Novel Allergen Discovery through Comprehensive De Novo Transcriptomic Analyses of Five Shrimp Species

Shellfish allergy affects 2% of the world's population and persists for life in most patients. The diagnosis of shellfish allergy, in particular shrimp, is challenging due to the similarity of allergenic proteins from other invertebrates. Despite the clinical importance of immunological cross-reactivity among shellfish species and between allergenic invertebrates such as dust mites, the underlying molecular basis is not well understood. Here we mine the complete transcriptome of five frequently consumed shrimp species to identify and compare allergens with all known allergen sources. The transcriptomes were assembled de novo, using Trinity, from raw RNA-Seq data of the whiteleg shrimp (Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), banana shrimp (Fenneropenaeus merguiensis), king shrimp (Melicertus latisulcatus), and endeavour shrimp (Metapenaeus endeavouri). BLAST searching using the two major allergen databases, WHO/IUIS Allergen Nomenclature and AllergenOnline, successfully identified all seven known crustacean allergens. The analyses revealed up to 39 unreported allergens in the different shrimp species, including heat shock protein (HSP), alpha-tubulin, chymotrypsin, cyclophilin, beta-enolase, aldolase A, and glyceraldehyde-3-phosphate dehydrogenase (G3PD). Multiple sequence alignment (Clustal Omega) demonstrated high homology with allergens from other invertebrates including mites and cockroaches. This first transcriptomic analyses of allergens in a major food source provides a valuable resource for investigating shellfish allergens, comparing invertebrate allergens and future development of improved diagnostics for food allergy

Exposure to Bioaerosols during Fish Processing on Board Norwegian Fishing Trawlers

Objectives: The main objective was to gain more knowledge on exposure to bioaerosols in the processing area on board fishing trawlers. Methods: Exposure sampling was carried out during the work shifts when processing fish in the processing area on board five deep-sea fishing trawlers (trawlers 1-5). Exposure samples were collected from 64 fishermen breathing zone and from stationary sampling stations on board five deep-sea fishing trawlers (1-5). Trawlers 2, 3, and 4 were old ships, not originally built for on board processing of the catch. Trawlers 1 and 5 were relatively new and built to accommodate processing machineries. On trawlers 1-4 round fish was produced; the head and entrails were removed before the fishes were frozen in blocks. Trawler 5 had the most extensive processing, producing fish fillets. Samples were analysed for total protein, trypsin activity, parvalbumin, and endotoxin. One side analysis of variance and Kruskal-Wallis H test were used to compare levels of exposure on the different trawlers. Results: Personal exposure to total protein were higher on the three oldest trawlers (2, 3, and 4) compared with the two new trawlers (1 and 5). Highest activity of trypsin was detected on the four trawlers producing round fish (1-4). Parvalbumin was detected in 58% of samples from the fillet-trawler (5) compared with 13% of samples from the four trawlers producing round fish. The highest level of endotoxin was detected when using high-pressure water during cleaning machines and floors in the processing area. Conclusions: Fishermen in the processing area on board Norwegian trawlers are exposed to airborne bioaerosols as proteins, trypsin, fish allergen parvalbumin, and endotoxin. Levels varied between trawlers and type of production

Variability of allergens in commercial fish extracts for skin prick testing

Background Commercial allergen extracts for allergy skin prick testing (SPT) are widely used for diagnosing fish allergy. However, there is currently no regulatory requirement for standardization of protein and allergen content, potentially impacting the diagnostic reliability of SPTs. We therefore sought to analyse commercial fish extracts for the presence and concentration of fish proteins and in vitro IgE reactivity using serum from fish-allergic patients. Methods Twenty-six commercial fish extracts from five different manufacturers were examined. The protein concentrations were determined, protein compositions analysed by mass spectrometry, followed by SDS-PAGE and subsequent immunoblotting with antibodies detecting 4 fish allergens (parvalbumin, tropomyosin, aldolase and collagen). IgE-reactive proteins were identified using serum from 16 children with confirmed IgE-mediated fish allergy, with focus on cod, tuna and salmon extracts. Results The total protein, allergen concentration and IgE reactivity of the commercial extracts varied over 10-fold between different manufacturers and fish species. The major fish allergen parvalbumin was not detected by immunoblotting in 6/26 extracts. In 7/12 extracts, five known fish allergens were detected by mass spectrometry. For cod and tuna, almost 70% of patients demonstrated the strongest IgE reactivity to collagen, tropomyosin, aldolase A or beta-enolase but not parvalbumin. Conclusions Commercial fish extracts often contain insufficient amounts of important allergens including parvalbumin and collagen, resulting in low IgE reactivity. A comprehensive proteomic approach for the evaluation of SPT extracts for their utility in allergy diagnostics is presented. There is an urgent need for standardized allergen extracts, which will improve the diagnosis and management of fish allergy

