Browse
Submit Data
Databases
API
Help

Dataset Information

30 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Identification of Cryptosporidium spp. oocysts in United Kingdom noncarbonated natural mineral waters and drinking waters by using a modified nested PCR-restriction fragment length polymorphism assay.

ABSTRACT: We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65 degrees C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.

SUBMITTER: Nichols RA

PROVIDER: S-EPMC165191 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Species-specific, nested PCR-restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts.

Project description:Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.

| S-EPMC92922 | biostudies-literature

Detection of cryptosporidium and identification to the species level by nested PCR and restriction fragment length polymorphism.

Project description:Cryptosporidiosis is an emerging protozoan disease associated with large waterborne outbreaks. Diagnosis relies on microscopic examination of stools, but this method cannot identify the infecting species of Cryptosporidium. We have developed a test based on nested PCR and restriction fragment length polymorphism (RFLP) that offers simple identification of Cryptosporidium hominis, Cryptosporidium parvum, and most other human infective species in stool samples. Purified C. parvum oocysts were used for PCR development. Extracted DNA was amplified by nested PCR targeting a 214-bp fragment of the 18S RNA gene. Enzymatic restriction sites were identified by bioinformatic analysis of all published Cryptosporidium 18S rRNA sequences. Experiments with spiked stool samples gave an estimated PCR detection limit of one oocyst. Specificity was assessed by testing 68 stool samples from patients with microscopically proven cryptosporidiosis and 31 Cryptosporidium-negative stools. Sixty-seven (98.5%) of the 68 stool samples from patients with microscopically proven cryptosporidiosis and 2 of the other stool samples were positive by PCR and could be genotyped. RFLP analysis identified 36 C. hominis, 19 C. parvum, 8 Cryptosporidium meleagridis, and 6 Cryptosporidium felis or Cryptosporidium canis samples. Species determination in 26 PCR-positive cases was in full agreement with DNA sequencing of the 18S rRNA hypervariable region. The excellent sensitivity of PCR, coupled with the accuracy of RFLP for species identification, make this method a suitable tool for routine diagnosis and genotyping of Cryptosporidium in stools.

| S-EPMC1081268 | biostudies-literature

Detection of Cryptosporidium molnari oocysts from fish by fluorescent-antibody staining assays for cryptosporidium spp. affecting humans.

Project description:Three direct fluorescent-antibody staining assay kits for the detection of zoonotic Cryptosporidium species were used to detect Cryptosporidium molnari from Murray cod, and the cryptosporidia were characterized by using small-subunit (SSU) ribosomal DNA (rDNA). To facilitate rapid diagnosis of infection, this study demonstrated that all three kits detected fresh C. molnari and two kits detected formalin-fixed oocysts.

| S-EPMC3067297 | biostudies-literature

Cryopreservation of infectious Cryptosporidium parvum oocysts.

Project description:Cryptosporidiosis in an enteric infection caused by Cryptosporidium parasites and is a major cause of acute infant diarrhea in the developing world. A major bottleneck to research progress is the lack of methods to cryopreserve Cryptosporidium oocysts, thus requiring routine propagation in laboratory animals. Here, we report a method to cryopreserve C. parvum oocysts by ultra-fast cooling. Cryopreserved oocysts exhibit high viability and robust in vitro excystation, and are infectious to interferon-? knockout mice. The course of the infection is comparable to what we observe with unfrozen oocysts. Oocyst viability and infectivity is not visibly changed after several weeks of cryogenic storage. Cryopreservation will facilitate the sharing of oocysts from well-characterized isolates and transgenic strains among different laboratories.

| S-EPMC6056504 | biostudies-other

Detection and resolution of Cryptosporidium species and species mixtures by genus-specific nested PCR-restriction fragment length polymorphism analysis, direct sequencing, and cloning.

Project description:Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.

| S-EPMC3131642 | biostudies-literature

Terminal restriction fragment length polymorphism for identification of Cryptosporidium species in human feces.

Project description:Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.

| S-EPMC2612217 | biostudies-literature

The transcriptome of Cryptosporidium oocysts and intracellular stages.

Project description:Human cryptosporidiosis is caused primarily by two species of apicomplexan parasites, Cryptosporidium parvum and C. hominis. Although infection of cell monolayers with sporozoites does not support the complete parasite life cycle, the in vitro system is used to study the asexual phase of multiplication, which consists of two generations of merogony. To better understand host-parasite interaction and to gain insight into gene regulatory processes driving the complex life cycle of Cryptosporidium parasites, we analyzed the transcriptome of C. parvum in oocysts, sporozoites and infected cell monolayers 2-48 h post-infection. Analysis of RNA-Seq data from replicate oocyst, sporozoite and intracellular samples revealed significant differences between transcriptomes expressed outside and inside the host cell. Compared to the transcriptome found in the host cell, the oocyst transcriptome is less diverse. Biological processes significantly over-represented intracellularly relate to biosynthetic processes. Genes significantly overexpressed in oocysts show evidence of specialized functions not found in other Apicomplexa. A more comprehensive view of gene regulation during the Cryptosporidium life cycle will require the analysis of later time points during the infection, particularly of the poorly studied sexual phase of the life cycle.

| S-EPMC6536522 | biostudies-literature

Identification of species and sources of Cryptosporidium oocysts in storm waters with a small-subunit rRNA-based diagnostic and genotyping tool.

Project description:The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of an immunofluorescent assay. In this study, we have used a small-subunit rRNA-based PCR-restriction fragment length polymorphism technique to identify species and sources of Cryptosporidium oocysts present in 29 storm water samples collected from a stream in New York. A total of 12 genotypes were found in 27 positive samples; for 4 the species and probable origins were identified by sequence analysis, whereas the rest represent new genotypes from wildlife. Thus, this technique provides an alternative method for the detection and differentiation of Cryptosporidium parasites in environmental samples.

| S-EPMC92489 | biostudies-literature

Immunomagnetic separation (IMS)-fluorescent antibody detection and IMS-PCR detection of seeded Cryptosporidium parvum oocysts in natural waters and their limitations.

Project description: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.

| S-EPMC123930 | biostudies-literature

A waterborne outbreak and detection of cryptosporidium oocysts in drinking water of an older high-rise apartment complex in seoul.

Project description:From May to June 2012, a waterborne outbreak of 124 cases of cryptosporidiosis occurred in the plumbing systems of an older high-rise apartment complex in Seoul, Republic of Korea. The residents of this apartment complex had symptoms of watery diarrhea and vomiting. Tap water samples in the apartment complex and its adjacent buildings were collected and tested for 57 parameters under the Korean Drinking Water Standards and for additional 11 microbiological parameters. The microbiological parameters included total colony counts, Clostridium perfringens, Enterococcus, fecal streptococcus, Salmonella, Shigella, Pseudomonas aeruginosa, Cryptosporidium oocysts, Giardia cysts, total culturable viruses, and Norovirus. While the tap water samples of the adjacent buildings complied with the Korean Drinking Water Standards for all parameters, fecal bacteria and Cryptosporidium oocysts were detected in the tap water samples of the outbreak apartment complex. It turned out that the agent of the disease was Cryptosporidium parvum. The drinking water was polluted with sewage from a septic tank in the apartment complex. To remove C. parvum oocysts, we conducted physical processes of cleaning the water storage tanks, flushing the indoor pipes, and replacing old pipes with new ones. Finally we restored the clean drinking water to the apartment complex after identification of no oocysts.

| S-EPMC3770878 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data