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In JoVE (1)
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Articles by Mary Jean See in JoVE
JoVE Immunology and Infection
Obtenção altamente purificada Toxoplasma gondii Oocistos por um gradiente de cloreto de césio descontínuos
1Dynamac, Inc., 2Department of Biological Sciences, University of Cincinnati, McMicken College of Arts and Science, 3Animal Parasitic Disease Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 4National Exposure Research Laboratory, US Environmental Protection Agency
Este estudo descreve o desenvolvimento de um método modificado CsCl que facilmente purifica oocistos T. gondii a partir de fezes de gatos infectados que são adequados para biológica molecular e manipulação de cultura de tecidos
Other articles by Mary Jean See on PubMed
Using Quantitative Reverse Transcriptase PCR and Cell Culture Plaque Assays to Determine Resistance of Toxoplasma Gondii Oocysts to Chemical Sanitizers
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.
Determining UV Inactivation of Toxoplasma Gondii Oocysts by Using Cell Culture and a Mouse Bioassay
The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated UV-irradiated oocysts by three assays: a SCID mouse bioassay, an in vitro T. gondii oocyst plaque (TOP) assay, and a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay. The results from the animal bioassay show that 1- and 3-log(10) inactivation is achieved with 4 mJ/cm(2) UV and 10 mJ/cm(2) low-pressure UV, respectively. TOP assay results, but not RT-qPCR results, correlate well with bioassay results. In conclusion, a 3-log(10) inactivation of T. gondii oocysts is achieved by 10-mJ/cm(2) low-pressure UV, and the in vitro TOP assay is a promising alternative to the mouse bioassay.
Evaluation of Hollow-fiber Ultrafiltration Primary Concentration of Pathogens and Secondary Concentration of Viruses from Water
Tangential flow hollow-fiber ultrafiltration (HFUF) was evaluated for virus and Cryptosporidium parvum concentration from water. Recovery of viruses at a low filtration rate was found to be significantly greater than at a higher filtration rate, with the recoveries of bacteriophage MS2 at high and low filtration rates shown to be 64.7% and 98.7%, respectively. Poliovirus recoveries from tap water were similar to MS2, with recoveries of 62.9% and 104.5% for high and low filtration rates, respectively. C. parvum, which was only tested at high filtration rates, had an average recovery was 105.1%. In addition to the optimization of the primary concentration technique, this study also compared several secondary concentration procedures. The highest recovery (89.5%) of poliovirus from tap water concentrates was obtained when a beef extract-celite method was used and the virus was eluted from the celite with phosphate buffered saline, pH 9.0. When HFUF primary concentration and the optimal secondary concentration methods were combined, an average recovery of 97.0 ± 35.6% or 89.3 ± 19.3%, depending on spike level, was achieved for poliovirus. This study demonstrated that HFUF primary concentration method is effective at recovering MS2, poliovirus and C. parvum from large volumes of water and that beef extract-celite method is an effective secondary concentration method for the poliovirus tested.
