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    Figure 1.

    Top view of hut box, lid open. Double-headed arrow shows the position of the wire grate separating chickens’ rooms. The 1.5-cm wire grates are also seen on the floor. Triatomines set free in the box can move from one room to another and hide safely in dwellings inaccessible to chicken beaks.

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    Figure 2.

    Natural immunization by repeated exposure of chickens to T. infestans bites shortens the insect’s probing time. A 40-min exposure was required for 90% of the insects to obtain their fill from non-immune chickens; however, a 15-min exposure was required for 50% of the insects to obtain their fill and to become engorged with blood from chickens that had undergone natural immunization with saliva injected through the insects’ bites.

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    Figure 3.

    Effect of immunization by repeated exposures to insect bites or by injections of crude saliva antigens in chickens. IgG antibody titers in 1:200 serum dilutions were detected by ELISA assays (see Materials and Methods). After the third immunizing procedure, antibodies in the serum of chickens that received saliva injections were approximately twofold higher than in the serum of chickens that had been immunized through insect bites. Each point represents the mean ± SD of six repeated experiments.

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    Figure 4.

    Profiles of IgG anti-saliva antibody titers detected by ELISA in immune and in non-immune, control chickens. At serum dilutions from 1:100 to 1:1600, the immune chickens, regardless of the route of immunization, showed significantly higher specific IgG antibodies titers (P < 0.001) than the control, non-immune birds. The bars indicate the mean ± SD of six repeated experiments.

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    Figure 5.

    Detection of T. infestans saliva protein bands with chicken serum IgG. Lanes 1 and 5, bands formed with sera from chickens immunized with crude saliva antigens injected in Loubel-Rouge and in congenic B12/B12 chickens, respectively; lanes 2 and 4, bands formed with heterophile antibodies from a non-immune Loubel-Rouge serum (note the 100-kDa band present in each lane); lane 3, bands formed with serum antibodies of a Loubel-Rouge chicken immunized by exposure to T. infestans bites; lane 6, control non-immune B12/B12 chicken serum.

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    Figure 6.

    Blood source detection. The immune chicken had fluorescein-labeled (apple green) blood leukocytes and the non-immune chicken had rhodamine-labeled (red) leukocytes. Leukocytes (A) in the immune chicken’s blood smears fluoresce under light stimuli (B), and leukocytes from the gut (C) of an insect that fed upon that chicken also stain apple-green (D). Similarly, leukocytes in the non-immune chicken’s blood smears (E) stain red (F), and leukocytes in the gut (G) of an insect that fed upon that chicken also stain red (H). This method allowed identification of the chicken that T. infestans had preyed upon. This figure appears in color at www.ajtmh.org. which accelerated growth of F1 and F2 progeny, appeared evident in the next generation.

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    Figure 7.

    Immune status influence upon T. infestans life cycle. (A) Survival ratios of triatomines feeding upon immune and non-immune chickens were similar in the three generations observed. (B) Fertility ratios observed in the three generations were not statistically significant (see text). (C) Fecundity measured by the ratios of eggs to hatching nymphs was also similar. (D) Length of time required for nymph (F1 and F2) development was 40 days shorter when the bugs fed upon the immune chicken as compared with the time required for development and molting of nymphs fed upon non-immune chickens (F1, P < 0.01; F2, P < 0.05).

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    Figure 8.

    Effect of a chicken immune status toward elicitation of delayed-type skin reactivity against T. infestans saliva proteins. (A, B) Congenic chicken normal skin 72 h after injection of 60 μg of saliva in 100 μL of 0.15 M saline solution. (C) Indurate skin lesion with central necrotic area is seen in an immune congenic chicken challenged with the same dose of saliva antigen. (D) Intense inflammatory infiltrates and dilated blood vessels in the dermis (arrows). This figure appears in color at www.ajtmh.org.

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TRIATOMA INFESTANS CHOOSES TO FEED UPON IMMUNE PREY

MARIANA M. HECHTChagas Disease Multidisciplinary Research Laboratory, Faculty of Medicine, University of Brasília, Federal District, Brazil

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ANA CAROLINA BUSSACOSChagas Disease Multidisciplinary Research Laboratory, Faculty of Medicine, University of Brasília, Federal District, Brazil

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SILENE P. LOZZIChagas Disease Multidisciplinary Research Laboratory, Faculty of Medicine, University of Brasília, Federal District, Brazil

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JAIME M. SANTANAChagas Disease Multidisciplinary Research Laboratory, Faculty of Medicine, University of Brasília, Federal District, Brazil

