STress Responses In the foodborne protozoan Parasites Toxoplasma gondii and Cryptosporidium spp. – STRIP

The proposed multidisciplinary approach will correlate structural changes, metabolic pathways and oocyst infectivity, following the application of stress, in laboratory conditions, but also at a pilot-scale in food matrices.

The project will study the stress response at the following levels:
i) Structure, mechanics and surface properties of the oocyst wall (WP2)
- permeability of the wall (fluorescent probes)
- adhesion rate of oocysts to host cells
- ultrastructure (electron microscopy)
- mechanical properties and adhesion (Atomic Force Microscopy)
- hydrophobicity (measurement of contact angles) and electrical charge (zetametry)

ii) Viability and infectivity of oocysts (WP3)
- viability: assessed by RT-qPCR (mRNA detection)
- infectivity: determined by infection of cells or in animal models.

iii) Metabolism (WP4)
- transcriptomic study by RNA seq.
- comparative differential label-free proteomic study (nanoLC-MS / MS)

In addition, the impact of oxidative stress (chlorine, ozone) on the survival of oocysts in food matrices will be evaluated at a pilot-scale. The viability and the infectivity of the oocysts will be determined after having stressed oocysts adhered to the surface of vegetables, under conditions miming those implemented in minimally-processed vegetables industries (pilot of 90L).

Expected results are:
1. To accurately characterize the oocyst wall of T. gondii and C. parvum for a better understanding of its role in the resistance and survival of oocysts, in their ability to adhere to food matrices and in their behavior.
2. To provide an overall view of the stress response.
3. To correlate structural, biophysical and metabolic changes with information on viability and infectivity of oocysts. These results will allow the identification of markers of the infective character of the oocysts.
4. To produce the first efficacy data for chlorine and ozone treatments on protozoan parasites in food.

- Stress response mechanisms: these fundamental data will contribute to a better understanding of the interactions and survival of oocysts in their environments (food, water). In the long term perspective, this knowledge will help to better understand the transmission of these pathogens to humans.
- Identification of markers of the infective character: such targets could be part of a decision support tools in risk assessment.
- Relationship between host and parasites: these works will allow the identification of new specific therapeutic targets that are still lacking for humans.
- Effects of treatments on the structure: the concentration / purification techniques of oocysts based on their surface properties will benefit from this new knowledge to increase efficiency and reliability.
- Efficacy of MPV usual (chlorine) and innovative (ozone) processes: these data will make it possible to identify critical points and to propose control measures. In a long term perspective, the characterization of efficient and safe processes will have a positive impact on the economy of agri-food companies (competitiveness, reduction of costs related to curative solutions, potential recalls of batches, etc.) and on the cost of healthcare.

The results obtained in this project will be disseminated through national / international conferences and through expert networks of the different partners. They will also be communicated through scientific publications in peer-reviewed journals.

The emergence of foodborne parasites, such as the protozoa Cryptosporidium spp. and Toxoplasma gondii, is greatly favored by changing in eating habits towards fresh and minimally processed food, the global trade of foodstuffs, changes in food production systems and the increased number of sensitive people. Foodborne transmission of Cryptosporidium spp. and Toxoplasma gondii greatly depends on the ability of their oocyst forms to remain infective in food matrices in spite of external stresses combined to food characteristics. We hypothesize that oocysts are able to remain infective in foods by adapting their biological functions (structure and metabolism) at the cellular and/or molecular levels, and that food matrices can modify this response and the ability of the parasites to survive. In this context, the first objective of the STRIP project is to characterize, following a multidisciplinary approach, the biological response of oocysts submitted to selected stresses encountered by these parasites in food matrices (pH variations, reducing conditions) or following exposure to chemical disinfectants (chlorine, ozone) as applied in minimally-processed vegetables (MPV) industries. The oocyst response will be evaluated regarding (i) the structure, mechanics, and biochemistry of the oocyst wall and its possible interaction with host cells, (ii) the ability of oocysts to remain viable and infective, and (iii) the potential of the oocysts to adapt their metabolism to retain their infectivity. A second objective is to evaluate the impact of oxidative stress, at a pilot-scale, on the survival of oocysts associated to food matrices (leafy green vegetables). To reach these objectives, we will implement innovative imaging, biophysical, and ‘omic’ technologies coupled to viability and infectivity assays (RT-qPCR techniques, animal and cell culture assays). This tools and methodologies will provide an unprecedented characterization of the molecular and cellular mechanisms by which Cryptosporidium and T. gondii oocysts resist to stresses and retain their infectivity in food. The new important basic knowledge generated by the STRIP project will benefit to the field of food parasitology by providing for the first time a response-wide overview to stresses of these parasites, and to the 'agro-food' sector, in particular MPV industries, in order to implement innovative control measures, such as ozone-based technologies. We expect that the STRIP project contribute to a better risk assessment and management of these emerging foodborne parasitic pathogens in a context of global trade.