The Journal of Allergy and Clinical Immunology
Volume 100, Issue 2 , Pages 192-198, August 1997

Cross-reactivity of skin and serum specific IgE responses and allergen analysis for three mosquito species with worldwide distribution☆☆☆★★★

Winnipeg, Manitoba, Canada

Received 16 July 1996; received in revised form 27 March 1997; accepted 28 March 1997.

Article Outline

Abstract 

Background: Allergic reactions to mosquito bites are a common problem worldwide. Cross-reactive immunologic responses have not been reported. Objective: For the three most common mosquito species, Aedes (Ae.) vexans, Ae. aegypti, and Culex (Cx.) quinquefaciatus, we investigated skin and serum specific IgE responses and analyzed salivary allergens. Methods: Locally, Ae. vexans is a major pest, but Ae. aegypti and Cx. quinquefaciatus are not found. We studied 41 subjects living in Manitoba, using (1) skin bite tests, (2) ELISA to measure serum mosquito saliva– or salivary gland–specific IgE, (3) ELISA inhibition tests, and (4) sodium dodecylsulfate–polyacrylamide gel electrophoresis and immunoblotting. Results: Twenty-nine of the 41 subjects had skin reactions to Ae. vexans bites. Twenty-two of the 29 also reacted to Ae. aegypti bites. Mean serum mosquito–IgE levels to each of the three species were significantly higher in the reactive subjects than in the nonreactive subjects. Significant intercorrelations were found among skin reactions and mosquito-specific IgE levels for the three species. The Ae. aegypti–IgE ELISA reaction could be inhibited by addition of each mosquito extract. The serum IgE and IgG from Manitobans reacted with the antigens of all three species. A 37 kd allergen in each of the three species was recognized by the antibody against a recombinant Ae. aegypti saliva protein. Conclusions: Strong cross-reactive skin and IgE responses and species-shared antigens exist among the three mosquito species studied. (J Allergy Clin Immunol 1997;100:192-8.)

Keywords:  Allergen, allergy, antigen, IgE, immunoblot, mosquito, Aedes vexans, Aedes aegypti, Culex quinquefaciatus

Abbreviations:  Ae., Aedes, Cx., Culex, Mosquito-specific IgE , Mosquito saliva– or salivary gland–specific IgE, PBS , Phosphate-buffered saline, SDS-PAGE , Sodium dodecylsulfate– polyacrylamide gel electrophoresis

 

Allergic reactions to mosquito bites are a global problem. Although the bites commonly cause cutaneous wheals and flares and delayed papules, severe local reactions and systemic reactions may also occur.1, 2, 3, 4 Despite this, there are few reports in which modern immunologic techniques have been applied to the study of mosquito allergy.

Previous studies have revealed that both IgE-mediated and T-lymphocyte–mediated hypersensitivities are involved in the development of mosquito allergy.2, 5, 6, 7, 8 Analogous to immunotherapy with venoms in Hymenoptera allergy, successful subcutaneous immunotherapy with crude mosquito whole-body extract has been reported.3, 9, 10 To improve diagnosis and immunotherapy of mosquito allergy, purified or recombinant mosquito saliva antigens should be used.11 In anticipation of this, it is important to determine whether there are any cross-reactive skin and IgE responses and species-shared antigens among various mosquito species, especially those with worldwide distribution.

Aedes (Ae.) vexans, Ae. aegypti, and Culex (Cx.) quinquefasciatus are the three most important mosquito species distributed globally. Ae. aegypti12 and Cx. quinquefasciatus13 are found throughout the tropical regions of the world within 20° C isotherms; and Ae. vexans is found in North America, Eurasia, Asia, and Africa.14 Comparison of the human immunologic response to the three species has never been made. In Canada where Ae. aegypti and Cx. quinquefasciatus are not found, Ae. vexans is a major pest, representing up to 80% of the local mosquito population. To determine whether the three mosquito species have cross-reactive immunologic responses and species-shared antigens, we performed skin bite tests and evaluated serum mosquito specific IgE for the three mosquito species in 41 Manitobans who had been exposed to Ae. vexans bites but not to Ae. aegypti and Cx. quinquefasciatus bites. We also analyzed species-shared allergens by immunoblotting, using the sera from subjects allergic to mosquitoes and an antibody to a recombinant Ae. aegypti salivary protein.

