Isolation Of Oocysts Research Articles

Factors Affecting Recovery Efficiency in Isolation of Cryptosporidium Oocysts and Giardia Cysts from Vegetables for Standard Method Development

While recently published techniques for recovering parasites from fruits and vegetables demonstrate a marked increase in efficiency and utility, there is still scope for further improvement in developing a standard method, particularly with difficult, but important, sample matrices such as bean sprouts. Herein, a number of parameters used in published techniques are investigated more closely. While sample size reduction may improve recovery efficiency because of a range of factors, it is important to keep the sample large enough for detection of low-level contamination. Age of sample is also important, and samples should be as fresh as possible. Elution procedures may contribute to losses of Giardia and should be more thoroughly investigated. Improved immunomagnetic separation techniques currently coming onto the market also have the potential to increase recovery efficiency substantially, even with difficult samples such as aged bean sprouts. However, merely increasing magnetic strength of the capturing magnet does not affect recovery efficiency, which must be reliant on a superior bead system, buffering system, or both.

Risk Assessment for Cryptosporidium: A Hierarchical Bayesian Analysis of Human Dose Response Data

Three dose–response studies were conducted with healthy volunteers using different Cryptosporidium parvum isolates (IOWA, TAMU, and UCP). The study data were previously analyzed for median infectious dose (ID 50) using a simple cumulative percent endpoint method (Reed and Muench, 1938). ID 50s were derived using two definitions of infection: one as subjects having oocysts detected in stool by direct fluorescence assay, and the other by a clinical finding of diarrhea with or without detected oocysts (Chappell et al., 1998; Okhuysen et al., 1999). In the present study, the data were analyzed using the broader definition of infection (i.e., presence of oocysts in stool and/or diarrheal illness characteristic of cryptosporidiosis). Maximum likelihood dose–response parameter estimates for UCP, IOWA, and TAMU were 2980, 190, and 17.5, respectively. Based on these estimates, the ID 50s of the three respective isolates were 2066, 132, and 12.1. The three oocyst isolates were considered representative of a larger population of human-infecting strains and analyzed as combined data using a hierarchical Bayesian model. Hyperparameters defined the distribution of dose–response parameters for the population of strains. Output from Markov Chain Monte Carlo analysis described posterior distributions for the hyperparameters and for the parameters of the IOWA, TAMU, and UCP strains. Point estimates of dose–response parameters produced by this analysis were similar to the maximum likelihood estimates. Finally, the utility of these results for probabilistic risk assessment was evaluated. The risk of infection from single oocyst doses was derived for a mixture of the three isolates (where IOWA, TAMU, or UCP are equally likely), and for an oocyst selected at random from the larger population of strains. These estimated risks of infection were 0.018 and 0.028, respectively.

Recovery of Cryptosporidium oocysts and Giardia cysts from source water concentrates using immunomagnetic separation

Immunomagnetic separation (IMS) procedures for the simultaneous isolation of Cryptosporidium oocysts and Giardia cysts have recently become available. We validated Dynal's GC-Combo IMS kit using source water at three turbidity levels (5000, 500 and 50 nephelometric turbidity units [ntu]) obtained from different geographical locations and spiked with approximately 9–11 (oo)cysts per ml. Mean recoveries of Cryptosporidium oocysts and Giardia cysts in deionized water were 62% and 69%, respectively. In turbid water matrices, mean recoveries of Cryptosporidium oocysts were between 55.9% and 83.1% while mean recoveries of cysts were between 61.1% and 89.6%. Marginally higher recoveries of the heat inactivated (oo)cysts were observed (119.4% Cryptosporidium oocysts and 90.9% Giardia cysts) in deionized water when compared with recoveries of viable (oo)cysts (69.7% Cryptosporidium oocysts and 79% Giardia cysts). Age of (oo)cysts on recoveries using the GC-Combo IMS kit demonstrated no effects up to 20 months old. Recovery of Giardia cysts was consistent for isolates aged up to 8 months (81.4%), however, a significant reduction in recoveries was noted at 20 months age. Recoveries of low levels (5 and 10 (oo)cysts) of Cryptosporidium oocysts and Giardia cysts in deionized water using IMS ranged from 51.3% to 78% and from 47.6% to 90.0%, respectively. Results of this study indicate that Dynal's GC-Combo IMS kit is an efficient technique to separate Cryptosporidium/ Giardia from turbid matrices and yields consistent, reproducible recoveries. The use of fresh (recently voided and purified) (oo)cysts, aged (oo)cysts, viable and heat-inactivated (oo)cysts indicated that these parameters do not influence IMS performance.

