IgE sensitization to the fish parasite Anisakis simplex in Norway

Abstract

Anisakis simplex is a fish parasite which infects many commercially important fish species worldwide. When ingested in contaminated raw or poorly prepared seafood, this parasite may lead to infection and/or allergic reaction in humans. During the last decade, cases of this food-borne infection and allergy have increased in countries with high fish consumption, causing an emerging public health problem. Norway is one of the high fishconsuming countries, but the situation of this parasite-related problem was still unknown. Therefore, we conducted the first pilot study in Norway to investigate the prevalence of IgE sensitization to A. simplex. In this epidemiologic survey, the anonymized serum samples from newly recruited blood donors (designated as “BDO”) and from patients with high serum IgE antibodies level ≥ 1000 kU\l (designated as “IGE+”) were collected at the Haukeland University Hospital, Bergen. The sera were analyzed by the ImmunoCAP® method for total IgE and IgE antibodies against A. simplex and other possible cross allergens such as house dust mite (HDM), shrimp and cod. The results showed 2.0% and 37% of IgE sensitization to A. simplex in BDO and IgE+ groups, respectively. But these prevalences might be overestimated owing to crosssensitization. Later on, we performed an extended study using larger population sizes and a recently developed more specific analysis method. This test is an enzyme-linked immunosorbent assay (ELISA) which utilizes 2 recombinant (r) major and specific allergens of A. simplex, namely rAni s 1 and rAni s 7 as target antigens. SDS-PAGE and Western immunoblotting analyses were also performed. As expected, the results of the extended study showed even lower prevalences of sensitization than that obtained in the previous study. Whereas the prevalences by the ImmunoCAP® method were 0.4% and 16.2%, analyses with the more specific test showed very low incidences of 0.0% and 0.2% in the BDO and IGE+ groups, respectively. Cross-reactivity analyses also suggested that most of the ImmunoCAP® positive sera were probably false-positive due to cross-sensitization to HDM and shrimp. The 0.0% prevalence in the BDO group may indicate an absence of sensitization against the fish parasite A. simplex in the healthy population. On the other hand, the frequency of 0.2% in the IGE+ group may either represent a certain degree of true sensitization in the “allergic” population or maybe still due to cross-sensitization. The low prevalence of A. simplex sensitization in Norway compared to other high-fish consuming countries might be partly explained by the relatively low consumption of raw or uncooked fish among the Norwegians and the low genetic susceptibility of Norwegian population to A. simplex allergens. With respect to food safety, certain regulations for storing and preparing the fish have been implemented to kill this commonly occurring nematode in fish. However, among Anisakis allergens, several heat- and pepsin-resistant proteins have been shown to remain active even after cooking or digestion. The ingestion or inhalation of or contact with A. simplex allergens has been claimed to evoke allergic symptoms in sensitized individuals. While directly contaminated fish were accounted for most allergic cases, indirect contamination of food via carry-over from contaminated feed has also been reported to be involved. For this reason, we conducted a zebrafish (Danio rerio) feeding trial to investigate the transmissibility of A. simplex allergens from feed to fish. Zebrafish was chosen as the study object owing to their rapid maturation and favorable small size affording less space and feed than edible fish such as salmon or trout. The results of feed analyses by both ELISA and immunostaining indicated that certain A. simplex allergens have been retained throughout feed processing, confirming the presence of heat and degradation-resistant proteins of this parasite as reported in other studies. The trace amounts of A. simplex peptides in the fish flesh were detected by both immunostaining and LCMSMS, thereby providing a positive proof for the transmissibility of A. simplex allergens from feed to fish. Nevertheless, considering the relatively higher effect dose of A. simplex allergens compared to other food allergens, the low amounts of Anisakis proteins transferred via contaminated feed to fish probably do not represent a major food safety problem for consumers with A. simplex allergy. In conclusion, our seroprevalence studies indicated a very low sensitization against the fish parasite A. simplex in the studied Norwegian populations as compared with other high fish-consuming countries. Our zebrafish feeding trial suggested the transmissibility of A. simplex allergens from feed to fish, but only the low amounts were detected. Given the results from our surveys, the fish parasite A. simplex is apparently not an immediate food safety and public health problem in Norway.