Detection of Cryptosporidium oocysts in water: techniques for generating precise recovery data

When determining the recovery efficiency of a procedure for the detection of Cryptosporidium or the removal efficiency of a treatment process, it is necessary to accurately enumerate a 'seed dose'. Conventional techniques for this are highly variable and consequently, can result in misleading data. In this study, a flow cytometric method was developed for the production of suspensions of Cryptosporidium oocysts in which the number of organisms could be precisely determined. A Becton Dickinson FACScalibur flow cytometer was employed to produce oocyst suspensions containing 100 oocysts. Analysis of these suspensions resulted in a mean dose of 99.5 oocysts (S.D. = 1.1, %cv = 1.1). These results indicate that the use of such suspensions to seed test systems generates far more accurate data than is presently possible using conventional techniques. In addition, the use of immunomagnetic separation (IMS) for the isolation of oocysts from three different water matrices, after seeding with oocysts counted using flow cytometry, was investigated. The recovery efficiency of the IMS procedure was found to be high, with the percentage recovery of oocysts ranging from 82.3 to 86.3%, and the use of precise numbers of oocysts allowed accurate recovery efficiency data to be generated. A laser scanning instrument (ChemScan RDI) was employed for the rapid detection and enumeration of oocysts after capture using membrane filtration. This technique was found to be faster and easier to perform than conventional epifluorescence microscopy. These findings demonstrate that the ChemScan RDI system may be used as alternative procedure for the routine examination of IMS supernatant fluids for the presence of Cryptosporidium.     

Comparative study between two laser scanning cytometers and epifluorescence microscopy for the detection of Cryptosporidium oocysts in water.

BACKGROUND: Cryptosporidium detection in water and environmental samples has increased during the last years, largely due to an increase in the number of reported waterborne outbreaks of cryptosporidiosis and the implementation of new regulations about Cryptosporidium monitoring in water supplies. The aim of this study was to validate and compare the capacity of two laser scanning cytometers commercially available (LSC and ChemScanRDI), against manual microscopic enumeration of Cryptosporidium oocysts in surface water and reference material samples. METHODS: Reference material and surface water samples were analysed by two laser scanning cytometers methodologies and by manual epifluorescence microscopy. Two mAbs from commercial suppliers were used to evaluate background reduction. RESULTS: Highly significant correlations were obtain between both cytometers (R(2) = 0.99) and with manual microscopy (R(2) = 0.98), showing that oocysts counts made by cytometers were equivalent to those obtained with conventional methods. We observed a variability in oocysts counts when different antibodies where used with laser scanning cytometers and manual microscopy. CONCLUSIONS: This study showed the efficacy of the laser scanning technology (LSC and ChemScanRDI), as an automated and a more standardized alternative to manual epifluorescence microscopy examination, for Cryptosporidium detection in water samples. High quality antibodies are needed for automated enumeration as well as for manual microscope observations.     

Computer-Assisted Laser Scanning and Video Microscopy for Analysis of Cryptosporidium parvum Oocysts in Soil, Sediment, and Feces

A computer-assisted laser scanning microscope equipped for confocal laser scanning and color video microscopy was used to examine Cryptosporidium parvum oocysts in two agricultural soils, a barnyard sediment, and calf fecal samples. An agar smear technique was developed for enumerating oocysts in soil and barnyard sediment samples. Enhanced counting efficiency and sensitivity (detection limit, 5.2 x 10(sup2) oocysts(middot)g [dry weight](sup-1)) were achieved by using a semiautomatic counting procedure and confocal laser scanning microscopy to enumerate immunostained oocysts and fragments of oocysts in the barnyard sediment. An agarose-acridine orange mounting procedure was developed for high-resolution confocal optical sectioning of oocysts in soil. Stereo images of serial optical sections revealed the three-dimensional spatial relationships between immunostained oocysts and the acridine orange-stained soil matrix material. In these hydrated, pyrophosphate-dispersed soil preparations, oocysts were not found to be attached to soil particles. A fluorogenic dye permeability assay for oocyst viability (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992) was modified by adding an immunostaining step after application of the fluorogenic dyes propidium iodide and 4(prm1),6-diamidino-2-phenylindole. Comparison of conventional color epifluorescence and differential interference contrast images on one video monitor with comparable black-and-white laser-scanned confocal images on a second monitor allowed for efficient location and interpretation of fluorescently stained oocysts in the soil matrix. This multi-imaging procedure facilitated the interpretation of the viability assay results by overcoming the uncertainties caused by matrix interference and background fluorescence.     

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. RESULTS: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. CONCLUSIONS: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.     

Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r2 = 0. 95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.     

Application of laser scanning cytometry followed by epifluorescent and differential interference contrast microscopy for the detection and enumeration of Cryptosporidium and Giardia in raw and potable waters

AIMS: The main goal of this study was to validate a new laser scanning cytometry method (ChemScanRDI) that couples immunofluorescence detection with differential interference contrast (DIC) confirmation, against manual microscopic enumeration of Giardia and Cryptosporidium (oo)cysts. This study also assessed the basic performance of the new Association Fran?aise de Normalisation (AFNOR) NF T 90-455 method for Giardia and Cryptosporidium (oo)cyst enumeration with respect to (oo)cyst yield, linearity, repeatability, influence of turbidity and detection limit in raw and potable waters. METHODS AND RESULTS: The new standard method relies on cartridge (Envirocheck) filtration, immunomagnetic separation purification, immunofluorescence staining and detection followed by DIC confirmation. The recovery was 30-50% for both parasites at seeding levels from 30 to 230 (oo)cysts. The method is linear from 0 to around 400 seeded (oo)cysts and the yield does not significantly vary for turbidity levels from 10 to 40 Formazin Nephelometric Units (FNU). The results were obtained using manual microscopic enumeration of the (oo)cysts. The ChemScanRDI yielded counts that were at least equivalent to those obtained using manual microscopy for both parasites in raw and potable water concentrates, for seeding levels of 10-300 or 10-100, respectively. The purification and labelling method proposed by the supplier of theChemScanRDI (Chemunex) reached very similar recoveries to the AFNOR protocol (70-86% in both cases). CONCLUSIONS: Laser scanning cytometry can be used as a more standardized alternative to manual enumeration as part of the new AFNOR standard method. SIGNIFICANCE AND IMPACT OF THE STUDY: By using laser scanning cytometry instead of manual microscopy, laboratories could circumvent the limitations of manual microscopy, namely: low sample throughput, operator subjectivity and operator fatigue. The study further supports the drive to incorporate laser scanning cytometry in the standard methods for Giardia and Cryptosporidium enumeration.     

Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus)

Bivalve molluscs concentrate Cryptosporidium oocysts from fecal-contaminated aquatic environments and are therefore useful in monitoring water quality. A real-time TaqMan polymerase chain reaction (PCR) system was developed to allow for large scale quantitative detection of Cryptosporidium spp. in mussels (Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) assays, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a 1 log(10) improvement when samples were first processed by IMS. For tank exposed mussels, TaqMan and conventional PCR methods detected C. parvum in <5% of hemolymph samples. No gill washings from these same mussels tested positive by TaqMan PCR or DFA analysis even with IMS concentration. All methods detected the highest prevalence of C. parvum-positive samples in digestive gland tissues of exposed mussels. In conclusion, the most sensitive method for the detection of C. parvum in oocyst-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive. TaqMan PCR was comparable to conventional PCR for detection of C. parvum oocysts in mussels and additionally allowed for automated testing, high throughput, and semi-quantitative results.     

Comparison of immunofluorescence assay and immunomagnetic electrochemiluminescence in detection of Cryptosporidium parvum oocysts in karst water samples

Immunofluorescence assay (IFA) and immunomagnetic electrochemiluminescence (IM-ECL) were used for comparison of the percent recovery of Cryptosporidium parvum in environmental water samples obtained from a spring draining a karst basin. The monoclonal antibodies to C. parvum, isotype IgG3 were used for optimization of the IM-ECL protocol. The combination of biotinylated and TAG-labeled anti-C. parvum antibodies with the streptavidin beads gave a linear regression slope for log ECL vs. log fresh oocysts of 0.79 (from 5 to 5,000 oocysts), which indicates a constant ECL signal per oocyst. Standard curves gave a dynamic range of 5 to 5,000 oocysts/ml (fresh) and 10 to 100,000 cells/ml (4-month-old oocysts) with the maximum limit of linear detection higher than 100,000. The linear slope of 4-month-old oocysts decreased to 0.62, which indicates that ECL signal is a function of oocyst age. The experiment associated with bead storage time shows that even after 4 months of storage of the biotinylated antibodies, the complex retains the ability for binding the oocysts and generating the ECL signal. Based on the IFA results in the experiment evaluating different protocols for oocysts recovery from karst water samples, the most efficient protocol involved dispersion, followed by flotation and immunomagnetic separation (IMS) (24% recovery). The ECL results obtained in that experiment were very similar to the results obtained in the IFA method, which indicates that the IM-ECL method is accurate. Results of the IFA in the study of the prevalence of C. parvum in the groundwater showed that oocysts were present in 78% of 1 L water samples with average number of oocysts of 6.4+/-5.5 and ranged from 0 (13 samples) to 23.3 (2 samples). The ECL signal generated from these water samples ranged from 3,771 to 622 (average 1,620+/-465). However, the background value estimated in groundwater samples with low number of oocysts detected by IFA was highly variable and elevated (from 3,702 to 272, average 1,503+/-475). The background value as a result of nonspecific binding to beads by unidentified organic components in the water can inhibit or even completely mask the signal generated by oocysts. Our investigations showed that the IM-ECL method appears to be promising for the qualitative and quantitative detection of C. parvum from the environmental water; however, the method requires further development to improve sensitivity and account for background signals.     

