey of similar experiments, to compute attachment (collision) efficiencies (α) used in colloid filtration theory (CFT) using three models for the single collector efficiency (η) across a wide range of experimental conditions. Most prior experiments have observed the transport of surface-treated, sterile C. parvum oocyst in porous media. Our column data confirm for freshly harvested oocysts that the presence of iron coatings on the sand medium and the presence of suspended illite clay drastically enhance oocyst deposition. Increasing ionic strength and decreasing pH also systematically enhance the attachment efficiency. Attachment efficiency decreases only at a very high ionic strength, most likely as a result of steric repulsion and possibly other changes in oocyst surface properties. Attachment efficiencies vary with fluid flow rate but without showing specific trends. We found that the computed attachment efficiency across all reported experiments could be reliably estimated using a regression model based on parameters related to ionic strength and pH. The regression model performed better with the Nelson-Ginn η model and Tufenkji-Elimelech η model than with the Rajagopalan-Tien η model. When CFT is used in environmental assessments, the proposed regression model provides a practical estimator for attachment efficiencies of C. parvum oocyst deposition in porous media for a variety of environmental conditions unfavorable to attachment.     

Inactivation of Cryptosporidium parvum oocysts in cider by flash pasteurization

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 degrees 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 degrees 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.     

Bulk Optical Properties of Potato Flesh in the 500–1900 nm Range

In this study, the optical properties of potato flesh tissue were estimated using double-integrating sphere (DIS) measurements combined with an inverse adding-doubling (IAD) light propagation model. Total reflectance, total transmittance, and unscattered transmittance were measured for the wavelength range 500–1900 nm with 5-nm resolution. From these measurements, the bulk optical properties (absorption coefficient, scattering coefficient, and anisotropy factor) of 53 potato tubers of the Hermes cultivar were estimated. The estimated absorption coefficient spectra were dominated by water and starch absorption bands, the main chemical components of potato tissue. Comparison of these values to those reported in literature for similar products showed comparable absorption profiles. The obtained scattering coefficient spectra showed a smooth decrease from 166 to 160 cm^?1 in the near-infrared (NIR) spectral range with increasing wavelength, which is common for biological tissues. The anisotropy factor spectra obtained for the full wavelength range studied ranged between 0.949 and 0.959 with a maximum variability of 0.009 among the set of samples used. The information obtained in this study is essential to understand the effects of absorption and scattering on the propagation of light through the potato tubers in order to design more efficient sensors for non-destructive quality evaluation.     

Direct detection of Cryptosporidium parvum oocysts by immunomagnetic separation-polymerase chain reaction in raw milk

Cryptosporidium parvum is an emerging protozoan parasite responsible for several serious outbreaks of cryptosporidiosis, an enteric infection characterized by severe intestinal distress. This parasite can be transmitted through contaminated water and raw food in the oocyst form, which is resistant to many environmental stresses and food processes. C. parvum is also commonly found on dairy farms and could be transmitted to humans through contaminated raw milk and dairy products. Thus, an immunomagnetic separation-polymerase chain reaction assay for direct detection of C. parvum oocysts in milk was developed. The procedure was able to detect < 10 C. parvum oocysts. Thus, it could be used for monitoring milk samples.     

Artificial UV-B and solar radiation reduce in vitro infectivity of the human pathogen Cryptosporidium parvum

The potential for solar ultraviolet (UV) radiation to act as a significant abiotic control of Cryptosporidium parvum oocysts in nature is unknown. Infectivity of C. parvum following exposure to artificial UV-B and natural solar radiation, with and without UV wavelengths, was tested under controlled pH and temperature conditions. Percent infectivity of exposed oocysts was determined by in vitro cell culture. Artificial UV-B exposures of 32 and 66 kJ/m2 significantly decreased oocyst infectivity by an average of 58 and 98%, respectively. Exposure of oocysts to approximately half and full intensity of full solar spectrum (all wavelengths) for a period of less than 1 day (10 h) in mid-summer reduced mean infectivity by an average of 67% and >99.99%, respectively. Exposure of the C. parvum oocysts to UV-shielded solar radiation (>404 nm) in early autumn reduced mean infectivity by 52%, while full spectrum solar radiation (exposure at all wavelengths) reduced mean infectivity by 97%. The data provide strong evidence that exposure to natural solar radiation can significantly reduce C. parvum infectivity. Direct effects of solar radiation on oocysts in nature will depend on the depth distribution of the oocysts, water transparency, mixing conditions, and perhaps other environmental factors such as temperature, pH, and stress.     