Back to Article Outline

Methods 

Subjects 

This project was approved by The University of Manitoba Faculty Committee on the Use of Human Subjects in Research, and the participants gave written, informed consent before study entry. Forty-one healthy subjects (21 men and 20 women), aged 19 to 57 years, with skin reactions to mosquito bites ranging from none to strongly positive were recruited during the summer of 1993. All the subjects had lived in Canada more than 2 years, and 71% had lived in Canada (mostly in Manitoba) since birth. Antihistamines and other medications that might suppress the skin bite test response were withheld for an appropriate length of time before the study.

Mosquitoes, mosquito saliva, and salivary gland extracts 

Female Ae. vexans mosquitoes were collected in local fields and identified by scientists in the Department of Entomology, University of Manitoba. The Ae. aegypti colony was obtained from the same department and maintained in our laboratory. The Cx. quinquefaciatus colony was imported from Dr. Robert J. Novak's laboratory (University of Illinois, Champaign) and maintained in our laboratory. Four- to twelve-day-old adult Ae. aegypti mosquitos were used for the bite tests and for saliva collection. Salivary glands were dissected from 4- to 12-day-old adult Cx. quinquefaciatus mosquitoes.

Mosquito saliva or salivary gland extracts were prepared for use in the ELISA and immunoblot. Saliva was collected from female adult Ae. aegypti mosquitoes by placing the proboscis of each mosquito into a capillary tube filled with water. Salivation was induced by applying a small amount of malathion-acetone solution to the thorax.16 One hour later, the contents of capillary tubes containing saliva were collected, pooled, and lyophilized. The saliva was reconstituted before use by dissolving the lyophilized proteins in 0.02 mol/L phosphate-buffered saline (PBS).

Salivary gland extracts of Ae. vexans and Cx. quinquefaciatus were prepared by the methods previously described7 from female Ae. vexans mosquitoes collected from the fields and female adult Cx. quinquefaciatus mosquitoes raised in our laboratory, respectively. The protein concentration was 0.6 mg/ml for Ae. vexans extract, 0.4 mg/ml for Cx. quinquefaciatus extract, and 0.3 mg/ml for Ae. aegypti extract as measured by a Bio-Rad Protein Assay kit (Bio-Rad Labs, Richmond, Calif.).

When we used the method applied to Ae. aegypti mosquitoes, Cx. quinquefasciatus mosquitoes did not salivate, and thus we had to use a salivary gland extract from this species. For Ae. vexans, we compared the salivary gland extract and the saliva in an ELISA and found that the salivary gland extract was better than saliva. Although both techniques are time-consuming and labor-intensive, dissection of salivary glands is relatively easier than saliva collection; therefore, we used salivary gland extract of Ae. vexans in the study.

Skin bite tests and blood samples 

Skin bite tests were performed with one Ae. vexans mosquito and one Ae. aegypti mosquito on the volar aspect of each subject's forearm, as previously described.8 The wheal-and-flare circumferences were traced at 20 minutes and 24 hours after the bite by using a felt-tipped pen. All wheal-and-flare tracings were transferred to transparent paper. The area of the wheal, flare, or induration was measured with an IBM-PC digitizer and stereometric measurement software (IBM Instruments, Inc., Danbury, Conn.).15

A wheal of less than 0.3 cm2 with no flare and no itching was considered to be a negative immediate reaction. An induration less than 0.3 cm2 was considered to be a negative delayed reaction.

Ten milliliters of blood was obtained for measurement of mosquito-specific IgE to the three species. Serum was separated and stored at –70° C until use.

Measurement of mosquito-specific IgE by ELISA 

Serum mosquito-specific IgE to Ae. vexans, Ae. aegypti, and Cx. quinquefaciatus was measured by an indirect ELISA as previously described.7 Optimal conditions for dilutions of the three mosquito extracts, serum samples, goat anti-human IgE, and conjugated rabbit anti-goat IgG were chosen by checkerboard titration. Standardization of ELISA results among assays and estimation of the relative amount of mosquito-specific IgE in each sample was accomplished by using reference sera. The reference serum used to measure Ae. aegypti-IgE and Cx. quinquefaciatus-IgE was obtained from a subject with systemic reactions to mosquito bites (kindly provided by Dr. Renata J. Engler, Walter Reed Army Medical Center, Washington, D.C.). Another reference serum used to measure Ae. vexans–IgE came from a Manitoban with severe skin reactions to mosquito bites (immediate wheal, 1.5 cm2; flare, 11.6 cm2). Both reference sera were defined as 1000 U/ml for mosquito-specific IgE. Microplates coated with mosquito saliva or salivary gland extract (0.02 to 0.05 μg/well) were sequentially incubated with serum samples (1:20) or reference serum (twofold dilutions from 1:20 to 1:10,240), 1:1000 goat anti-human IgE (P.S. myeloma-affinity purified, a gift from Dr. N. Franklin Adkinson, Jr., The Johns Hopkins Allergy and Asthma Center), and 1:1000 alkaline phosphatase–conjugated rabbit anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.). The values of mosquito-specific IgE in the tested samples were calculated by interpolation from the dilution curve of the reference serum. The sensitivity of the ELISAs was 0.8 U/ml.

ELISA inhibition tests 

To study the cross-reactivity among the three species, ELISA inhibition tests were performed. A serum sample with high mosquito-specific IgE (final dilution, 1:20) was incubated with serially diluted Ae. vexans, Ae aegypti, and Cx. quinquefaciatus extracts with final dilutions of 1:4 and 1:20 at room temperature for 1 hour and then at 4° C overnight. Incubation of the serum with ELISA buffer served as a positive control. These incubated materials were then assessed for Ae. aegypti–IgE by means of ELISA.

Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis 

Among the 41 subjects studied, sera from six subjects with large immediate skin wheal responses (>1.0 cm2) in the bite tests and high Ae. vexans–specific IgE levels (>1000 U/ml) were pooled and used for immunoblotting.

SDS-PAGE and immunoblotting were performed according to the method previously described.17 In the analysis of IgE and IgG antigens, 2 μg of the proteins from each mosquito extract were loaded and electrophoresed in 12% acrylamide SDS-PAGE under reducing conditions. Proteins separated by SDS-PAGE were electrophoretically transferred onto nitrocellulose membranes. Free binding sites on the membranes were blocked by incubation for 2 hours with 3% bovine serum albumin in 0.02 mol/L PBS, pH 7.4, containing 0.05% (vol/vol). After washing three times with PBS–Tween-20, the membranes were incubated overnight with the pooled serum (1:10 dilution for IgE and 1:50 for IgG) and washed again. Incubation of the membranes with PBS–Tween-20 served as a negative control. This was followed by sequential incubations with 1:15,000 monoclonal anti-human IgE (clone 7.12, from Dr. Andrew Saxon's laboratory, University of California) or 1:15,000 monoclonal anti-human IgG (PharMingen, San Diego, Calif.), and then horseradish peroxidase–conjugated goat anti-mouse IgG (1:5000 for IgE, 1:10,000 for IgG; Calbiochem Corp., La Jolla, Calif.). After washing, the membranes were finally incubated with ECL detecting reagents (Amersham Life Science, Buckinghamshire, England) and then exposed to film (X-Omat, Kodak). Prestained SDS-PAGE standards (Bio-Rad Labs) were used to determine the relative molecular weights of the electrophoresed components.

