title: Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizers.
abstract: Toxoplasma gondii oocysts are highly resistant to many chemical sanitizers. Methods used to determine oocyst infectivity have relied primarily on mouse, chicken, and feline bioassays. Although considered gold standards, they only provide a qualitative assessment of oocyst viability. In this study, two alternative approaches were developed to quantitate viable T. gondii oocysts following treatment with several common sanitizers. The first is a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay targeting the ACT1 and SporoSAG genes to enumerate viable T. gondii oocysts. RT-qPCR C(T) values between Wescodyne(R), acidified ethanol, or heat treated oocysts were not significantly different as compared with untreated controls. By contrast, treatment with formalin or Clorox(R) resulted in a 2-log(10) reduction in C(T) values. An in vitro T. gondii oocyst plaque assay (TOP-assay) was also developed to measure oocyst viability. This assay used a combination of bead milling and bile digestion, followed by culturing the excysted sporozoites in a confluent fibroblast cell monolayer. Results showed that no significant reduction in sporozoite viability was detected in acidified ethanol or Wescodyne(R) treated oocysts while at least a 2-log(10) reduction in plaques formed was observed with Clorox(R) treated oocysts. Moreover, formalin or heat treatment of oocysts resulted in at least a 5-log(10) reduction in plaques formed. This study demonstrates that an mRNA-based PCR viability assay targeting the ACT1 or SporoSAG genes is a relatively rapid technique compared to in vitro and in vivo assays. In addition, the TOP-assay proved very effective and sensitive at quantifying oocyst viability when compared with animal bioassays.
authors.affiliation: National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268, USA. villegas.eric@epa.gov
authors.forename: Eric N
authors.initials: EN
authors.lastname: Villegas
authors.forename: Swinburne A J
authors.initials: SA
authors.lastname: Augustine
authors.forename: Leah Fohl
authors.initials: LF
authors.lastname: Villegas
authors.forename: Michael W
authors.initials: MW
authors.lastname: Ware
authors.forename: Mary Jean
authors.initials: MJ
authors.lastname: See
authors.forename: H D Alan
authors.initials: HD
authors.lastname: Lindquist
authors.forename: Frank W
authors.initials: FW
authors.lastname: Schaefer
authors.forename: J P
authors.initials: JP
authors.lastname: Dubey
mesh_terms: D001681:Biological Assay / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004202:Disinfectants / Q000494:pharmacology*; D005347:Fibroblasts / Q000469:parasitology; D006801:Humans; D033661:Oocysts / Q000187:drug effects* / Q000502:physiology; D010274:Parasitology / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000187:drug effects* / Q000502:physiology
chemical_list: D004202:Disinfectants