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

1998 ◽  
Vol 64 (11) ◽  
pp. 4495-4499 ◽  
Author(s):  
Z. Bukhari ◽  
R. M. McCuin ◽  
C. R. Fricker ◽  
J. L. Clancy
Keyword(s):  
Quality Assurance ◽  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Immunomagnetic Separation ◽  
Deionized Water ◽  
Source Water ◽  
Geographical Locations

ABSTRACT 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.

scholarly journals Detection and Discrimination of Cryptosporidium parvum and C. hominis in Water Samples by Immunomagnetic Separation-PCR

2005 ◽  
Vol 71 (2) ◽  
pp. 898-903 ◽  
Author(s):  
Yoshitsugu Ochiai ◽  
Chieko Takada ◽  
Mitsugu Hosaka
Keyword(s):  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Detection Method ◽  
Fluorescent Antibody ◽  
Immunomagnetic Separation ◽  
Antibody Assay ◽  
Pcr Products ◽  
Genetic Detection

ABSTRACT 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.

scholarly journals Effects of pH and Magnetic Material on Immunomagnetic Separation of Cryptosporidium Oocysts from Concentrated Water Samples

2002 ◽  
Vol 68 (4) ◽  
pp. 2066-2070 ◽  
Author(s):  
Ryan C. Kuhn ◽  
Channah M. Rock ◽  
Kevin H. Oshima
Keyword(s):  
Magnetic Material ◽  
Water Samples ◽  
Environmental Water ◽  
Immunomagnetic Separation ◽  
Deionized Water ◽  
Ph Values ◽  
Cryptosporidium Oocysts ◽  
Optimum Ph ◽  
Effects Of Ph ◽  
Optimal Ph

ABSTRACT In this study, we examined the effect that magnetic materials and pH have on the recoveries of Cryptosporidium oocysts by immunomagnetic separation (IMS). We determined that particles that were concentrated on a magnet during bead separation have no influence on oocyst recovery; however, removal of these particles did influence pH values. The optimal pH of the IMS was determined to be 7.0. The numbers of oocysts recovered from deionized water at pH 7.0 were 26.3% higher than those recovered from samples that were not at optimal pH. The results indicate that the buffers in the IMS kit did not adequately maintain an optimum pH in some water samples. By adjusting the pH of concentrated environmental water samples to 7.0, recoveries of oocysts increased by 26.4% compared to recoveries from samples where the pH was not adjusted.

scholarly journals Immunomagnetic Separation (IMS)-Fluorescent Antibody Detection and IMS-PCR Detection of Seeded Cryptosporidium parvum Oocysts in Natural Waters and Their Limitations

2002 ◽  
Vol 68 (6) ◽  
pp. 2991-2996 ◽  
Author(s):  
Gregory D. Sturbaum ◽  
Patricia T. Klonicki ◽  
Marilyn M. Marshall ◽  
B. Helen Jost ◽  
Brec L. Clay ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Natural Waters ◽  
Fluorescent Antibody ◽  
Pcr Amplification ◽  
Immunomagnetic Separation ◽  
Pcr Detection ◽  
Antibody Detection ◽  
Rrna Gene ◽  
Pcr Targeting

ABSTRACT Detection and enumeration of Cryptosporidium parvum in both treated and untreated waters are important to facilitate prevention of future cryptosporidiosis incidents. Immunomagnetic separation (IMS)-fluorescent antibody (FA) detection and IMS-PCR detection efficiencies were evaluated in two natural waters seeded with nominal seed doses of 5, 10, and 15 oocysts. IMS-FA detected oocysts at concentrations at or below the three nominal oocyst seed doses, illustrating that IMS-FA is sensitive enough to detect low oocyst numbers. However, the species of the oocysts could not be determined with this technique. IMS-PCR, targeting the 18S rRNA gene in this study, yielded positive amplification for 17 of the 18 seeded water samples, and the amplicons were subjected to restriction fragment length polymorphism digestion and DNA sequencing for species identification. Interestingly, the two unseeded, natural water samples were also PCR positive; one amplicon was the same base pair size as the C. parvum amplicon, and the other amplicon was larger. These two amplified products were determined to be derived from DNA of Cryptosporidium muris and a dinoflagellate. These IMS-PCR results illustrate that (i) IMS-PCR is able to detect low oocyst numbers in natural waters, (ii) PCR amplification alone is not confirmatory for detection of target DNA when environmental samples are used, (ii) PCR primers, especially those designed against the rRNA gene region, need to be evaluated for specificity with organisms closely related to the target organism, and (iv) environmental amplicons should be subjected to appropriate species-specific confirmatory techniques.