The same method, with some modifications, was used in the study of a 37 kd recombinant Ae. aegypti saliva protein, which was kindly provided by Dr. Anthony James at the University of California, Irvine.18 One microliter of the baculovirus medium containing the recombinant protein and 2 μg of each mosquito extract were loaded onto different wells and electrophoresed. Medium from cells infected with wild-type baculovirus was used as a control. The membranes containing separated proteins were then incubated with rabbit antibody to the recombinant protein (1:5000, from Dr. Anthony James) followed by incubation with horseradish peroxidase–conjugated goat anti-rabbit IgG (1:5000). The remaining steps were the same as those for the immunoblot with human serum.

Statistical analysis 

Analysis of the data was performed by using the “Number Crunch Statistical System” software (published by Dr. Jerry L. Hintze, Kaysville, Utah). Unpaired t tests were used for between-group comparisons. Linear regressions were used for the analysis of correlations among skin reactions and serum mosquito-specific IgE levels.

Back to Article Outline

Results 

Skin bite reactions 

No systemic reactions were noted after the bite tests. Immediate and delayed skin reactions to mosquito bites were observed. The immediate reaction consisted of a pruritic wheal with a surrounding flare or erythema appearing within several minutes, reaching a peak at 20 to 30 minutes and then subsiding. The delayed reaction consisted of an indurated papule, which appeared several hours later, peaked at 24 to 36 hours, and diminished over several days after the bite. In five subjects with severe delayed reactions, vesicles were found in the centers of the indurated areas, precisely in the area where the immediate wheal had been.

Twenty-nine of the 41 subjects had positive immediate skin reactions to Ae. vexans bites. Twenty-two of the 29 also reacted to Ae. aegypti bites. Seven subjects reacted to Ae. vexans bites only. The size of the immediate reactions ranged from 0 to 3.1 cm2 for the wheal and 0 to 23.0 cm2 for the flare, and the size of delayed reactions ranged from 0 to 29.5 cm2.

In the immediate reaction, wheals correlated significantly with flares in both species (r = 0.72, p < 0.00 for Ae. vexans; r = 0.88, p  < 0.00 for Ae. aegypti). Also, immediate wheal-and-flare reactions showed significant correlation with the delayed reactions in each species as well (r values between 0.43 and 0.61, p values < 0.00). More interestingly, significant correlations of skin reactions were found between the two species as shown in Table I and Fig. 1, especially between the immediate wheal sizes of the two species (r  = 0.84, p < 0.00) and the delayed induration reactions of the two species (r = 0.77, p < 0.00).

Table I. Coefficients of correlation among skin bite reactions and mosquito-specific IgE levels
Ae. aegypti immediate whealAa. aegypti delayed indurationAe. vexans immediate whealAe. vexans delayed indurationAe. vexans–IgEAe. aegypti–IgE
Ae. aegypti delayed induration0.43*
Ae. vexans immediate wheal0.84*0.26
Ae. vexans delayed induration0.68*0.77*0.61*
Ae. vexans–IgE0.42*0.45*0.37*0.45*
Ae. aegypti–IgE0.34*0.42*0.260.43*0.60*
Cx. quinquefasciatus–IgE0.280.180.34*0.41*0.57*0.35*

*p < 0.05.

Mosquito-specific IgE levels 

As shown in Fig. 2 (top), the geometric mean Ae. vexans–IgE, Ae. aegypti–IgE, and Cx. quinquefaciatus–IgE levels were all significantly higher in the subjects with immediate skin reactions to Ae. vexans bites than in those with no immediate skin reaction to the bites (p < 0.05).

Similar results were found for mean mosquito-specific IgE levels of the three species in the subjects with or without immediate skin reactions to Ae. aegypti bites (Fig. 2, bottom). Significant correlations were also found among the IgE levels of the three species (r values between 0.35 to 0.60, p values < 0.03) (Table I).

