Detection of Infectious Cryptosporidium parvum Oocysts in Surface and Filter Backwash Water Samples by Immunomagnetic Separation and Integrated Cell Culture-PCR

A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable C. parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious C. parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C. parvum KSU-1 hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C. parvum were detected.     

Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.     

Method for Detection and Enumeration of Cryptosporidium parvum Oocysts in Feces, Manures, and Soils

Eight concentration and purification methods were evaluated to determine percentages of recovery of Cryptosporidium parvum oocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 10², 10³, 10⁴, and 10⁵ oocysts g of bovine feces⁻¹. The percentages of recovery ranged from 10.8% (25 oocysts g⁻¹) to 17.0% (10⁴ oocysts g⁻¹) (r² = 0.996). A conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces⁻¹. Percentages of recovery were determined for six different types of animal feces (cow, horse, pig, sheep, deer, and chicken feces) at a single oocyst concentration (10⁴ oocysts g⁻¹). The percentages of recovery were highest for bovine feces (17.0%) and lowest for chicken feces (3.2%). Percentages of recovery were determined for bovine manure after 3 to 7 days of storage. The percentages of recovery ranged from 1.9 to 3.5% depending on the oocyst concentration, the time of storage, and the dispersing solution. The percentages of oocyst recovery from soils were evaluated by using different flotation solutions (NaCl, cold sucrose, ZnSO₄), different dispersing solutions (Triton X-100, Tween 80, Tris plus Tween 80), different dispersion techniques (magnetic stirring, sonication, blending), and different dispersion times (5, 15, and 30 min). Twenty-five-gram soil samples were used to reduce the spatial variability. The highest percentages of recovery were obtained when we used 50 mM Tris–0.5% Tween 80 as the dispersing solution, dispersion for 15 min by stirring, and saturated NaCl as the flotation solution. The percentages of oocyst recovery from freshly spiked sandy loam, silty clay loam, and clay loam soils were ca. 12 to 18, 8, and 6%, respectively. The theoretical detection limits were ca. 1 to 2 oocysts g of soil⁻¹ depending on the soil type. The percentages of recovery without dispersant (distilled H₂O or phosphate-buffered saline) were less than 0.1%, which indicated that oocysts adhere to soil particles. The percentages of recovery decreased with storage time, although the addition of dispersant (Tris-Tween 80) before storage appeared to partially prevent adhesion. These data indicate that the NaCl flotation method is suitable for routine detection and enumeration of oocysts from feces, manures, soils, or soil-manure mixtures.     

Influence of Pretreatment and Experimental Conditions on Electrophoretic Mobility and Hydrophobicity of Cryptosporidium parvum Oocysts

Surface properties of Cryptosporidium parvum oocysts were investigated by using electrophoretic mobility and hydrophobicity measurements. Oocysts purified from calf feces by several sucrose flotation steps and deionized water (DI) washes (DIS method) had an electrophoretic mobility (neutral surface charge) near 0.0 m² V⁻¹ s⁻¹ over a pH range of 2 to 10. The mean electrophoretic mobility of oocysts stored in DI containing a mixture of antibiotics had a lower standard deviation (ς = 0.36) than that of oocysts stored in DI without antibiotics (ς = 0.53); their electrophoretic mobility remained unchanged up to 121 days after collection. The electrophoretic mobility of oocysts purified on a cold Percoll-sucrose gradient after the feces was defatted with ethyl acetate (EAPS method) varied linearly with pH from 0.0 m² V⁻¹ s⁻¹ at pH 2.4 to −3.2 × 10⁻⁸ m² V⁻¹ s⁻¹ at pH 10 (ς = 0.52), thus displaying the negative surface charge at neutral pH observed by other researchers. The hydrophobicity of oocysts and two types of polystyrene beads was measured as a function of ionic strength by adhesion to polystyrene. Oocysts were purified by the DIS method. The ionic strength of the suspending solution was varied from 0 to 95 mmol liter⁻¹. Two-week-old oocysts exhibited strong adhesion (~85%) at ionic strengths of 0 to 10 mmol liter⁻¹ and moderate adhesion (~20%) at ionic strengths of 20 to 95 mmol liter⁻¹. Two-month-old oocysts exhibited high adhesion (~60 to 80%) at all ionic strengths. These results show that adhesion properties governed by the electrophoretic mobility of purified C. parvum oocysts can be altered by the method of purification and that hydrophobicity can change as oocysts age.     

Decrease in Cryptosporidium parvum Oocyst Infectivity In Vitro by Using the Membrane Filter Dissolution Method for Recovering Oocysts from Water Samples

Exposure of Cryptosporidium parvum oocysts to solutions used for cellulose acetate membrane (CAM) dissolution filtration reduced their infectivity in HCT-8 cells. Ethanol (95% [vol/vol] and 70% [vol/vol]) alone and short exposure times to acetone decreased infectivity. These findings contrast with similar experiments using excystation assays and infectivity in mice.     

