Anaphylactic Reactions to Novel Foods: Case Report of a Child With Severe Crocodile Meat Allergy
Availability of “exotic” foods is steadily increasing. In this report, we describe the first case of anaphylaxis to crocodile meat. The patient was a 13-year-old boy with severe immunoglobulin E–mediated allergy to chicken meat. When tasting crocodile meat for the first time, he developed an anaphylactic reaction. Cross-reactivity between chicken and crocodile meat was suspected to have triggered this reaction. Basophil activation and immunoglobulin E testing confirmed the boy’s allergic reaction to crocodile meat proteins. Molecular analysis identified a crocodile α-parvalbumin, with extensive sequence homology to chicken α-parvalbumin, as the main cross-reactive allergen. We conclude that crocodile meat can be a potent food allergen and patients with allergy to chicken meat should be advised to avoid intake of meat from crocodile species. Both foods and people travel around the world and accessibility to exotic foods is steadily growing. As a result, novel allergic cross-reactivities are likely to become a challenge in the management of food allergy and, as our report illustrates, cross-reactivity has to be considered even between foods that might not intuitively be perceived as related.
- CD-sens —
- basophil allergen threshold sensitivity
- ELISA —
- enzyme-linked immunosorbent assay
- IgE —
- immunoglobulin E
- MS —
- mass spectrometry
- sIgE —
- serum immunoglobulin E
Food allergy is an increasing problem in the pediatric population and is estimated to affect 8% of children with a high negative impact on quality of life.1–3 Allergy to chicken meat has been reported both in children and adults and is estimated to be rare, but reliable prevalence data are missing.4–8 Cross reactivity between chicken and duck, turkey, and goose has been described.4 Little is known about the causative allergens for chicken meat allergy. There are no reports of allergic reactions to crocodile meat. Although crocodiles belong to the reptile group, they are also the closest living relatives of birds.9
A 13-year-old boy presented to the pediatric emergency department at Sachs’ Children and Youth Hospital in Stockholm, Sweden, due to an anaphylactic reaction to crocodile meat. He had been a regular outpatient at the hospital’s pediatric allergy department since 5 years of age when he was diagnosed with chicken meat allergy (specific immunoglobulin E [IgE], 19 kUA/L). He was otherwise healthy and ate all foods except poultry. At 7 years of age, an adrenaline autoinjector was prescribed after an anaphylactic reaction due to accidental consumption of turkey. Although strict avoidance of chicken and turkey meat was recommended, he continued to experience anaphylactic reactions due to accidental consumption of foods contaminated by chicken or turkey. As a consequence, he was reluctant to eat foods outside of his home, which had a significant impact on his social life. More recently, his father, a professional chef, bought and prepared crocodile meat to create a novel meal for his son. The boy reacted at first bite with itch in the mouth and throat, facial urticaria, conjunctivitis, angioedema, chest tightness, and breathing difficulties. Intramuscular adrenaline and β-2 agonist inhalation was administered at home by the parents. At the pediatric emergency department, he presented with facial urticaria, angioedema around the eyes, bilateral redness of the sclera, and heavy breathing but no bronchoconstriction. After 4 hours of observation, he was free of symptoms and discharged.
Reviewing the literature, we found no reports of allergic reactions to crocodile. We had previously identified α-parvalbumin as a relevant chicken meat allergen7 and because another report showed that parvalbumin is expressed in the tail muscle of Alligator mississippiensis,10 we hypothesized that the reaction to crocodile was due to IgE cross-reactivity between α-parvalbumins in chicken and crocodile meat.
Written informed consent was obtained from the patient and his parents.
Crocodile filet was purchased from the same store that sold the crocodile meat that caused the allergic reaction. It was analyzed by the certified laboratory of the Swedish National Food Agency (Livsmedelsverket; Uppsala, Sweden) for content of DNA and protein from chicken and turkey. In addition, the crocodile filet was used to prepare a protein extract.11 Briefly, the muscle tissue was grinded in liquid nitrogen followed by extraction in lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, and 1% Triton X-100). Extract supernatants were dialyzed against phosphate-buffered saline (pH 7.2) before basophil activation assay and specific IgE–binding analysis. For the basophil activation assay and measurements of allergen-specific IgE, muscle tissue was homogenized followed by extraction in phosphate-buffered saline (pH 7.4).