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.     

An immunomagnetic separation-real-time PCR method for quantification of Cryptosporidium parvum in water samples

The protozoan parasite Cryptosporidium parvum is known to occur widely in both raw and drinking water and is the cause of waterborne outbreaks of gastroenteritis throughout the world. The routinely used method for the detection of Cryptosporidium oocysts in water is based on an immunofluorescence assay (IFA). It is both time-consuming and nonspecific for the human pathogenic species C. parvum. We have developed a TaqMan polymerase chain reaction (PCR) test that accurately quantifies C. parvum oocysts in treated and untreated water samples. The protocol consisted of the following successive steps: Envirochek capsule filtration, immunomagnetic separation (IMS), thermal lysis followed by DNA purification using Nanosep centrifugal devices and, finally, real-time PCR using fluorescent TaqMan technology. Quantification was accomplished by comparing the fluorescence signals obtained from test samples with those from standard dilutions of C. parvum oocysts. This IMS-real-time PCR assay permits rapid and reliable quantification over six orders of magnitude, with a detection limit of five oocysts for purified oocyst solutions and eight oocysts for spiked water samples. Replicate samples of spiked tap water and Seine River water samples (with approximately 78 and 775 oocysts) were tested. C. parvum oocyst recoveries, which ranged from 47.4% to 99% and from 39.1% to 68.3%, respectively, were significantly higher and less variable than those reported using the traditional US Environmental Protection Agency (USEPA) method 1622. This new molecular method offers a rapid, sensitive and specific alternative for C. parvum oocyst quantification in water.     

Evaluation of an alternative IMS dissociation procedure for use with Method 1622: detection of Cryptosporidium in water

U.S. EPA Methods 1622 and 1623 are used to detect and quantify Cryptosporidium oocysts in water. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluorescent antibody and confirmation by the demonstration of 1-4 sporozoites or nuclei after staining with 4',6-diamidino-2-phenyl indole dihydrochloride (DAPI). The purpose of this study was to evaluate a new IMS dissociation, a 10-min incubation at 80 degrees C. Heat dissociation improved the average oocyst recovery from 41% to 71% in seeded reagent water, and from 10% to 51% in seeded river samples. The average DAPI confirmation rate improved from 49% to 93% in reagent water, and from 48% to 73% in river samples. This modification improved both oocyst recovery and confirmation.     

Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623

The U.S. Environmental Protection Agency has developed method 1623 for simultaneous detection of Cryptosporidium oocysts and Giardia cysts in water. Method 1623 includes four major steps: filtration, immunomagnetic separation (IMS), fluorescent antibody (FA) staining and microscopic examination. It was noted that the recovery levels following IMS-FA and FA staining were high, averaging more than 92.0% and 89.0% for C. parvum oocysts and G. lamblia cysts, respectively. In contrast, when the filtration step was incorporated, the recovery level of C. parvum oocysts declined significantly to 18.1% in seeded tap water, while a relatively high recovery level of 77.2% for G. lamblia cysts could still be achieved. Further study indicated that the recovery level of C. parvum oocysts could be enhanced significantly when an appropriate amount of silica particles was added to a water sample. The recovery level of C. parvum oocysts was affected by particle size and concentration. The optimal silica particle size was determined to be within the range of 5-40 microm, and the corresponding optimal silica concentration was 1.42 g for 10-l tap water. When both G. lamblia cysts and C. parvum oocysts were spiked into the tap water sample containing the optimum amount of silica particles, the average recovery levels of oocysts and cysts were 82.7% and 75.4%, respectively. The results obtained clearly suggested that addition of an appropriate amount of silica particles could improve the recovery level of C. parvum oocysts significantly and yet there was no noticeable deleterious effect on the recovery level of G. lamblia cysts. Further study indicated that the rotation time in the IMS procedure using the Dynal GC-Combo IMS kit (which was recommended in method 1623) was important for G. lamblia cyst detection. In contrast, the recovery level of C. parvum oocysts was not affected by the rotation time. Furthermore, it was found that the recovery levels of C. parvum oocysts using methods 1622 and 1623 were quite close although different IMS kits were used in the two methods.     

