Abstract
A serological survey was conducted in Macao, China, in 753 individuals, with the objective of looking for antibodies to the mosquito, Aedes albopictus (Skuse 1894) (Diptera: Culicidae), and to dengue, before the occurrence of any autochthonous dengue cases. Blood samples were collected at several public health services, a questionnaire was answered, and enzyme-linked immunosorbant assay (ELISA) and Western blot techniques were performed with extracts of mosquito head and thorax (HT). Anti-Aedes albopictus IgG antibodies were present in titres 1:102-1:103 in 9%, and in titres 1:104-1:105 in 42% of the sera tested. This reactivity was more frequent (59%) in the population which had resided only in Macao in the 2 years previous to the survey, as opposed to those that had also resided in other areas (50%). From the 230 reactive sera selected for immunoblot, 48 (21%) reacted with a wide range of proteins from above 224 kDa to 21 kDa, with different patterns according to individual sera. Proteins in the intervals 35.3-28.7 kDa and 28.7-21.1 kDa were labelled by the greatest number of sera, 15 and 19 respectively. The presence of anti-Aedes albopictus antibodies presented a statistical relation to skin reaction to mosquito bites, but immunoblot patterns did not. Anti-dengue IgG antibodies were found in 48% of the subjects, with a higher proportion in people who had resided out of Macao, or who were nationals from dengue-endemic neighboring countries. Anti-dengue reactivity was in agreement with anti-mosquito reactivity in half of the population. It would be interesting to see if this proportion has changed since dengue became endemic in Macao in 2001.
Female mosquitoes need vertebrate blood for optimal development of their eggs (10), and in tropical climates are well-known vectors of many important diseases, such as dengue, malaria and yellow fever (37). Mosquito bites can cause cutaneous reactions that can be divided into five different stages ranging from immediate wealing and delayed bite papules to non-reactivity (16), and rarely anaphylaxis, Arthus-type reactions, or serum sickness-like syndromes, with systemic symptoms (6, 11, 12, 14). These reactions are considered to be provoked by an allergic response to mosquito saliva injected into the host tissue during the blood meal. Mosquito saliva, like that of many other haematophagous arthropods, not only carries essential proteins for the success of blood-feeding, such as vasodilators, inhibitors of the blood-coagulation cascade and inhibitors of platelet aggregation, but it also has a role in parasite transmission (41).
The injection of mosquito antigens induces antibody production, the presence of which has long been correlated with skin reactions (16, 24, 42). Anti-mosquito antibodies, both immunoglobulin (Ig) G and IgE classes have been demonstrated in sera from mosquito-allergic patients, and those with a history of reactivity to mosquito bites, and in human and animal populations exposed continuously to mosquito bites (4, 7, 19, 25, 29, 31, 32, 33, 35, 43).
Antigens recognized in extracts of whole mosquitoes ranged in molecular weight from 14 kDa to over 200 kDa. Human sera recognized a common antigen of 62 kDa in the salivary glands of Aedes, Culex and Anopheles mosquitoes (32). In head/thorax extracts and saliva of Aedes communis, a 21.5 kDa antigen was recognized by IgG from rabbits and humans. In humans, this antigen was recognized by 50% of adults and children, but not by infants (7). A 36 kDa antigen was recognized by IgE and IgG4 from adults (8). Aedes albopictus antigens of several molecular masses have been reported to be recognized by immunoglobulins from patients allergic to mosquito bites (19, 25, 29, 38, 39, 43).
Macao, situated in south-eastern China, borders endemic areas for dengue viruses but did not register transmission of this virus until 2001 (21, 22), despite harbouring Aedes albopictus (Skuse 1894), one of the most competent vectors, and ferocious biters, with particularly high human-biting rates (314 bites per person per day in the wet season) (3). Therefore, it was our objective in this study to analyze anti-mosquito reactivity in the human population exposed to such high biting rates, determine antibody IgG titres, analyze antigen reactivity profiles, as well as compare these with mosquito bite reactions, in order to look for possible associations. As this study was conducted in 1997 and was prior to the registry of any dengue cases in Macao, an epidemiological survey was carried out. It consisted of a questionnaire and a seroprevalence study for dengue in order to understand the immunity status of the population, considering that (i) dengue cases had not been reported until then, despite the proximity of this territory to dengue-endemic areas, (ii) there was a large migrant population from countries were dengue was endemic and (iii) there has been an increase in travelling brought about by the construction of an international airport in 1996. Another objective was to analyse whether the serological reactivity to dengue and to mosquitoes were in any way related.
Materials and Methods
Human sera. Human sera were collected from 753 individuals from Macao's population, aged between 16 and 97 years. Five hundred and twenty-eight sera were from males and 225 were from females, collected at several public health services, namely: Primary Care Health Centres (PCHCs), Blood Transfusion Centres (BTCs), and the Laboratory for Public Health (LPH), during the month of September 1997. Donors were users for routine blood tests or blood donors. The population from the LPH were mainly foreigners from neighbouring countries, working in Macao for variable periods of time. Informed consent was obtained, and a questionnaire was performed addressing factors such as country of origin and residence for the previous two years, clinical history of dengue and of mosquito bite reactions. Sera were stored at −80°C until tested. Procedures were performed according to the regulations of the Ethics Committee of the Instituto de Higiene e Medicina Tropical (Institute for Hygiene and Tropical Medicine) and the named health centres of Macao.
Mosquito extracts. Aedes albopictus mosquitoes of both sexes were originally from a colony from the Center for Diseases Control and Prevention, USA, courtesy of the late Dr C. Mitchell. Mosquitoes were killed and sent to our laboratory frozen, from which head and thorax (HT) extracts were prepared. Wings and legs were removed from female mosquitoes then HTs were separated from the remaining carcass and kept in isotonic medium (100 mM Tris pH 7.2, 300 mM mannitol) with protease inhibitors and at −80°C.
HTs were homogenized at 4°C in a glass-glass homogenizer. Homogenates were sonicated (Vibra Cell, Newtown, CT, USA) on ice twice for 30 s at 6 μm, and then centrifuged at 3000 ×g for 10 min. Protein concentration of the extracts was measured by the Bio-Rad™ (Hemel Hempstead, Hertfordshire, UK) protein assay, based on Bradford dye-binding procedure, yielding 1 mg/ml.
Production of anti-mosquito immune serum. Three female CD-1 mice were each injected intraperitoneally with 500 μg protein of HT extract per mouse, emulsified in an equal volume of Freund's complete (first immunization) or incomplete (subsequent boosts) adjuvant. Three immunizations were performed at 15-day intervals. Control mice were inoculated with adjuvant and homogenization medium. Before the first immunization, blood samples were obtained through tail bleeding and after the last immunization by cardiac puncture. Blood was centrifuged at 1000 ×g for 10 min at 4°C, and sera were stored at −80°C until used. Procedures were performed according to the animal experimentation regulations of the Ethics Committee of the Instituto de Higiene e Medicina Tropical (Institute for Hygiene and Tropical Medicine).
Anti-mosquito ELISA. Indirect enzyme-linked immunosorbant assay (ELISA) against mosquito HT extract was used to detect the presence of anti-mosquito antibodies. Extracts were diluted in bicarbonate buffer (0.015 M NaCO3, 0.035 M NaHCO3, pH 9.6) and coated onto PVC microtitre (Titertek-Merck, Darmstadt, Germany) plates at 500 ng protein per well overnight at 4°C, as a standardized concentration in earlier assays (2). Plates were blocked with 10% (w/v) dried skimmed milk (DSM) in phosphate-buffered saline (PBS) at room temperature (RT) for 2 h. Plates were rinsed with PBS-Tween 20 (PBS-T) and used immediately or stored at −80°C. Sera, serially diluted in PBS-T, 50 μl/ well, were incubated for 1 h at RT. Plates were washed 3×5 min in PBS-T and binding was detected using horseradish peroxidase (HRP)-conjugated goat anti-human IgG or HRP-conjugated anti-mouse IgG, diluted 1:1000 in PBS-T. Absorbance at 492 nm was read in an Anthos 2010 microplate reader (Biochrom Ltd., Cambridge, UK). Sera were serially diluted and used to determine the antibodies titre, against equivalent dilutions of standard human immunoglobulin G as negative control (Human IgG; Sigma-Aldrich Company Ltd., Gillingham, Dorset, UK), all used in triplicate replicas. Positivity cut-offs were determined for each dilution as two standard deviations above the mean of the negative controls (9).
Anti-mosquito Western blot. Mosquito extracts prepared as above were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a discontinuous buffer system (20) on 5-20% gradient gels, using a BioRad Mini-PROTEAN II apparatus. Samples were mixed (1:1) with sample buffer (80 mM Tris pH 6.8, 2% SDS, 20% glycerol, 0.24% bromophenol blue), containing 100 mM β-mercaptoetanol as reducing agent. Samples were heated at 100°C for 5 min, and loaded on to the gels at a concentration of 5 μg protein per well as previously standardized (2). For Western blotting, proteins were transferred from gels onto a 0.45 μm pore size nitrocellulose membrane, in a semidry system after which non-specific binding was blocked with 10 % (w/v) DSM in PBS overnight, 4°C. Human sera were diluted 1:100 in 1% (w/v) DSM in PBS; standard human immunoglobulin G was used as negative control (Human IgG, Sigma), and immune mouse serum was used as a positive control in the same dilution. Strips were washed 6 times, 5-10 min each in 0.5% (w/v) DSM in PBS-T with constant agitation at RT. HRP-conjugated goat anti-human IgG and HRP-conjugated goat anti-mouse IgG diluted 1:1000 in 1% (w/v) DSM in PBS was incubated for 90 min at RT, then strips were washed as above. 4-Chloronaphtol (0.3 mg/ml) in PBS, with fresh 30 % (v/v) H2O2 (10 μl/ml) was added and after 45 min the reaction was stopped in running water.
Immunoblot analysis of head and thorax (HT) extract of Aedes albopictus with human sera from Macao population, negative control human standard immunoglobulin G (NC) and positive control imunized mice sera (PC). Mosquito antigens were separated by 5-20% SDS-PAGE under reducing conditions, transferred to nitrocellulose membranes and probed with sera (respective numbers underneath) diluted 1:100.
Anti-dengue Western blot. Anti-dengue antibodies were detected using a commercial kit, Dengue Blot® (Genelabs Diagnostics, Singapore), which detects IgG antibodies against dengue viruses types 1, 2, 3 and 4 in the sera being studied, according to the instructions of the manufacturers.
Statistical analysis. Data from questionnaires, and tests performed were analyzed using the statistical packages Nanostat (London School of Hygiene and Tropical Medicine, University of London, UK) and SPSS 14 for Windows (SPSS, Chicago, IL, USA). Chi-squared tests were performed for independence of different variables, and McNemar's chi-squared tests were performed for paired variables (18).
Results
In total, 753 sera were collected from the human population from Macao. However, not all had the corresponding fully answered questionnaire, reducing the effective sample size which varied according to the parameters being analysed.
Anti-mosquito reactivity. Sera from the study population did not contain anti-Aedes albopictus antibodies against HT extract in 49% of the cases (372/753), presented anti-mosquito antibody titres 1:102-1:103 in 9% (68/753), and exhibited antibody titres 1:104-1:105 in 42% of the sera (313/753). Positive control mice immunized with HT Aedes albopictus extracts presented titres ≥1:104.
As to the area of residence in the two years previous to the survey, the population which had resided only in Macao had a higher percentage of anti-mosquito positivity at 59% (309/521) than the population which had resided in other areas too, 50% (117/232) (χ2=4.79 (1 degree of freedom (df)), p=0.0285). Likewise, anti-Aedes albopictus reactivity was more frequent in the sera collected in the PCHC and BTC (63%), than in the sera collected in the LPH (43%) (χ2=28.17 (1 df) p<0.0001).
A random sub-sample of the sera, with endpoint titre ≥1:104 against mosquito extracts by ELISA, i.e. 230 sera, were assayed by Western blot against Aedes albopictus HT extracts. Labelling of protein bands was detected in 48 sera (21%), from above 224 kDa to 21 kDa, with varying patterns of reactivity (Table I and Figure 1). Proteins in the intervals 35.3-28.7 kDa and 28.7-21.1 kDa were labelled by the greatest number of sera, namely 15 and 19 respectively.
Reactivity to mosquito bites and anti-mosquito antibodies. Four hundred questionnaires were completed for the data on reactivity to mosquito bites. Forty-six percent (183/400) of people stated they suffered a reaction to mosquito bites. Anti-mosquito antibodies were found in more individuals who reacted to mosquito bites, 73% (134/183), than in individuals who denied reacting to mosquito bites, 47% (101/217), (χ2=28.07 (1 df) p<0.0001). However, no differences were observed between the immunoblot pattern of sera from people who referred no reaction to mosquito bites, small (<10 mm) or large (>20 mm) wheal or papule size, as antigens of identical molecular masses were recognized.
Anti-dengue reactivity of sera. From the total 753 sera collected in the territory of Macao in 1997, prior to any dengue case occurrence, anti-dengue IgG 121 dot blot tests gave inconclusive results, reducing the eligible sample to 632 sera. Anti-dengue antibodies were detected in 306 (48%), and were not detected in 326 (52%). The presence of anti-dengue IgG antibodies was more frequent in samples from the users of the LPH (161/252, 64%), than in those of the HC and BTC (145/380, 38%), (χ2=39.14 (1 df) p<0.0001).
The proportion of subjects reactive for anti-dengue was larger (147/200, 74%) in people who had resided out of Macao for some time during the two years previous to the survey than in the population which had resided only in Macao (159/432, 37%) (χ2=72.24 (1 df) p<0.0001). In fact, anti-dengue antibodies were more common in people from neighbouring countries where dengue is endemic, such as, Thailand (128/167, 77%), Cambodia (11/14, 79%), Vietnam (13/18, 72%), and the Philippines (12/13, 92%).
Half of the sera positive for anti-dengue were also positive for anti-mosquito antibodies (164/306, 54%), and half of the sera negative for anti-dengue were also negative for anti-mosquito (179/326, 55%), revealing some concordance between the presence of anti-dengue and anti-mosquito antibodies (χ2=6.5 (1 df) p=0.0108).
Discussion
A total of 753 sera from the human population of the territory of Macao, China, were analysed for the presence of anti-mosquito Aedes albopictus antibodies, which were found in 51%. Furthermore, high titre anti-Aedes albopictus antibodies were more common in the population which had resided only in Macao in the two years previously to the survey (59% versus 50%), as well as in the population from the local health centres (63% positive), than in the users from the LPH (43% positive), whose donors were mainly foreigners from neighbouring countries (96%). This high anti-mosquito reactivity in local population is understandable as Aedes albopictus in Macao had, at the time, a high human biting rate which reached 314 mosquito bites per person per day in the wet season (3). Antibodies against this mosquito have also been found in subjects from eastern USA and south east Asian countries where it abounds (15), such as Taiwan, Japan, and China, (19, 29, 39). The fact that half of the tested human sera were negative (49%) may be due to the dilution effect of the foreign population, non-responders, or possible desensitization with low antibody titres, as previously observed elsewhere (1). On the other hand, the high human biting rates measured in Macao were in a rural setting (Seak Pai Van natural park) where mosquito density was higher than in the city of Macao, and therefore not all of the individuals might have been exposed to such high biting rates. However, it is also possible that this result was due to the low amount of salivary proteins in the crude HT extracts used for the ELISA assays.
