Clinical monosensitivity to salmon and rainbow trout: a case report

To the Editor, Seafood plays an important role in human nutrition; the increasing consumption of certain fishes due to their healthy effects has led to a raising in the number of cases related to allergic reactions (1). The major fish allergen is parvalbumin (2), an acidic, calcium-binding 12 kDa protein resistant to heat and gastrointestinal digestion. Fish allergic patients are often sensitized to more than one species due to the cross-reactivity of their betaparvalbumins. However, until now, only few cases of monosensitivity to fish have been described (3–7). We report the case of a 14-year-old boy with a history of food allergy (milk) from the first year of life. At age 4, he presented his first allergic reaction to fish, after ingesting a few appetizers containing smoked salmon. In a few minutes, he suffered from rush cutaneous and lip edema, which regressed after oral administration of corticosteroids. Skin prick tests, performed with commercial extracts (Lofarma, Milano, Italy), and prick by prick tests showed negative results to mollusks, crustaceans, and fishes, including salmon. Two years later, few minutes after the ingestion of broiled salmon, he showed a new allergic reaction including lip and nose edema, which regressed after cortisone treatment. At the age 11, a small quantity of broiled rainbow trout (Salmonidae) determined a sudden and severe intense throat itching; this reaction regressed spontaneously in a few minutes. After this new event, the boy stopped consuming salmon, rainbow trout, and other freshwater fishes. He had safely consumed codfish, halibut, anchovies, tuna fish, and mackerel, apart from a case of reaction to fish sticks (containing only cod fish) where a cross-contamination with salmon in the production chain was hypothesized. As monosensitization to fish is quite unusual, the patient was tested at molecular level to confirm the specific pattern of reactivity by immunoblotting. IgE multiplex test for the most usual allergens showed a positive response only for Gadus callarias (cod fish) muscle IU = 0.03. Further investigation by ISAC test showed the presence of serum-specific IgEs versus the allergens Onc m from rainbow trout (Oncorhynchus mykiss) ISU = 2.10 and Sal s from salmon (Salmon salar) ISU = 2.22. ISAC tests that were reported negative were obtained with fishes from the families of Gadidae (cod fish, Gad m), Soleidae (sole, Sol so), and Scombridae (tuna and similar fishes, Thu a). Finally, a negative result was found for Anisakis simplex (Ani s). Proteins from codfish (Gadus morhua), salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), sole (Solea vulgaris), and tuna fish (Thunnus albacares) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) under reducing conditions. Fish species were from a local supermarket and were analyzed as such (raw) and cooked (boiled in water at 100°C for 20 min) to simulate the usual domestic preparation. All the samples were freeze-dried, ground, and the resulting flour was suspended in sample buffer (containing 0.063 M TRIS–HCl pH 6.8, 1.88% glycerol, 0.5% SDS, 2.5% b-mercaptoethanol) at a final concentration of 7.5 mg/ml (w/v), corresponding to 4 mg/ml of proteins approximately. A pre-stained molecular weight standard solution (broad range; Bio-Rad, Richmond, CA, USA) was run in parallel to the samples. SDS-PAGE and immunoblotting were performed according to Ballabio et al. (8). Briefly, after SDS-PAGE, proteins were transferred to a PVDF membrane by Western blotting, blocked, and washed with gelatin solutions to prevent non-specific adsorption of the immunological reagents. The membrane was then immersed in 10 ml of 0.25% gelatin solution containing 0.3 ml of the patient’s serum. Antigen–IgE complexes were detected using 10 ll of goat antihuman IgE antibodies labeled with alkaline phosphatase in the presence of the specific substrates. To verify the specificity of immunoreactivity, immunoblotting was performed under identical conditions using the serum of two children highly allergic to foods different from fish (results not shown) with faint unspecific immunoreactivity. Figure 1 shows the electrophoretic profiles of fishes included in the study, tested as such (left panel) and after boiling (right panel). All the samples yielded a complex protein pattern, with some differences from the qualitative and quantitative point of view. Raw codfish, rainbow trout, and salmon showed some protein bands having low molecular weight (MW); in particular, two bands (L and M) were less abundant or absent in sole and tuna fish. Parvalbumin was only visible in codfish and rainbow trout (band L, MW 12–14 kDa). Cooked samples presented several differences in their electrophoretic pattern when compared to raw fish; in particular, cooked samples showed a larger number of protein bands in the MW region from 6 kDa to 30 kDa. A band having a MW of about 12–14 kDa, corresponding to parvalbumins, was clearly evident in all the samples analyzed, although the intensity of this band in the sole was very low (band L). Immunoblotting analysis (Fig. 2) showed that serum allergen-specific IgEs were mainly reactive for two protein bands having a molecular weight of approximately 35 kDa (G) and 75 kDa (C) in raw salmon. These two protein bands were also immunoreactive in the other fish species tested, with the exception of codfish, in which the IgE recognized the 35 kDa (G) protein and a second band having a MW>100 kDa (B). Several bands were recognized by serum IgEs in rainbow trout after cooking: A (170 kDa), B