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 2min. 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.
  
  
  CONCLUSIONS
  Results indicate that the CC-PCR assay presents a valid and cost-effective alternative to the mouse infectivity assay.
authors.affiliation: Department of Civil & Environmental Engineering, Arizona State University, Tempe, Arizona, USA.
authors.forename: Absar
authors.initials: A
authors.lastname: Alum
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Rubino
authors.forename: M
authors.initials: M
authors.lastname: Khalid Ijaz
mesh_terms: D000818:Animals; D000891:Anti-Infective Agents, Local / Q000633:toxicity*; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D003457:Cryptosporidiosis / Q000469:parasitology*; D016785:Cryptosporidium parvum / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D016054:DNA, Protozoan / Q000032:analysis; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D051379:Mice; D015335:Molecular Probes; D033661:Oocysts / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000891:Anti-Infective Agents, Local; D015415:Biomarkers; D016054:DNA, Protozoan; D018840:HSP70 Heat-Shock Proteins; D015335:Molecular Probes; D006861:Hydrogen Peroxide; D005087:Glucan 1,4-alpha-Glucosidase