Simple and rapid measurement of Cryptosporidium excystation using flow cytometry

Vesey, Graham; Griffiths, Katherine; Gauci, Mark; Deere, Daniel; Williams, Keith L.; Veal, Duncan

doi:10.1016/s0020-7519(97)00085-4

Cited by 31 publications

(37 citation statements)

References 18 publications

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“…C. parvum oocyst walls were purified from excysted oocysts using immunomagnetic separation (IMS). Freshly purified oocysts were excysted (16), and the percentage of excystation was determined by flow cytometry (25). Only samples with Ͼ99.5% empty oocysts were processed further.…”

Section: Methodsmentioning

confidence: 99%

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An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts

Weir¹,

Vesey²,

Slade

et al. 2000

Clin Diagn Lab Immunol

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The detection of Cryptosporidium oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to Cryptosporidium oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of Cryptosporidium oocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG 1 monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies.

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Section: Methodsmentioning

confidence: 99%

“…All currently available Cryptosporidium-specific MAbs are either of the immunoglobulin M (IgM) or IgG3 subclasses (25), which have been developed for detection in fecal material rather than for water analysis. IgM MAbs are known to be larger and more "sticky" than MAbs of the IgG1 subclass (18).…”

mentioning

confidence: 99%

An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts

Weir¹,

Vesey²,

Slade

et al. 2000

Clin Diagn Lab Immunol

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“…Flow cytometry, a rapid and simple alternative to microscopy, was used to detect viable oocysts and to document experimental parasite loads (2,25,26). In this model, high-yield parasite amplification was useful for evaluation of the maximal infectivity risk, since the estimated sensitivity was as low as 1 to 10 viable oocysts.…”

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confidence: 99%

Quantitative Flow Cytometric Evaluation of Maximal Cryptosporidium parvum Oocyst Infectivity in a Neonate Mouse Model

Delaunay

Gargala

Li³

et al. 2000

Appl Environ Microbiol

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The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.Cryptosporidium parvum is presently identified as a common cause of diarrhea in immunocompetent individuals. In immunodeficient individuals, cryptosporidiosis may lead to lifethreatening chronic diarrhea, and, because of the incidence of AIDS, the disease poses a significant public health problem in developing countries where AIDS is endemic (8,14,17,22).Recent outbreaks of cryptosporidiosis support the concern about C. parvum oocyst contamination of treated and surface water (15). In Sydney, Australia, from July to September 1998, contamination of the drinking water supply involved over 3,000,000 residents (16).Water oocyst count is a commonly used parameter to evaluate the infectious risk. However, the significance of oocyst numbers is questionable, since storage duration and environmental conditions, such as pH, temperature, and/or the presence of oxidants, are likely to influence oocyst viability (5, 10, 18). Moreover, factors such as salinity, temperature, or storage duration may not decrease the infectivity enough to prevent infection in susceptible individuals (11,12).Oocyst viability is currently estimated by the quantitation of in vitro excystation rates or by incorporation of nucleic acid dyes (7). However, dyeing is influenced by the degree of oocyst permeabilization and may not reflect parasite infectivity (3,21). Oocyst infectivity can be evaluated by monitoring in vitro parasite development in highly permissive cells (9,13,24). In vivo, C. parvum infection is usually investigated using susceptible animal models such as immunocompromised or neonatal mice (19).The aim of this work was to assess C. parvum oocyst infectivity using a suckling mouse model (6). Flow cytometry, a rapid and simple alternative to microscopy, was used to detect viable oocysts and to docume...

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“…Flow cytometry with fluorescence-activated cell sorting (FACS) is used increasingly as an alternative method for separation and enumeration, but difficulties are encountered when autofluorescent algae are present and cross-reactions of the MAbs lead to unspecific binding (2,8,48). Furthermore, expensive and difficult-to-operate cell sorters are required, which inhibits wide application.…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection and Enumeration of Giardia lamblia Cysts in Water Samples by Immunomagnetic Separation and Flow Cytometric Analysis

Keserue

Füchslin

Egli

2011

Appl Environ Microbiol

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Giardia lamblia is an important waterborne pathogen and is among the most common intestinal parasites of humans worldwide. Its fecal-oral transmission leads to the presence of cysts of this pathogen in the environment, and so far, quantitative rapid screening methods are not available for various matrices, such as surface waters, wastewater, or food. Thus, it is necessary to establish methods that enable reliable rapid detection of a single cyst in 10 to 100 liters of drinking water. Conventional detection relies on cyst concentration, isolation, and confirmation by immunofluorescence microscopy (IFM), resulting in low recoveries and high detection limits. Many different immunomagnetic separation (IMS) procedures have been developed for separation and cyst purification, so far with variable but high losses of cysts. A method was developed that requires less than 100 min and consists of filtration, resuspension, IMS, and flow cytometric (FCM) detection. MACS MicroBeads were used for IMS, and a reliable flow cytometric detection approach was established employing 3 different parameters for discrimination from background signals, i.e., green and red fluorescence (resulting from the distinct pattern emitted by the fluorescein dye) and sideward scatter for size discrimination. With spiked samples, recoveries exceeding 90% were obtained, and false-positive results were never encountered for negative samples. Additionally, the method was applicable to naturally occurring cysts in wastewater and has the potential to be automated.

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Simple and rapid measurement of Cryptosporidium excystation using flow cytometry

Cited by 31 publications

References 18 publications

An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts

An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts

Quantitative Flow Cytometric Evaluation of Maximal Cryptosporidium parvum Oocyst Infectivity in a Neonate Mouse Model

Rapid Detection and Enumeration of Giardia lamblia Cysts in Water Samples by Immunomagnetic Separation and Flow Cytometric Analysis

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