Anisakis simplex (s.l.) resistance to the action of gastric enzymes depends upon previous treatments applied to infected fish mince and affects antigen release

Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives that determine endpoints such as the bioaccessibility of nutrients and non-nutrients or the digestibility of macronutrients (e.g. lipids, proteins and carbohydrates) are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.

Infection of humans with the nematode worm parasite Anisakis simplex was first described in the 1960s in association with the consumption of raw or undercooked fish. During the 1990s it was realized that even the ingestion of dead worms in food fish can cause severe hypersensitivity reactions, that these may be more prevalent than infection itself, and that this outcome could be associated with food preparations previously considered safe. Not only may allergic symptoms arise from infection by the parasites ("gastroallergic anisakiasis"), but true anaphylactic reactions can also occur following exposure to allergens from dead worms by food-borne, airborne, or skin contact routes. This review discusses A. simplex pathogenesis in humans, covering immune hypersensitivity reactions both in the context of a living infection and in terms of exposure to its allergens by other routes. Over the last 20 years, several studies have concentrated on A. simplex antigen characterization and innate as well as adaptive immune response to this parasite. Molecular characterization of Anisakis allergens and isolation of their encoding cDNAs is now an active field of research that should provide improved diagnostic tools in addition to tools with which to enhance our understanding of pathogenesis and controversial aspects of A. simplex allergy. We also discuss the potential relevance of parasite products such as allergens, proteinases, and proteinase inhibitors and the activation of basophils, eosinophils, and mast cells in the induction of A. simplex-related immune hypersensitivity states induced by exposure to the parasite, dead or alive.

Polymerase-chain-reaction-based restriction fragment length polymorphism analysis was performed to establish genetic markers in rDNA, for the identification of the three sibling species of the Anisakis simplex complex and morphologically differentiated Anisakis species, i.e. Anisakis physeteris, Anisakis schupakovi, Anisakis typica and Anisakis ziphidarum. Different restriction patterns were found between A. simplex sensu stricto and Anisakis pegreffii with two of the restriction endonucleases used (HinfI and TaqI), between A. simplex sensu stricto and A. simplex C with one endonuclease (HhaI), and between A. simplex C and Aniskis pegreffii with three endonucleases (HhaI, HinfI and TaqI), while no variation in patterns was detected among individuals within each species. The species A. physeteris, A. schupakovi, A. typica and A. ziphidarum were found to be different from each other and different from the three sibling species of the A. simplex complex by distinct fragments using 10-12 of the endonucleases tested. The polymorphisms obtained by restriction fragment length polymorphisms have provided a new set of genetic markers for the accurate identification of sibling species and morphospecies.