Determination of the Recovery Efficiency of Cryptosporidium Oocysts and Giardia Cysts from Seeded Bivalve Mollusks

2013 ◽  
Vol 76 (1) ◽  
pp. 93-98 ◽  
Author(s):  
FRANCISKA M. SCHETS ◽  
HAROLD H. J. L. van den BERG ◽  
ANA MARIA de RODA HUSMAN
Keyword(s):  
Quantitative Assessment ◽  
Intestinal Parasites ◽  
Cryptosporidium Oocyst ◽  
Immunomagnetic Separation ◽  
Giardia Cyst ◽  
Bivalve Mollusks ◽  
Recovery Efficiency ◽  
Risk Of Infection ◽  
Cryptosporidium Oocysts

The intestinal parasites Cryptosporidium and Giardia are transmitted by water and food and cause human gastroenteritis. Filter-feeding bivalve mollusks, such as oysters and mussels, filter large volumes of water and thus concentrate such pathogens, which makes these bivalves potential vectors of disease. To assess the risk of infection from consumption of contaminated bivalves, parasite numbers and parasite recovery data are required. A modified immunomagnetic separation (IMS) procedure was used to determine Cryptosporidium oocyst and Giardia cyst numbers in individually homogenized oysters (Crassostrea gigas) and mussels (Mytilus edulis). About 12% of the commercial bivalves were positive, with low (oo)cyst numbers per specimen. The recovery efficiency of the IMS procedure was systematically evaluated. Experiments included seeding of homogenized bivalves and whole animals with 100 to 1,000 (oo)cysts. Both seeding procedures yielded highly variable recovery rates. Median Cryptosporidium recoveries were 7.9 to 21% in oysters and 62% in mussels. Median Giardia recoveries were 10 to 25% in oysters and 110% in mussels. Giardia recovery was significantly higher than Cryptosporidium recovery. (Oo)cysts were less efficiently recovered from seeded whole animals than from seeded homogenates, with median Cryptosporidium recoveries of 5.3% in oysters and 45% in mussels and median Giardia recoveries of 4.0% in oysters and 82% in mussels. Both bivalve homogenate seeding and whole animal seeding yielded higher (oo)cyst recovery in mussels than in oysters, likely because of the presence of less shellfish tissue in IMS when analyzing the smaller mussels compared with the larger oysters, resulting in more efficient (oo)cyst extraction. The data generated in this study may be used in the quantitative assessment of the risk of infection with Cryptosporidium or Giardia associated with the consumption of raw bivalve mollusks. This information may be used for making risk management decisions.

Comparison of methods for the concentration ofCryptosporidiumoocysts andGiardiacysts from raw waters

2004 ◽  
Vol 50 (9) ◽  
pp. 675-682 ◽  
Author(s):  
Christobel Ferguson ◽  
Christine Kaucner ◽  
Martin Krogh ◽  
Daniel Deere ◽  
Malcolm Warnecke
Keyword(s):  
Internal Control ◽  
Membrane Filtration ◽  
Immunomagnetic Separation ◽  
The Other ◽  
Recovery Efficiency ◽  
Comparison Of Methods ◽  
Raw Water ◽  
High Recovery ◽  
Cryptosporidium Oocysts ◽  
Giardia Cysts

