Comparison of molecular markers for determining the viability and infectivity of Cryptosporidium oocysts and validation of molecular methods against animal infectivity assay

Absar Alum, Joseph R. Rubino, M. Khalid Ijaz

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Background: Globally, disinfectants are widely used to intervene in the dissemination of Cryptosporidium oocysts. However, extensive investigations of oocyst inactivation by various disinfectants are not feasible due to the limitations imposed by animal infectivity methods. Molecular techniques provide an alternative strategy; however, non-metabolic genes have been used as markers for determining viability/infectivity. Methods: In this study we used amyloglucosidase (AG) - a metabolic protein - as a marker to determine viability/infectivity of Cryptosporidium. Oocysts were exposed to 6% hydrogen peroxide for 2. min. Samples were analyzed by cell culture polymerase chain reaction (CC-PCR) using PCR primers specific for heat shock protein 70 (hsp70) and AG. Both target genes were amplified with the same level of intensity. Results: Based on the results it can be concluded that AG is a valid target for the study of environmental survival and for the evaluation of the efficacy of microbicides against Cryptosporidium using molecular and cellular assays. Comparison of the CC-PCR assay and mouse infectivity assay showed a fairly good correlation under these test conditions. Conclusion: Results indicate that the CC-PCR assay presents a valid and cost-effective alternative to the mouse infectivity assay.

Original languageEnglish (US)
JournalInternational Journal of Infectious Diseases
Volume15
Issue number3
DOIs
StatePublished - Mar 1 2011

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Cryptosporidium
Oocysts
Glucan 1,4-alpha-Glucosidase
Polymerase Chain Reaction
Cell Culture Techniques
Disinfectants
HSP70 Heat-Shock Proteins
Anti-Infective Agents
Hydrogen Peroxide
Genes
Costs and Cost Analysis
Proteins

Keywords

  • Animal infectivity assay
  • Cryptosporidium
  • Molecular markers
  • Molecular methods
  • Viability and infectivity of oocysts

ASJC Scopus subject areas

  • Infectious Diseases
  • Microbiology (medical)

Cite this

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title = "Comparison of molecular markers for determining the viability and infectivity of Cryptosporidium oocysts and validation of molecular methods against animal infectivity assay",
abstract = "Background: Globally, disinfectants are widely used to intervene in the dissemination of Cryptosporidium oocysts. However, extensive investigations of oocyst inactivation by various disinfectants are not feasible due to the limitations imposed by animal infectivity methods. Molecular techniques provide an alternative strategy; however, non-metabolic genes have been used as markers for determining viability/infectivity. Methods: In this study we used amyloglucosidase (AG) - a metabolic protein - as a marker to determine viability/infectivity of Cryptosporidium. Oocysts were exposed to 6{\%} hydrogen peroxide for 2. min. Samples were analyzed by cell culture polymerase chain reaction (CC-PCR) using PCR primers specific for heat shock protein 70 (hsp70) and AG. Both target genes were amplified with the same level of intensity. Results: Based on the results it can be concluded that AG is a valid target for the study of environmental survival and for the evaluation of the efficacy of microbicides against Cryptosporidium using molecular and cellular assays. Comparison of the CC-PCR assay and mouse infectivity assay showed a fairly good correlation under these test conditions. Conclusion: Results indicate that the CC-PCR assay presents a valid and cost-effective alternative to the mouse infectivity assay.",
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AU - Rubino, Joseph R.

AU - Khalid Ijaz, M.

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N2 - Background: Globally, disinfectants are widely used to intervene in the dissemination of Cryptosporidium oocysts. However, extensive investigations of oocyst inactivation by various disinfectants are not feasible due to the limitations imposed by animal infectivity methods. Molecular techniques provide an alternative strategy; however, non-metabolic genes have been used as markers for determining viability/infectivity. Methods: In this study we used amyloglucosidase (AG) - a metabolic protein - as a marker to determine viability/infectivity of Cryptosporidium. Oocysts were exposed to 6% hydrogen peroxide for 2. min. Samples were analyzed by cell culture polymerase chain reaction (CC-PCR) using PCR primers specific for heat shock protein 70 (hsp70) and AG. Both target genes were amplified with the same level of intensity. Results: Based on the results it can be concluded that AG is a valid target for the study of environmental survival and for the evaluation of the efficacy of microbicides against Cryptosporidium using molecular and cellular assays. Comparison of the CC-PCR assay and mouse infectivity assay showed a fairly good correlation under these test conditions. Conclusion: Results indicate that the CC-PCR assay presents a valid and cost-effective alternative to the mouse infectivity assay.

AB - Background: Globally, disinfectants are widely used to intervene in the dissemination of Cryptosporidium oocysts. However, extensive investigations of oocyst inactivation by various disinfectants are not feasible due to the limitations imposed by animal infectivity methods. Molecular techniques provide an alternative strategy; however, non-metabolic genes have been used as markers for determining viability/infectivity. Methods: In this study we used amyloglucosidase (AG) - a metabolic protein - as a marker to determine viability/infectivity of Cryptosporidium. Oocysts were exposed to 6% hydrogen peroxide for 2. min. Samples were analyzed by cell culture polymerase chain reaction (CC-PCR) using PCR primers specific for heat shock protein 70 (hsp70) and AG. Both target genes were amplified with the same level of intensity. Results: Based on the results it can be concluded that AG is a valid target for the study of environmental survival and for the evaluation of the efficacy of microbicides against Cryptosporidium using molecular and cellular assays. Comparison of the CC-PCR assay and mouse infectivity assay showed a fairly good correlation under these test conditions. Conclusion: Results indicate that the CC-PCR assay presents a valid and cost-effective alternative to the mouse infectivity assay.

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