EU-FORA 2024/25: Assessing allergenic potential, exposure, and risk of Anisakis spp.

The program aims to enable a risk assessment of the allergenic potential of Anisakis spp. a foodborne parasite of increasing importance. This will be achieved by (i) providing thoroughly validated analytical tools for species and allergen detection in the relevant matrices, (ii) enabling an exposure assessment by generating a high quality dataset comprising sample collections from both participating institutions, (iii) collecting relevant food consumption data available for the European Union and (iv) assessing the allergenic potential of Anisakis spp. All data will be combined to start a risk assessment process to evaluate the emerging risks of sensitization and allergenic reactions to Anisakis spp. in Europe.

A DNA-based detection method (real-time PCR) was successfully developed and extensively validated (Task 1). However, low specificity has been observed for the primers and probes published to date, hence requiring them to be newly designed, and an entirely new method to be developed, rather than merely establishing and verifying a pre-existing method. Validation was pursued according to guidelines and proved the detection of all tested Anisakis species to be feasible in as low as 10^(-9) Ct in both raw and processed fish patties and with no interfering results from > 50 species relevant for food matrices. In a small market survey dedicated to assessing Anisakis prevalence, 3 out of 12 feed samples declaring fish ingredients were detected positive for Anisakis spp., whereas none were traceable in the 70 food-grade fish (filets) and 22 feed-grade fish meal samples studied to date. As a result of the more extensive work required for Task 1 and for the sake of focusing on not only the exposure side of risk assessment, mass-spectrometric method development and validation were abandoned (Task 2). Instead, the expression of two allergenic Anisakis proteins was pursued later to assess their relative allergenicity (Task 3). The two custom-synthesised plasmid constructs did not express the desired proteins after being transfected into E. coli cells, therefore further and ongoing work is dedicated to firstly transferring the constructs into different expression vectors, followed by test expressions to identify conditions that provide maximum yields of the target proteins. Risk assessment (Task 4) will be possible only after completing the hazard assessment, i.e., allergy assessment, and is currently postponed until after the programme’s end, as already outlined in the proposal.The fellow participated in all EFSAshort forEuropean Food Safety Authority training courses and worked through the associated training materials provided by EFSAshort forEuropean Food Safety Authority. In addition, a comprehensive literature review was conducted, incorporating both independent research and key publications recommended by the supervisors. The BfRshort forGerman Federal Institute for Risk Assessment coordinator provided the relevant laboratory protocols, and all supervisors jointly agreed on a step-by-step approach for the real-time PCR validation process. The fellow learned to work in a DIN EN ISO/IEC 17025-accredited laboratory and about international guidelines for PCR method validation required before attempting their standardisation. Furthermore, the fellow learned and executed protocols for cloning and protein expression in E. coli. Regular project meetings have been held to monitor progress, and the BfRshort forGerman Federal Institute for Risk Assessment / BCAS partners continue to meet biweekly to proactively address any emerging issues related to the project’s activities.