Exploring tumourigenic potential of the parasite Anisakis: a pilot study.

Anisakiasis is a global disease caused by consumption of raw or lightly cooked fish parasitised with Anisakis spp. third-stage larvae. Cases in the literature show colocalised anisakiasis and colorectal cancer, and the incidental finding of Anisakis larvae at the tumour site was reported. Data from our group suggested an epidemiological link between previous infection and gastrointestinal cancer. Furthermore, it has recently been reported that Anisakis products lead to inflammation and DNA damage. Based on these facts, the aim was to investigate whether Anisakis antigens are able to induce changes in the proliferation of epithelial cells in vitro or in the expression of serum microRNA (miRNA) in Sprague-Dawley rats. Anisakis complete extract (CE) induced increases in cell proliferation and decreases in apoptosis compared with nontreated cells, which resulted in a significant increase in the absolute number of viable cells at 48 h of exposure (P < .05). Furthermore, the miRNAs mmu-miR-1b-5p and mmu-miR-10b-5p (a cancer-related miRNA) were significantly decreased (P < .05) in sera from the rats inoculated with Anisakis CE, compared with control rats inoculated with saline. Additionally, based on their relative quantification values, four other cancer-related miRNAs were considered to be differently expressed, rno-miR-218a-5p and mmu-miR-224-5p (decreased) and rno-miR-125a-3p and rno-miR-200c-3p (increased). Anisakis CE was able to induce changes both in epithelial cells in vitro and in an animal model. The results obtained with Anisakis CE, in terms of increasing cell proliferation, decreasing apoptosis and inducing changes in the expression of serum cancer-related miRNAs in rats, suggest that Anisakis could have tumourigenic potential.

Antigenicity of Anisakis simplex s.s. L3 in parasitized fish after heating conditions used in the canning processing.

BACKGROUND: Some technological and food processing treatments applied to parasitized fish kill the Anisakis larvae and prevent infection and sensitization of consumers. However, residual allergenic activity of parasite allergens has been shown. The aim here was to study the effect of different heat treatments used in the fish canning processing industry on the antigen recognition of Anisakis L3. Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) were experimentally infected with live L3 Anisakis. After 48 h at 5 ± 1 °C, brine was added to the muscle, which was then canned raw (live larvae) or heated (90 °C, 30 min) (dead larvae) and treated at 113 °C for 60 min or at 115 °C for 90 min. Anisakis antigens and Ani s 4 were detected with anti-crude extract and anti-Ani s 4 antisera respectively. RESULTS: Ani s 4 decreased in all lots, but the muscle retained part of the allergenicity irrespective of the canning method, as observed by immunohistochemistry. Dot blot analysis showed a high loss of Ani s 4 recognition after canning, but residual antigenicity was present. CONCLUSION: The results indicate that heat treatment for sterilization under the conditions studied produces a decrease in Ani s 4 and suggest a potential exposure risk for Anisakis-sensitized patients.

Antigenicity and viability of Anisakis larvae infesting hake heated at different time-temperature conditions.

Heat treatments (40 to 94 degrees Celsius, 30 s to 60 min) were applied to different batches of Anisakis simplex L3 larvae isolated from hake ovaries and viscera to study the effect of heat on the viability of the larvae measured as mobility, emission of fluorescence under UV light, and changes in color after staining with specific dyes, and on A. simplex antigenic proteins. The aim was to determine the lowest time-temperature conditions needed to kill the larvae to avoid anisakiasis in consumers, and to evaluate whether high temperature modifies the antigenicity of A. simplex extracts. Heating at 60 degrees Celsius for 10 min (recommended by some authors) was considered unsafe, as differences in viability between batches were found, with some larvae presenting spontaneous movements in one batch. At higher temperatures (> or = 70 degrees Celsius for > or = 1 min), no movement of the larvae was observed. Antigenic protein Ani s 4 and A. simplex crude antigens were detected in the larvae heated at 94 + or - 1 degrees Celsius for 3 min. This indicates that allergic symptoms could be provoked in previously sensitized consumers, even if the larvae were killed by heat treatment.

Allergenic properties and cuticle microstructure of Anisakis simplex L3 after freezing and pepsin digestion.

This article examines the viability of and the alterations to the larval cuticle and the pattern of the antigens released when live or frozen Anisakis simplex larvae were treated with acid and pepsin. The results showed that freezing did not greatly alter the larva body. If ruptures were observed, the antigen release to the incubation media was not enhanced, and most of the antigenic content was retained inside the bodies of the larvae. The immunoblotting assay demonstrated that most of the antigens released, including the allergen Ani s 4, were resistant to pepsin. Freezing killed the larvae, but their survival was not compromised by acid treatment or pepsin digestion when kept chilled. All these findings support recommendations about freezing fish for consumption raw or undercooked to prevent human infection by A. simplex larvae. However, our data show that the antigenicity of the larvae is preserved after freezing and may explain why some sensitized patients develop symptoms after ingestion of infested frozen fish.

Scanning electron microscopy of Anisakis larvae following different treatments.

Ingestion of fish parasitized with Anisakis larvae can produce infestation and/or allergy in consumers. Technological and food processing treatments have been applied to parasitized fish in order to kill the larvae and avoid the infestation; however, their influence on allergenicity has not been studied. Four lots of hake (Merluccius merluccius) steaks artificially parasitized with Anisakis larvae were subjected to two storage chilling (5 degrees C +/- 1 degrees C) and freezing (-20 degrees C +/- 1 degrees C) treatments and two food processing treatments of heat (final temperature 86.3 degrees C) and microwave (final temperature 66.9 degrees C) and studied by scanning electron microscopy, environmental scanning electron microscopy (ESEM) (acid [pH = 2] and water preparations), and emission of fluorescence. Anisakis larvae were resistant to acid conditions, remaining alive after treatment. Larvae in the heat- and microwave-treated lots presented coagulated and disrupted zones in the cuticle with release of fluids. The cylindrical shape changed to a dehydrated appearance mainly observed by ESEM. Fluorescence was only noticeable in the frozen larvae. Larvae without apparent changes, together with dehydrated ones, were observed by ESEM in the frozen lot; nevertheless, no disruptions in the cuticle were perceptible. Further studies are needed in order to elucidate if the changes observed in the cuticle reduce the resistance of the parasites to the action of gastric enzymes in the gastrointestinal tract and to determine the release of allergens to the flesh by the live larvae during chilled storage of the fish.

Viability and antigenicity of anisakis simplex after conventional and microwave heating at fixed temperatures.

Inactivation of parasites in food by microwave treatment may vary due to differences in the characteristics of microwave ovens and food properties. Microwave treatment in standard domestic ovens results in hot and cold spots, and the microwaves do not penetrate all areas of the samples depending on the thickness, which makes it difficult to compare microwave with conventional heat treatments. The viability of Anisakis simplex (isolated larvae and infected fish muscle) heated in a microwave oven with precise temperature control was compared with that of larvae heated in a water bath to investigate any additional effect of the microwaves. At a given temperature, less time was required to kill the larvae by microwaves than by heated water. Microwave treatment killed A. simplex larvae faster than did conventional cooking when the microwaves fully penetrated the samples and resulted in fewer changes in the fish muscle. However, the heat-stable allergen Ani s 4 was detected by immunohistochemistry in the fish muscle after both heat treatments, even at 70°C, suggesting that Ani s 4 allergens were released from the larvae into the surrounding tissue and that the tissues retained their allergenicity even after the larvae were killed by both heat treatments. Thus, microwave cooking will not render fish safe for individuals already sensitized to A. simplex heat-resistant allergens.