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

1999 ◽  
Vol 65 (8) ◽  
pp. 3427-3432 ◽  
Author(s):  
George D. Di Giovanni ◽  
F. Helen Hashemi ◽  
Nancy J. Shaw ◽  
Felicia A. Abrams ◽  
Mark W. LeChevallier ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Dna Sequence ◽  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Immunomagnetic Separation ◽  
The United States ◽  
Dna Sequence Analysis ◽  
Raw Water ◽  
Nucleotide Substitutions ◽  
Filter Backwash Water

ABSTRACT A new strategy for the detection of infectiousCryptosporidium 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-1hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70fragments, suggesting that several different strains of infectiousC. parvum were detected.

Campylobacter in waterborne epidemics in Finland

2004 ◽  
Vol 4 (2) ◽  
pp. 39-45 ◽  
Author(s):  
M.-L. Hänninen ◽  
R. Kärenlampi
Keyword(s):  
Campylobacter Jejuni ◽  
Causative Agent ◽  
Water Samples ◽  
Fecal Coliforms ◽  
Risk Level ◽  
Source Water ◽  
Indicator Organisms ◽  
Faecal Contamination ◽  
E Coli ◽  
July August September

The sources for drinking water in Finland are surface water, groundwater or artificially recharged groundwater. There are approximately 1400 groundwater plants in Finland that are microbiologically at a high risk level because in most cases they do not use any disinfection treatment. Campylobacter jejuni has caused waterborne epidemics in several countries. Since the middle of the 1980s, C. jejuni has been identified as the causative agent in several waterborne outbreaks in Finland. Between 1998 and 2001, C. jejuni or C. upsaliensis caused seven reported waterborne epidemics. In these epidemics approximately 4000 people acquired the illness. Most of the outbreaks occurred in July, August , September or October. In four of them source water and net water samples were analysed for total coliforms or fecal coliforms, E. coli and campylobacters. We showed that large volumes of water samples in studies of indicator organisms (up to 5000 ml) and campylobacters (4000–20,000 ml) increased the possibility to identify faecal contamination and to detect the causative agent from suspected sources.

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 Evaluation of Minerals Content of Drinking Water in Malaysia

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Azrina Azlan ◽  
Hock Eng Khoo ◽  
Mohd Aizat Idris ◽  
Amin Ismail ◽  
Muhammad Rizal Razman
Keyword(s):  
Drinking Water ◽  
Water Samples ◽  
Mineral Water ◽  
Tap Water ◽  
Fluoride Concentration ◽  
Daily Intake ◽  
Peninsular Malaysia ◽  
Mineral Water Sample ◽  
Geographical Locations ◽  
Iron And Manganese

The drinking and mineral water samples obtained from different geographical locations had concentrations of the selected minerals lower than the standard limits, except for manganese, arsenic, and fluoride. The concentrations of manganese and arsenic in two mineral water samples were slightly higher than the standard international recommended limits. One mineral water sample had a fluoride concentration higher than the standard limits, whereas manganese was not detected in nine drinking and mineral water samples. Most of the selected minerals found in the tap water samples were below the international standard limits, except for iron and manganese. The concentrations of iron and manganese in the tap water samples were higher than the standard limits, which were obtained from one and three of the studied locations, respectively. The potable water obtained from various manufacturers and locations in Peninsular Malaysia is safe for consumption, as the minerals concentrations were below the standard limits prescribed by the Malaysian Food Regulations of 1985. The data obtained may also provide important information related to daily intake of these minerals from drinking water.

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