Cryptosporidium parvum genotype 2 infections in free-ranging mountain gorillas (Gorilla gorilla beringei) of the Bwindi Impenetrable National Park, Uganda.

For behavioral research and due to growing ecotourism, some populations of free-ranging mountain gorillas (Gorilla gorilla beringei) have become habituated to humans. Molecular analysis of two Cryptosporidium sp. oocyst isolates originating from two human-habituated gorilla groups and two oocyst isolates from non-habituated gorillas yielded positive identification of C. parvum Genotype 2 (G2; i.e., "cattle", "animal-adapted", or "zoonotic"). As G2 is cross-transmissible between humans and animals, C. parvum infections can be propagated in the habitats of human-habituated, free-ranging gorillas through both zoonotic and anthroponotic transmission cycles.

Polymorphic microsatellite markers identified in individual Plasmodium falciparum oocysts from wild-caught Anopheles mosquitoes.

The oocysts of Plasmodiom falciparam that form on the midgut wall of anopheline vectors represent the meiotic products of the sexual cycle of the parasite and are useful for genetic analyses. Recognizing the limitations of current markers for the analysis of genetic variation, we describe a protocol for the DNA isolation and PCR amplification of microsatellite loci from individual oocysts. We present a simple method for the storage and isolation of individual Plasmodium oocysts from wild-caught mosquitoes and demonstrate that it is possible to extract sufficient oocyst DNA for approximately 50 amplifications. Genotyping was successful for 10 microsatellite loci and, using Genescan technology, we found extensive microsatellite polymorphism in an analysis of a dozen oocysts from each of 2 Anopheles gambiae female specimens collected within 6 months at the same location in northern Tanzania. All of the loci exhibited at least 2 alleles, with a maximum of 5. Homozygous and heterozygous oocysts could easily be discerned, but most of the oocysts were homozygous. The procedure has great potential for shedding light on genetic mechanisms operating during meiosis in mosquito vectors and the influence this may have on the genetic structure of natural populations of P. falciparum.

Isolation and Enumeration of Giardia Cysts, Cryptosporidium Oocysts, and Ascaris Eggs from Fruits and Vegetables

Published techniques for recovering parasites from fruit and vegetables are generally inadequate, with low and variable recovery efficiencies. Here we describe an improved methodology for analyzing fruit and vegetables for Giardia cysts, Cryptosporidium oocysts, and Ascaris eggs. The method includes washing procedures, sonication, and, for Giardia and Cryptosporidium, immunomagnetic separation. Identification is by immunofluorescence (Giardia and Cryptosporidium) or brightfield microscopy (Ascaris). Recovery efficiencies from lettuce, Chinese leaves, and strawberries were found to be approximately 67% for Giardia, 42% for Cryptosporidium, and 72% for Ascaris. Recovery efficiencies from bean sprouts tended to be more variable and lower. This could be due to material removed with the parasites during the washing procedures, which, in turn, appeared related to the age of the bean sprouts. It is therefore recommended that fruit and vegetables should be as fresh as possible when analyzed for parasites.