This study compared the recovery of Cryptosporidium oocysts and Giardia cysts ((oo)cysts) from raw waters using 4 different concentration–elution methods: flatbed membranes, FiltaMaxTMfoam, EnvirochekTMHV capsules, and Hemoflow ultrafilters. The recovery efficiency of the combined immunomagnetic separation and staining steps was also determined. Analysis of variance of arcsine-transformed data demonstrated that recovery of Cryptosporidium oocysts by 2 of the methods was statistically equivalent (flatbed filtration 26.7% and Hemoflow 28.3%), with FiltaMaxTMand EnvirochekTMHV recoveries significantly lower (18.9% and 18.4%). Recovery of Giardia cysts was significantly higher using flatbed membrane filtration (42.2%) compared with the other 3 methods (EnvirochekTMHV 29.3%, FiltaMaxTM29.0%, and Hemoflow 20.9%). All methods were generally acceptable and are suitable for laboratory use; 2 of the methods are also suitable for field use (FiltaMaxTMand EnvirochekTMHV). In conclusion, with recoveries generally being statistically equivalent or similar, practical considerations become important in determining which filters to use for particular circumstances. The results indicate that while low-turbidity or "finished" waters can be processed with consistently high recovery efficiencies, recoveries from raw water samples differ significantly with variations in raw water quality. The use of an internal control with each raw water sample is therefore highly recommended.Key words: catchments, EnvirochekTMHV, Hemoflow, FiltaMaxTM, flatbed filtration.

Analysis of Cured Meat Products for Cryptosporidium Oocysts following Possible Contamination during an Extensive Waterborne Outbreak of Cryptosporidiosis

2012 ◽  
Vol 75 (5) ◽  
pp. 982-988 ◽  
Author(s):  
LUCY J. ROBERTSON ◽  
QIRONG HUANG
Keyword(s):  
Cryptosporidium Oocyst ◽  
Meat Products ◽  
Recovery Efficiency ◽  
Northern Sweden ◽  
High Fat ◽  
Simple Method ◽  
Waterborne Outbreak ◽  
Cryptosporidium Oocysts ◽  
Cured Meat ◽  
Food Matrices

An outbreak of waterborne cryptosporidiosis in a town in northern Sweden during winter 2010 resulted in the potential exposure of cured meat products to Cryptosporidium oocysts during their manufacture. The purpose of this work was to develop a method for analyzing cured meat products for contamination with Cryptosporidium oocysts and use this method to analyze potentially contaminated product samples. A simple method of elution, concentration, separation, and detection was used, based on work with other food matrices but adapted for the relatively high fat content of cured meat surfaces. Using spiking experiments, the recovery efficiency of this method was found to be over 60%. In the analysis of the potentially contaminated products, only one putative Cryptosporidium oocyst was detected, and this was sufficiently deformed so that it could not be confirmed as an oocyst; if it was an oocyst, it was considered to have been probably deformed and inactivated prior to analysis. Based on the results of the analyses, together with data on the probable extent of contamination of the products and on our knowledge of factors, such as water activity, which affect oocyst survival, the products were safely released to the market.

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

2001 ◽  
Vol 64 (11) ◽  
pp. 1799-1805 ◽  
Author(s):  
L. J. ROBERTSON ◽  
B. GJERDE
Keyword(s):  
Sample Size ◽  
Standard Method ◽  
Fruits And Vegetables ◽  
Method Development ◽  
Immunomagnetic Separation ◽  
Recovery Efficiency ◽  
Factors Affecting ◽  
Cryptosporidium Oocysts ◽  
Magnetic Strength ◽  
Bean Sprouts

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.

scholarly journals Removal of bacteria, protozoa and viruses through a multiple-barrier household water disinfection system

2013 ◽  
Vol 12 (1) ◽  
pp. 94-104 ◽  
Author(s):  
A. C. Espinosa-García ◽  
C. Díaz-Ávalos ◽  
R. Solano-Ortiz ◽  
M. A. Tapia-Palacios ◽  
N. Vázquez-Salvador ◽  
...  
Keyword(s):  
Mexico City ◽  
Cryptosporidium Oocyst ◽  
Water Disinfection ◽  
Giardia Cyst ◽  
E Coli ◽  
Water Characteristics ◽  
Removal Capacity ◽  
Household Water ◽  
Cryptosporidium Oocysts ◽  
Barrier Device