Characterization of Ras k 1 a novel major allergen in Indian mackerel and identification of parvalbumin as the major fish allergen in 33 Asia-Pacific fish species

Background: Fish is a well-recognized cause of food allergy and anaphylaxis. The evolutionary and taxonomic diversity of the various consumed fish species pose a challenge in the identification and characterization of the major fish allergens critical for reliable diagnostics. Globally, fish is a rising cause of food allergy complicated by a large under-investigated variety of species as well as increasing global tourism and trade. This is the first comprehensive study on allergen profiles of heat-processed fish from Vietnam. Objective: The aim of this study was to identify the major heat-stable allergens from frequently exported Asia-Pacific freshwater and marine fish and to characterize the major allergen parvalbumin (PV) from one of the most consumed and exported fish species from Asia, the Indian mackerel (Rastrelliger kanagurta). Methods: Heated protein extracts from 33 fish species were separated by gel electrophoresis. PV isoforms were identified by immunoblotting utilizing 3 different PV-specific monoclonal and polyclonal antibodies and further characterized by mass spectrometry. IgE reactivity was investigated using sera from 21 patients with confirmed fish allergy. Results: Heat-stable IgE-reactive PVs, with up to 5 isoforms per species, were identified in all 33 analysed fish species. In the Indian mackerel, 7 PV isoforms were identified by 2D-gel electrophoresis combined with mass spectrometric analyses. The amino acid sequence deduced from cDNA of the most expressed isoform showed a high identity (>90%) to PVs from 2 other mackerel species. Conclusions and Clinical Relevance: Different PVs were identified as the major heat-stable allergens in all 33 analysed freshwater and marine fish species from Vietnam, many of which are exported world-wide and 21 species that have never been investigated before. The Indian mackerel PV represents a novel fish allergen, now officially registered as Ras k 1. Improved diagnostics for fish allergy against Asia-Pacific species should be developed with focus on PV

Reconstruction of fish allergenicity from the content and structural traits of the component β-parvalbumin isoforms

12 pages, 7 figures.-- This article is licensed under a Creative Commons Attribution 4.0 International LicenseMost fish-allergic patients have anti-β-parvalbumin (β-PV) immunoglobulin E (IgE), which cross-reacts among fish species with variable clinical effects. Although the β-PV load is considered a determinant for allergenicity, fish species express distinct β-PV isoforms with unknown pathogenic contributions. To identify the role various parameters play in allergenicity, we have taken Gadus morhua and Scomber japonicus models, determined their β-PV isoform composition and analyzed the interaction of the IgE from fish-allergic patient sera with these different conformations. We found that each fish species contains a major and a minor isoform, with the total PV content four times higher in Gadus morhua than in Scomber japonicus. The isoforms showing the best IgE recognition displayed protease-sensitive globular folds, and if forming amyloids, they were not immunoreactive. Of the isoforms displaying stable globular folds, one was not recognized by IgE under any of the conditions, and the other formed highly immunoreactive amyloids. The results showed that Gadus morhua muscles are equipped with an isoform combination and content that ensures the IgE recognition of all PV folds, whereas the allergenic load of Scomber japonicus is under the control of proteolysis. We conclude that the consideration of isoform properties and content may improve the explanation of fish species allergenicity differencesThis work was supported by grants from the Spanish AEI/EU-FEDER SAF2014-52661-C3 (MG) and BFU2015- 72271-EXP (MG), Angulas Aguinaga (MG, RRP) contract and the Ramón Areces Foundation (MC). MC is a Ramón y Cajal fellowPeer reviewe

Diagnosis and management of shellfish allergy: current approach and future needs

Purpose of review: Shellfish allergy is an increasing health concern worldwide with over 2% of the population affected and higher rates in countries with high consumption. Shellfish includes both crustaceans and mollusks and constitutes one of the major food groups triggering allergic reactions. Recent findings: Shrimp is the best-studied crustacean, in which the major shellfish allergen, tropomyosin, was initially characterized. Nevertheless, several other allergens have been identified and should be considered despite prevalence of sensitization being lower than tropomyosin (e.g., arginine kinase, myosin light chain, and sarcoplasmic calcium-binding protein). Summary: Diagnosis is not always straightforward; due to the conserved nature of most allergens, there is extensive cross-reactivity between different species which hampers proper diagnosis and management. Clinical symptoms can range from mild local ones to life-threatening anaphylaxis, sometimes with cofactor involvement. Currently, there is no available curative treatment besides diet avoidance and treatment of symptoms in case of accidental exposure