Measurement of Allergen-Specific IgE
IgE antibodies against chicken (f83), turkey (f284), and crocodile meat proteins (5 µg of biotinylated crocodile extract coupled to Streptavidin ImmunoCAP) and total IgE were measured by ImmunoCAP (Phadia AB/Thermo Fisher Scientific, Uppsala, Sweden).
Basophil Activation Assay
Basophil allergen threshold sensitivity (CD-sens) was performed as previously described.12,13 Briefly, cells were stimulated with increasing concentrations of crocodile or chicken extracts (0.5–500 ng protein/mL) or negative controls (peanut, birch, casein) and then analyzed by flow cytometry.
Allergen Analysis by IgE Immunoblot and Enzyme-Linked Immunosorbent Assay
Chicken and crocodile meat extracts were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).11 Patient serum was diluted 5× in 3% bovine serum albumin (Sigma-Alderich, Bornem, Belgium) for immunoblot and 5–10× for IgE enzyme-linked immunosorbent assay (ELISA).11 For IgE cross-inhibition, patient serum was preincubated with chicken or crocodile extract at a concentration of 1 mg inhibitor/mL for immunoblot and 200 μg inhibitor/mL for ELISA. Parvalbumins were detected by using an antiparvalbumin IgG antibody mix (Swant, Marly, Switzerland; Abcam, Cambridge, MA).
Biomolecular Allergen Characterization
Chicken and crocodile parvalbumins were isolated by ion exchange and gel filtration chromatography as previously reported.7,14 Protein purity of chicken and crocodile parvalbumins was confirmed by silver-stained, two-dimensional SDS-PAGE.11 Crocodile parvalbumin isoforms were additionally separated by isoelectric focusing (Offgel, Agilent, Diegem, Belgium) before an intact mass measurement by mass spectrometry (MS) analysis. Parvalbumins were trypsinized for MS peptide mass fingerprint analysis,11 which revealed the complete sequence of 1 crocodile parvalbumin isoform. Unidentified peptides of the other isoform were sequenced de novo on a second MALDI TOF/TOF instrument (AB Sciex 5800, Framingham, MA), identifying 92% of the protein sequence. Protein structures were calculated using Modeller 9v2 software,15,16 and chicken parvalbumin (P43305; 2KYF) was used to establish the surface model.
No contamination of the crocodile meat by chicken or turkey residues was demonstrated. The boy’s serum immunoglobulin E (sIgE) levels were 40 kUA/L to chicken, 14 kUA/L to turkey, and 6 kUA/L to crocodile meat extract, whereas total serum-IgE was 190 kU/L. CD-sens was clearly positive to both crocodile and chicken meat extracts (Fig 1A). In immunoblot analysis, the patient’s IgE bound to chicken (14 kDa, 19/22 kDa, 30 kDa) and crocodile meat proteins (18–50 kDa) (Fig 1B). IgE-binding to chicken allergens was not inhibited by preincubation with crocodile extract, whereas IgE-binding was inhibited by preincubation with chicken extract (Fig 1B). Because chicken is never consumed raw, an extract from heated chicken meat was also included in the immunoblot analysis. The patient’s sIgE recognized a single 14-kDa chicken protein only, which was confirmed to be parvalbumin by using an antiparvalbumin antibody. Homologous crocodile parvalbumins were detected as 2 bands at 6 and 14 kDa (potentially 2 isoforms, denoted “6-kDa” and “14-kDa parvalbumin”). The patient’s sIgE levels were 108 kUA/L to purified chicken α-parvalbumin and 25 kUA/L to crocodile 14-kDa parvalbumin. The selective IgE reactivity to crocodile parvalbumins was confirmed by IgE immunoblot, where the patient’s IgE antibodies only detected the 14-kDa but not the 6-kDa isoform (Fig 1B). After preincubation of serum with the respective homolog, IgE reactivity to chicken α-parvalbumin and 14-kDa crocodile parvalbumin was efficiently inhibited (94% and 100%, respectively). Amino acid sequencing revealed the complete sequence of the 14-kDa (108/108 amino acids) and most of the 6-kDa crocodile parvalbumin (100/108 amino acids). The protein identity of the 14-kDa crocodile parvalbumin compared with the chicken homolog was 94%, and a molecular model comparison visualized extensive surface identity of both allergens (Fig 2).
