Quantitative detection of Toxoplasma gondii in tissues of experimentally infected turkeys and in retail turkey products by magnetic-capture PCR

Koethe, Martin; Straubinger, Reinhard K.; Pott, Susan; Bangoura, Berit; Geuthner, Anne-Catrin; Daugschies, Arwid; Ludewig, Martina

doi:10.1016/j.fm.2015.06.005

Cited by 28 publications

(17 citation statements)

References 31 publications

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“…Indirect detection methods for T. gondii , such as bioassays, also have limitations [24]. Cat bioassays, the gold standard for T. gondii detection, require up to 500 grams of tissue in feeding trials although this also has the advantage of increasing the possibility of detecting a tissue cyst.…”

Section: Introductionmentioning

confidence: 99%

“…However, kit-based DNA extraction methods from small tissue quantities (on the order of 25–100 mg) limit detection since T. gondii tissue cysts are not uniformly distributed in tissues [26, 27]. As a result, a magnetic-capture DNA extraction and real-time PCR method (MC-PCR) has been developed for testing up to 100 grams of tissue, allowing for improved detection and quantification of parasite DNA [24, 25, 28, 29]. Briefly, sequence-specific DNA fragments bound to magnetic beads help to capture low concentrations of parasite DNA against high backgrounds of host DNA and inhibitory PCR products [28].…”

Section: Introductionmentioning

confidence: 99%

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Serological and molecular detection of Toxoplasma gondii in terrestrial and marine wildlife harvested for food in Nunavik, Canada

et al. 2019

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Background Toxoplasma gondii, a zoonotic protozoan parasite, infects mammals and birds worldwide. Infection in humans is often asymptomatic, though illnesses can occur in immunocompromised hosts and the fetuses of susceptible women infected during pregnancy. In Nunavik, Canada, 60% of the Inuit population has measurable antibodies against T. gondii. Handling and consumption of wildlife have been identified as risk factors for exposure. Serological evidence of exposure has been reported for wildlife in Nunavik; however, T. gondii has not been detected in wildlife tissues commonly consumed by Inuit. Methods We used a magnetic capture DNA extraction and real-time PCR protocol to extract and amplify T. gondii DNA from large quantities of tissues (up to 100 g) of 441 individual animals in Nunavik: 166 ptarmigan ( Lagopus lagopus ), 156 geese ( Branta canadensis and Chen caerulescens ), 61 ringed seals ( Pusa hispida) , 31 caribou ( Rangifer tarandus ) and 27 walruses ( Odobenus rosmarus ). Results DNA from T. gondii was detected in 9% (95% CI: 3–15%) of geese from four communities in western and southern Nunavik, but DNA was not detected in other wildlife species including 20% (95% CI: 12–31%) of ringed seals and 26% (95% CI: 14–43%) of caribou positive on a commercial modified agglutination test (MAT) using thawed heart muscle juice. In geese, tissue parasite burden was highest in heart, followed by brain, breast muscle, liver and gizzard. Serological results did not correlate well with tissue infection status for any wildlife species. Conclusions To our knowledge, this is the first report on the detection, quantification, and characterization of DNA of T. gondii (clonal lineage II in one goose) from wildlife harvested for food in Nunavik, which supports the hypothesis that migratory geese can carry T. gondii into Nunavik where feline definitive hosts are rare. This study suggests that direct detection methods may be useful for detection of T. gondii in wildlife harvested for human consumption and provides data needed for a quantitative exposure assessment that will determine the risk of T. gondii exposure for Inuit who harvest and consume geese in Nunavik.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Serological and molecular detection of Toxoplasma gondii in terrestrial and marine wildlife harvested for food in Nunavik, Canada

et al. 2019

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“…When performing the PCR test for tissue cyst detection, false‐negatives can result from an insufficient sample size due to the inhomogeneous distribution of T. gondii cysts in animal meat tissue (Esteban‐Redondo et al, ). Conventional DNA extraction methods that use as little as 25 mg of tissue as the starting material for T. gondii detection and quantification may be flawed (Koethe et al, ). On the basis of our development of the boiling plus chemical lysis method for meat sample processing (Jiang et al, ), a 10 g meat sample can be used instead of a 25 mg sample to extract T. gondii DNA, increasing the probability of detection by the developed PCR assays.…”

Section: Discussionmentioning

confidence: 99%

Assessment of self‐produced PCR methods for the detection of Toxoplasma gondii DNA in meat

