Anisakis simplex larvae parasitize animals used as seafood and can producea specific imtnune response in man. The ingestion of seafood contaminatedwith stage three oiA. simplex larvae can induce a specific IgE response withclinical symptoms, usually urticaria, even if the fish is cooked beforeingestion and the invasive infestation power destroyed by heating. Ourprehminary studies showed a strong association of A. simplex sensitizationwith Ascaris lumbricoides, Daphnia, chironomid spp,, Atlantic shrimp(Pandaltts borealis), and German cockroach {Blattella germanica). Weconducted the cross-reaetivity study with cockroach, a ubiquitous insect, andChironomidae (red mosquito larvae), a work-related allergen, without anypossibility of Anisakis contamination. Serum samples were collected from60 pediatric patients, with serum specific IgE to A. simplex. Both specific-IgE and itnmunoblot-inhibition studies, with a serum pool from 18 patients,were performed to determine whether the association of sensitizations tonemalodes and arthropods was due to immunologic cross-reactivity. Inaddition, serum samples from 2f of 60 patients who showed also sensitizationto German cockroach were used for individual immunoblot studies. In theserum pool, dose-dependent inhibition of B, germanica and Chironomtts spp,was observed after preincubation with the A. simplex extract, Immunoblotof Anlsaids, inhibited with Chironomus and German cockroach, yielded apartial blot inhibition but mainly on bands below 41 kDa, Blot inhibition ofGerman cockroach and Chironomus with Anisakis was dose related. Theband patterns in individual blots were heterogeneous, but most of them hadbands of 30-43 kDa, None of these sera recognized allergens in the 14-kDaarea. In our study, CAP-inhibition and immunoblot-inhibition analysis ofAnisakis showed that several IgE-binding components could be shared bythe three allergens.
C. Y, PascuaP, J. F. Crespo^S, San Marti^^ N. Ornia\N. Ortega\ T, Caballero\M, Munoz-Pereira\M. Martin-Esteban^'Hospital Universitario "LA PAZ", Madrid;^Hospital Universitario "12 de Octubre", Madrid,Spain
Cristina PascualLaboratorio InmunoalergiaServicio Alergia PediatricaHospital Universitario "La Paz"Castellana 261, Madrid 20046Spain
Accepted for publication 4 December 1996
IgE-mediated hypersensitivity to fish is a elinicallyrelevant problem, particularly iti certain Europeancountries. In Spain, it is the second food in pre-valence, after egg, in children's food-itiduced aller-gic reactions (1), Fish hypersensitivify has beenreported to cause fatal or near-fatal allergic reac-tions. The most prevalent clinical feature in fishallergy is immediate urticaria and angioedema (2),Since 1990, another factor has been imphcatedin fish-related anaphylaxis, the fish parasites ofthe genus Anisakis (3-6), It is well known thatanisakiasis is a parasitic disease caused by ingestionof mainly third stage Anisakidae larvae from raw
or improperly prepared sea fish and squid, Manis an accidental host in which the parasites donot mature or reproduce. Adult worms are foundin the stomachs of sea mammals (seals, dolphins,and whales), and eggs are passed from the host'sgut into the ocean. The first-stage Anisakis larvaeare eaten by crustaceans, which act as transporthosts. Fish are intermediate hosts, and marinemammals are the definitive hosts in the life cycleof these netnatodes (7), Tlie ingestion of seafoodcontaminated with third-stage Anisakis larvae caninduce a specific IgE response with clinical symp-toms, usually urticaria, even if the fish is cooked
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before ingesfion and the invasive infestation powerdestroyed by heating (8),
In a previous study, we found a strong associationof Anisakis sensitization with Ascaris, Daphnia.ehironomids, Atlantic shrimp, and German cock-roach (9), The close relationship with Ascaris iswell known (10), both being parasitic netnatodesof the same superfamily, Ascaridoidea, Daphnia, agenus of crustacean used as food for fish in cap-tivity, may be contaminated by Anisakis larvae, andthe same contamination may be suspected inshrimp. Therefore, we conducted the cross-reactivitystudy with cockroach, a ubiquitous insect, andehironomids (red mosquito larvae), source of awork-related allergen, without any possibility ofAnisakis larvae contamination.