Correlations among skin reactions and the IgE levels 

Table I summarizes the intercorrelations among skin reactions and IgE levels to the three species. As expected, Ae. vexans–IgE values correlated significantly with skin reactions to Ae. vexans bites, and Ae. aegypti–IgE values correlated significantly with skin reactions to Ae. aegypti bites. Ae. vexans–IgE also significantly correlated with skin reactions to Ae. aegypti bites. The same correlation was found between Ae. aegypti–IgE and the delayed skin reactions to Ae. vexans and between Cx. quinquefaciatus–IgE and both immediate and delayed reactions to Ae. vexans bites.

ELISA inhibition tests 

The cross-reactive immunologic responses among the three species were further confirmed by ELISA inhibition tests. Ae. aegypti–specific IgE reactions could be inhibited by incubation of the serum with all three extracts in a dose-dependent manner, supporting the existence of species-shared antigens among the extracts of the three species (Fig. 3).

  • View full-size image.
  • Fig. 3. 

    ELISA inhibition tests. Pooled serum from subjects allergic to mosquitoes was incubated with Ae. vexans extract (left), Ae. aegypti extract (middle), and Cx. quinquefaciatus extract (right) overnight and then assayed for Ae. aegypti–IgE by ELISA as described in Methods.

SDS-PAGE and immunoblot analysis 

Immunoblot analysis further revealed the existence of species-shared antigens. When the pooled serum from Manitobans allergic to mosquitos was used, the IgE and IgG antibodies not only bound to Ae. vexans antigens but also to the antigens of the two species that are not found in Manitoba (Fig. 4, left and middle).

  • View full-size image.
  • Fig. 4. 

    Immunoblot analysis of antigens of the three mosquito species. SDS-PAGE and immunoblotting were performed as described in Methods. Proteins of saliva or salivary gland extracts separated by SDS-PAGE were transferred to nitrocellulose membranes. Membranes were sequentially incubated with pooled serum from subjects allergic to mosquito bites, monoclonal anti-human IgE (left) or monoclonal anti-human IgG (middle), and conjugated goat anti-mouse IgG. Membranes were also incubated with the rabbit antibody to a recombinant 37 kd Ae. aegypti saliva protein (right), followed by incubation with conjugated goat anti-rabbit IgG.

There were no antigen bands found in the PBS control strips. Species-shared antigens are indicated by arrows a, b, c, and f; and Ae. vexans–specific antigens are indicated by arrows d and e. Further, as shown on the right of Fig. 4, two bands in Ae. aegypti and Cx. quinquefaciatus extracts (equivalent to bands b and c) and one band in Ae. vexans extract (equivalent to band b) were recognized by the rabbit antibody to the recombinant protein, suggesting that the 37 kd protein of Ae. aegypti is also present in the salivary glands of Cx. quinquefaciatus and Ae. vexans. Although the molecular weight of band b was higher in Ae. vexans than those in the other two species and the molecular weights of bands b and c were obviously different, they all were recognized by the rabbit antibody, suggesting that these polypeptides share some common antigenic structure.

Back to Article Outline

Discussion 

Skin reactions to mosquito bites consisted of an immediate wheal and flare and a delayed induration. The elapsed time of the immediate reaction was compatible with that of an IgE-mediated hypersensitivity. Further, the mean serum IgE to the three species was significantly higher in subjects with positive skin bite test results than in those with negative bite test results, and the sizes of skin immediate wheals correlated significantly with the values of mosquito-specific IgE for the same species and also with other species. Serum mosquito-specific IgE was previously identified in human beings by using the Prausnitz-Küstner test,19, 20 ELISA,5, 7, 21 RAST,22 and Western blot techniques.6, 17, 23, 24 Two studies reported that mosquito-specific IgE correlated with immediate skin reactions.5, 8 In agreement with previous studies, our results confirm that IgE is involved in the development of skin immediate reactions in mosquito-allergic subjects.