Depletion of Cryptosporidium parvum Oocysts from Contaminated Sewage by Using Freshwater Benthic Pearl Clams (Hyriopsis schlegeli)

The freshwater benthic pearl clam, Hyriopsis schlegeli, was experimentally exposed to Cryptosporidium parvum oocysts, and it was verified that the oocysts were eliminated predominantly via the fecal route, retaining their ability to infect cultured cells (HCT-8). The total fecal oocyst elimination rate was more than 90% within 5 days after exposure to the oocysts. H. schlegeli was able to survive in the final settling pond of a sewage plant for long periods, as confirmed by its pearl production. In the light of these findings, the clam was placed in the final settling pond in a trial to test its long-term efficacy in depleting oocysts contaminating the pond water. The number of clams placed was set to ensure a theoretical oocyst removal rate of around 50%, and the turbidity and the density of feed microbes in the overflow trough water of the pond were about 35% and 40 to 60% lower, respectively, than in the control water throughout the year. It was found that the clam feces containing oocysts were sufficiently heavy for them to settle to the bottom of the pond, despite the upward water flow. From these results, we concluded that efficient depletion of oocysts in the sewage water of small or midscale sewage treatment plants can be achieved by appropriate placement of H. schlegeli clams.     

Impact of Zooplankton Grazing on the Excystation, Viability, and Infectivity of the Protozoan Pathogens Cryptosporidium parvum and Giardia lamblia

Very little is known about the ability of the zooplankton grazer Daphnia pulicaria to reduce populations of Giardia lamblia cysts and Cryptosporidium parvum oocysts in surface waters. The potential for D. pulicaria to act as a biological filter of C. parvum and G. lamblia was tested under three grazing pressures (one, two, or four D. pulicaria grazers per 66 ml). (Oo)cysts (1 × 10⁴ per 66 ml) were added to each grazing bottle along with the algal food Selenastrum capricornutum (6.6 × 10⁴ cells per 66 ml) to stimulate normal grazing. Bottles were rotated (2 rpm) to prevent settling of (oo)cysts and algae for 24 h (a light:dark cycle of 16 h:8 h) at 20°C. The impact of D. pulicaria grazing on (oo)cysts was assessed by (i) (oo)cyst clearance rates, (ii) (oo)cyst viability, (iii) (oo)cyst excystation, and (iv) oocyst infectivity in cell culture. Two D. pulicaria grazers significantly decreased the total number of C. parvum oocysts by 52% and G. lamblia cysts by 44%. Furthermore, two D. pulicaria grazers significantly decreased C. parvum excystation and infectivity by 5% and 87%, respectively. Two D. pulicaria grazers significantly decreased the viability of G. lamblia cysts by 52%, but analysis of G. lamblia excystation was confounded by observed mechanical disruption of the cysts after grazing. No mechanical disruption of the C. parvum oocysts was observed, presumably due to their smaller size. The data provide strong evidence that zooplankton grazers have the potential to substantially decrease the population of infectious C. parvum and G. lamblia in freshwater ecosystems.     

Occurrence of Cryptosporidium and Giardia in Wild Ducks along the Rio Grande River Valley in Southern New Mexico

Fecal samples were taken from wild ducks on the lower Rio Grande River around Las Cruces, N. Mex., from September 2000 to January 2001. Giardia cysts and Cryptosporidium oocysts were purified from 69 samples by sucrose enrichment followed by cesium chloride (CsCl) gradient centrifugation and were viewed via fluorescent-antibody (FA) staining. For some samples, recovered cysts and oocysts were further screened via PCR to determine the presence of Giardia lamblia and Crytosporidium parvum. The results of this study indicate that 49% of the ducks were carriers of Cryptosporidium, and the Cryptosporidium oocyst concentrations ranged from 0 to 2,182 oocysts per g of feces (mean ± standard deviation, 47.53 ± 270.3 oocysts per g); also, 28% of the ducks were positive for Giardia, and the Giardia cyst concentrations ranged from 0 to 29,293 cysts per g of feces (mean ± standard deviation, 436 ± 3,525.4 cysts per g). Of the 69 samples, only 14 had (oo)cyst concentrations that were above the PCR detection limit. Samples did test positive for Cryptosporidium sp. However, C. parvum and G. lamblia were not detected in any of the 14 samples tested by PCR. Ducks on their southern migration through southern New Mexico were positive for Cryptosporidium and Giardia as determined by FA staining, but C. parvum and G. lamblia were not detected.     

Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts

Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.     

Patterns of Cryptosporidium Oocyst Shedding by Eastern Grey Kangaroos Inhabiting an Australian Watershed

The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 × 10⁶ oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum “bovine” genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium “marsupial” genotype I or “marsupial” genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.     

Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy

To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ·cm⁻² doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ·cm⁻². With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ·cm⁻² of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ·cm⁻² of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.     

Chlorine Dioxide Inactivation of Cryptosporidium parvum Oocysts and Bacterial Spore Indicators

Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number–cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg · min/liter were needed to inactivate approximately 0.5 log₁₀ and 2.0 log₁₀ units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg · min/liter were required to achieve approximately 2.0 log₁₀ units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity.     

Risk of Handling as a Route of Exposure to Infectious Waterborne Cryptosporidium parvum Oocysts via Atlantic Blue Crabs (Callinectes sapidus)

Commercial Atlantic blue crabs (Callinectes sapidus) were exposed to 2.0 × 10⁴ infectious waterborne oocysts of Cryptosporidium parvum. The study demonstrated that blue crabs can transfer C. parvum oocysts to persons involved in handling or preparing crabs and that they may contaminate other surfaces or products during storage.     

Improved Quantitative Estimates of Low Environmental Loading and Sporadic Periparturient Shedding of Cryptosporidium parvum in Adult Beef Cattle

Our primary goal was to generate an accurate estimate of the daily environmental loading rate of Cryptosporidium parvum oocysts for adult beef cattle, using immunomagnetic separation coupled with direct immunofluorescence microscopy for a highly sensitive diagnostic assay. An additional goal was to measure the prevalence and intensity of fecal shedding of C. parvum oocysts in pre- and postparturient cows as an indicator of their potential to infect young calves. This diagnostic method could detect with a ≥90% probability oocyst concentrations as low as 3.2 oocysts g of feces⁻¹, with a 54% probability of detecting just one oocyst g of feces⁻¹. Using this diagnostic method, the overall apparent prevalence of adult beef cattle testing positive for C. parvum was 7.1% (17 of 240), with 8.3 and 5.8% of cattle shedding oocysts during the pre- and postcalving periods, respectively. The mean intensity of oocyst shedding for test-positive cattle was 3.38 oocysts g of feces⁻¹. The estimated environmental loading rate of C. parvum ranged from 3,900 to 9,200 oocysts cow⁻¹ day⁻¹, which is substantially less than a previous estimate of 1.7 × 10⁵ oocysts cow⁻¹ day⁻¹ (range of 7.7 × 10⁴ to 2.3 × 10⁵ oocysts cow⁻¹ day⁻¹) (B. Hoar, E. R. Atwill, and T. B. Farver, Quant. Microbiol. 2:21-36, 2000). Use of this highly sensitive assay functioned to detect a greater proportion of low-intensity shedders in our population of cattle, which reduced the estimated mean intensity of shedding and thereby reduced the associated environmental loading rate compared to those of previous studies.     

Method Detection Limits of PCR and Immunofluorescence Assay for Cryptosporidium parvum in Soil

We determined and compared the method detection limits (MDL_α) of a PCR and an immunofluorescence assay (IFA) for detection of Cryptosporidium parvum oocysts in soils. Based on the MDL_α and the quantitative nature and stability of the IFA, PCR analysis is not a useful screening step for soil studies of oocyst transport.     

Recovery and Enumeration of Cryptosporidium parvum from Animal Fecal Matrices

Accurate quantification of Cryptosporidium parvum oocysts in animal fecal deposits on land is an essential starting point for estimating watershed C. parvum loads. Due to the general poor performance and variable recovery efficiency of existing enumeration methods, protocols were devised based on initial dispersion of oocysts from feces by vortexing in 2 mM tetrasodium pyrophosphate, followed by immunomagnetic separation. The protocols were validated by using an internal control seed preparation to determine the levels of oocyst recovery for a range of fecal types. The levels of recovery of 10² oocysts from cattle feces (0.5 g of processed feces) ranged from 31 to 46%, and the levels of recovery from sheep feces (0.25 g of processed feces) ranged from 21% to 35%. The within-sample coefficients of variation for the percentages of recovery from five replicates ranged from 10 to 50%. The ranges for levels of recovery of oocysts from cattle, kangaroo, pig, and sheep feces (juveniles and adults) collected in a subsequent watershed animal fecal survey were far wider than the ranges predicted by the validation data. Based on the use of an internal control added to each fecal sample, the levels of recovery ranged from 0 to 83% for cattle, from 4 to 62% for sheep, from 1 to 42% for pigs, and from 40 to 73% for kangaroos. Given the variation in the levels of recovery of oocysts from different fecal matrices, it is recommended that an internal control be added to at least one replicate of every fecal sample analyzed to determine the percentage of recovery. Depending on the animal type and based on the lowest approximate percentages of recovery, between 10 and 100 oocysts g of feces⁻¹ must be present to be detected.     