Reactivity to HT extracts is natural since the salivary glands and secretions, to which people bitten by mosquitoes are bound to get an immune response, are situated in the thorax. In fact, most authors detect antibodies to salivary components in mosquito-allergic patients (7, 8, 11, 17, 19, 29, 30, 31), although Shen et al. (39) found antibodies against Aedes albopictus whole-body extract in several individuals, but only one had antibodies against salivary gland extracts.
Comparing the methods of analysis of anti-mosquito reactivity, there was no concordance between the reactivity of sera by ELISA and immunoblot methods. A smaller proportion (21%) of the highly reactive sera were also positive by immunoblot (McNemar χ2=131 (1df), p<0.0001). Antigens used for ELISA were closer to the natural proteins to which individuals are exposed during mosquito biting than those used in immunoblots, where the antigens have been denatured and reduced, thus potentially preventing the recognition of conformational epitopes.
Salivary gland extracts would have been a better antigen to use in our assays. However, we did not have the logistics to breed Aedes albopictus in our insectary due to security considerations, and therefore frozen specimens from USA were used, preventing the preparation of such extracts. This is a limitation of our study, as salivary gland proteins will be present in small quantities in HT extracts, preventing their proper recognition. Furthermore, protein degradation inherent to the freezing and thawing procedure has likely reduced their content, and together could have been the justification for the weak reactivity evident in the immunoblots. Aedes aegypti recombinant salivary proteins were identified only after our study had been conducted (30, 31), bringing clear advantages to the diagnosis of anti-mosquito immune responses.
The patterns of reactivity in immunoblot ranged from non-reactivity to various combinations of bands. Reactivity with proteins of molecular mass of 68 kDa, and in the range of 30 and 27 kDa have been identified in salivary gland extracts of Aedes albopictus by sera of subjects allergic to mosquito bites (29). Although anti-mosquito antibodies were more common in people who referred reaction to mosquito bites, no differences were observed between the immunoblot patterns of people reacting to mosquito bites and those not. Serum IgE antibodies directed against salivary proteins are the markers for mosquito allergy and exist in very small amounts (25, 29, 31, 43). This would preclude their detection with our assays as we used HT extracts likely containing very small amounts of salivary proteins. In fact, both ELISA and immunoblot with a sample of 35 highly positive sera gave negative results for IgE antibodies (results not shown). Therefore, we did not reach a conclusion to whether there were people allergic to mosquito bites in our survey. However, IgG antibodies have also been found in those allergic to mosquito bites, and in some cases to Aedes albopictus (11, 19, 26, 27, 28, 32, 34, 35, 38, 39). The fact that the studied population did not include young children might have been another reason for these results, as it is known that the intensity of the IgG response to mosquito salivary proteins decreases with age and with exposure to mosquito bites. Measurement of anti-mosquito IgG4 might have been informative as it has been found in sensitised individuals in other studies (7, 8).
Reactivity pattern of 48 sera in immunoblot against Aedes albopictus HT extract*.