The delayed reaction appeared to be consistent with lymphocyte-mediated hypersensitivity, which appears only after several hours and peaks between 24 and 48 hours.25 In guinea pigs, delayed skin reaction to mosquito bites has been successfully transferred with spleen cells but not with serum.26 In vitro studies, including our previous investigation8 and another study,5 showed that the size of the delayed papules correlated with lymphocyte proliferation response to mosquito antigens.

Another mechanism may also be involved in the early stage of the delayed reaction. Cutaneous late-phase IgE-mediated allergic reactions are characterized by erythema and induration, peaking between 4 and 8 hours27 and can be transferred from a sensitized to a nonsensitized individual with serum.28 In our study five subjects had a vesiculated area in the center of the delayed induration exactly mirroring the area in which the immediate wheal had occurred. This suggests that an IgE-mediated late-phase reaction may be involved in the delayed reaction. The suggestion is supported by the observation of other investigators that eosinophils and neutrophils are recruited in mosquito-bite lesions after the wheals have formed.2

Cross-reactivity with respect to bite reactions caused by different mosquito species has been reported previously.29, 30 Sensitization of animals by mosquito bites or injection of antigenic extracts from one species can confer reactivity against another species.29, 30 Desensitization of Ae. aegypti–allergic rabbits can be achieved by injections of Ae. atropalpus, Ae. pionips, or Cx. pipiens extracts.31 Although cross-reactive IgE responses have not been previously reported in mosquito allergy, cross-reactive IgE responses were reported for Hymenoptera allergy in wasps, yellow jackets, and hornets.32, 33 Unlike other studies of cross-reactivity to insect bites or stings, we selected a specific location where Ae. aegypti and Cx. quinquefasciatus are not present, whereas Ae. vexans is the major pest, representing up to 80% of the indigenous mosquito population. This allowed us to exclude the sensitization caused by the other two mosquito species.

The immunologic basis for the reactive skin and IgE responses among different mosquito species is the existence of species-shared antigens, which are based on their identical protein sequences. Salivary secretions have been demonstrated to be directly responsible for skin reactions to mosquito bites.34 In this study, immunoblot analysis with saliva or salivary gland extracts, a number of species-shared antigens and several Ae. vexans–specific antigens were found (Fig. 4). The number of antigens and their molecular weights in the three species will be reported in a subsequent article. Both species-specific and species-shared antigens have been previously reported in various mosquito species.17, 35, 36 A 62 kd antigen was found in the salivary gland extracts of five mosquito species (Ae. aegypti, Ae. taeniorhynchus, Cx. nigripalpus, Cx. quinquefaciatus, and Anopheles quadrimaculatus)36 and in the head and thorax extracts of two other species.17 In our previous study of antigens in Ae. vexans, Cx. tarsalis, and Culiseta inornata, nine antigens were shared by three species, six antigens were shared by two species, and only three antigens were species-specific.17 Whether these antigens from various species with identical molecular weights are immunologically identical remains to be confirmed.

Recently, the gene encoding a 37 kd salivary gland protein of Ae. aegypti has been cloned and expressed, and the specific antibody to the protein has been raised.18 In this study, using the antibody to the recombinant protein, we found the 37 kd Ae. aegypti saliva protein in Ae. vexans and Cx. quinquefaciatus salivary gland extracts (Fig. 4, left), confirming the existence of species-shared antigens in the three species. These species-shared antigens may well explain the cross-reactivity of skin reactions and IgE responses among different mosquito species.

Back to Article Outline

Acknowledgements 

We thank Dr. Reinhart Brust, Dr. Anthony James, Dr. Renata J. M. Engler, Dr. N. Franklin Adkinson, Jr., Dr. Jian Yang, Hong Li, RT, Andrew Mackay, MSc, and Andrew Fox, MSc, for their contributions to this work. This study was supported by the Children's Hospital Research Foundation, Winnipeg, Manitoba, Canada.