Detection of Cryptosporidium parvum oocysts on fresh vegetables and 1 herbs using antibodies specific for a Cryptosporidium parvum viral antigen

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: 10(6), 10(2), and 10(1). 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.     

Optimization of DNA extraction and molecular detection of Cryptosporidium oocysts in natural mineral water sources

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 degrees 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 degrees 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.     

Evaluation of microspheres as surrogates for Cryptosporidium parvum oocysts in filtration experiments

The size and surface characteristics of a surrogate particle and Cryptosporidium parvum oocysts are important in determining the ability of the particle to mimic the behavior of C. parvum oocysts in filtration and particle transport experiments. The zeta potential, hydrophobicity, and filterability of a surrogate particle, 5 microm carboxylated latex microspheres, and oocysts were compared for a variety of solution conditions. C. pervum oocysts had a slightly negative zeta potential (-1.5 to -12.5 mV) at pH 6.7 over a wide range of calcium concentration (10(-6)-10(-1) M), while the fluorescent microspheres were more negatively charged under the same conditions (-7.4 to -50.2 mV). After exposure to 5 mg of C/L of Suwanee River natural organic matter (NOM), the ; potentials of both particles became significantly more negative, with the microspheres consistently maintaining a more negative zeta potential than the oocysts. Alum was able to neutralize the negative zeta potentials of both particles when in the presence of NOM, but nearly twice the dosage was required for the microspheres. NOM also affected the hydrophobicity of the particles by increasing the hydrophobicity of the relatively hydrophilic oocysts and decreasing the hydrophobicity of the relatively hydrophobic microspheres. A bench-scale filtration system removed less microspheres (40.3 +/- 1.5%) than oocysts (49.7 +/- 2.9%) when 0.01 M CaCl2 was supplied as coagulant. After preexposure to 5 mg of C/L of NOM, the removals of both particles declined significantly, and the removals of microspheres (13.7 +/- 1.5%) and oocysts (16.3 +/- 1.5%) were similar. Finally, the removal efficiencies of microspheres and oocysts in the presence of NOM increased to 69.3 +/- 3.5% and 67.7 +/- 6.4%, respectively, when alum was supplied as coagulant at the optimum dosage needed to destabilize the oocysts. These experimental results suggest that microspheres can be used to provide a conservative estimate of oocyst removal in filters containing hydrophilic negatively charged filter media.     

Simultaneous prediction of Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of synthetic waters

A model was developed to simultaneously assess Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of synthetic solutions in batch and flow-through reactors. The model incorporated 65 elementary chemical reactions involved in the decomposition of ozone and the oxidation of bromine species and their corresponding rate or equilibrium constants reported in the literature. Ozonation experiments were performed with a laboratory-scale batch reactor to evaluate the model with respect to the rate of ozone decomposition and bromate formation. The model was found to provide a good representation of experimental results when the ozone decomposition initiation reaction with hydroxide ion was assumed to produce superoxide radical instead of the alternatively proposed product hydrogen peroxide. The model was further developed to simulate the performance of a flow-through bubble-diffuser reactor with an external recirculation line. Each compartment of the reactor (bubble column and recirculation line) was assumed to behave as a plug flow reactor as supported by tracer test results, and an empirical correlation was used to represent the rate of ozone gas transfer in the bubble column. Model predictions of the performance of the flow-through ozone bubble-diffuser contactor were in good agreement with experimental results obtained for bromate formation and C. parvum oocyst inactivation under all conditions investigated. Additional model simulations revealed that hydrodynamic conditions had a more pronounced effect on C. parvum oocyst inactivation than on bromate formation. In contrast, pH had a strong effect on bromate formation without affecting the inactivation efficiency of C. parvum oocysts for a given level of exposure to ozone. These findings suggested that bromate formation could be minimized while achieving target inactivation levels for C. parvum oocysts by designing ozone reactors with hydrodynamic conditions approaching that of an ideal plug flow reactor and by lowering the pH of the target water.     

