Molecular and immunological characterisation of proteins from Anisakis pegreffii and their immune stimulatory effect on the human health system

With the fast growth of seafood trading worldwide, the potential health risks of eating contaminated seafood have greatly increased. Biological effects of contaminated seafood are associated and caused by a variety of bacteria, viruses, and parasites; this various groups of pathogens results in an extensive diversity of clinical syndromes, each with its own epidemiological characteristics. According to a World Health Organization investigation, more than 1 billion people worldwide are infected with different species of the Ascaris lumbricoides worm parasite (282), which causes serious conditions ranging from mild to lethal. Anisakis parasites in marine fish have imposed a significant economic burden, reducing productivity and requiring elaborate and expensive control methods. Nematodes of the family Anisakidae is a major group parasitise fish, mammals, birds and reptiles, with the larval stages of some species. Several species of Anisakis have been found to be parasitic in marine mammals such as whales and dolphins in their adult stages, and their larvae are found in a variety of fish species. Anisakid nematodes have complex life cycles that include invertebrate and vertebrate hosts at various developmental stages during their life. Most reports of anisakiasis infections are associated with eating raw or undercooked fish that contain larval Anisakis. Humans can also be accidental hosts for larval Anisakis, however they cannot progress their life cycles, but they can frequently cause hypersensitivity IgE-mediated reactions with or without several gastrointestinal manifestations ranging from urticaria to angioedema, called anisakiasis diseases. The development of an allergic reaction mediated by IgE may be against ES and somatic allergens of larval Anisakis. Nuclear ribosomal DNA (rDNA) provides suitable genetic markers for the identification of larval Anisakis. Therefore, the sequences of internal transcribed spacers (ITS-1 and ITS-2) are a powerful approach to identify and distinguish anisakid nematodes (at any developmental stage) for diagnostic or taxonomic purposes, for exploring the genetic composition of larval anisakid populations and for investigating their ecology. Although several allergens have been identified in Anisakis simplex, little information is available for other parasite species infecting fish. Therefore, this project aims to investigate the allergenicity of tropomyosin (Ani p 3) and a cysteine protease inhibitor (Ani p 4) from the sibling species, Anisakis pegreffii, using biochemical, genetic and immunological approaches. Larvae (L3) were harvested manually from tiger flathead fish and identified morphologically using a light microscope. For molecular characterisation and genetic diversity, total genomic DNA was extracted from individual samples and verified by species-specific PCR amplification using the sequences of the first and/or second internal transcribed spacers (ITS-1 and/or ITS-2) of ribosomal DNA (rDNA) as the species-specific genetic markers. For molecular characterisation of Anisakis allergens, RNA of Anisakis pegreffii was extracted, then cDNA synthesised and amplified using designed primers and used to amplify tropomyosin and cysteine protease inhibitor genes from Anisakis pegreffii. Whole muscle proteins from Anisakis larvae were extracted directly with extraction solution and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by immunoblotting assays. Tropomyosin protein was detected using anti-tropomyosin and anti-crustacean polyclonal antibodies raised in rabbits while a cysteine protease inhibitor was detected using anti-cysteine protease inhibitor polyclonal antibodies raised in rabbit. The allergenicity of the nematode tropomyosin and cysteine protease inhibitor was evaluated using human serum of atopic patient with a shellfish allergy. The result of SDS-PAGE and immunoblotting assays of Anisakis pegreffii extracts indicates the presence of tropomyosin and cysteine protease inhibitor proteins of 44 and 12.8 kDa molecular weight, respectively. These results are similar to the allergenic tropomyosin and cysteine protease inhibitor of Anisakis simplex. Also, strong reactivity with human serum designated these proteins as allergens in Anisakis pegreffii spp. The molecular sequencing was successful for tropomyosin and cysteine protease inhibitor genes from Anisakis pegreffii, and demonstrated that they are closely related to the Anisakis simplex proteins previously sequenced and submitted in the GenBank database. Our results indicated that, the cDNA sequence similarity searches using the BLAST program and phylogenetic analysis (Clustal W programme) revealed that tropomyosin of A. pegreffii L3 has the highest homology to α tropomyosins isoform from A. simplex L3 (98%) and A. lumbricoides (84%), respectively. In addition, cysteine-protease inhibitor of A. pegreffii L3 has the highest identity to cysteine-protease inhibitor isoform from A. simplex L3 (98%) using the BLAST program and 100% using phylogenetic analysis (Clustal W programme). This analysis highlights that this nematode tropomyosin and cysteine protease inhibitor share high sequence identity with other parasitic nematodes, house dust and crustacean tropomyosin, respectively