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In-vitro Cell Culture and Real-time Reverse Transcriptase PCR-based Assays to Detect Infective Toxoplas gondii Oocysts
Citation:
AUGUSTINE, S. J., L. F. VILLEGAS, M. W. WARE, S. L. HAYES, M. J. See, J. P. Dubey, AND E. VILLEGAS. In-vitro Cell Culture and Real-time Reverse Transcriptase PCR-based Assays to Detect Infective Toxoplas gondii Oocysts. Presented at 2008 American Society for Microbiology Annual Meeting, Boston, MA, June 01, 2008.
Impact/Purpose:
1) Refine new, practical methods for the detection of CCL-related and emerging waterborne human protozoa. 2) Evaluate new technologies for their use in method development. 3) Evaluate the efficacy of disinfection procedures on protozoans in collaboration with NRMRL scientists.
Description:
Toxoplasma gondii is an obligate intracellular, apicomplexan parasite that infects humans. It is ubiquitous in nature and seroprevalence in the United States and in Europe ranges from 25->70%. Although typically associated with causing foodborne outbreaks, recent studies in Canada and Brazil have shown this parasite to also cause waterborne outbreaks of toxoplasmosis, suggesting its presence and persistence in drinking and recreational waters. To date, little is known about the prevalence of T. gondii oocysts in water and its resistance to disinfection. In this study, we developed a quantitative reverse-transcriptase PCR (RT-qPCR) assay using four constitutive and inducible gene targets (gra7, act1, sporosag, and tgowp genes) to detect and quantify viable T. gondii oocysts. Additionally, an in vitro cell culture assay was also developed to determine viability of T. gondii. Preliminary results revealed that although sporosag is specifically expressed in the infective sporulated oocyst stage, levels of sporosag mRNA did not correlate with viability. Similar results were obtained with gra7, act1, and Tgowp gene targets. In contrast, our in vitro cell culture assay provided a sensitive and quantitative assay to measure disinfection treatment efficacies commonly used to inactivate waterborne parasites. Initial results indicated that oocysts treated with 10% formalin or heat inactivation for 1 hour at 80oC were effectively killed. Other treatments that had marked effects include sodium hypochlorite, Wescodyne, ethanol, and UV. This study reveals that mRNA-based PCR viability assay using gra7, act1, sporosag, and tgowp genes may not be useful gene candidates to measure potential infectivity of T. gondii oocysts. Other mRNA species that have a shorter half-life may prove more useful. We also present a novel in vitro cell culture assay that can be used to quantify treatment efficacies commonly used in drinking and wastewater industries.