To the best of our knowledge, this is the first report of a food-allergic reaction to crocodile meat. We demonstrate that this anaphylactic reaction, confirmed by the positive basophil challenge (CD-sens) with crocodile meat, was due to cross-reactivity of a highly homologous α-parvalbumin found in both chicken and crocodile meat. Our hypothesis of clinical cross-reactivity via highly conserved B cell epitopes on these parvalbumins could be confirmed by IgE cross-inhibition assays using the purified chicken and crocodile allergens. Furthermore, we showed extensive sequence identity representing putative linear epitopes and high structural homology representing putative conformational epitopes. Although chicken was assumed to be the primary sensitization source, the patient had experienced anaphylactic reactions to turkey as well. These reactions can be explained by our previous finding that chicken and turkey α-parvalbumin are 100% identical,7 which reflects their close phylogenetic relationship. In another recent publication, we reported on 36 patients (including 17 children <14 years old) with chicken meat allergy and showed that 61% had specific IgE against the allergen Gal d 8, chicken parvalbumin.17 Therefore, most patients with chicken meat allergy might also be at risk for allergic reactions to crocodile meat. We believe that chicken meat allergic individuals should be informed about the potential risk of cross-reactivity and advised to avoid meat from crocodile species.
Both foods and people travel around the world and accessibility to “exotic” foods is steadily growing. Food safety is an important aspect to be addressed before the introduction of these new products to the market.18 Assessment of potential allergenicity of novel foods is highly challenging because it is not well understood what makes a food protein a potent allergen. However, as this case illustrates, additional research concerning allergic cross-reactivities is needed so that clinicians will be able to give accurate advice to food-allergic patients intending to broaden their diet.
Crocodile meat can be a potent food allergen. Because patients with allergy to chicken meat are at risk for reacting to crocodile meat, clinical testing with crocodile meat may need to be included in the diagnostic workup of these individuals. Novel allergic cross-reactivities are likely to become a challenge in the management of food allergy and, as our report illustrates, cross-reactivity has to be considered even between foods that might not intuitively be perceived as related.
We thank M. Sandberg and Y. Sjögren Bolin at the Swedish National Food Agency (Uppsala, Sweden) for analysis of crocodile meat for (1) detection of poultry protein by use of ELISA and (2) detection of chicken and turkey DNA by real time PCR. We thank D. Revets (Luxembourg Institute of Health) for excellent technical support with MS analysis. We also thank Dr K. Arumugam (Luxembourg Institute of Health) for establishing the structural model of chicken and crocodile parvalbumin.
- Accepted September 22, 2016.
- Address correspondence to Natalia Ballardini, MD, PhD, Institute of Environmental Medicine, Karolinska Institutet, Nobels väg 13, SE-171 77 Stockholm, Sweden. E-mail:
FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.
FUNDING: This work was supported by research grants from the Swedish Research Council, the Stockholm County Council, the Swedish Asthma and Allergy Association’s Research Foundation, the Swedish Cancer and Allergy Foundation, the Hesselman Foundation, Karolinska Institutet, the Karin and Sten Mörtstedt Initiative on Anaphylaxis, and the Ministry of Higher Education and Research in Luxembourg.
POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.
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- Copyright © 2017 by the American Academy of Pediatrics