The study aimed to detennine whether theassociation of sensitizations to Anisakis simplexand several arthropods (Gertnan cockroach andchironomid spp,) was due to immunologic cross-reactivity, and to determine the IgE-binding patternto Anisakis components in patietits with imtiiediatehypersensitivity to arthropods.
Material and methodsSera
Serum samples were collected from 60 pediatricpatients, with serum specific IgE to A. simplex(AK), These patients had been diagnosed as havingacute urticaria and/or seafood hypersensitivity (40seafood hypersensitivity and 20 acute urticaria notfood-related) by clinical history, skin prick testing,in vitro specific IgE, and oral challenges. Eachserum was tested also for specific IgE to Ascarislumbricoides, Blattella germanica (BG), Pandalusborealis (PB), and Chironomus spp, (CD). Serawere stored at -70°C until use. Sera from 18/60patients, by means of similar IgE to AK, BG, andCD higher than 10 kU/1, but total IgE less than1000 kU/1, were pooled and stored at -70°C, to beused in the inhibition assay and immunoblotting.
Serum samples from the 21/60 patients with specificIgE to both AK and BG higher than 3,5 kUI/1 wereused for individual immunoblot studies.
Allergen extracts
AK extracts were prepared from the third larvalstage. The larvae were obtained from hake fleshprovided by a local fishmarket. After idetitificationby the microbiology and parasitology departtnent,the larvae were homogenated in liquid nitrogen for5 min with a pestle, Tlie powdered material wasthen resuspended with phosphate-buffered saline(PBS) (10% w/v). The mixture was stirred for 4 h
and centrifuged at 15 000 g for 30 tnin at 4°C, Tliesupernatant was dialyzed (Spectrapor, SpectrumMedical Industries, Los Angeles, CA, USA, molec-ular weight cutoff >3,5 kDa) at 4°C for 24 h against10 mM glycine, centrifuged at 30 000 g for 30 minat 4°C, and passed through a 0,45-|im Milliporefilter (Millipore, Bedford, MA, USA), Finally, thematerial was lyophilized. The protein content wasdetermined by the Lowry method (11),
BG were collected at our insect farm and killedby freezing. Frozen CD {Chironomus) were pur-chased from a local aquarium shop. Both allergenswere processed in the same way as Anisakis. Tliethree lyophilized allergens were stored at -20°C indesiccator until needed.
Allergen-specific IgE levels
AK-, BG-, and CD-specific IgE levels in all 60 indi-vidual sera and in the serum pool were determinedby an enzymatic immunoassay using Immuno-CAP (cellulose sponge) as solid phase for aller-gens (CAP-FEIA), performed according to thetnanufacturer's instructions (Pharmacia Diagnostics,Uppsala, Swedeti), Results were expressed in kU/1,Significant reactivity was determined to be higheror equal to 0,35 kU/1, according to the manu-facturer's instructions, Antihake, antisquid, anti-Ascaris, antishrinip, and antiovalbumin IgE werealso determined by the CAP-FEIA method in thepooled serum,
CAP-EEIA inhibition
The CAP-inhibifion procedure was a modificationof the RAST-inhibition method of Gleich et al,(12), Several concetitrations of cotnpeting fluid-phase antigens (1, 10, 100, and 500 |ag/nil finaldilution of AK, BG, and CD), diluted with PBS,were preincubated with an equal volume of pooledserum for 1 h with intermittent shaking at roomtemperature. Positive controls were diluted andpreincubated in PBS alone. After preincubation,the mixtures were centrifuged, and 50-jul duplicatedaliquots were incubated at rootn temperature withImmuno-CAP, with either the same or anotherextract, for measurement of AK-, BG-, and CD-specific IgE levels. Percent inhibition of AK-, BG-,and CD-specific IgE by soluble antigen was plottedon semilog graph paper, with the y-axis as per-cent itihibition and the x-axis as log concentration(|ig/ml) of the competing fluid-phase antigen (13),
For assessment of the specificity of AK inhibi-tory capacity, a pool sera of known ovalbuminvalue was inhibited with AK in the same way,Ovalbumin was also used as negative control ofCAP inhibition of the three allergens.