Municipal water disinfection systems in some areas are not always able to meet water consumer needs, such as ensuring distributed water quality, because household water management can be a contributing factor in water re-contamination. This fact is related to the storage options that are common in places where water is scarce or is distributed over limited time periods. The aim of this study is to assess the removal capacity of a multiple-barrier water disinfection device for protozoa, bacteria, and viruses. Water samples were taken from households in Mexico City and spiked with a known amount of protozoa (Giardia cyst, Cryptosporidium oocyst), bacteria (Escherichia coli), and viruses (rotavirus, adenovirus, F-specific ribonucleic acid (FRNA) coliphage). Each inoculated sample was processed through a multiple-barrier device. The efficiency of the multiple-barrier device to remove E. coli was close to 100%, and more than 87% of Cryptosporidium oocysts and more than 98% of Giardia cysts were removed. Close to 100% of coliphages were removed, 99.6% of the adenovirus was removed, and the rotavirus was almost totally removed. An effect of site by zone was detected; this observation is important because the water characteristics could indicate the efficiency of the multiple-barrier disinfection device.

scholarly journals Enumeration of Cryptosporidium spp. in Water with U.S. EPA Method 1622

2000 ◽  
Vol 83 (5) ◽  
pp. 1108-1114 ◽  
Author(s):  
James L Sinclair
Keyword(s):  
Environmental Protection Agency ◽  
Fluorescent Antibody ◽  
Cryptosporidium Oocyst ◽  
Immunomagnetic Separation ◽  
Dapi Staining ◽  
Cell Structures ◽  
Cryptosporidium Oocysts ◽  
Additional Cell ◽  
Sparse Methods ◽  
Made In

Abstract The occurrence of Cryptosporidium parvum or other pathogenic Cryptosporidium species in water must be known in order to assess risk and determine the treatment needed to reduce Cryptosporidium oocysts to acceptable levels in finished drinking water. Because Cryptosporidium oocyst occurrence may be sparse, methods must concentrate a large volume of water and correctly identify oocysts in the concentrate. The U.S. Environmental Protection Agency Information Collection Rule (ICR) protozoan method gives low and variable recoveries of Cryptosporidium oocysts, making risk assessment difficult. Therefore, a method giving better oocyst recovery and more consistent results was needed. Method 1622 was developed with existing materials and procedures, and improvements were made in filtration, cleanup, and detection. Absolute porosity filters were used, with cleanup by immunomagnetic separation and detection by direct fluorescent antibody stain with 4′,6-diamidino-2-phenylindole (DAPI) staining for additional cell structures. Both the level and consistency of oocyst recovery were improved compared to recovery with the ICR method.

Immunomagnetic separation of Cryptosporidium oocysts from water samples: round robin comparison of techniques

1997 ◽  
Vol 35 (11-12) ◽  
pp. 397-401 ◽  
Author(s):  
A. Campbell ◽  
H. Smith
Keyword(s):  
Water Samples ◽  
Immunomagnetic Separation ◽  
Clean Water ◽  
Recovery Efficiency ◽  
Blue Book ◽  
Negative Results ◽  
Cryptosporidium Oocysts ◽  
Comparison Of Techniques ◽  
User Friendly ◽  
Multiple Samples

A prototype immunomagnetic separation (IMS) technique was tested in five laboratories, which undertake routine analysis of water samples for Cryptosporidium oocysts, by comparing the recovery efficiency of the IMS technique with techniques in current use (the “Blue Book” Standing Committee of Analysts, SCA, method and flow cytometry, FCM). In very low turbidity samples (clean waters) of both 1ml and 10ml volumes the IMS demonstrated significantly better results than both SCA and FCM methods. Not only were higher oocyst recovery efficiencies reported but variation in recovery efficiency was reduced and fewer negative results were reported from oocyst-seeded samples than with the other two techniques. In trials with clean water or low turbidity water, FCM was the technique which most consistently reported negative results in oocyst-seeded samples and for clean water this difference was found to be statistically significant. When the water sample was turbid the recovery efficiency of the IMS technique diminished. The results suggest that the IMS technique is affected to different extents by different material constituents in water concentrates and that FCM is apparently less affected by interfering particulate matter. Despite the potential difficulties with the IMS method with turbid water samples, the results from these trials indicate that this technique would be a very useful addition to the armoury of methods for the concentration of oocysts from water samples and was considered by the trial participants to be simple, user-friendly and applicable to the processing of multiple samples simultaneously.

