In vitro assay to determine inactivation of Toxoplasma gondii in meat samples

Kód výsledku v IS VaVaI

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RIV/62157124:16810/22:43880128

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

In vitro assay to determine inactivation of Toxoplasma gondii in meat samples

Popis výsledku v původním jazyce

Consumption of raw and undercooked meat is considered as an important source of Toxoplasma gondii infections in Europe. However, most non-heated meat products are processed by salting and addition of additives. NaCl affects T. gondii viability, but published experiments show variable results and are not always performed in line with industrial processing. Currently, mouse bioassay is the standard for determining T. gondii viability, but the use of experimental animals limits possibilities for large scale testing. It was our aim to develop an in vitro method to substitute the bioassay and to determine the effect of salting on T. gondii viability. Two sheep were orally inoculated with 6.5×104 oocysts, and infection confirmed by the detection of anti-T. gondii antibodies in combination with qPCR on tissue samples collected 10 weeks post inoculation. Grinded meat samples of 50 g were prepared from heart, diaphragm, four meat cuts (thick flank, strip loin, topside and silverside), and a pooled sample made from those meat cuts. The pooled meat samples were either kept untreated (positive control), frozen (negative control) or supplemented with 0.6 %, 0.9 %, 1.2 % or 2.7 % NaCl. All samples were digested in pepsin-HCl solution. For each, 1 ml of digest was inoculated in duplicate onto monolayers of RK13 (a rabbit kidney cell line). Cells were maintained for 4 weeks, replacing 50 % of the cell culture medium two times per week. Parasite growth was monitored by assessing the Cq-values in T. gondii qPCR for the cell-culture supernatant in intervals of one week. Decreasing Cq-values (i.e. a positive ?Cq) indicated multiplying parasites. To confirm viability of parasites in untreated meat samples, 1 ml of each digest for the individual meat cuts, heart and diaphragm were inoculated in duplicate in IFN? KO mice. Mice were euthanized when developing signs of toxoplasmosis or kept for six weeks, and tested serologically and by qPCR. Both sheep developed an antibody response and tissue samples contained similar concentrations of T. gondii DNA. For all untreated meat samples positive ?Cq values were detected in cell culture. These results indicated viability of parasites in individual meat samples; this was in line with mouse bioassay, with the exception of a negative mouse bioassay for the heart of the second sheep. Samples supplemented with 0.6 %-1.2 % NaCl showed positive ?Cq values over time. The frozen sample and sample supplemented with 2.7 % NaCl remained qPCR positive but with high Cq-values which indicated no growth. In conclusion, the in vitro method has successfully been used to detect viable T. gondii in tissues of experimentally infected sheep, and a clear difference in T. gondii viability was observed between the samples supplemented with 2.7 % NaCl and those with 1.2 % NaCl or less. In future, more NaCl concentrations or additives will be tested to obtain better insight in the risk of T. gondii infection via nonheated processed meat products.

Název v anglickém jazyce

In vitro assay to determine inactivation of Toxoplasma gondii in meat samples

Popis výsledku anglicky

Consumption of raw and undercooked meat is considered as an important source of Toxoplasma gondii infections in Europe. However, most non-heated meat products are processed by salting and addition of additives. NaCl affects T. gondii viability, but published experiments show variable results and are not always performed in line with industrial processing. Currently, mouse bioassay is the standard for determining T. gondii viability, but the use of experimental animals limits possibilities for large scale testing. It was our aim to develop an in vitro method to substitute the bioassay and to determine the effect of salting on T. gondii viability. Two sheep were orally inoculated with 6.5×104 oocysts, and infection confirmed by the detection of anti-T. gondii antibodies in combination with qPCR on tissue samples collected 10 weeks post inoculation. Grinded meat samples of 50 g were prepared from heart, diaphragm, four meat cuts (thick flank, strip loin, topside and silverside), and a pooled sample made from those meat cuts. The pooled meat samples were either kept untreated (positive control), frozen (negative control) or supplemented with 0.6 %, 0.9 %, 1.2 % or 2.7 % NaCl. All samples were digested in pepsin-HCl solution. For each, 1 ml of digest was inoculated in duplicate onto monolayers of RK13 (a rabbit kidney cell line). Cells were maintained for 4 weeks, replacing 50 % of the cell culture medium two times per week. Parasite growth was monitored by assessing the Cq-values in T. gondii qPCR for the cell-culture supernatant in intervals of one week. Decreasing Cq-values (i.e. a positive ?Cq) indicated multiplying parasites. To confirm viability of parasites in untreated meat samples, 1 ml of each digest for the individual meat cuts, heart and diaphragm were inoculated in duplicate in IFN? KO mice. Mice were euthanized when developing signs of toxoplasmosis or kept for six weeks, and tested serologically and by qPCR. Both sheep developed an antibody response and tissue samples contained similar concentrations of T. gondii DNA. For all untreated meat samples positive ?Cq values were detected in cell culture. These results indicated viability of parasites in individual meat samples; this was in line with mouse bioassay, with the exception of a negative mouse bioassay for the heart of the second sheep. Samples supplemented with 0.6 %-1.2 % NaCl showed positive ?Cq values over time. The frozen sample and sample supplemented with 2.7 % NaCl remained qPCR positive but with high Cq-values which indicated no growth. In conclusion, the in vitro method has successfully been used to detect viable T. gondii in tissues of experimentally infected sheep, and a clear difference in T. gondii viability was observed between the samples supplemented with 2.7 % NaCl and those with 1.2 % NaCl or less. In future, more NaCl concentrations or additives will be tested to obtain better insight in the risk of T. gondii infection via nonheated processed meat products.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

40301 - Veterinary science

Projekt

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Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů