Consumption of raw and undercooked meat is considered as an important source of infections. However, most non-heated meat products contain salt and additives, which affect viability. It was our aim to develop an in vitro method to substitute the mouse bioassay for determining the effect of salting on viability. Two sheep were experimentally infected by oral inoculation with 6.5 × 10 oocysts. Grinded meat samples of 50 g were prepared from heart, diaphragm, and four meat cuts. Also, 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, and digests were inoculated in duplicate onto monolayers of RK13 (a rabbit kidney cell line). Cells were maintained for up to four weeks and parasite growth was monitored by assessing Cq-values using the qPCR on cell culture supernatant in intervals of one week and ΔCq-values determined. Additionally, 500 μL of each digest from the individual meat cuts, heart and diaphragm were inoculated in duplicate in IFNγ KO mice. Both sheep developed an antibody response and tissue samples contained similar concentrations of DNA. From all untreated meat samples positive ΔCq-values were obtained in the in vitro assay, indicating presence and multiplication of viable parasites. This was in line with the mouse bioassay, with the exception of a negative mouse bioassay on one heart sample. Samples supplemented with 0.6 %–1.2 % NaCl showed positive ΔCq-values over time. The frozen sample and the 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 in tissues of experimentally infected sheep, and a clear difference in viability was observed between the samples supplemented with 2.7 % NaCl and those with 1.2 % NaCl or less.

中文翻译：

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体外测定肉类样品中弓形虫的灭活情况

食用生肉和未煮熟的肉被认为是重要的感染源。然而，大多数非加热肉制品含有盐和添加剂，这会影响活力。我们的目标是开发一种体外方法来替代小鼠生物测定法来确定盐腌对活力的影响。两只羊通过口服接种6.5×10个卵囊进行实验感染。 50 克绞肉样品由心脏、膈肌和四块切肉制成。此外，混合的肉样品要么未经处理（阳性对照），要么冷冻（阴性对照），要么补充 0.6%、0.9%、1.2% 或 2.7% 氯化钠。所有样品均在胃蛋白酶-HCl 溶液中消化，并将消化物一式两份接种到单层 RK13（兔肾细胞系）上。将细胞维持长达 4 周，并通过每隔一周对细胞培养物上清液使用 qPCR 评估 Cq 值来监测寄生虫生长，并确定 ΔCq 值。此外，将来自各个肉块、心脏和隔膜的每种消化物各 500 μL 一式两份接种到 IFNγ KO 小鼠中。两只羊都产生了抗体反应，组织样本中含有相似浓度的 DNA。在体外测定中，所有未经处理的肉类样品均​​获得了阳性 ΔCq 值，表明活寄生虫的存在和繁殖。这与小鼠生物测定结果一致，除了一份心脏样本的小鼠生物测定结果为阴性之外。补充了 0.6%–1.2% NaCl 的样品随着时间的推移显示出正的 ΔCq 值。冷冻样品和补充了 2.7% NaCl 的样品仍保持 qPCR 阳性，但 Cq 值较高，表明没有生长。总之，体外方法已成功用于检测实验感染羊组织中的存活率，并且在添加 2.7% NaCl 的样品和添加 1.2% NaCl 或更少的样品之间观察到存活率的明显差异。