Cryptosporidium spp. and Giardia spp. in surface water supply of Campinas, Southeast Brazil

2010 ◽  
Vol 62 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Romeu Cantusio Neto ◽  
Luciana Urbano dos Santos ◽  
Maria Ines Zanoli Sato ◽  
Regina Maura Bueno Franco
Keyword(s):  
Surface Water ◽  
Membrane Filtration ◽  
Cryptosporidium Oocyst ◽  
Treatment Plant ◽  
Water Treatment Plant ◽  
Immunomagnetic Separation ◽  
Immunofluorescence Assay ◽  
Giardia Cyst ◽  
Filtration Method ◽  
Cryptosporidium Spp

Surface water contaminated by domestic sewage discharges is a potential source of pathogens, including protozoa. During 2005–2006, the source water (Atibaia River) of the Surface Water Treatment Plant (WTP) of Campinas city, São Paulo, Brazil was sampled to obtain an assessment of Cryptosporidium oocyst and Giardia cyst concentrations. Calcium carbonate flocculation (CCF) and membrane filtration (MF) concentration techniques, with and without purification by immunomagnetic separation (IMS) were evaluated. The cysts and oocysts were detected by immunofluorescence assay (IFA) and confirmed by differential interference contrast (DIC). Membrane filtration method generally produced higher recovery efficiency. Giardia spp. was detected in 87.5% of the water samples analyzed with densities ranging from 2.5 to 120 cysts per L. Cryptosporidium spp were detected in 62.5% and the concentrations ranged from 15 to 60 oocysts per L. Cryptosporidium oocyst and Giardia cyst concentrations detected in this study were elevated and are associated with discharge of untreated sewage in Atibaia River. Measures should be taken to protect surface water from sources of contamination.

Quantitative Assessment of Soluble Fibrin in Plasma by Affinity Chromatography – A Comparative Study with desAA-Fibrin, desAABB-Fibrin and Fibrinogen

1985 ◽  
Vol 54 (02) ◽  
pp. 533-538 ◽  
Author(s):  
Wilfried Thiel ◽  
Ulrich Delvos ◽  
Gert Müller-Berghaus
Keyword(s):  
Affinity Chromatography ◽  
Calcium Ions ◽  
Quantitative Assessment ◽  
Fluid Phase ◽  
Soluble Fibrin ◽  
Binding Characteristics ◽  
Different Temperatures ◽  
Immobilized Proteins ◽  
Sepharose 4B

SummaryA quantitative determination of soluble fibrin in plasma was carried out by affinity chromatography. For this purpose, desAA-fibrin and fibrinogen immobilized on Sepharose 4B were used at the stationary side whereas batroxobin-induced 125I-desAA-fibrin or thrombin-induced 125I-desAABB-fibrin mixed with plasma containing 131I-fibrinogen represented the fluid phase. The binding characteristics of these mixtures to the immobilized proteins were compared at 20° C and 37° C. Complete binding of both types of fibrin to the immobilized desAA-fibrin was always seen at 20° C as well as at 37° C. However, binding of soluble fibrin was accompanied by substantial binding of fibrinogen that was more pronounced at 20° C. Striking differences depending on the temperature at which the affinity chromatography was carried out, were documented for the fibrinogen-fibrin interaction. At 20° C more than 90% of the applied desAA-fibrin was bound to the immobilized fibrinogen whereas at 37° C only a mean of 17% were retained at the fibrinogen-Sepharose column. An opposite finding with regard to the tested temperature was made with the desAABB-fibrin. Nearly complete binding to insolubilized fibrinogen was found at 37° C (95%) but only 58% of the desAABB-fibrin were bound at 20° C. The binding patterns did not change when the experiments were performed in the presence of calcium ions. The opposite behaviour of the two types of soluble fibrin to immobilized fibrinogen at the different temperatures, together with the substantial binding of fibrinogen in the presence of soluble fibrin to insolubilized fibrin in every setting tested, devaluates affinity chromatography as a tool in the quantitative assessment of soluble fibrin in patients’ plasma.

