ABSTRACT: Detection and characterisation of putative allergens in Anisakis food-borne parasites using advanced transcriptomic and bioinformatic technologies
Project description:Protein extracts from Anisakis simplex L3 larvae were autoclaved at 121C for 60 min. Subsequently, prepared samples were analyzed in LC-MS/MS to identify autoclave-resistant proteins (also allergens).
Project description:The total proteomes of Anisakis simplex s.s., A. pegreffii and their hybrid genotype have been compared by quantitative proteomics (iTRAQ approach), which considers the level of expressed proteins. A total of 1,976 proteins have been identified using public databases. One hundred ninety six proteins were found significantly differentially expressed; results of pairwise Log2 ratio comparisons among them were statistically treated and supported in order to convert them into discrete character states. This comparison selected thirty six proteins as discriminant biomarkers among A. simplex, A. pegreffii and their hybrid genotype; eighteen of these biomarkers, encoded by nine loci, are specific allergens of Anisakis (Ani s7, Ani s8, Ani s12 and Ani s14) and other (Ancylostoma secreted) is a common nematodes venom allergen.
Project description:The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius.
Project description:Two hundred ventricles were manually dissected from L3 stage larvae of Aniskakis simplex s.s. to allow direct protein analysis. Denaturing gel electrophoresis followed by monochromatic silver staining revealed the presence of differential (enriched) proteins when compared to total nematode extracts. Such comparison was performed by means of 1D and 2D electrophoresis. Pooled antisera from Anisakis spp allergic patients were used in western blots revealing the presence of 13 immunoreactive bands in the ventricular extracts in 1D, while 82 spots in 2D. The corresponding protein bands and spots were excised from the silver stained gel and protein assignation was made by MALDI-TOF/TOF. A total of eleven proteins (including isoforms) were unambiguosly identified. The majority of these proteins are known to be secreted by nematodes into the external environment, of which three are described as being major allergens in other organisms with different phylogenetic origin.