Transport of Cryptosporidium parvum oocysts in a silicon micromodel

Effective removal of Cryptosporidium parvum oocysts by granular filtration requires the knowledge of oocyst transport and deposition mechanisms, which can be obtained based on real time microscopic observation of oocyst transport in porous media. Attachment of oocysts to silica surface in a radial stagnation point flow cell and in a micromodel, which has 2-dimensional (2-D) microscopic pore structures consisting of an array of cylindrical collectors, was studied and compared. Real time transport of oocysts in the micromodel was recorded to determine the attached oocyst distributions in transversal and longitudinal directions. In the micromodel, oocysts attached to the forward portion of clean collectors, where the flow velocity was lowest. After initial attachment, oocysts attached onto already attached oocysts. As a result, the collectors ripened and the region available for flow was reduced. Results of attachment and detachment experiments suggest that surface charge heterogeneity allowed for oocyst attachment. In addition to experiments, Lattice-Boltzmann simulations helped understanding the slightly nonuniform flow field and explained differences in the removal efficiency in the transversal direction. However, the hydrodynamic modeling could not explain differences in attachment in the longitudinal direction.     

Double integrating sphere measurements for estimating optical properties of pig subcutaneous adipose tissue

Optical properties of pig subcutaneous adipose tissue were estimated using double integrating sphere (DIS) measurements in combination with an inverse adding doubling (IAD) light propagation model. Total reflectance, total transmittance and unscattered transmittance were measured for the wavelength range 1150–2250 nm with 10 nm resolution. These measurements were used to estimate the bulk optical properties (absorption coefficient, scattering coefficient and anisotropy factor) of 10 Iberian pig adipose tissue samples. The estimated absorption coefficient spectra were dominated by water and lipid absorption bands, the main chemical components of the adipose tissue. These values showed a suitable match with the theoretical values calculated based on the average composition of the tissue. The obtained scattering coefficient spectra showed a smooth decrease with increasing wavelength, which is typical for biological tissues. The anisotropy factor spectra of adipose tissue were obtained, which describe the angular scattering pattern, instead of using a fixed value for the estimation procedure. The optical characterization described in this paper is crucial for a better understanding of the impact of pure absorption and scattering on the NIR spectra collected by a single measurement in the laboratory, process line at the slaughterhouse or on the farm. Moreover, the estimated data reported in this study would promote a more efficient sensor design and data processing optimization.Industrial relevanceThis paper provides an optical characterization (absorption, scattering and angular scattering distribution) of pig adipose tissues in a wide near-infrared wavelength range (1150–2250 nm) based on a novel spectroscopic setup. The estimated data reported are relevant to promote the design of innovative NIRS sensors and the development of new modeling procedures in order to optimize the non-destructive analysis of intact adipose tissues. This can have an important impact on the implementation of NIRS quality control systems in the agro-industry.     

Cryptosporidium oocysts in mussels (Mytilus edulis) from Normandy (France)

Cultured mussels (Mytilus edulis) were collected seasonally during one year from three sites on the Northwestern coastal area of Normandy (France). Flesh, gills and innerwater were examined for Cryptosporidium oocyst detection using immunomagnetic separation and immunofluorescence assay. Oocysts were present in all samples for all sites and seasons and flesh was the most contaminated part. Oocyst rates were apparently related with seasonal rain precipitation variations. Molecular analysis revealed that oocysts belonged to the species Cryptosporidium parvum (formerly genotype 2 or ). Oocyst infectivity was assessed by oral administration to suckling NMRI-mice, and developmental stages were observed in only one mouse infected with oocysts from one location. The detection of potentially infectious C. parvum oocysts of likely cattle-breeding origin in cultured edible mussels confirms their resistance to sea environments, and underlines the potential risk of food-borne infection. This work reports for the first time the presence of infectious Cryptosporidium oocysts in shellfish from France.     

