Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizers

Villegas, Eric N.; Augustine, Swinburne A. J.; Villegas, Leah Fohl; Ware, Michael W.; See, Mary Jean; Lindquist, Heather; Schaefer, Frank W.; Dubey, J. P.

doi:10.1016/j.mimet.2010.03.023

Cited by 23 publications

(23 citation statements)

References 23 publications

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“…Infections lead to possible deleterious ocular and neurological complications and even death (7). In this context, a global effort has emerged to decipher the basic structures (8,9) and biological processes of the oocyst (10)(11)(12)(13) that allow the parasite to survive different environmental conditions and disinfectants (14)(15)(16)(17)(18).…”

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confidence: 99%

Mechanics of the Toxoplasma gondii oocyst wall

Dumètre

Dubey

Ferguson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The ability of microorganisms to survive under extreme conditions is closely related to the physicochemical properties of their wall. In the ubiquitous protozoan parasite Toxoplasma gondii, the oocyst stage possesses a bilayered wall that protects the dormant but potentially infective parasites from harsh environmental conditions until their ingestion by the host. None of the common disinfectants are effective in killing the parasite because the oocyst wall acts as a primary barrier to physical and chemical attacks. Here, we address the structure and chemistry of the wall of the T. gondii oocyst by combining wall surface treatments, fluorescence imaging, EM, and measurements of its mechanical characteristics by using atomic force microscopy. Elasticity and indentation measurements indicated that the oocyst wall resembles common plastic materials, based on the Young moduli, E, evaluated by atomic force microscopy. Our study demonstrates that the inner layer is as robust as the bilayered wall itself. Besides wall mechanics, our results suggest important differences regarding the nonspecific adhesive properties of each layer. All together, these findings suggest a key biological role for the oocyst wall mechanics in maintaining the integrity of the T. gondii oocysts in the environment or after exposure to disinfectants, and therefore their potential infectivity to humans and animals.oocyst structure | oocyst integrity | oocyst resistance | Toxoplasma infectivity | transmission

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confidence: 99%

Mechanics of the Toxoplasma gondii oocyst wall

Dumètre

Dubey

Ferguson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…RT-PCR detects mRNA, which is only produced by metabolically active parasites and could be a valuable strategy for the screening of viable tachyzoites in complex matrices to benchmark inactivation potential [27]. The in vivo mouse model is still considered the gold standard for T. gondii vitality and infectivity but is laborious and expensive [28] and there are ethical concerns associated with it [29]. The in vitro cell culture system, an alternative to mouse bioassay, must be optimized for assessment of vitality in complex food matrices, such as cheeses, and may require a long time to reduce assay risk [27,29].…”

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confidence: 99%

Absence of Viable Toxoplasma gondii in Artisanal Raw-Milk Ewe Cheese Derived from Naturally Infected Animals

et al. 2020

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The presence of viable Toxoplasma gondii was investigated in artisanal cheeses made from milk of naturally infected ewes. Ewe milk was analyzed beforehand for the presence and vitality of T. gondii by loop-mediated isothermal amplification (LAMP) and reverse-transcriptase PCR (RT-PCR), respectively. Cheeses were prepared from raw milk following a traditional cheesemaking process. The cheese obtained from T. gondii-positive milk was analyzed by LAMP to detect Toxoplasma DNA-positive samples. RT-PCR was then carried out to assess the viability of the parasites in T. gondii-positive milk samples and fresh cheese, after 5 and 15 days of ripening. Physical-chemical parameters of cheeses were also investigated. All cheese samples derived from T. gondii-positive milk were positive according to LAMP, at both 5 and 15 days of ripening, while none of the samples were positive according to RT-PCR. Thus, while the presence of the parasite was demonstrated by the detection of specific DNA, the absence of detectable T. gondii RNA supports the hypothesis that changes in the chemical and physical characteristics occurring during the cheesemaking process and ripening period, could be sufficient to inactivate viable T. gondii in milk, minimizing the risk of human infection through consumption of raw sheep milk cheese.

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“…RT-qPCR was performed to detect the ACT1 and SporoSAG mRNA as previously described (60). Each sample analyzed contained total RNA extracted from 1,000 oocysts as described above.…”

Section: Vol 76 2010 Inactivation Of T Gondii Oocysts By Uv Disinfmentioning

confidence: 99%

“…Oocyst excystation as reported by Villegas et al was used for this study (60). Briefly, oocysts at a concentration of 2 ϫ 10 6 in 1 ml of 1ϫ PBS were added to 2-ml microcentrifuge screw cap conical tubes containing 0.5 g of 0.5-mm glass beads (Biospec Products, Inc., Bartlesville, OK) and mechanically disrupted using a mini-BeadBeater (BioSpec Products, Inc.) for 20 s at 250 rpm.…”

Section: Vol 76 2010 Inactivation Of T Gondii Oocysts By Uv Disinfmentioning

confidence: 99%

“…The cell monolayers were exposed to various dilutions (1 to 1 ϫ 10 6 ) of excysted T. gondii oocysts and incubated at 37°C and in 5% CO 2 for 10 days. At the end of the 10-day period, the cell monolayers were stained with crystal violet for 30 min, washed, and analyzed microscopically by counting of plaques as previously described (52,60).…”

Section: Vol 76 2010 Inactivation Of T Gondii Oocysts By Uv Disinfmentioning

confidence: 99%

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Determining UV Inactivation of Toxoplasma gondii Oocysts by Using Cell Culture and a Mouse Bioassay

Ware

Augustine²,

Erisman³

et al. 2010

Appl Environ Microbiol

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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.

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Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizers

Cited by 23 publications

References 23 publications

Mechanics of the Toxoplasma gondii oocyst wall

Mechanics of the Toxoplasma gondii oocyst wall

Absence of Viable Toxoplasma gondii in Artisanal Raw-Milk Ewe Cheese Derived from Naturally Infected Animals

Determining UV Inactivation of Toxoplasma gondii Oocysts by Using Cell Culture and a Mouse Bioassay

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