Back to Article Outline

References 

  1. Gluck JC, Pacin MP. Asthma from mosquito bites: a case report. Ann Allergy. 1986;56:492–493
  2. Reunala T, Brummer-Korvenkontio H, Palosuo T. Are we really allergic to mosquito bites?. Ann Allergy. 1994;26:301–306
  3. McCormack DR, Salata KF, Hershey JN, Carpenter GB, Engler RJ. Mosquito bite anaphylaxis: immunotherapy with whole body extracts. Ann Allergy Asthma Immunol. 1995;74:39–44
  4. Fan P-C, Chang H-N. Hypersensitivity to mosquito bites: a case report. Kaohsiung J Med Sci. 1995;11:420–424
  5. Oka K. Correlation of Aedes albopictus bite reaction with IgE antibody assay and lymphocyte transformation test to mosquito salivary antigens. J Dermatol. 1989;16:341–347
  6. Brummer-Korvenkontio H, Lappalainen P, Reunala T, Palosuo T. Detection of mosquito saliva-specific IgE and IgG4 antibodies by immunoblotting. J Allergy Clin Immunol. 1994;93:551–555
  7. Peng Z, Yang M, Simons FER. Measurement of mosquito Aedes vexans salivary gland-specific IgE and IgG antibodies and the distribution of the antibodies in human sera. Ann Allergy Asthma Immunol. 1995;74:259–264
  8. Peng Z, Yang M, Simons FER. Immunological mechanisms in mosquito allergy: correlations of skin reactions with specific IgE and IgG antibodies and lymphocyte proliferation response to mosquito antigens. Ann Allergy Asthma Immunol. 1996;77:238–244
  9. Lelong M. Severe local reactions to mosquito bites. Apropos of 20 cases. Allerg Immunol (Paris). 1986;18:21–22
  10. Benaim-Pinto C, Fassrainer A. Intradermal immunotherapy in children with severe skin inflammatory reactions to Aedes aegypti and Culex quinquefasciatus mosquito bites. Int J Dermatol. 1990;29:600–601
  11. Peng Z, Simons FER. Comparison of proteins, IgE and IgG binding antigens, and skin reactivity in commercial and laboratory-made mosquito extracts. Ann Allergy Asthma Immunol. 1996;77:371–376
  12. Christophers SR. Aedes aegypti (L.) the yellow fever mosquito: its life history, bionomics and structure. In: London: : Cambridge University Press; 1960;p. 739
  13. Knight KL, Stone A. A catalog of the mosquitoes of the world. In: 2nd ed. Washington (DC): : Entomology Society of America (Thomas Say Foundation); 1977;p. 611
  14. Wood DM, Dang PT, Ellis RA. The insects and arachnids of Canada. Part 6. The mosquitoes of Canada (Diptera: Culicidae). Agric Can Publ. 1979;1686:390
  15. Simons FER, McMillan JL, Simons KJ. A double-blind, single-dose, crossover comparison of cetirizine, terfenadine, loratadine, astemizole, and chlorpheniramine versus placebo: suppressive effects on histamine-induced wheals and flares during 24 hours in normal subjects. J Allergy Clin Immunol. 1990;86:540–547
  16. Boorman J. Induction of salivation in biting midges and mosquitoes, and demonstration of virus in the saliva of infected insects. Med Vet Entomol. 1987;1:211–214
  17. Peng Z, Li H, Simons FER. Allergens and antigens in extracts of Aedes vexans, Culiseta inornata, and Culex tarsalis. Int Arch Allergy Immunol. 1996;110:46–51
  18. James AA, Blackmer K, Marinotti O, Ghosn CR, Racioppi JV. Isolation and characterization of the gene expressing the major salivary gland protein of the female mosquito, Aedes aegypti. Mol Biochem Parasitol. 1991;44:245–254
  19. Brown A, Griffitts THD, Erwin S, Dyrenforth LY. Arthus's phenomenon from mosquito bites. S Med J. 1938;31:590–596