Parasitism and fitness of invaders: oligochaete Chaetogaster limnaei produces gill damage and increased respiration rates in freshwater Asian clams

Biological interactions between invasive and resident species are perceived as a key factor mediating impacts in invaded ecosystems, although symbiotic interactions have received relatively little attention. To evaluate the association between the invasive clam Corbicula fluminea and its oligochaete symbiont Chaetogaster limnaei, we conducted laboratory assessments of prevalence and intensity of infection, external and internal macroscopic alterations, and histological analyses of the gills of uninfected and infected clams collected from the shores of the Río de la Plata estuary in Argentina. To evaluate potential ramifications of physical alterations on physiological traits and host fitness, we conducted respiration experiments on the same clams. Worm prevalence was higher in smaller than large clams. While no macroscopical alterations were detected, several histopathological changes were observed in the gills of infected clams. Infected clams had significantly higher metabolic rates than uninfected conspecifics and smaller clams had higher metabolic rates than larger conspecifics. Increased host metabolic rates, even at very low infections intensities, were likely a compensatory response to the stress caused by gill damaged inflicted by the oligochaetes. The relationship between C. limnaei and invasive C. fluminea in Río de la Plata best fits as parasitism.

     

Evaluation of Two DNA Template Preparation Methods for Post-Immunomagnetic Separation Detection of Cryptosporidium parvum in Foods and Beverages by PCR

Cryptosporidium parvum oocysts were recovered by immunomagnetic separation from six artificially contaminated foods. Two DNA isolation methods were subsequently evaluated by PCR. The FTA Concentrator-PS filter provided rapid and reproducible detection, although variability increased at lower inoculum levels (88% and 15% detection in high- and low-inoculum-level samples, respectively). Total DNA extraction generated consistent results at all oocyst levels but resulted in longer analysis time (100% and 59% detection in high- and low-inoculum-level samples, respectively). Also reflected in this study was that the matrix played an important role in the ability to recover oocysts, as sample turbidity, pH, and PCR inhibitors all influenced detection.     

Insect herbivory on native and exotic aquatic plants: phosphorus and nitrogen drive insect growth and nutrient release

The invasive clam Corbicula fluminea causes severe environmental and economic impacts in invaded sites and fouled water-dependent industries. The biological control of invasive species has potential as an effective, safe and low-cost tool. The potential of using direct (clam consumption) and indirect approaches (predator-avoidance behaviour) to control the pest was evaluated, aiming at establishing a proof-of-principle regarding the suitability of such strategies in confined settings (e.g. pipelines, channels, raw water tanks and other water-dependent facilities). Invertebrate (Procambarus clarkii) and vertebrate (Lepomis gibbosus, Luciobarbus bocagei) predators were examined for their control potential. In feeding experiments, barbels foraged upon C. fluminea but ingested very few clams, and clam consumption by crayfish was only observed in the smallest size class (<1 cm). Additional experiments linked these results to the protective role of the clam’s shell. In practical terms, the potential of the fish species as C. fluminea predators is limited, but crayfish can be important predators of small clams. In a second set of experiments, putative predator avoidance by clams was assessed in dual-choice aquaria. Refuge availability, predator diet and light conditions were taken into account. Clam avoidance was observed under particular conditions, but the amplitude of dislocation was limited.     

Immunomagnetic Separation of Cryptosporidium parvum from Source Water Samples of Various Turbidities

Immunomagnetic separation (IMS) procedures which specifically capture Cryptosporidium oocysts and have the potential to isolate oocysts from debris have become commercially available. We compared two IMS kits (kit DB [Dynabeads anti-Cryptosporidium; product no. 730.01; Dynal A.S., Oslo, Norway] and kit IC1 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC, Portland, Maine]) and a modification of kit IC1 (kit IC2 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC]) at three turbidity levels (50, 500, and 5,000 nephelometric turbidity units [ntu]) by using water matrices obtained from different geographical locations. In deionized water, kit DB yielded recoveries between 68 and 83%, whereas the recoveries obtained with kits IC1 and IC2 were more variable and ranged from 0.2 to 74.5%. In water matrices with turbidity levels up to 500 ntu, the oocyst recoveries were more variable with kit DB; however, the recoveries were similar to those obtained in deionized water. In contrast, there were notable reductions in oocyst recoveries in the turbid matrices with kits IC1 and IC2, and the highest recovery (8.3%) was obtained with a 50-ntu sample. An examination of the effects of age on oocyst recovery with kit DB revealed that oocysts up to 16 weeks old yielded recoveries similar to the recoveries observed with fresh oocysts. These data indicate that all IMS kits do not perform equally well, and it is important to conduct in-house quality assurance work before a commercially available IMS kit is selected to replace flotation procedures for recovery of Cryptosporidium oocysts.