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.     

Microwave inactivation of Cyclospora cayetanensis sporulation and viability of Cryptosporidium parvum oocysts

The efficacy of microwave heating on the viability of Cryptosporidium parvum oocysts and on the sporulation of Cyclospora cayetanensis oocysts for various periods of cooking times (0, 10, 15, 20, 30, and 45 s) at 100% power was determined. Cyclospora oocysts were stored in 2.5% dichromate at 23 degrees C for 2 weeks, and sporulation rates were then determined. The 4',6-diamidino-2-phenylindole and propidium iodide vital stain and the neonate animal infectivity assay determined Cryptosporidium oocyst viability. Cryptosporidium oocysts could be completely inactivated with as little as 20 s of cooking time, whereas Cyclospora sporulation was observed up to 45 s. Two of the examined microwave ovens were more effective at reducing sporulation and viability than the third one. Because of the variability of temperature achieved by the various ovens, cooking time was not an accurate parameter for parasite inactivation. Cryptosporidium oocysts could be inactivated only when temperatures of 80 degrees C or higher were reached in the microwave ovens.     

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.     

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.     

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.     

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.     

Interaction force profiles between Cryptosporidium parvum oocysts and silica surfaces

The interaction force profile between single Cryptosporidium parvum oocysts and silica particles was measured in aqueous solutions using an atomic force microscope. The oocysts were immobilized during the measurements by entrapment in Millipore polycarbonate membranes with a 3 microm pore size. Experiments were performed in both NaCl and CaCl2 solutions at ionic strengths ranging from 1 to 100 mM. For both electrolytes the decay length of the repulsive force profile, obtained via the slope of a plot of the logarithm of interaction force versus separation, was found to be essentially independent of the ionic strength and always much larger than the theoretical Debye length of the system. In addition, the magnitude of the force was found to be essentially the same for both electrolytes, suggesting that the long-range repulsive forces are primarily steric in nature. Fitting the force to an expression for the steric repulsive force between two grafted brush layers yields a layer thickness of approximately 115 nm. These results support the idea that the oocysts are covered by a relatively thick layer of uncharged (or weakly charged) carbohydrates, possibly mixed with a thinner layer of charged protein.     

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.     

Seasonal temperature fluctuations induces rapid inactivation of Cryptosporidium parvum

This study measured the inactivation rate of bovine genotype A Cryptosporidium parvum oocysts attributable to diurnal oscillations of ambient temperature and solar radiation typical of California rangelands and dairies from spring through autumn. We first measured the relationship between air temperature and the internal temperature of bovine feces exposed to sunlight on commercial operations throughout California. Once maximum air temperature exceeded the mid 20 degrees C, diurnal thermal regimes of bovine fecal material exhibited peaks of over 40, 50, 60, and 70 degrees C. These diurnal thermal regimes were emulated using a thermocycler, with oocysts suspended in distilled water or fecal-water mix. Using oral inoculations of 10(5) C. parvum oocysts per neonatal Balb/c mouse (>1000-fold the ID50), no infections were observed using 1 to 5-day cycles of these thermal regimes. Loss of infectivity induced bythese thermal regimes was primarily due to partial or complete in vitro excystation during the first 24-h diurnal cycle and secondarily to thermal inactivation of the remaining intact or partial oocysts. These results suggest that as ambient conditions generate internal fecal temperatures > or = 40 degrees C via conduction, radiation, and convection, rapid environmental inactivation occurs at a rate of > or = 3.27 log reduction d(-1) for C. parvum oocysts deposited in the feces of cattle.     

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.     

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.     

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.     

Effect of a commercial freeze/tempering process on the viability of Cryptosporidium parvum oocysts on lean and fat beef trimmings

Lean and fat beef trimmings (25 cm⁻²) were inoculated with approximately 250,000 Cryptosporidium parvum oocysts, placed in commercial packages (28 kg boxes) and subjected to normal commercial processes i.e. blast frozen (to −20 °C within 60 h), stored (−20 °C, 21 days), tempered (48 h at −3 °C), and held at 0 °C for 10 h. Inoculated areas were then excised, pulsified (30 s in 50 ml PBST), and centrifuged (2500×g, 15 min). The resultant pellet was resuspended in 10 ml water and subjected to immunomagnetic separation and viability dye assay. Following the commercial freeze/tempering process the viability of the oocysts had decreased from 90.6% viable in the working stock suspension to 7.17% and 9.46% viable on lean and fat trimmings, respectively. The results of this study indicate that if C. parvum oocysts were present on beef trimmings their viability would be substantially reduced as a result of the freeze/tempering process.     