  20. Reunala T, Brummer-Korvenkontio H, Räsänen l, François G, Palosuo T. Passive transfer of cutaneous mosquito-bite hypersensitivity by IgE anti-saliva antibodies. J Allergy Clin Immunol. 1994;94:902–906
  21. Konishi E. Distribution of immunoglobulin G and E antibody levels to salivary gland extracts of Aedes albopictus (Diptera: Culicidae) in several age groups of a Japanese population. J Med Entomol. 1990;27:519–522
  22. Suzuki S, Negishi K, Tomizawa S, Shibaski M, Kuroume T, Matsumura T. A case of mosquito allergy. Acta Allergol. 1976;31:428–441
  23. Shen HD, Chen CC, Chang HN, Chang LY, Tu WC, Han SH. Human IgE and IgG antibodies to mosquito proteins detected by the immunoblot technique. Ann Allergy. 1989;63:143–146
  24. Wu CH, Lan JL. Immunoblot analysis of allergens in crude mosquito extracts. Int Arch Allergy Appl Immunol. 1989;90:271–273
  25. Roitt IM. Hypersensitivity. Essential immunology. In: 6th ed. Boston: : Blackwell Scientific Publications; 1988;p. 193–214
  26. Allen JR. Passive transfer between experimental animals of hypersensitivity to Aedes aegypti bites. Exp Parasitol. 1966;19:132–137
  27. Lemanske RF, Kaliner MA. Late phase allergic reactions. In:  Middleton E,  Reed CE,  Ellis EF,  Adkinson NF,  Yunginger JW,  Busse WW editor. Allergy: principles and practice. St. Louis: Mosby; 1993;p. 320–361
  28. Solley GO, Gleich GJ, Jordan RE, Schroeter AL. The late phase of the immediate wheal and flare skin reaction: its dependence upon IgE antibodies. J Clin Invest. 1976;58:408–420
  29. Hudson A, McKiel JA, West AS, Bourns TKR. Reactions to mosquito bites. Mosquito News. 1958;18:249–252
  30. Killby VA, Silverman PH. Hypersensitive reactions in man to specific mosquito bites. Am J Trop Med Hyg. 1967;16:374–380
  31. McKiel JA, West AS. Effects of repeated exposures of hypersensitive humans and laboratory rabbits to mosquito antigens. Can J Zool. 1961;39:597–603
  32. Panzani R, Blanca M, Sanchez F, Juarez C. Sensitivity to European wasps in a group of allergic patients in Marseille: preliminary results. J Investig Allergol Clin Immunol. 1994;4:42–46
  33. Puyana J, Diez-Gomez ML, Cuevas M, Quirce S, Fernandez-Rivas M, Hinojosa M. Stinging insect allergy: sensitization to vespids in Madrid and surroundings. Cross-reactivity study. Allergy. 1990;45:126–129
  34. Hudson A, Bowman L, Orr CWM. Effects of absence of saliva on blood feeding by mosquitoes. Science. 1960;131:1730–1731
  35. Penneys NS, Nayar JK, Bernstein H, Knight JW. Circulating antibody detection in human serum to mosquito salivary gland proteins by the avidin-biotin-peroxidase technique. J Am Acad Dermatol. 1988;18:87–92
  36. Penneys NS, Nayar JK, Bernstein H, Knight JW, Leonardi C. Mosquito salivary gland antigens identified by circulating human antibodies. Arch Dermatol. 1989;125:219–222

☆ From athe Section of Allergy and Clinical Immunology, Department of Pediatrics and Child Health and bDepartment of Immunology, Faculty of Medicine, University of Manitoba.

☆☆ Supported by the Children's Hospital Research Foundation, Winnipeg, Manitoba, Canada.

★ Reprint requests: Zhikang Peng, MD, MS746A-820 Sherbrook St., Winnipeg, Manitoba, Canada R3A 1R9.

★★ 1/1/82538

PII: S0091-6749(97)70224-0

The Journal of Allergy and Clinical Immunology
Volume 100, Issue 2 , Pages 192-198, August 1997

Article Tools

* Image Usage & Resolution