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SDS-PAGE and immunoblotThe electrophoresis procedure was a modificationof the method of Sutton et al, (14), SDS-PAGEwas performed with a 12% polyacrylamide sepa-rating gel and a stacking gel of 4%, Twenty micro-liters of a 1 mg/ml solution of each allergenicextract was applied to each well, Electrophoresiswas performed for 1 h at 130 V per gel for the8 cm by 7,5 cm gels (Mini-Protean II system,Bio-Rad Laboratories), Silver stainitig (Bio-RadLaboratories) was done on gels to ensure properprotein separation and visualization. Proteins wereelectrophoretically transferred from the separatinggel to a PDVF (Millipore, USA) membrane in atransfer buffer (Tris-glycine) with 20% methanol(13), This procedure was dotie in a minitransferapparatus (Bio-Rad Laboratories),
After removal from the transblot apparatus, themolecular weight standard lane and an AK lanewere dyed with amido black (1% in ethanol-aceticacid solution) as controls. The rest of the mem-brane was placed in blocking solution (1% gelatin/TBS Tween) for 1 h at room temperature. ThePDVF blot was then washed four times with TTBS(TBS with 0,1% Tween 20) and incubated with thepooled serutn (1 : 3 v/v dilution in TTBS) for 3 hat room temperature with rocking. The PDVFblot was again washed with TBS four times, andalkaline phosphatase-conjugated goat antihumanIgE (1 :500 v/v of TBS, Caltag Laboratory,San Francisco, CA, USA) was added and incubatedat room temperature with rocking for 3 h. Afteran additional wash with TBS three times, the blotwas developed with an alkaline phosphatase con-jugate substrate kit (Bio-Rad) of nitro-blue tetra-zolium and 5-bromo-4-chloro-3-indolyl-phospliateat room temperature for 15 min. The reaction wasstopped by incubating the PDVF blot for 10 tninwith distilled water, Tlie blot was then air-dried.The three blotted allergens were also incubatedwith a negative pool sera of pollinic patients. Theimmunodetection of specific IgE dLnii-Anisakis inthe 21 individual sera was done in the same wayexcept for the dilution rate of the implicated serum(estabhshed at 3-6 IgE kU/1),
Immunoblot inhibition
Aliquots (1,5 ml) of the serum pool (1 : 1 v/vdilution in TTBS) were incubated overnight withequal volumes of AK, BG, and CD extracts ata final concentration of 0,01, 0,1, and 1 mg/ml.Mixtures were then centrifuged and each super-natant incubated with AK-, CD-, or GC-blotted PDVF(Millipore, USA) membranes, followed by immuno-detection as described above. The immunoblot was
also inhibited with ovalbumin (1 mg/ml finalconcentration) (Sigma Chemical Co, St Louis, MO,USA) as negative control of inhibition.
ResultsCAP-EEIA inhibition studies
Specific IgE values of the serum pool were asfollows: AK 33,4, Ascaris 21, CD 15,5, BG 13,5,shrimp 25, and ovalbumin 16,7 kU/1, Tlie total IgEvalue was 420 kU/1,
CAP-FEIA inhibition experimetits are shown inFig, 1, Dose-dependetit inhibition was observedafter preincubation with AK or hotnologous (BGor CD) extracts, CD-specific IgE was inhibitedafter preincubation of the serum pool with AKextract (maximum dose of 500 jig/tnl) at 60%, andBG-specific IgE only 47%, To exclude the possi-bility that the observed cross-inhibition was due totionspecific binding of IgE, similar experiments(with ovalbumin as solid phase) were performedwith a serum pool with a known level of specificIgE to ovalbumin. None of these IgE reactionswere affected by preincubation of the sera withAK, BG, and CD {daia not shown),
Immunoblot and immunoblot inhibition (serumpool)
