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.     

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.     

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.     

Cooking mussels (Mytilus galloprovincialis) by steam does not destroy the infectivity of Cryptosporidium parvum

The consumption of shellfish has increased considerably worldwide, with an associated increase in foodborne illnesses. Among the bivalves, the mussels are usually cooked by steam, which constitutes a typical dish in several regions. In this article, we demonstrate that this preparation is not sufficient to destroy completely the infectivity of Cryptosporidium parvum. Oocysts recovered from experimentally contaminated mussels (Mytilus galloprovincialis) were infectious to neonatal mice after cooking. Although, to date, no official cases of cryptosporidiosis linked to shellfish consumption have been reported, we recommend that people with reduced immunity avoid this type of food because they are at high risk of being infected with Cryptosporidium spp. after eating raw or undercooked contaminated bivalves.     

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.     

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.     

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.     

Temporal occurrence of Cryptosporidium in the Manila clam Ruditapes philippinarum in northern Adriatic Italian lagoons

In order to evaluate the temporal occurrence of Cryptosporidium oocysts in Ruditapes philippinarum clams bred along the northeastern Italian Adriatic coast and molecularly characterize the isolates, 2,160 specimens (180 clams per month) were collected from three clam farms from January to December 2004. Two farms (sites A and B) were located in Venice (Chioggia, Veneto region) and one (site C) in the Marano Lagoons (Friuli Venezia Giulia region). Clams from 36 pools (i.e., one pool of 60 clams per month per site) were subjected to a high-sensitivity seminested PCR assay specific for a 360-bp diagnostic region internal to the Cryptosporidium spp. outer wall protein gene. Positive amplicons were sequenced and analyzed. Cryptosporidium DNA was found in clams from seven pools (sites A and B) during 1 month of sampling at site A and 6 months of sampling at site B, with Cryptosporidium hominis and Cryptosporidium parvum being detected. The expected infection rate of the clams was 0.36%. Site B showed a significantly higher expected infection rate (1.15%) than did the other sites (A = 0.14% and C = 0%). Given its high sensitivity and specificity, this seminested PCR assay can be considered a reliable tool for detecting and distinguishing species within the Cryptosporidium genus. The seasonal pattern of contamination and the related public health risks are of particular concern.     

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.     

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.     

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.     

Effect of lot variability on ultraviolet radiation inactivation kinetics of Cryptosporidium parvum oocysts

Numerous studies have demonstrated the efficiency of ultraviolet (UV) radiation for the inactivation of oocysts of Cryptosporidium parvum. In these studies inactivation is measured as reduction in oocysts. A primary goal is to estimate the UV radiation required to achieve a high degree of inactivation. Different lots of Cryptosporidium parvum oocysts are used in these studies, and the inactivation rate may vary depending on the lot of oocysts used. The goal of this paper is to account for the error in estimating the amount of inactivation after exposure to UV radiation, and for the effect of lot variability in determining the required UV radiation. A Bayesian approach is used to simultaneously model the logistic dose-response model and the UV inactivation kinetic model. The oocysts lot variability is incorporated using a hierarchical Bayesian model. Posterior distributions using Markov Chain Monte Carlo method is used to obtain estimates and Bayesian credible interval for the required UV radiation to achieve a given inactivation level of Cryptosporidium parvum oocysts.     

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.     

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.     

Use of a common laboratory glassware detergent improves recovery of Cryptosporidium parvum and Cyclospora cayetanensis from lettuce, herbs and raspberries

The success of any protocol designed to detect parasitic protozoa on produce must begin with an efficient initial wash step. Cryptosporidium parvum and Cyclospora cayetanensis oocysts were seeded onto herbs, lettuces and raspberries, eluted with one of four wash solutions and the recovered number of oocysts determined via fluorescent microscopy. Recovery rates for fluorescein thiosemicarbazide labeled C. parvum oocysts seeded onto spinach and raspberries and washed with de-ionized water were 38.4 ± 10.1% and 34.9 ± 6.2%, respectively. Two alternative wash solutions viz. 1M glycine, pH 5.5 and a detachment solution were tested also using labeled C. parvum seeded spinach and raspberries. No statistically significant difference was noted in the recovery rates. However, a wash solution containing 0.1% Alconox, a laboratory glassware detergent, resulted in a significant improvement in oocyst recovery. 72.6 ± 6.6% C. parvum oocysts were recovered from basil when washed with 0.1% Alconox compared to 47.9 ± 5.8% using detachment solution. Also, C. cayetanensis oocysts were seeded onto lettuces, herbs and raspberries and the recovery using de-ionized water were compared to 0.1% Alconox wash: basil 17.5 ± 5.0% to 76.1 ± 14.0%, lollo rosso lettuce 38.3 ± 5.5% to 72.5 ± 8.1%, Tango leaf lettuce 45.9 ± 5.4% to 71.1 ± 7.8% and spring mix (mesclun) 39.8 ± 0.7% to 80.2 ± 11.3%, respectively. These results suggest that the use of Alconox in a wash solution significantly improves recovery resulting in the detection of these parasitic protozoa on high risk foods.     

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.     

Microbial characterization and population changes in nonpotable reclaimed water distribution systems

Changes in the microbial quality of nonpotable reclaimed water distribution systems in seven metropolitan areas of the southwestern United States were investigated by performing pathogen monitoring and bacterial growth. Water samples were collected from tertiary-treated effluents at point of entry and point of use in the distribution systems. The samples were analyzed for Cryptosporidium oocysts, Giardia cysts, enteroviruses, microbial indicators, and assimilable organic carbon (AOC). Cryptosporidium and Giardia were detected in 16% (12/77) and 43% (33/77) of nonpotable reclaimed water samples, whereas no infectious Cryptosporidium parvum were detected in any of the samples. No infectious enteroviruses were detected in any sample (0/27). At point of entry total coliform and fecal coliforms ranged from 0.7 to 280 and from 0 to 1.9 colony-forming units (cfu)/100 mL, respectively. Increases in the number of coliforms were observed as water passed through the reclaimed water distribution systems. However, no such increase in the number of coliphages was found. The chlorination practices at some utilities were not sufficient to inactivate coliforms and coliphages, but supplemental ultraviolet disinfection resulted in lower numbers of these microbial indicators. AOC levels decreased by 3-fold as water passed through the distribution systems, which inversely correlated with bacterial regrowth.     

Contamination of bivalve molluscs by Cryptosporidium oocysts: the need for new quality control standards

A yearlong study was carried out to investigate the presence and viability of Cryptosporidium oocysts in 203 samples of cultured shellfish from Galicia (NW Spain) and 38 samples imported from other European Union (EU) countries. Shellfish samples included mussels, oysters, clams and cockles. Cryptosporidium oocysts were detected, using a direct immunofluorescence antibody test (IFAT), in 34.4% of the samples analyzed; use of the fluorogenic dye propidium iodide (PI) revealed viable potentially infective oocysts in 53.0% of these samples. There was no relation between the presence of Cryptosporidium oocysts and the microbiological contamination detected in the samples expressed as Most-Probable-Number (MPN) of fecal coliforms, the different species of mollusc, or the month of sampling. One important finding was that the depuration process was ineffective in totally removing oocyst contamination. Furthermore, the existence of viable oocysts in samples with microbiological contamination levels lower than 300 fecal coliforms/100 g, which in accordance with current legislation are considered suitable for human consumption, suggests the need to include parasitological analyses in the quality control for these molluscs.