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.

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

2001 ◽  
Vol 67 (3) ◽  
pp. 1123-1127 ◽  
Author(s):  
Otto D. Simmons ◽  
Mark D. Sobsey ◽  
Christopher D. Heaney ◽  
Frank W. Schaefer ◽  
Donna S. Francy
Keyword(s):  
Surface Water ◽  
Surface Waters ◽  
Hollow Fiber ◽  
Water Samples ◽  
Environmental Protection Agency ◽  
Fluorescent Antibody ◽  
Immunomagnetic Separation ◽  
Recovery Rates ◽  
Microscopic Evaluation ◽  
Surface Water Samples

ABSTRACT The protozoan parasite Cryptosporidium parvumis 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. parvumoocyst 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.

scholarly journals Failure To Differentiate Cryptosporidium parvum from C. meleagridis Based on PCR Amplification of Eight DNA Sequences

1998 ◽  
Vol 64 (4) ◽  
pp. 1454-1458 ◽  
Author(s):  
Dominique Champliaud ◽  
Philippe Gobet ◽  
Muriel Naciri ◽  
Odile Vagner ◽  
José Lopez ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Dna Sequences ◽  
Target Genes ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Restriction Enzyme Digestion ◽  
Human Pathogens

ABSTRACT In order to determine the specificities of PCR-based assays used for detecting Cryptosporidium parvum DNA, eight pairs of previously described PCR primers targeting six distinct regions of theCryptosporidium genome were evaluated for the detection ofC. parvum, the agent of human cryptosporidiosis, andC. muris, C. baileyi, and C. meleagridis, three Cryptosporidium species that infect birds or mammals but are not considered to be human pathogens. The four Cryptosporidium species were divided into two groups: C. parvum and C. meleagridis, which gave the same-sized fragments with all the reactions, and C. muris and C. baileyi, which gave positive results with primer pairs targeting the 18S rRNA gene only. In addition to being genetically similar at each of the eight loci analyzed by DNA amplification, C. parvum and C. meleagridiscouldn’t be differentiated even after restriction enzyme digestion of the PCR products obtained from three of the target genes. This study indicates that caution should be exercised in the interpretation of data from water sample analysis performed by these methods, since a positive result does not necessarily reflect a contamination by the human pathogen C. parvum.

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 First report and molecular characterization of Cryptosporidium spp. in humans and animals in Khartoum state, Sudan

2019 ◽  
Vol 12 (1) ◽  
pp. 183-189 ◽  
Author(s):  
Kaltoum Yagoub Adam ◽  
A. A. Ismail ◽  
M. A. Masri ◽  
A. A. Gameel
Keyword(s):  
Nested Pcr ◽  
Mammalian Species ◽  
Pcr Amplification ◽  
Health Concern ◽  
Economic Losses ◽  
Staining Procedure ◽  
Rrna Gene ◽  
Nested Polymerase Chain Reaction ◽  
Cryptosporidium Spp ◽  
Pcr Targeting

Background and Aim: Cryptosporidium is recognized to infect several mammalian species as well as humans, causing substantial economic losses and serious public health concern. Infected animals can be a source of environmental contamination and human infections. In general, the occurrence of Cryptosporidium species in animals and human in Sudan and zoonotic importance is not well documented. This study aimed to identify Cryptosporidium spp. infecting different animal species and humans and to compare between different isolates obtained. Materials and Methods: To provide molecular information about Cryptosporidium in animals and humans, both modified Ziehl-Neelsen (MZN) specific stain and molecular assay were used. Concentration techniques followed by three protocols of DNA extraction were carried out. After microscopic screening of 263 fecal samples (goats [n=197], cattle [n=12], sheep [n=12], and human [n=42]), 61 positive and 30 negative, randomly selected samples were used in nested polymerase chain reaction (PCR) targeting part of the 18S RNA. Results: Nested PCR amplification confirmed 91.8% (56/61) of microscopic-positive samples. 8.2% (5/61) of negative samples by PCR (positive by microscopy) were considered false negatives. Sequencing followed by alignment of the 14 isolates indicated that all samples were identical (100%) and belonged to Cryptosporidium parvum. Conclusion: MZN staining procedure is reliable for the routine diagnosis of Cryptosporidium; cetyltrimethylammonium bromide extraction buffer and nested PCR targeting 18S rRNA gene are reliable and useful in epidemiological studies of this parasite.