Cryptosporidium parvum studies with dairy products

Cryptosporidium parvum is a protozoan parasite capable of causing massive waterborne outbreaks. This study was conducted to model the transfer of C. parvum oocysts from contaminated water via food contact surfaces into yogurt and ice-cream, as well as to examine oocyst survival. Propidium iodide staining, combined with a direct immunofluorescence assay, was used for oocyst viability determination. Oocysts were recovered from milk products by a sucrose flotation-based procedure, with average recoveries of 82.3, 60.7, and 62.5% from low (1%) fat milk, 9% fat ice-cream, and 98% fat-free yogurt, respectively. Oocysts were also recovered, by rinsing with tap water, from stainless steel surfaces inoculated with oocyst suspension, with average recoveries of 93.1% when the surface was still wet and 69.0% after the surface had air-dried at room temperature. Viability of oocysts on the surface was significantly affected by desiccation; 5% of the oocysts remained viable after 4 h of air-drying at room temperature, while the proportion of viable oocysts was 81, 69, and 45% after air-drying for 10 min, 1 h, and 2 h, respectively. In contrast, oocyst viability only dropped from 82 to 75% after 30 min contact at room temperature with 5% bleach solution (equivalent to 0.26% NaOCl). Transfer of oocysts from milk and stainless steel surfaces into yogurt, and oocyst survival during the process were analyzed. Yogurt was made from pasteurized low fat milk and live yogurt starter by incubating at 37 degrees C for 48 h and then stored at 4 degrees C. Oocyst viability decreased from 83% (80%) to approximately 60% after 48 h at 37 degrees C and to approximately 58% following 8 days of storage, similar to oocyst survival in the controls using pasteurized milk without the addition of live yogurt. Oocyst survival in ice-cream was investigated by inoculating oocysts into ice-cream mix, and mixing and freezing in an ice-cream freezer, and hardening at -20 degrees C. Although approximately 20% (25 and 18%) of oocysts were viable before hardening, none were viable after 24 h at -20 degrees C. Control samples of oocysts suspended in distilled water and stored at -20 degrees C were taken at the same time intervals and 8% of the oocysts were still viable after 24 h.     

Survival of Cryptosporidium parvum oocysts after prolonged exposure to still natural mineral waters

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 degrees C, whereas a progressive decline in the viability of both oocyst isolates was observed when immersed in mineral waters at 20 degrees C. At 20 degrees 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 degrees C in our studies indicates that they could survive for prolonged periods of time in U.K. groundwaters (average temperature approximately 10 degrees C) and thus represent a potential public health hazard if contamination of mineral water sources by viable oocysts were to occur.     

Adhesion kinetics of viable Cryptosporidium parvum oocysts to quartz surfaces

The transport and deposition (adhesion) kinetics of viable Cryptosporidium parvum oocysts onto ultrapure quartz surfaces in a radial stagnation point flow system were investigated. Utilizing an optical microscope and an image-capturing device enabled real time observation of oocyst deposition behavior onto the quartz surface in solutions containing either monovalent (KCl) or divalent (CaCl2) salts. Results showed a significantly lower oocyst deposition rate in the presence of a monovalent salt compared to a divalent salt. With a monovalent salt, oocyst deposition rates and corresponding attachment efficiencies were relatively low, even at high KCl concentrations where Derjaguin-Landau-Verwey-Overbeek (DLVO) theory predicts the absence of an electrostatic energy barrier. On the other hand, in the presence of a divalent salt, oocyst deposition rates increased continuously as the salt concentration was increased over the entire range of ionic strengths investigated. The unusually low deposition rate in a monovalent salt solution is attributed to "electrosteric" repulsion between the Cryptosporidium oocyst and the quartz surface, most likely due to proteins on the oocyst surface that extend into the solution. It is further proposed that specific binding of calcium ions to the oocyst surface functional groups results in charge neutralization and conformational changes of surface proteins that significantly reduce electrosteric repulsion.     

