An in-vitro method and predictive model to quantify Toxoplasma gondii inactivation by salting

Consumption of raw meat products is considered an important source of T. gondii infections. These products are usually processed with NaCl and other additives. Such additives affect the viability of T. gondii, but results described in literature are variable. Mouse bioassay is currently the standard method for T. gondii inactivation experiments, but ethically undesirable, costly and time consuming. Moreover, mouse bioassays provide very limited quantitative information on actual reductions in the concentration of viable parasites in tested meat products. Therefore it was our aim to validate and compare an in-vitro method as a quantitative alternative to the mouse bioassay for determining the effect of salting on T. gondii viability.

Muscle tissue of sheep experimentally infected with T. gondii was used to prepare 50 g portions of minced meat supplemented with 0.6 - 2.7% NaCl and sodium lactate or sodium acetate. Portions were stored overnight at 4°C. Pepsin-HCl digestion was performed and digests were inoculated onto RK13 cell monolayers or into IFNγ-KO mice, to evaluate the presence of infective parasites. In addition, a four-fold dilution series ranging from 4 to 256 bradyzoites per ml of tissue digest or culture medium was tested to evaluate and compare the detection limit of these methods.

Both, the in-vitro and the in-vivo method gave positive results for samples spiked with 4 or 16 bradyzoites and were consistently positive when a minimum of 64 bradyzoites was added. Viable parasites were detected in portions of experimental sheep tissue supplemented with up to 1.5% NaCl, but not in samples supplemented with both 1.2% NaCl and 1.4% sodium lactate. A statistical model was fitted, using actual and previous results with cell culture and, as an example, indicates a 5-log reduction in infective parasites after supplementation with 2.5% NaCl.

In conclusion, the in-vitro method was successfully used to obtain data on T. gondii inactivation by salting. The model provides more insight in the possibilities to use salt for controlling the risk of T. gondii in raw meat products. Replacement of the mouse bioassay opens up possibilities to investigate other potential inactivation methods. In the future, we hope to also overcome the need for experimental infection to obtain positive muscle tissue and we look forward to discuss possibilities with in-vitro cultivated tissue cysts.