Inter-laboratory comparison of the CD-1 neonatal mouse logistic dose-response model for Cryptosporidium parvum oocysts.

cryptosporidium parvum oocyst viability can be determined by vital dyes, in vitro excystation, and cell culture; however, neonatal mouse infectivity assays are the reference method. Unfortunately, there have been few efforts to standardize methods for infectivity assays thus casting a veil of uncertainty over the significance and comparability of results. In order to address this issue, two laboratories proficient in measuring oocyst infectivity conducted independent dose titration studies with neonatal CD-1 mice using standardized protocols and a well-characterized isolate of Cryptosporidium parvum. The resulting independent logistic dose-response models derived by regression analysis were compared with each other and with a published model. The comparisons showed these dose-response functions to be reproducible under standardized conditions. It is important to standardize mouse strain, age of mice at inoculation and necropsy, oocyst isolate, and age of oocysts. However, other factors, including methods used to detect infectivity and to count oocyst doses, appear less critical. Adopting a standardized assay for oocyst infectivity will provide both a basis for comparing data from various oocyst disinfection studies and a suitable platform for evaluating new or existing in vitro viability surrogates such as excystation, vital dyes or cell culture.

A novel two-color flow cytometric assay for the detection ofCryptosporidium in environmental water samples

Cryptosporidium is an important waterborne pathogen. Detection of Cryptosporidium in concentrated water samples depends on oocyst isolation using immunomagnetic separation (IMS) and/or fluorescence-activated cell sorting (FACS), followed by confirmation using immunofluorescence staining (IFA) and fluorescence microscopy. These methods require highly trained microscopists for oocyst identification and confirmation. Analysis is hampered due to the presence of autofluorescent particles coupled with particles binding nonspecifically with the monoclonal antibodies (mAbs) used for detection. Flow cytometry (FCM) has the potential to be a more specific method for oocyst detection, but such a system would require more than one selection parameter. Various mAbs from commercial suppliers were paired with CRY104-PE and evaluated. The mAb combination that best discriminated stained oocyst from detritus was optimized and compared to Cryptosporidium detection utilizing one-color IFA/FACS. A highly specific two-color assay employing the IgG(1) mAb CRY104 was developed. The assay resulted in reductions, up to 20-fold, in the number of non-Cryptosporidium particles detected. The addition of a second selection parameter improved microscopic analysis times and simplified oocyst confirmation by microscopists. A two-color assay employing competing surface mAbs reduces the number of fluorescent particles sorted, thus improving FCM detection methods for Cryptosporidium.

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.

PCR detection of Cryptosporidium parvum in environmental samples--a review of published protocols and current developments

Since 1991 more than 30 PCR protocols have been published, which show a potential to replace the current microscopic detection method for Cryptosporidium parvum in environmental samples and food. This review provides a synoptic comparison of these protocols with respect to the following features: isolation and purification of oocysts from tested matrices, elimination of free DNA, viability and infectivity assessment, release of nucleic acids, nucleic acid extraction, type of PCR (PCR, RT-PCR, internal-standard-PCR, in situ PCR, TaqMan-PCR), primary product detection, additional specificity control, secondary product detection, reported sensitivity, cross-reaction with other Cryptosporidium species, and target and sequence information such as amplicon length, primer sequences, multiple copy target, presence of strain-specific differences in the amplicon, GenBank accession numbers and gene function. The results demonstrate that problems like PCR inhibition, viability assessment, and the requirement of an extreme sensitivity have been solved. PCR assays would be most valuable to control presence-absence standards in defined matrix volumes, and the setup of such standards would very much contribute to a rapid introduction of this awaited technology into routine monitoring of environmental, water and food samples, and to a further standardization of the various protocols. It can be expected that satisfactory solutions for quantification will be found for a growing number of PCR-based assays. Systematic field evaluation and interlaboratory studies will complement our present knowledge of these methods in the near future.

Waterborne Cryptosporidiosis: Current Status

In the past ten years Cryptosporidium oocysts have been shown to be common contaminants of water, causing at least 19 waterborne outbreaks of cryptosporidiosis which have affected more than 427 000 individuals. Recommended methods for oocyst isolation and enumeration are time-consuming and inefficient and experts state that the absence of Cryptosporidium oocysts in drinking water can never be guaranteed. In the UK alone, a National Research Programme costing pound3 million has been undertaken. Here, Huw Smith and Joan Rose review the current status of knowledge and identify approaches taken by UK and USA Government regulatory bodies in order to reduce the likelihood of waterborne transmission.