Comparative detection of Cryptosporidium parvum oocysts from apple juice

Drinking unpasteurized apple juice (or cider) has been associated with cryptosporidiosis, the diarrheal disease caused by the small protozoan parasite, Cryptosporidium parvum. This report compares detection of C. parvum oocysts from apple juice by acid-fast staining (AFS), direct immunofluorescence assay (DIFA), and polymerase chain reaction (PCR), following sample concentration by formalin-ethyl acetate sedimentation or sucrose flotation. Flotation was more efficient than sedimentation in recovering oocysts, and DIFA consistently detected lower numbers of oocysts than AFS. In combination, flotation-AFS could detect 3000 to 10,000 oocysts inoculated into 100 ml of apple juice while flotation-DIFA was able to detect as few as 100 oocysts. The highest sensitivity, 10 to 30 oocysts per 100 ml of apple juice, was achieved by DIFA following immunomagnetic capture (IC) of oocysts from samples concentrated by the flotation method. The detection limit of PCR following flotation or flotation IC was 30 to 100 oocysts; sequence analysis of the amplicon demonstrated that the PCR amplicon was C. parvum-specific.     

POTATO CRISP MOISTURE ESTIMATION USING NEAR INFRARED SPECTROSCOPY

Near infrared spectral analysis is a widely used tool for non-destructive determination of moisture content in food. Moisture in potato crisps is often measured using near infrared analysis, however shape contours and curvatures in samples causes scattering of light, which reduces the accuracy of the measurement. The piecewise multiple scatter correction algorithm is commonly used to reduce the effect of light scattering from the sample. Implementation of the piecewise multiple scatter correction algorithm requires careful selection of scatter correction window size and combinations of spectral wavelength predictors. This paper considers different search heuristics for the purpose of piecewise multiple scatter correction window size selection and optimal spectral wavelength predictor selection. The search heuristics under consideration are a genetic algorithm, hill climbing, feature selection and full spectrum modeling. Calibration models using partial least square regression are formed from the scatter corrected data. The standard error of a cross-validated calibration data set was used to compare the performance of the different techniques. It has been found that a genetic algorithm produced the lowest cross validated standard error from the techniques considered. This suggests that the use of a genetic algorithm resulted in more judicious selection of near infrared model parameters.     

Infectivity of Cryptosporidium parvum oocysts after storage of experimentally contaminated apples

Irrigation water and washing water have been inferred to be associated with contamination of fresh fruits and vegetables with pathogenic microorganisms infectious for humans. The objective of the present study was to determine whether apples experimentally contaminated with Cryptosporidium oocysts represent a food safety concern. Laser scanning confocal microscopy revealed no morphological changes in Cryptosporidium parvum oocysts attached to apples after 6 weeks of cold storage, suggesting that oocysts might remain viable and possibly infectious during prolonged storage. Mice were fed apple peels from experimentally contaminated apples to determine whether oocysts had remained infectious on apples stored for 4 weeks. All mice developed cryptosporidiosis. To evaluate the strength of oocyst attachment to apples, washing methods that have been reported to be helpful for recovery of oocysts from various foodstuffs were evaluated, except that the intensity of washing was increased in the present study. None of the tested washing methods succeeded in completely removing oocysts from the apple peel. The most efficient removal (37.5%) was achieved by rigorous manual washing in water with a detergent and by agitation in an orbital shaker with Tris-sodium dodecyl sulfate buffer. Glycine and phosphate-buffered saline buffers had no effect on oocyst removal. Scanning electron microscopy revealed that some oocysts were attached in deep natural crevices in the apple exocarp and others were attached to the smooth surface of the peel. Some oocysts were closely associated with what appeared to be an amorphous substance with which they might have been attached to the apple surface.