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- Material and methods
Background: The nematode Anisakis simplex is a common parasite on fish and other seafood. It is considered to be a food allergen and to induce IgE-mediated reactions. Allergenic cross-reactivity between A. simplex and other nematodes has been reported, as has cross-reactivity with arthropods: red mosquito larvae and German cockroach. We have here studied the allergenic relationship between A. simplex and four different dust-mite species.
Methods: Serum samples collected from 69 farmers allergic to dust mites were analyzed for IgE to A. simplex by CAP FEIA. Allergenic cross-reactivity between A. simplex and dust mites was studied in two of the sera by CAP FEIA and immunoblotting inhibition.
Results: We found that 14/69 farmers had detectable levels of IgE antibodies to A. simplex. The IgE response in CAP FEIA to A. simplex was inhibited to various degrees in the two studied sera by extracts of the dust mites Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae, and Dermatophagoides pteronyssinus. In the reverse inhibition experiment, extract of A. simplex inhibited the response in both sera to A. siro and T. putrescentiae, but not to L. destructor. The IgE binding to D. pteronyssinus was inhibited in one of the two sera. In blotting inhibition experiments, the IgE binding to several allergens in A. simplex was inhibited by each of the four mite extracts, especially by A. siro and T. putrescentiae, which completely inhibited the IgE binding to several allergens.
Conclusions: The results show allergenic cross-reactivity between several allergens in A. simplex and four dust-mite species. The clinical significance of this cross-reactivity remains to be evaluated.
The nematode Anisakis simplex commonly parasitizes marine invertebrates as well as fish and sea mammals. Its life cycle comprises the egg, several larval stages, and the adult nematode. Encapsulated larvae of the third stage are common in the viscera and musculature of many fishes (1). The larva infects fish of many species and over a broad geographic area (2); thus, eating raw or undercooked infected fish can lead to infestation with the larva and cause severe gastrointestinal symptoms (anisakiasis) (3). Several studies have suggested that A. simplex can act as a food allergen and induce IgE-mediated reactions, mostly urticaria and angioedema, but also anaphylactic reactions (4–8). Two case reports have shown A. simplex to be also an occupational allergen causing asthma and conjunctivitis (9, 10). It has been assumed that even properly cooked infected fish can cause allergic symptoms, as some of the A. simplex allergens have been demonstrated to be thermostable (11). However, recent studies indicate that allergic clinical symptoms can be induced only after ingestion of a live larva, which can then secrete antigens affecting the gastric mucosa (12–15).
IgE antibodies to the parasite have been detected in high frequency among individuals without symptoms, including blood donors, controls, and atopic children (2). The incidence of IgE to A. simplex in 169 sera from blood donors at the Karolinska Hospital, Stockholm, Sweden, was 13% (not published). However, the cause of sensitization to A. simplex is not fully understood. It has been suggested that the occurrence of IgE to A. simplex in individuals not displaying allergic symptoms is due to an earlier infestation with the larvae, probably without symptoms, and is a consequence of the normal immune response to parasites (16, 17). Cross-reactivity may also be a possible explanation. Antigenic and allergenic cross-reactivity between A. simplex and other nematodes is a well-known phenomenon (18, 19). Surprisingly, allergenic cross-reactivity has also been described between A. simplex and arthropods (20). CAP FEIA and immunoblotting inhibition analysis have shown that German cockroach and red mosquito larvae (Blattella germanica and Chironomus spp.) share IgE-binding components with A. simplex (20). In a preliminary study, we found an association between sensitization to A. simplex and yet another arthropod, the nonpyroglyphid dust mite Lepidoglyphus destructor. Several species of nonpyroglyphid dust mites are important occupational allergens in rural environments (21, 22), but recent data show that IgE-mediated sensitization to these mites is found also in urban populations (23–25), suggesting that the nonpyroglyphid dust mites are of greater clinical importance than previously known. L. destructor is one of the most common species in Europe, but Acarus siro and Tyrophagus putrescentiae are also common (26–28).
