Investigating contamination of ready-to-eat salads with Toxoplasma gondii oocysts in a European-wide multicenter survey

The majority of horizontal Toxoplasma gondii infections in humans are assumed to be foodborne. The relative contribution of the meat-borne (tissue cysts) versus environmental (oocyst-driven) routes of infections is largely unknown. The consumption of unwashed raw fruits or vegetables contaminated with T. gondii oocysts has been identified as a putative risk factor. Important methodological inconsistencies and limitations have been identified in the surveys carried out worldwide in fresh produce. The present study aimed to unravel the occurrence of T. gondii oocysts in commercially available ready-to-eat (RTE) salad mixes in ten European countries. For that purpose, the detection methodology was first validated in an interlaboratory ring trial. Next, a harmonized sampling strategy and the validated methodology were implemented by all participants.

In this multicentre study, weekly sampling (from October 2021 to October 2022) was set to detect seasonal variations. Two categories of RTE salad mixes (baby leaves and cut leaves mixes) were sampled to explore potential associations between oocyst contamination and cultivation and growth conditions. In the study period, 3,329 RTE salad samples were tested using a standard operating procedure (SOP) that allows molecular detection of T. gondii oocysts in such matrices with a detection limit of 10 oocysts per 30 g of salad. Robustness and sensitivity of this SOP as well as the comparability of results among the laboratories had been confirmed in the ring trial before the start of the study. The key analytical steps include oocysts recovery by sample washing followed by pelleting of the eluate by centrifugation, DNA extraction and DNA detection by triplex qPCR, targeting two multicopy fragments (529RE, B1 gene) and an internal amplification control. Positive samples were confirmed by ITS-1 single tube nested PCR followed by Sanger sequencing. This is the first European-wide study to estimate the prevalence of T. gondii oocysts in RTE leafy green salads using a validated and standardized procedure to assess the associated potential risk for human infections.

This work was done as part of TOXOSOURCES project, EU Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme.