Effect of meat processing on viability of Toxoplasma gondii: Towards replacement of mouse bioassay by in vitro testing

Felines are the definitive hosts of T. gondii and primary infection results in fecal shedding of infectious oocysts. Infected intermediate hosts will develop tissue cysts, which are infective to both cats and intermediate hosts. Meat containing viable tissue cysts is considered one of the main sources of human infection. In contrast to fresh meat, raw meat products usually undergo processing, including salting and mixing in additives such as acetate and lactate, which affects the viability of T. gondii. However, the experiments currently described in literature, are not always performed in line with the processing methods applied in industry. Therefore we aimed to study the effect of salting and additives according to the recipes used by commercial producers. Mouse or cat bioassay is the gold standard to demonstrate the presence of viable T. gondii. However, it is costly, time consuming and for ethical reasons not preferred for large-scale studies. Therefore, our second aim was to develop an alternative for mouse bioassay that can be used to determine the effect of processing on the viability of T. gondii tissue cysts. We focused on a tissue culture method to determine the parasite's ability to multiply, and a PMA-based assay to selectively detect DNA from live cells. Results with the PMA-based method were inconsistent and did not sufficiently discriminate between live and dead parasites. The tissue culture method showed promising results, but further optimization is needed before it can replace or reduce the number of mouse bioassays needed. Small scale experiments with minced meat incubated for 20h with low concentrations of salt, lactate and acetate showed a large but incomplete reduction of the number of infected mice. In future, in vitro methods are needed to allow more extensive testing of product-specific processing methods, thereby providing a better indication of the risk of T. gondii infection for consumers.