Jiang

Zhang

et al. 2016

Journal of Food Safety

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The objective was to develop a highly sensitive and specific polymerase chain reaction (PCR) method for the detection of Toxoplasma gondii DNA in meat. To increase the capacity for the processing of meat samples by improving DNA extraction, four PCR assays were developed and compared using the B1 gene as the target gene for the detection of T. gondii DNA in meat. The results show high specificity and reproducibility for all four PCR assays in clearly detecting DNA samples from four strains of T. gondii while producing no amplification signal for ten other parasites or in healthy meat. Three real‐time quantitative PCR assays were 10‐fold more sensitive than the conventional PCR assay, with a detection limit of 4.5 × 101 copies of T. gondii DNA being equivalent to 1.3 bradyzoites. The most sensitive method was SYBR Green I qPCR because it uses special fluorescent dyes directly binding to the amplified double‐stranded DNA sequences and the fluorescence signal is proportional to the amount of PCR products that observed by nearly 1,000‐fold increase in fluorescence intensity. This assay has another advantages over other PCR assays, including less expense (without probe), ease of operation and quicker operation of the test. Practical applications The traditional method for detecting T. gondii in meat samples involves the use of a bioassay that is laborious, time‐consuming and not sensitive. The SYBR Green I qPCR assay developed in this study provides a rapid, sensitive, and specific technique and is applicable to surveillance measures for foodborne toxoplasmosis pathogens in meat or meat products as an alternative detection method.

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“…Toxoplasmosis in adults with normal immune function is generally asymptomatic however it can lead to a wide range of clinical manifestations in fetus and immune-compromised patients, such as those with acquired immuno-deficiency syndrome, immunosuppressed cancer patients and transplant recipients (Koethe et al 2015;Masatani et al 2016).…”

Section: Introductionmentioning

confidence: 99%

The use of Toxoplasma gondii tachyzoites produced in HeLa cells adhered to Cytodex 1 microcarriers as antigen in serological assays: an application of microcarrier technology

et al. 2019

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Toxoplasma gondii can infect nearly all warm-blooded animals, including humans. In the laboratory diagnosis of toxoplasmosis, serological tests have importance in detecting antibody response. Traditionally T. gondii tachyzoites grown in vivo are being used as an antigen source in serological assays. Currently, tachyzoites produced in vitro are being tested as an antigen source in order to decrease animal use. Microcarrier technology allowed us to grow anchorage-dependent host cells on microcarrier suspension in short time and approximately 10 times more than traditional flask technique. The ability of T. gondii tachyzoites to grow in host cells adhered to microcarriers has not been analyzed yet. In this study, we aimed to develop a novel in vitro culture method to produce T. gondii tachyzoites abundantly using HeLa cells adhered to Cytodex 1 microcarriers. Initially, the growth of HeLa cells adhered to Cytodex 1 was analyzed using RPMI 1640, DMEM, and EMEM. Next, HeLa cells with a concentration of 1 9 10 5cells/ml and 2 9 10 5 cells/ml were adhered to Cytodex 1 and grown in spinner flasks. Then, T. gondii tachyzoites were inoculated with 1:1 and 2:1 cell:tachyzoite ratios to HeLa cells adhered to microcarriers in spinner flaks. During continuous production in spinner flasks, tachyzoites were harvested at the 2nd, 4th, and 7th day of culture and the quality of antigens produced from these tachyzoites were tested in ELISA and Western Blotting using sera of patients with toxoplasmosis. The optimization studies showed that finest HeLa inoculation value was 2 9 10 5 cells/ml using RPMI 1640, and the cell:tachyzoite ratio to obtain the highest tachyzoite yield (17.1 9 10 7) was 1:1 at the 4th day of inoculation. According to the results of ELISA comparing HeLa cell and mouse derived antigens, the highest correlation with mouse antigen was achieved at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio (P \ 0.0001). The sensitivity and specificity ratios of ELISA were 100%. In addition, Western blotting banding patterns Saime İsmet Deliloglu Gürhan, Mert Döşkaya were the advisors of the BS student Pelin Saglam Metiner.

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Quantitative detection of Toxoplasma gondii in tissues of experimentally infected turkeys and in retail turkey products by magnetic-capture PCR

Cited by 28 publications

References 31 publications

Serological and molecular detection of Toxoplasma gondii in terrestrial and marine wildlife harvested for food in Nunavik, Canada

Serological and molecular detection of Toxoplasma gondii in terrestrial and marine wildlife harvested for food in Nunavik, Canada

Assessment of self‐produced PCR methods for the detection of Toxoplasma gondii DNA in meat

The use of Toxoplasma gondii tachyzoites produced in HeLa cells adhered to Cytodex 1 microcarriers as antigen in serological assays: an application of microcarrier technology

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