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.     

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.     

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.     

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.     

Phylogenetic analysis implicates birds as a source of Cryptosporidium spp. oocysts in agricultural watersheds

Cryptosporidium parvum and C. hominis are protozoan parasites responsible for cryptosporidiosis, an acute gastrointestinal illness that can be life-threatening for immunocompromised persons. Sources and genotypes of Cryptosporidium oocysts were investigated in two agricultural areas within the Wachusett Reservoir watershed, a drinking water source for Boston, Massachusetts. Two brooks (denoted Brook SF and Brook JF, respectively), each downgradient from a dairy farm, were chosen as sample sites. For one year, Brooks SF and JF were sampled monthly; oocysts were detected in 6 (50%) out of 12 samples from Brook JF, and no oocysts were detected in Brook SF. Oocyst genotypes from agricultural surface waters were compared to oocyst genotypes from Genbank, as well as fecal samples of cattle and birds, using phylogenetic analysis of a hypervariable region of the 18S rRNA gene by both neighbor-joining and parsimony methods. Results show extensive heterogeneity among Cryptosporidium spp. 18S rRNA sequences, and also suggest that birds are an oocyst source in this watershed. Principal components analysis showed oocyst presence correlating strongly with seasonal factors, and oocysts in surface waters were only detected in the summer through late fall, co-incident with the presence of migratory birds in this watershed. If birds are confirmed to be an important source of oocysts infectious to humans, the data suggest that protection of raw drinking water supplies in some agricultural areas may depend upon management and control of resident and migratory bird populations.     

Detection of Cryptosporidium and Giardia in molluscan shellfish by multiplexed nested-PCR

A multiplexed nested-PCR procedure (ABC-PCR) previously developed to detect Cryptosporidium spp. and Giardia duodenalis assemblages A and B in whole human faeces was applied to DNA extracted from filter-feeding molluscs. Species of Cryptosporidium and G. duodenalis were identified by restriction fragment analysis of the PCR products and by DNA sequencing. The extraction and ABC-PCR procedures were shown to be suitable for application to shellfish by amplification of specific target sequences using DNA from Cryptosporidium parvum genotype 2 and G. duodenalis assemblages A and B which were spiked into DNA extracted from mussels. Using 49 molluscan shellfish specimens (18 clam, 22 mussel and 9 oyster samples) from Spain, cryptosporidial oocysts were detected in 56% by immunofluorescence microscopy, and in 44% by ABC-PCR. For detection of Cryptosporidium, there was a significant association, but not total agreement, between the results of microscopy and PCR. G. duodenalis assemblage B was detected from one oyster sample by PCR. Amongst 38 specimens (20 mussel and 18 cockle samples) collected in the UK and tested by the ABC-PCR, G. duodenalis was not detected, and Cryptosporidium was detected in 11% of the samples. Overall, the 26 samples where Cryptosporidium was detected, C. hominis/C. parvum genotype 1 was detected in 1, C. parvum genotype 2 in 22, and the remaining three samples contained either sequences similar to C. parvum genotype 2 or heterogeneous mixtures of Cryptosporidium species. There was no significant association between the level of Escherichia coli detected by conventional microbiological methods and the presence of Cryptosporidium detected by ABC-PCR.     

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.     

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.     

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.     

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.     

Analysis of cured meat products for cryptosporidium oocysts following possible contamination during an extensive waterborne outbreak of Cryptosporidiosis

An outbreak of waterborne cryptosporidiosis in a town in northern Sweden during winter 2010 resulted in the potential exposure of cured meat products to Cryptosporidium oocysts during their manufacture. The purpose of this work was to develop a method for analyzing cured meat products for contamination with Cryptosporidium oocysts and use this method to analyze potentially contaminated product samples. A simple method of elution, concentration, separation, and detection was used, based on work with other food matrices but adapted for the relatively high fat content of cured meat surfaces. Using spiking experiments, the recovery efficiency of this method was found to be over 60%. In the analysis of the potentially contaminated products, only one putative Cryptosporidium oocyst was detected, and this was sufficiently deformed so that it could not be confirmed as an oocyst; if it was an oocyst, it was considered to have been probably deformed and inactivated prior to analysis. Based on the results of the analyses, together with data on the probable extent of contamination of the products and on our knowledge of factors, such as water activity, which affect oocyst survival, the products were safely released to the market.     