Occurrence of Giardia cysts and Cryptosporidium oocysts in raw and treated water from two water treatment plants in Selangor, Malaysia

The occurrence of Giardia cysts and Cryptosporidium oocysts was determined both in the raw water and the treated waters from two water treatment plants in Selangor, Malaysia between July 1994 and January 1995. At each treatment plant, raw and treated water samples were collected fortnightly on 10 separate occasions. Physical parameters, including dissolved oxygen, pH, temperature, conductivity, turbidity and free and total chlorine were measured, and samples were also taken for faecal coliform analyses. The isolation and enumeration of cysts and oocysts was according to UK standard methods. Whilst Giardia cysts were detected in 90% of the raw water samples (range 0–60 cysts per litre) no Cryptosporidium oocysts were detected. Higher concentrations of cysts were detected in the raw water of the first treatment plant. All raw water samples were faecally contaminated, as faecal coliform counts ranged from 4.6 × 10 4 to 1.3 × 10 5 colony forming units per 100 ml. No correlation between the concentration of Giardia cysts and the concentration of faecal coliforms could be demonstrated statistically, nor was there any correlation between the concentration of Giardia cysts and the physical parameters tested for any of the raw waters. No cysts, oocysts or faecal coliforms were detected in samples of treated water.

The status of UK methods for the detection of cryptosporidium spp oocysts and giardia spp cysts in water concentrates

Current UK methods for the isolation and enumeration of Cryptosporidium spp oocysts and Giardia spp cysts in water concentrates are time-consuming and inefficient, providing minimal information regarding the biology of the organism. Minimising the portion of sample concentrate examined can reduce the level of confidence in the result obtained and this issue requires appreciation by the water regulators. Alternative methods for isolation and enumeration require corroboration with the established method using a variety of water types. Developments that increase information regarding the biology of these organisms should be encouraged. Not only does method performance (recovery efficiency, reproducibility etc), but also method significance (integrity of health, operational and regulatory assessments etc) require to be addressed. For Cryptosporidium the fluorogenic viability assay can not only assist in the decisions taken by health officials when oocysts are found in water supplies but also in designing catchment assessment and sampling policies for individual treatment plants, yet few laboratories are experienced in this assay.

A self-filling micropipette for the isolation of single coccidial oocysts

A disposable, self-filling micropipette is described for the isolation of single oocysts of chicken coccidia from laboratory stocks. A sterile 1 μl capillary tube is mounted, using sealing wax, into the narrower end of a broken-off Pasteur pipette. This micropipette is pushed into one end of a 60 cm length of flexible plastic tubing and a 5 cm glass tube is pushed into the other end. The apparatus is used by holding the glass tube in one's mouth, maintaining a positive pressure in the buccal cavity, while the tip of the capillary tube in the micropipette is juxtaposed with a single oocyst in a diluted stock on a cavity slide, observed under a microscope objective. A sudden release of the positive pressure causes the chosen oocyst to be instantaneously taken up into the capillary tube. Using the tube again, the oocyst is then blown out onto a piece of ‘Cellophane’ which is placed in a gelatine capsule. The capsule is administered to a chick in a plastic-film isolator.

The status of UK methods for the detection of Cryptosporidium spp oocysts and Giardia spp cysts in water concentrates

Current UK methods for the isolation and enumeration of Cryptosporidium spp oocysts and Giardia spp cysts in water concentrates are time-consuming and inefficient, providing minimal information regarding the biology of the organism. Minimising the portion of sample concentrate examined can reduce the level of confidence in the result obtained and this issue requires appreciation by the water regulators. Alternative methods for isolation and enumeration require corroboration with the established method using a variety of water types. Developments that increase information regarding the biology of these organisms should be encouraged. Not only does method performance (recovery efficiency, reproducibility etc), but also method significance (integrity of health, operational and regulatory assessments etc) require to be addressed. For Cryprosporidium the fluorogenic viability assay can not only assist in the decisions taken by health officials when oocysts are found in water supplies but also in designing catchment assessment and sampling policies for individual treatment plants, yet few laboratories are experienced in this assay.