scholarly journals Molecular Evidence of Chlamydia-Like Organisms in the Feces of Myotis daubentonii Bats

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
K. Hokynar ◽  
E. J. Vesterinen ◽  
T. M. Lilley ◽  
A. T. Pulliainen ◽  
S. J. Korhonen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Sequence Similarity ◽  
Pcr Amplification ◽  
Rrna Genes ◽  
23S Rrna ◽  
Molecular Evidence ◽  
Rrna Gene ◽  
16S Rrna Sequence ◽  
Content Type ◽  
Pcr Targeting

ABSTRACT Chlamydia-like organisms (CLOs) are recently identified members of the Chlamydiales order. CLOs share intracellular lifestyles and biphasic developmental cycles, and they have been detected in environmental samples as well as in various hosts such as amoebae and arthropods. In this study, we screened bat feces for the presence of CLOs by molecular analysis. Using pan-Chlamydiales PCR targeting the 16S rRNA gene, Chlamydiales DNA was detected in 54% of the specimens. PCR amplification, sequencing, and phylogenetic analysis of the 16S rRNA and 23S rRNA genes were used to classify positive specimens and infer their phylogenetic relationships. Most sequences matched best with Rhabdochlamydia species or uncultured Chlamydia sequences identified in ticks. Another set of sequences matched best with sequences of the Chlamydia genus or uncultured Chlamydiales from snakes. To gain evidence of whether CLOs in bat feces are merely diet borne, we analyzed insects trapped from the same location where the bats foraged. Interestingly, the CLO sequences resembling Rhabdochlamydia spp. were detected in insect material as well, but the other set of CLO sequences was not, suggesting that this set might not originate from prey. Thus, bats represent another potential host for Chlamydiales and could harbor novel, previously unidentified members of this order. IMPORTANCE Several pathogenic viruses are known to colonize bats, and recent analyses indicate that bats are also reservoir hosts for bacterial genera. Chlamydia-like organisms (CLOs) have been detected in several animal species. CLOs have high 16S rRNA sequence similarity to Chlamydiaceae and exhibit similar intracellular lifestyles and biphasic developmental cycles. Our study describes the frequent occurrence of CLO DNA in bat feces, suggesting an expanding host species spectrum for the Chlamydiales. As bats can acquire various infectious agents through their diet, prey insects were also studied. We identified CLO sequences in bats that matched best with sequences in prey insects but also CLO sequences not detected in prey insects. This suggests that a portion of CLO DNA present in bat feces is not prey borne. Furthermore, some sequences from bat droppings not originating from their diet might well represent novel, previously unidentified members of the Chlamydiales order.

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.

scholarly journals First detection of Cryptosporidium spp. in red-bellied tree squirrels (Callosciurus erythraeus) in China

Parasite ◽  
2019 ◽  
Vol 26 ◽  
pp. 28 ◽  
Author(s):  
Yijun Chai ◽  
Lei Deng ◽  
Haifeng Liu ◽  
Jingxin Yao ◽  
Zhijun Zhong ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Pcr Amplification ◽  
Small Subunit ◽  
Rrna Gene ◽  
Opportunistic Pathogens ◽  
Cryptosporidium Spp ◽  
Tree Squirrels ◽  
Callosciurus Erythraeus ◽  
Animal Hosts ◽  
Genotype Ii

Cryptosporidium spp. are opportunistic pathogens that cause diarrhea in a variety of animal hosts. Although they have been reported in many animals, no information has been published on the occurrence of Cryptosporidium spp. in red-bellied tree squirrels (Callosciurus erythraeus). A total of 287 fecal specimens were collected from Sichuan province in China; the prevalence of Cryptosporidium spp., measured by nested-PCR amplification of the partial small-subunit (SSU) rRNA gene, was 1.4% (4/287). Three different Cryptosporidium species or genotypes were identified: Cryptosporidium parvum (n = 1), Cryptosporidium wrairi (n = 1), and Cryptosporidium rat genotype II (n = 2). The present study is the first report of Cryptosporidium infection in red-bellied tree squirrels in China. Although there is a relatively low occurrence of Cryptosporidium, the presence of C. parvum and C. wrairi, which were previously reported in humans, indicates that red-bellied tree squirrels may be a source of zoonotic cryptosporidiosis in China.

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