AK, BG, and CD extracts were run on SDS-PAGE, transferred to an Itnniobilon membrane,and immunoprobed with the serum pool to identifyIgE-binding components, Itnmunoblot from SDS-PAGE extracts in PDVF yielded a well-definedband pattern of proteitis by atnido-black staiti, Tlieband pattern of IgE anti-AK could be divided intothree groups. The first group comprised thoseabove 40 kDa with four double bands (78, 72, 58,and 41 kDa), Tlie second group comprised threebands between 40 and 30 kDa (34, 33, atid 32 kDa),The third group comprised three double batids (26,24, and 23 kDa), A double band at the 14-kDa levelcould hardly be identified (Fig, 2, lane 3), Theimmunoblot from SDS-PAGE of BG showed twobands above 94 kDa and three between 94 and 43kDa (86, 67, and 54 kDa) and five bands below 43kDa (41, 36, 33, 31, and 30 kDa), being moreintense in the area of 36-33 kDa (Fig, 3, lane 3),The band pattern of IgE anti-CD had one bandabove 94 kDa and four bands above 43 kDa (76,71,58, and 48 kDa), The area below 43 kDa showedthree bands (41, 39, and 36 kDa), Below 30 kDa,there were three more bands (29, 26, and 24 kDa),
Homologous immunoblot inhibition of AKextract with the highest dose (1 mg/ml) was com-plete (Fig, 2, lane 4), but the inhibition with the
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Ant\-Blattella germanica IgE100%
75% -
co
50%-
25% -
0%
10 100
tluid-phase inhibitor (|jg/ml)1.000
Anti-chironomid spp. IgE100%
75% -
co5 50% -
25% -
100
fluid-phase inhibitor (|jg/mi)
1.000
^g. 1. Top; inhibition ot specific anti-6, germanica IgE (solid phase) with A. simplex (triangles) and B. germanica (squares) extracts,DOttom; inhibition of specific anti-chironomid spp, IgE (solid phase) with A. simpiex (triatigles) and chironotnid SDD I'sauares")extracts f f \ ^ fextracts,
same amount of CD (Fig, 2, lane 5) and BG (Fig, 2,lane 6) yielded a partial blot inhibition, mainly ofbands below 41 kDa, Blot itihibition of BG withAK extract was dose related, being altnost total with1 mg/ml (Fig, 3), Blot inhibition of CD with AKWas also dose related, but the band fading was lessintense than the inhibition obtained on BG (Fig, 3),
SDS-PAGE immunoblot with individual sera
From the 60 selected sera with sensitization to AK,21 had specific IgE to BG above 3,5 kUI/1, Wetested the 21 sera with a strip of PDVF transblotfrom AK extract, Tlie band patterns were hetero-
geneous, but most of them had bands of 30-43 kDa(Fig, 4), Tlie band of 71 kDa was recognized by 13/21(61,9%) of the sera. The predominant band of58 kDa was recognized by 16/21 (76,19%) sera. Inthese sera, the bands below 30 kDa were presentin 19/21 (90,47%), and bands of 30-40 kDa weredetected in 18/21 (85,7%) sera. No bands in the14-KDa area of the AK extract were detected withindividual sera.
DiscussionAfter the Scandinavian countries, Spain has thehighest fish intake per inhabitant in Europe, The
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kDa
9467
43
30
2014
I 3
Fig. 2. SDS-PAGE immunoblot analysis of A. simplex extract.Lane 1) molecular weight standards: lane 2) AK stained withamido black: lane 3) immunodetection with specific IgE (serumpool): lane 4) homologous inhibition (1 mg/ml): lane 5) hetero-logous inhibition by BG extract (1 mg/ml); lane 6) heterologousinhibition by CD extract (1 mg/ml): lane 7) negative control.
kDa9467
43
30
2014
1 3
Fig. 3. SDS-PAGE immunoblot analysis of B. germanica andchironomid spp, inhibited with A. simplex (fluid phase). Lane1) MW standards: lane 2) BG in amido black: lane 3) BGextract (serum pool): lane 4) BG inhibition with AK (tnaximumdose 1 mg/ml): lane 5) CD in amido black; lane 6) CD withserum pool: lane 7) CD inhibition with AK extract (maximumdose 1 mg/ml).