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.     

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.     

THE POTENTIAL FOR CRYPTOSPORIDIUM PARVUM OOCYST SURVIVAL IN BEVERAGES ASSOCIATED WITH CONTAMINATED TAP WATER

Cryptosporidium parvum is an enteric coccidian protozoan which produces an environmentally stable oocyst that is excreted in the feces of infected individuals. There have been ten documented water borne outbreaks in North America. If food or beverages were prepared from contaminated water, that food or beverage would also be a hazard. The objective of this study was to evaluate the survival of Cryptosporidium parvum in beverages. Viability of oocysts, as determined by morphology decreased over 24 h exposure in carbonated beverages. Uptake of vital dyes indicated a loss of >85% of oocyst viability in beer or cola stored at 4C. Loss of viability in tap water, orange juice or infant formula was ± 35%. It is likely that the low pH of the carbonated beverages was involved in the loss of oocyst viability and premature excystation of the sporozoites .     

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.     

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.     

Towards standard methods for the detection of Cryptosporidium parvum on lettuce and raspberries. Part 1: development and optimization of methods

No standard method is available for detecting protozoan parasites on foods such as soft fruit and salad vegetables. We report on optimizing methods for detecting Cryptosporidium parvum on lettuce and raspberries. These methods are based on four basic stages: extraction of oocysts from the foodstuffs, concentration of the extract and separation of the oocysts from food materials, staining of the oocysts to allow their visualization, and identification of oocysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Oocyst staining is also performed using proprietary reagents. The performance parameters of the extraction steps were extensively optimized, using artificially contaminated samples. The fully developed methods were tested several times to determine their reliability. The method to detect C. parvum on lettuce recovered 59.0+/-12.0% (n=30) of artificially contaminated oocysts. The method to detect C. parvum on raspberries recovered 41.0+/-13.0% (n=30) of artificially contaminated oocysts.     

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.     

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.     

Effect of organic acids and hydrogen peroxide on Cryptosporidium parvum viability in fruit juices

Cryptosporidium parvum has historically been associated with waterborne outbreaks of diarrheal illness. Foodborne cryptosporidiosis has been associated with unpasteurized apple cider. Infectious oocysts are shed in the feces of common ruminants like cattle and deer in and near orchards. In this study, the ability of organic acids and hydrogen peroxide (H2O2) added to fruit juice to inhibit the survival of C. parvum was analyzed. Oocyst viability was analyzed by a cell culture infectivity assay with the use of a human ileocecal cell line (HCT-8) whose infectivity pattern is similar to that for human oral infectivity. Cell monolayers were infected with 10(6) treated oocysts or a series of 10-fold dilutions. Parasitic life stages were visualized through immunohistochemistry with 100 microscope fields per monolayer being counted. In vitro excystation assays were also used to evaluate these treatments. Organic acids and H2O2 were added to apple cider, orange juice, and grape juices on a weight/volume basis. Malic, citric, and tartaric acids at concentrations of 1 to 5% inhibited C. parvum's infectivity of HCT-8 cells by up to 88%. Concentrations ranging from 0.025 to 3% H2O2 were evaluated. The addition of 0.025% H2O2 to each juice resulted in a >5-log reduction of C. parvum infectivity as determined with a most-probable-number-based cell culture infectivity assay. As observed with differential interference contrast and scanning electron microscopy, reduced infectivity may be mediated through effects on the oocyst wall that are caused by the action of H2O2 or related oxygen radicals. The addition of low concentrations of H2O2 can represent a valuable alternative to pasteurization.