Effect of ferric oxyhydroxide grain coatings on the transport of bacteriophage PRD1 and Cryptosporidium parvum oocysts in saturated porous media

To test the effect of geochemical heterogeneity on microorganism transport in saturated porous media, we measured the removal of two microorganisms, the bacteriophage PRD1 and oocysts of the protozoan parasite Cryptosporidium parvum, in flow-through columns of quartz sand coated by different amounts of a ferric oxyhydroxide. The experiments were conducted over ranges of ferric oxyhydroxide coating fraction of lambda = 0-0.12 for PRD1 and from lambda = 0-0.32 for the oocysts at pH 5.6-5.8 and 10(-4) M ionic strength. To determine the effect of pH on the transport of the oocysts, experiments were also conducted over a pH range of 5.7-10.0 at a coating fraction of lambda = 0.04. Collision (attachment) efficiencies increased as the fraction of ferric oxyhydroxide coated quartz sand increased, from alpha = 0.0071 to 0.13 over lambda = 0-0.12 for PRD1 and from alpha = 0.059 to 0.75 over lambda = 0-0.32 for the oocysts. Increasing the pH from 5.7 to 10.0 resulted in a decrease in the oocyst collision efficiency as the pH exceeded the expected point of zero charge of the ferric oxyhydroxide coatings. The collision efficiencies correlated very well with the fraction of quartz sand coated by the ferric oxyhydroxide for PRD1 but not as well for the oocysts.     

Use of multiwavelength transmission spectroscopy for the characterization of Cryptosporidium parvum oocysts: quantitative interpretation

Combined scattering and absorption properties of suspended particles can be obtained as a function of wavelength by measuring the complete ultraviolet-visible (UV-vis) spectrum. This research reports on the quantitative interpretation of measured UV-vis spectra of Cryptosporidium parvum oocyst suspensions obtained from several commercial sources and evaluated using two different purification techniques. The reproducibility of the measured spectral data was assessed, and the quantitative interpretation of the oocyst spectra in terms of the particle size and the chemical composition of the particles are reported herein. The interpretation model of the spectra is based on light scattering theory, spectral deconvolution techniques, and on the approximation of the wavelength-dependent optical properties of the basic constituents of living organisms. A characteristic set of optical properties for C. parvum oocysts has been determined as a function of wavelength and used for the quantitative interpretation of UV-vis spectra. The results from the spectral deconvolution show quantitative differences among oocyst preparations. These results represent the first step in establishing a set of critical parameters (e.g., oocyst size and chemical composition) necessary for the detection and identification of C. parvum oocysts in water using spectroscopy.     

Effect of high hydrostatic pressure on Cryptosporidium parvum infectivity

The incidence of foodborne disease outbreaks caused by contaminated low-pH fruit juices is increasing. With recent mandatory pasteurization of apple juice and the industry's concerns of food safety, fruit juice processors are showing more interest in alternative nonthermal technologies that can kill >99.99% of microbial pathogens present in foods. The association of the coccidian protozoan, Cryptosporidium, with diarrheal disease outbreaks from contaminated tap water and fruit juice raises a safety concern in the food and beverage industries. The objective of this study was to evaluate the effects of high hydrostatic pressure (HHP) on C. parvum oocysts. Oocysts were suspended in apple and orange juice and HHP treated at 5.5 x 10(8) Pa (80,000 psi) for 0, 30, 45, 60, 90, and 120 s. Oocyst viability was assessed by excystation using bile salts and trypsin while the cell culture foci detection method was used to assess infectivity. Results indicated that HHP inactivated C. parvum oocysts by at least 3.4 log10 after 30 s of treatment. No infectivity was detected in samples exposed to > or =60 s of HHP and >99.995% inactivation was observed. This study demonstrated that HHP efficiently rendered the oocysts nonviable and noninfectious after treatment at 5.5 x 10(8) Pa.     

A histological study of the transit of Cryptosporidium parvum oocysts through clams (Tapes decussatus)

A histological study was carried out to investigate the transit of Cryptosporidium parvum oocysts through the clam Tapes decussatus. Spat of approximately 5-7 mm shell length were maintained in a tank of natural sea water contaminated with purified C. parvum oocysts. The experiment lasted 240 h and, every 24 h, five specimens were killed, placed in Bouin's fixative, and processed routinely for histological examination. Sections (3 mum) cut from the all body tissues were stained with modified Gomori's trichrome for their accurate identification; the oocysts were detected by a direct immunofluorescence procedure. Oocysts were detected in siphons, gills, stomach, digestive diverticula, and intestine. The oocysts present in the intestine were free or mixed with the intestinal contents; therefore release of these oocysts with the feces should favour dissemination of contamination. Oocysts were found in branchial mucus and within the interfilamentary spaces, which suggests the occurrence of repeated filtrations and the possibility that the retained oocysts maintain their infective capacity.     

Modeling Cryptosporidium parvum oocyst inactivation and bromate formation in a full-scale ozone contactor

The inactivation of Cryptosporidium parvum oocysts and the formation of bromate were assessed simultaneously by performing experiments with a full-scale ozone bubble-diffuser contactor used for drinking water disinfection. Fluorescence-dyed polystyrene microspheres were used as surrogates for C. parvum oocysts. Semi-batch ozonation experiments were performed to determine the fluorescence-intensity decay of individual microspheres, which was measured by flow cytometry. The results obtained with the microspheres were correlated to the inactivation kinetics of C. parvum oocysts by choosing an appropriate threshold fluorescence intensity below which microspheres were considered to be equivalent to nonviable oocysts. A mathematical model was then used to predict the inactivation efficiency and bromate formation. The contactor hydrodynamics were characterized by running tracer tests, and the kinetic parameters for ozone decomposition and bromate formation were obtained by performing batch experiments. Model predictions were in good agreement with full-scale experimental results. Additional model simulations revealed that ozone contactors should be designed with the lowest possible backmixing so that the target inactivation efficiency can be achieved with the lowest possible formation of bromate.     

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.