incidence of Anisakis sensitization is unknown inthe fish-eating European communities. The fishinfestation by this parasite has grown by 63% inthe last 15 years, especially in the North Sea (7), Theconcern about public health is great, and specificlaws have been passed (i,e,, in The Netherlands)forbidding the consumption of raw fish withoutdeep-freezing for 48 h (15), This treatment canprevent anisakiasis, but has no effect on IgE-mediated hypersensitivity, Japan has the highestanisakiasis prevalence, due to the habitual con-sumption of raw fish by the population. Since 1989,Japanese researchers have been discussing theimmunobiologic significance of antigens for anisa-kiasis, Tanaka & Torisu, suggested that acute anisa-kiasis is mainly due to a hypersensitivity reactionto specific Anisakis antigens, mainly of the secre-tory type (16), A 14-kDa band has been describedas one of the principal causes of antigenic homo-logy among Ascaroidea (10), For in vitro diagnosisof anisakiasis, micro-ELISA systems have beenused to detect antibodies to raw Anisakis antigensof IgG, IgA, IgM, and IgE isotypes, with contro-versial results (17), In an epidemiologic study ofanisakiasis, 63% of the general healthy populationhad a positive intradermal reaction to Anisakis(18), RAST has been used for the serologic diag-nosis of anisakiasis (19), and cross-reactivity withAscaris and Schistosoma has been found, but notwith the Toxocara canis antigen. In two Spanishreports of urticaria in, respectively, 10 and 12patients due to Anisakis sensitization, all of themalso had specific IgE to Ascaris, without parasiticinfestation (5, 20),
In a previous study, we quantified specific IgEto A. simplex, Ascaris lumbricoides, T. canis.
Equinococcus spp,, Dermatophagoides pteronyssinus,B. germanica (German cockroach), P. borealis(Atlantic shrimp), Merluccius merlucius (hake),Loligo spp, (squid), Chironomus spp,, atid Daphnia,in a group of pediatric patients with urticaria,sensitized to seafood, and in a control group ofatopic patients with elevated IgE and withouturticaria (9), AK-specific IgE antibodies weremore frequently foutid in patients with shellfishallergy or parasitic diseases, sensitized to severalarthropods and/or Ascaris. It is already estabhshedthat there is antigenic cross-reaction among asca-roid nematodes that infect man (10), But we stilldo not know whether a previous infestation withanother nematode could enhance the chnicalhypersensitivity to Anisakis upon exposure.
Cross-reactivity exists between shrimp, an impor-tant food allergen, and German cockroach, whichhas an increasing role as an indoor aeroallergen inallergic asthma (21), Immunologic studies haveshown that cross-reactivity exists between ehirono-mids and shrimp (22), Tropomyosin is claimed tobe the connecting panallergen (23-25), Recently,it has been demonstrated that mite immunotherapycan induce IgE against food such as shrimp or snail;in particular, IgE reactive to tropomyosin (26),
Our study focused on evaluating the possibilityof cross-reactivity between arthropods and nema-todes. In view of the possible contamination ofshrimp with Anisakis larvae, we chose two otherarthropods without possibihty of contaminationwith Anisakis, German cockroach (B. germanica),and Chironomus spp, larvae, source of a work-relatedallergen unusual in a pediatric population. In ourstudy, CAP-inhibition and immunoblot-inhibitionanalysis of Anisakis showed that several IgE-binding
Fig. 4. SDS-PAGE immunoblot of A. simpiex extract with 21 individual sera with specific IgE to A. simpiex and B. gennanica.
components could be shared by the three allergens.The specificity of these results is supported by propercontrols. The imtnunoblot inhibition of AK by GCand CD was partial but mainly in those protein bandsbelow 40 kDa, The opposite, immunoblot of GC andCD inhibited by AK, also showed a partial inhibi-tion mostly in bands of 30-40 kDa, One of the GCbands inhibited by AK was the 36-kDa tropo-myosin, described as the GC major allergen (27),
The 21 individual sera sensitized to GC andP. borealis had a heterogeneous band pattern, but19/21 (90,5%) recognized proteitis in the area of30-40 kDa, It may be that tropomyosin is the cross-sensitization link between these allergens of suchdifferent origin {Anisakis, a genus of nematode thatcontaminates seafood, and two well-characterizedarthropod aeroallergens). None of these 21 serarecognized bands in the 14-kDa area that is claimedto be specific for Ascaroidea sensitization. Furtherstudies are needed to determine whether a pos-sible cross-reactivity through a panallergen could playa role in the immediate clinical hypersensitivity toAnisakis that is becoming a health problem inSpain due to the high fish intake.
AcknowledgmentsThis paper is dedicated to Dr Zhara Bensabat, an outstandingcolleague, who died a few days before submission of this paper,We thank Ms Sofia Sanchez-Pastor, Ms Begona Varga, andMs Carmen Bernal for excellent technical assistance, andMs Mary McMacken for help in improving our EngUsh,
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