Evaluation of Commercial Enzyme Immunoassay (EIA) and Immunofluorescent Antibody (IFA) Test Kits for Detection of Cryptosporidium Oocysts of Species Other than Cryptosporidium parvum

A commercial enzyme immunoassay (EIA) (ProSpect Rapid Assay), a direct immunofluorescence antibody (IFA) test for stool testing (MERIFLUOR Cryptosporidium/Giardia), and an indirect IFA test for environmental testing (Hydrofluor-Combo Cryptosporidium/Giardia) were evaluated for detection of low public health risk Cryptosporidium oocyst isolates, and for C. parvum oocyst isolates from human and bovine feces that represent a high public health risk. There was no cross-reactivity of EIA with ova of eight medically important helminths, three Eimeria species oocysts, Sarcocystis cruzi sporocysts, and two Candida sp. isolates. All nine snake oocyst isolates (C. serpentis), two of seven lizard oocyst isolates, one turtle oocyst isolate, two avian oocyst isolates (turkey, C. meleagridis), one C. wrairi oocyst isolate from guinea pigs, one C. muris oocyst isolate from hyrax, one heifer C. muris isolate, and two C. muris-like oocyst isolates from a camel were positive by both IFA tests; six of these 19 oocyst isolates were EIA-positive. There was no difference in the sensitivity and specificity between direct and indirect IFA tests. The sensitivity of the EIA and both IFA tests to the C. parvum oocysts was 100%. The EIA showed less cross-reactivity with the non-C. parvum oocysts (24%) than direct or indirect IFA (76%), and was less sensitive to those isolates (20%) than both IFA tests (63%). A simulated sampling model for high and low public health risk Cryptosporidium oocysts showed that the low risk oocyst isolates may constitute up to 35% of all positive environmental samples by direct or indirect IFA determination, and up to 12% of all EIA positive samples. This study indicates a superiority of direct and indirect IFA and EIA for screening of human-or-bovine-origin fecal specimens, whereas testing of environmental samples may lead to misidentification of medically important isolates. The results demonstrated that the EIA kit can more accurately identify environmental samples containing oocytes pathogenic for humans than both IFA tests. The specificity of commercially available diagnostic kits to C. parvum should be critically examined for cross-species identification before they are recommended or adopted for use in testing environmental samples.

Studies evaluating the applicability of utilising the same concentration techniques for the detection of protozoan parasites and viruses in water

In this study concentration techniques regularly used for viral detection, i.e. flat-bed ultrafiltration and Filterite cartridge filtration, were evaluated for their efficacies in the recovery of protozoan parasites from water. Recovery of cysts was studied using tap water seeded with Giardia muris cysts and compared to methods designed for the detection of protozoan parasites. Recovery of cysts utilizing 1.2µm membrane filters was 11.1% (4.5-23%) compared to 11.6% (2.7-25.5%) with ultrafiltration (pore size 46-50 Å, with a molecular cut off of 50 000 daltons). Comparison of these methods for the isolation of Giardia cysts and Cryptosporidium oocysts from environmental water samples also indicated a similar efficacy. The recovery of cysts from 1001 of seeded samples using a Cuno wynd cartridge filter was 12.2% (1.6-46%) compared to 13.4% (5-24.2%) using a Filterite cartridge filter. Although the results indicated similar recovery efficacies for these two methods, use of Filterite resulted in a more consistent recovery rate. This study also indicated that the use of cartridge filters for the processing of large volume water samples (1001) showed a slightly better recovery efficacy than the flat-bed filtration technique which limits sample volume to about 101. This study shows that concentration techniques utilised for the isolation of enteric viruses can also be applied for the detection of protozoan parasites from water. This procedure allows for co-analysis of both viruses and protozoan parasites and provides a more rapid and cost-effective evaluation of water quality.