Enumeration of Cryptosporidium oocysts from surface water concentrates by laser-scanning cytometry

2000 ◽  
Vol 41 (7) ◽  
pp. 197-202 ◽  
Author(s):  
F. Zanelli ◽  
B. Compagnon ◽  
J. C. Joret ◽  
M. R. de Roubin
Keyword(s):  
Surface Water ◽  
Water Samples ◽  
Laser Scanning ◽  
Immunomagnetic Separation ◽  
Entire Surface ◽  
Cryptosporidium Oocysts ◽  
Different Types ◽  
Laser Scanning Cytometry ◽  
Direct Microscopic Observation

The utilization of the ChemScan® RDI was tested for different types of water concentrates. Concentrates were prepared by cartridge filtration or flocculation, and analysed either without purification, or after Immunomagnetic separation (IMS) or flotation on percoll-sucrose gradients. Theenumeration of the oocysts was subsequently performed using the ChemScan® RDI Cryptosporidium application. Enumeration by direct microscopic observation of the entire surface of the membrane was carried out as a control, and recoveries were calculated as a ratio between the ChemScan® RDI result and the result obtained with direct microscopic enumeration. The Chemscan enumeration technique proved reliable, with recoveries yielding close to 100% in most cases (average 125%, range from 86 to 467%) for all the concentration/purification techniques tested. The quality of the antibodies was shown to be critical, with antibodies from some suppliers yielding recoveries a low as 10% in some cases. This difficulty could, however, be overcome by the utilization of the antibody provided by Chemunex. These data conclusively prove that laser scanning cytometry, which greatly facilitates the microscopic enumeration of Cryptosporidium oocysts from water samples and decreases the time of observation by four to six times, can be successfully applied to water concentrates prepared from a variety of concentration/purification techniques.

scholarly journals Use of Oxidative Stress Responses to Determine the Efficacy of Inactivation Treatments on Cryptosporidium Oocysts

2021 ◽  
Vol 9 (7) ◽  
pp. 1463
Author(s):  
Tamirat Tefera Temesgen ◽  
Kristoffer Relling Tysnes ◽  
Lucy Jane Robertson
Keyword(s):  
Oxidative Stress ◽  
Stress Responses ◽  
Target Genes ◽  
Cryptosporidium Oocyst ◽  
Drinking Water Treatment ◽  
Proof Of Concept ◽  
Environmental Matrices ◽  
Oocyst Wall ◽  
Novel Approach ◽  
Cryptosporidium Oocysts

Cryptosporidium oocysts are known for being very robust, and their prolonged survival in the environment has resulted in outbreaks of cryptosporidiosis associated with the consumption of contaminated water or food. Although inactivation methods used for drinking water treatment, such as UV irradiation, can inactivate Cryptosporidium oocysts, they are not necessarily suitable for use with other environmental matrices, such as food. In order to identify alternative ways to inactivate Cryptosporidium oocysts, improved methods for viability assessment are needed. Here we describe a proof of concept for a novel approach for determining how effective inactivation treatments are at killing pathogens, such as the parasite Cryptosporidium. RNA sequencing was used to identify potential up-regulated target genes induced by oxidative stress, and a reverse transcription quantitative PCR (RT-qPCR) protocol was developed to assess their up-regulation following exposure to different induction treatments. Accordingly, RT-qPCR protocols targeting thioredoxin and Cryptosporidium oocyst wall protein 7 (COWP7) genes were evaluated on mixtures of viable and inactivated oocysts, and on oocysts subjected to various potential inactivation treatments such as freezing and chlorination. The results from the present proof-of-concept experiments indicate that this could be a useful tool in efforts towards assessing potential technologies for inactivating Cryptosporidium in different environmental matrices. Furthermore, this approach could also be used for similar investigations with other pathogens.

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