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United States Department of Agriculture

Agricultural Research Service

Research Project: EPIDEMIOLOGY AND CONTROL OF NEOSPORA CANINUM AND RELATED PROTOZOA Title: Using Quantitative Reverse Transcriptase PCR and Cell Culture Plaque Assays to Determine Resistance of Toxoplasma gondii Oocysts to Chemical Sanitizers

Authors
item Villegas, Eric -
item Augustine, S -
item Villegas, L -
item Ware, M -
item See, Mary Jane -
item Lindquist, Alan -
item Schaefer, Frank -
item Dubey, Jitender

Submitted to: Journal of Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 1, 2010
Publication Date: May 10, 2010
Citation: Villegas, E.N., Augustine, S.A., Villegas, L.F., Ware, M.M., See, M., Lindquist, A.H., Schaefer, F.W., Dubey, J.P. 2010. Using Quantitative Reverse Transcriptase PCR and Cell Culture Plaque Assays to Determine Resistance of Toxoplasma gondii Oocysts to Chemical Sanitizers. Journal of Microbiology. 81:219-225.

Interpretive Summary: Toxoplasma gondii is a single-celled parasite of all warm-blooded hosts worldwide. It causes mental retardation and loss of vision in children, and abortion in livestock. Cats are the main reservoir of T. gondii because they are the only hosts that can excrete the resistant stage (oocyst) of the parasite in the feces. Humans become infected by eating undercooked meat from infected animals and food and water contaminated with oocysts. This paper reports on methods to detect oocysts in the environment. The results will be of interest to biologists, parasitologists, public health workers, and veterinarians.

Technical Abstract: Toxoplasma gondii oocysts are highly resistant to many chemical sanitizers. Current methods used to determine oocyst infectivity have relied exclusively on mouse, chicken, and feline bioassays. Although considered gold standards, they only provide a qualitative assessment of oocyst infectivity. In this study, we present two alternative approaches to quantitate infectious T. gondii oocysts following treatment with several common sanitizers. The first approach is a quantitative reverse-transcriptase real-time PCR (RT-qPCR) assay targeting the ACT1 and SporoSAG genes to enumerate infectious T. gondii oocysts. RT-qPCR CT values between Wescodyne®, ethanol, or heat treated oocysts as compared with untreated controls were not significantly different. By contrast, a 2-log10 reduction in CT values was detected in 10% formalin or 10% Clorox® treated oocysts as compared to untreated controls. These results suggest limited application for assessing viability following treatment with strong oxidizing sanitizers. An in vitro T. gondii oocyst plaque assay (TOP-assay) was also developed to measure oocyst infectivity. This assay involves oocyst excystation using a combination of bead milling plus bile digestion, followed by culturing released sporozoites in a confluent fibroblast cell monolayer. Results showed that no significant reduction in sporozoite viability was detected in ethanol or Wescodyne® treated oocysts, and at least a 2-log10 reduction in plaques formed was observed with Clorox® treated oocysts. Moreover, formalin and heat treatment of oocysts resulted in at least a 5-log10 reduction in plaques formed. This study demonstrates that an mRNA-based PCR viability assay targeting the ACT1 or SoroSAG genes is a rapid technique to measure disinfection efficacies, but may be limited to detecting effects of strong oxidizing chemicals. By contrast, the TOP-assay proved very effective and more sensitive at determining oocyst viability.

Last Modified: 11/28/2014
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