Sensitive Detection of Escherichia coli O157:H7 in Food and Water by Immunomagnetic Separation and Solid-Phase Laser Cytometry

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r² = 0.95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r² = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.     

Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.     

Performances of the immunomagnetic separation method for Cryptosporidium in water under various operation conditions.

Immunomagnetic separation (IMS) has been specified as a standard method for the measurement of Cryptosporidium in some countries. In this study, the IMS method was evaluated on the basis of the recovery efficiencies of Cryptosporidium oocysts at various IMS operation conditions. The average recovery for different Cryptosporidium concentrations in deionized water was 82.6 +/- 18.2% (n = 52). No significant change in recovery was observed by altering the debris ratio of the water samples. The efficiency was increased by prolonging the reaction time, and by increasing the amount of immunomagnetic beads. The recoveries of oocysts seeded in an Eppendorf with a small reaction volume were similar to those seeded in glass tubes with 10 times the reaction volume. The recovery efficiency of oocysts was reduced significantly when the reaction buffer was replaced by PBS. In conclusion, this method has good reproducibility and high recovery.     

2′,7′-Bis-(2-carboxyethyl)-5(6)-carboxyfluorescein as a dual-emission fluorescent indicator of intracellular pH suitable for argon laser confocal microscopy

The widely used fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) serves as a pH-sensitive indicator in classical microscopy. Characteristics of BCECF were studied and a way of employing the probe in a confocal laser scanning microscope equipped with an argon laser at 488 nm was developed, based on the fact that the emission fluorescence spectra are pH-dependent with spectral maximum shift from 518 to 529 nm. Optical filters for the dual-emission ratio method were set to 506 and 529 nm. pH values measured inside a single cell of Saccharomyces cerevisiae were similar to those obtained with other pH estimation methods.     

Clams (Corbicula fluminea) as bioindicators of fecal contamination with Cryptosporidium and Giardia spp. in freshwater ecosystems in California

This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank exposure experiments to evaluate clams that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting Cryptosporidium parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.     

Methods for the recovery, isolation and detection of Cryptosporidium oocysts in wastewaters

This correspondence describes the successful development of methods for the recovery, isolation and detection of Cryptosporidium oocysts in wastewater and biosolids. Wastewater from one plant was used to optimize methods in raw influent as well as primary, secondary and tertiary effluents. Raw influents and primary effluents were concentrated using centrifugation followed by isolation of Cryptosporidium oocysts using immunomagnetic separation (IMS) and detection of recovered organisms using epifluorescence microscopy. Mean oocyst recovery in raw influent was 29.2+/-12.8% and 38.8+/-27.9% in primary effluent at three sample volumes tested. Secondary and tertiary effluents were analyzed using a modified Method 1622 resulting in mean oocyst recoveries of 53.0+/-19.2% and 67.8+/-4.4%, respectively. In biosolids with approximately 10% total solids, mean oocyst recovery was 43.9+/-10.1% using IMS with a 5 g (wet weight) sample size. Due to the variability in these matrices, an internal microbiological standard was incorporated to serve as a tool for method performance.     

Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns

We evaluated the MACS immunomagnetic separation (IMS) system for concentrating Cryptosporidium parvum. Oocysts were first labeled with fluorescein isothiocyanate (FITC) or rabbit anti-C. parvum antibodies, then linked to MicroBeads coated with anti-FITC or anti-rabbit IgG, and separated through a high gradient separation column. Results indicated that over 95% of oocysts were recovered and their fluorescence and infectivity were retained. The presence of MicroBeads showed no effect on genomic DNA extraction and subsequent polymerase chain reaction (PCR)-based analyses, as sensitivity of PCR (10 oocysts) and the band pattern of randomly amplified polymorphic DNA (RAPD) were identical to those using DNAs extracted from normally purified oocysts. IMS-PCR consistently detected as few as 10 oocysts from 100 ml of apple juice or homogenized milk and IMS-IFA could detect 100 oocysts from 1 g of deer manure, demonstrating the efficiency of IMS in recovering oocysts from environmental and food samples. Our results suggest that the MACS IMS system could be used for multiple applications in Cryptosporidium research.     

Detection and discrimination of Cryptosporidium parvum and C. hominis in water samples by immunomagnetic separation-PCR

Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.