Several proteins of molecular masses similar to the ones detected in this study have previously been described. Proteins with relative molecular masses between 70-60 kDa, such as labelled by these sera, have been detected in the saliva of Aedes albopictus, an α-glucosidase (67 kDa) and apyrase (61 kDa) (23), a 68 kDa allergen (29), and a 68 kDa salivary apyrase in Aedes aegypti (36). A 40 kDa protein was labelled by 10 sera and similarly, a 43 kDa protein of Culex pipiens was labelled by IgG from a patient with serum sickness-like syndrome to mosquito bite (11). Antigens of 32.5, 40, and 50 kDa from Aedes vexans have been detected by IgE and IgG antibodies from individuals with severe skin reactions (28). A 30 kDa protein from Culex pipiens has been detected by IgE antibodies from a patient with systemic anaphylaxis to mosquito bites (12). Most sera labelled a 22 kDa protein, and similarly in the extracts of head and thorax or whole-body of Aedes communis, a protein of 21.5-22 kDa was detected and considered the most immunogenic protein in their saliva (7, 8, 34, 35). A 22 kDa antigen was also identified in whole-body extract of C. pipiens by IgE antibodies of a patient with serum sickness-like syndrome in reaction to mosquito bites (11). Obviously, similarities in molecular mass may have no significance whatsoever as to the identity of the proteins being recognized. However, this could be an indicator of protein similarity, as species-shared antigens have been documented (29). As a matter of fact, several other mosquito species with a highly aggressive behaviour were detected in Macau at the time of our survey, namely Aedes (Stegomyia) w-albus (Theobald, 1905), Armigeres (Armigeres) subalbatus (Coquillett, 1898), Armigeres (Leicesteria) magnus (Theobald, 1908), Coquillettidia (Coquillettidia) crassipes Van der Wulp, 1881, Culex (Culex) bitaeniorhynchus Giles, 1901, Culex (Culex) quinquefasciatus Say, 1823, Culex (Culex) sitiens Wiedemann, 1828, Culex (Culex) tritaeniorhynchus Giles, 1901 (3), which could account for some antigenic cross-reactivity.
Over half (63%) of the population which had resided only in Macao for the two years previous to the survey was non-reactive to dengue virus, in agreement with the lack of register of dengue cases previous to 2001 (21, 22). This was reflected in that the users from the local health centres and blood transfusion centre, mainly the resident population from Macao, were mostly negative for dengue antibodies. However, the users from the LPH, the majority of which (96%) were migrants from neighbouring south east Asian countries where dengue is endemic, and which had at least for some time resided out of Macao in the two years previous to the survey, had the highest dengue positivity rate. Such was the case of the population original from Thailand, Cambodia, Vietnam, and Philippines, which had a markedly higher anti-dengue reactivity than those from other origins such as Macao and mainland China.
Half of the population positive for anti-dengue antibodies were also anti-mosquito positive. It is only natural that dengue, being a mosquito-borne disease, is positively correlated with anti-mosquito antibodies in endemic areas. Anti-Anopheles saliva antibodies have been positively correlated with the risk of developing malaria in children in an endemic area of Senegal (33). However, it can be argued that the presence of anti-mosquito antibodies depends on repeated exposure to mosquito bites, and that theoretically a single infective bite can be sufficient for dengue viral transmission (13). This survey was prior to any autochthonous dengue cases in Macao, therefore, it would be interesting to see if the proportion of anti-dengue and anti-mosquito positivity in the population, and their correlation, has changed since dengue became endemic after the 2001 outbreak (40).
Acknowledgements
This work was partially financed by the Project Grant “Aedes albopictus in Macao: Systematics, molecular biology and receptivity of the Territory to dengue and other arboviroses” n° PRAXIS XXI/2/2.1/SAL/1388/95, Coordinated by H. Ribeiro, and also UPMM, FCT, Portugal and FEDER. We thank the authorities in Macao that cooperated with us, namely the Health Centers, the Laboratory of Public Health, the Saint January Hospital, Municipality of the Islands. We are also indebted to Fundação Oriente and Fundação Macau, Lisbon, Portugal.
We thank all the members of the team that participated in this work, particularly the late Dr Carl Mitchell (CDC). For technical assistance we thank M. L. Anselmo, L. Pinto, R. Merelo-Lobo, S. Silva. We acknowledge Dr P. Billingsley for the critical review of the manuscript and suggestions.
Footnotes
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↵† Present address: Isabel Lopes de Carvalho, Centro de Estudos de Vectores e Doenças Infecciosas/INSA (Centre for the Study of Vectors and Infectious Diseases, National Health Institute Doutor Ricardo Jorge), Av. Padre Cruz, 1649-016 Lisboa, Portugal.
- Received February 4, 2011.
- Revision received March 8, 2011.
- Accepted March 10, 2011.
- Copyright © 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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