Differentiation between human and animal strains ofCryptosporidium parvumusing isoenzyme typing

Isoenzyme typing was used to study a number of oocyst isolates of Cryptosporidium parvum from different geographical locations and of human or animal origin. All isolates showed identical enzyme motility when glucose phosphate isomerase (GPI; 23 isolates tested) or lactate dehydrogenases (LDH; 20 isolates tested) was assayed. However, two isoenzyme forms were observed with phosphoglucomutase (PGM; 9 animal isolates showed one form, while 8/9 human isolates showed a second form) and hexokinase (HK; 4 human isolates showed one form and 6 animal isolates showed a second form). Thus, PGM and HK each exhibit 2 isoenzymes corresponding to 2 parasite populations associated with separate hosts. The data from this study, plus supportive evidence obtained by different methods and by independent researchers, lend support to the hypothesis that separate cycles of transmission of C. parvum may exist within human and animal hosts.

Studies evaluating the applicability of utilising the same concentration techniques for the detection of protozoan parasites and viruses in water

In this study concentration techniques regularly used for viral detection, i.e. flat-bed ultrafiltration and Filterite cartridge filtration, were evaluated for their efficacies in the recovery of protozoan parasites from water. Recovery of cysts was studied using tap water seeded with Giardia muris cysts and compared to methods designed for the detection of protozoan parasites. Recovery of cysts utilizing 1.2µm membrane filters was 11.1% (4.5–23%) compared to 11.6% (2.7–25.5%) with ultrafiltration (pore size 46–50 A, with a molecular cut off of 50 000 daltons). Comparison of these methods for the isolation of Giardia cysts and Cryptosporidium oocysts from environmental water samples also indicated a similar efficacy. The recovery of cysts from 1001 of seeded samples using a Cuno wynd cartridge filter was 12.2% (1.6–46%) compared to 13.4% (5–24.2%) using a Filterite cartridge filter. Although the results indicated similar recovery efficacies for these two methods, use of Filterite resulted in a more consistent recovery rate. This study also indicated that the use of cartridge filters for the processing of large volume water samples (1001) showed a slightly better recovery efficacy than the flat-bed filtration technique which limits sample volume to about 101.This study shows that concentration techniques utilised for the isolation of enteric viruses can also be applied for the detection of protozoan parasites from water. This procedure allows for co-analysis of both viruses and protozoan parasites and provides a more rapid and cost-effective evaluation of water quality.

Isolation and identification of cryptosporidium parvum oocysts with continuous percoll gradients and combined alcian blue-giemsa staining

The development of special techniques for the detection of Cryptosporidium oocysts in feces contributed significantly to our understanding of human and animal parasitoses by this organism. Garcia et al. (1983) who compared several techniques for the detection of Cryptosporidium oocysts, reported that the most effective method of concentration in their lab was flotation on a Sheather's solution (Sheather, 1923); material thus prepared did not require staining if observed with phase contrast microscopy. Few techniques for the purification of Cryptosporidium oocysts have been developed subsequently. To date, the most widely used of the newer approaches include separation on discontinuous Percoll gradients (Waldman et al., 1986; Arrowood and Sterling, 1987; Kilani and Sekla, 1987) and the use of the parasite concentrator device (FPC) (Evergreen Scientific, Los Angeles) developed by Zierdt (1984). These techniques did not yield good results due to high concentrations of contaminants in the oocyst suspensions. By testing new approaches to oocyst isolation, we found the use of Concanavalin A-Sepharose 4B to be costly, whereas separation with continuous Percoll gradients was faster, and recovered up to 80% of all oocysts present in the initial sample. Of the staining techniques tried to date, the most effective for diagnosis in fecal samples is the modified Zielh-Neelsen method by Bronsdon (1984). Fecal samples containing Cryptosporidium parvum oocysts were obtained from spontaneusly infected calves. For transport to the laboratory, the samples were mixed with an equal volume of 2.5% potassium dichromate. In the laboratory, gross remains were filtered out with nylon nets of decreasing mesh sizes (100 gm, 75 gm,