The aim of this study was to investigate a possible allergenic cross-reactivity between A. simplex and A. siro, L. destructor, and T. putrescentiae and also between A. simplex and the pyroglyphid house-dust mite Dermatophagoides pteronyssinus.
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- Material and methods
In this study, we used CAP FEIA and immunoblotting inhibition to evaluate allergenic cross-reactivity between A. simplex and the dust mites A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus. We found that 20% (14/69) of dust-mite-sensitized subjects had IgE to A. simplex. Taken together with the fact that of the 50 control subjects nonsensitized to dust mites, only one was positive to A. simplex, this points to an association between A. simplex and dust mites, probably one of allergenic cross-reactivity. However, as comparison of IgE values between A. simplex and each mite species showed only weak correlation, the highest being to A. siro (rs=0.47, P=0.0014), the implication is that IgE antibodies to A. simplex among these subjects are not solely dependent on sensitization to dust mites.
When we assessed the allergenic cross-reactivity in CAP FEIA inhibition, all four mite species inhibited the IgE binding to A. simplex, but A. siro and T. putrescentiae were the most potent inhibitors in the two investigated sera. Conversely, A. simplex was able partly to inhibit the IgE binding to the dust mites. About 50% of the IgE binding to A. siro, T. putrescentiae, and D. pteronyssinus was inhibited in at least one of the sera. However, the nematode could not inhibit the IgE binding to the L. destructor allergens in any of the serum samples. The results clearly show that A. simplex and each of the mite species A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus have common IgE-binding epitopes.
Allergenic cross-reactivity between A. simplex and other organisms has been reported by Fernández-Caldas et al. (19), who used ELISA inhibition to assess cross-reactivity between A. simplex and the anisakid nematode Hysterotylacium aduncum. The results suggest that the two species share allergenic epitopes. Pascual et al. (20) also described allergenic cross-reactivity between A. simplex and the two arthropods German cockroach and red mosquito larvae. By immunoblotting inhibition, they showed that several allergens, mainly of 30–40 kDa in all three species, were involved in the cross-reactions. They speculated that the cross-sensitization link between these disparate species was the 36-kDa muscle protein tropomyosin. Indeed, the highly conserved tropomyosin protein has been considered an important cross-sensitizing invertebrate panallergen (30). This allergen has been found in several organisms, among them the two (house) dust mites D. pteronyssinus (Der p 10) and D. farinae (Der f 10) (30). Different mite species have been shown to contain several groups of allergens with extensive homology between them (31). Therefore, we suspect that tropomyosin may also be a part of the allergen spectrum of all four mite species investigated in this study and involved in the cross-reactivity with A. simplex. In the immunoblotting inhibition experiment, we obtained inhibition to at least three different allergens in A. simplex with molecular mass close to that of tropomyosin. For example, an allergen at 36 kDa was in various degrees inhibited by all four mite extracts. A. simplex also inhibited allergens around 36 kDa in two of the mite extracts, T. putrescentiae and D. pteronyssinus. However, further inhibition experiments using native tropomyosin are needed to determine whether this protein is a cross-reacting allergen in A. simplex and dust mites.
According to the molecular mass of the inhibited allergens in A. simplex and in the dust mites, this study indicates that the allergenic cross-reactivity involves several allergens other than tropomyosin. For example, we recognized several allergens in A. simplex with high molecular mass that were inhibited by the dust mites. This is in accordance with the results of Lorenzo et al. (32), who recently have shown that a sugar epitope, an O-linked glycan on high-molecular-mass glycoproteins (139/154 kDa) in A. simplex, is an IgE-binding cross-reactive molecule.
In conclusion, we have shown that the nematode A. simplex and the dust mites A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus have common allergenic epitopes. The allergenic cross-reactivity was demonstrated both by CAP FEIA and immunoblotting inhibition, identifying at least 12 different allergens that were involved. Fourteen of the 69 dust-mite-sensitized subjects were shown to have IgE antibodies in serum to A. simplex. The clinical relevance of these probably cross-reacting antibodies is not known and needs to be further investigated.