Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe

Smith, James J.; Gunasekera, Thusitha S.; Barardi, Célia Regina Monte; Veal, Duncan; Vesey, Graham

doi:10.1046/j.1365-2672.2004.02150.x

Cited by 46 publications

(37 citation statements)

References 33 publications

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“…One option to degrade DNA in inactivated eggs would be to pretreat the eggs with proteinase and nucleases before PCR. This technique has been used effectively to decrease false-positive signals from inactivated viruses and Cryptosporidium oocysts (26,32).…”

Section: Discussionmentioning

confidence: 99%

A Real-Time PCR Method for Quantifying Viable Ascaris Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA

Pecson

Barrios

Johnson

et al. 2006

Appl Environ Microbiol

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Worldwide, 1.4 billion people are infected with the intestinal worm Ascaris lumbricoides. As a result, Ascaris eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable Ascaris eggs is time-and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable Ascaris eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to Ascaris egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications.

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Section: Discussionmentioning

confidence: 99%

A Real-Time PCR Method for Quantifying Viable Ascaris Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA

Pecson

Barrios

Johnson

et al. 2006

Appl Environ Microbiol

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“…Aliquots (10 ml) containing UV treated or mock treated samples were spotted onto poly-L-lysine coated coverslips and air-dried overnight at ambient temperature. Unexcysted samples were heated at 808C for 10 min to permeabilize oocysts [Smith et al, 2004]. Oocyst or sporozoite preparations were extracted with cold methanol, blocked in 10% BSA-PBS for 1 h, incubated individually with CpRPA1A and CpRPA1B polyclonal antibodies and TRITC-conjugated secondary antibodies as described previously [Millership and Zhu, 2002].…”

Section: Detection Of Cprpa1 Changesmentioning

confidence: 99%

Differential expression of the two distinct replication protein A subunits from Cryptosporidium parvum

Rider

Zhu

2008

J of Cellular Biochemistry

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Apicomplexan parasites differ from their host by possessing at least two distinct types (long and short) of replication protein A large subunits (RPA1). Different roles for the long and short types of RPA1 proteins have been implied in early biochemical studies, but certain details remained to be elucidated. In the present study, we have found that the Cryptosporidium parvum short-type RPA1 (CpRPA1A) was highly expressed at S-phase in parasites during the early stage of merogony (a cell multiplication process unique to this group of parasites), but otherwise present in the cytosol at a much lower level in other cell-cycle stages. This observation indicates that CpRPA1A is probably responsible for the general DNA replication of the parasite. On the other hand, the long-type CpRPA1B protein was present in a much lower level in the early life cycle stages, but elevated at later stages involved in sexual development, indicating that CpRPA1B may play a role in DNA recombination. Additionally, CpRPA1B could be up-regulated by UV exposure, indicating that this long-type RPA1 is probably involved in DNA repair. Collectively, our data implies that the two RPA1 proteins in C. parvum are performing different roles during DNA replication, repair and recombination in this parasite.

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“…Other more recent approaches utilize molecular biology-based techniques to assess oocyst viability. Fluorescence in situ hybridization (5,10,20,26,40,47), nucleic acid sequence-based amplification (4), reverse transcriptase PCR (RT-PCR) (17,22,50), and an integrated cell culture/ PCR assay (12,25) all show promise in providing a rapid and relatively inexpensive viability assay for Cryptosporidium. However, it still remains to be determined if these methods can detect all Cryptosporidium species and genotypes found in the environment.…”

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confidence: 99%

Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping of Viable Cryptosporidium Oocysts

Brescia¹,

Griffin

Ware³

et al. 2009

Appl Environ Microbiol

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Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

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Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe

Cited by 46 publications

References 33 publications

A Real-Time PCR Method for Quantifying Viable Ascaris Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA

A Real-Time PCR Method for Quantifying Viable Ascaris Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA

Differential expression of the two distinct replication protein A subunits from Cryptosporidium parvum

Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping of Viable Cryptosporidium Oocysts

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