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ANTONIO R.L. TEIXEIRAChagas Disease Multidisciplinary Research Laboratory, Faculty of Medicine, University of Brasília, Federal District, Brazil

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Blood-feeding Triatoma infestans obtained its fills from immune chickens in 15 min, but it needed 40 min for feeding upon non-immune chickens. High-titer specific IgGs and skin reactivity against T. infestans saliva antigens were elicited in immune chickens. Fluorescence-labeled leukocytes from non-immune or immune chickens were used to determine sources of blood drawn by equal numbers of triatomines distributed in separate compartments of a hut-like box. It was shown that 64.4 ± 4.7% of the reduviids were captured in the immune chicken room; 35.6 ± 4.5% were present in the non-immune chicken dwelling, and these differences were statistically significant (P < 0.001). Furthermore, T. infestans feeding upon immune birds reached the adult stage 40 days before those feeding upon non-immune birds, and differences were statistically significant. These results appear to have a broad epidemiologic significance as for spreading enzootics; hence, the immunologic status of vertebrate host populations appears to favor T. infestans as the main transmitter of Trypanosoma cruzi.

INTRODUCTION

Strictly hematophagous reduviids, true bugs belonging to the subfamily Triatominae, are the main vectors of the protozoan Trypanosoma cruzi, the agent of Chagas disease.1 The two main tribes of triatomines are Rhodniini and Triatomini, which are respectively involved in silvatic and peridomestic cycles of transmission of the disease.2 Whereas Rhodniini are involved in transmission of T. cruzi in the major humid tropical forests, Triatomini dwells in dry shrub savanna and cerrado ecosystems. In these ecosystems, Triatoma infestans developed anthropophylia and colonized peridomiciles and domiciles, and it has become a main transmitter of T. cruzi infections.3 T. infestans has been considered responsible for the transmission of approximately 10 million human cases of Chagas disease mainly in South America.4

The difficulties of achieving effective immunoprophylaxis of T. cruzi infections with currently available biotechnologies are double-fold. Firstly, full natural immunization, present in immune-competent hosts showing both innate and acquired responses, leads to a chronic, life-long, persisting infection.4 However, the cryptic infection can burst out when chagasic hosts undergo immunosuppressant therapy or immunodeficiency stemming from infectious or a proliferative disease. Secondly, acquired humoral and cellular immune mechanisms that curtail T. cruzi infections cannot be separated from an auto-immune component of Chagas disease pathology.5,6 In the absence of a conventional vaccine to prevent Chagas disease, some approaches have been considered, aiming at preventing transmission of T. cruzi infections.

Some investigations have suggested the possibility to prevent the transmission of T. cruzi infections through the insect bite.710 The saliva of blood-sucking cone-nosed triatomine has a large number of active bioamines, which have made it capable of circumventing the hemostatic mechanisms of its prey.11 A main group of proteins involved in the insect’s feeding capability are anti-platelet aggregators, anti-coagulants, vasodilators, and anesthetics. These proteins comprise the insect’s saliva cocktail injected in a host during blood feeding.12 Repeated inoculations of a gamut of saliva proteins in the body of individuals living in endemic areas for Chagas disease could be a potent natural immunizing procedure. In fact, specific reactions associate the saliva antigens of the triatomine with specific humoral antibodies and cell-mediated immune responses in individuals subjected to insect bites.1318 These observations raise questions on whether the immune status of a host interferes with the triatomine’s feeding pattern. Certainly, interference with the insect’s feeding pattern would suggest a role for natural or artificial immunization in the dynamics of T. cruzi infections transmission.

In this work, we immunized poultry with proteins from the saliva of T. infestans. Specific immune responses, which were detected and characterized, mounted by chickens against the saliva proteins established parameters for evaluating the effect of these procedures in the insect’s life cycle. Herein we describe the use of different fluorochromes to separately label immune and non-immune chickens and the notable preference of T. infestans toward feeding upon a fully immunized host.

MATERIALS AND METHODS

Chickens and triatomines.

Six- to 10-month-old Loubel-Rouge chickens weighing 1.8 ± 0.3 kg were purchased from a local breeder and maintained in an open-air pen next to our laboratory. Additionally, we performed immunizing experiments and skin testing in 6-month-old congenic B12/B12 chickens, a gift from Jiri Plachy, Czeck Academy of Sciences, Prague. This specific pathogen-free chicken lineage has been raised indoors, in rooms with positive pressure filtered air and constant temperature of 26°C, without access of any kind of hematophagous insects. Maintenance and care of experimental animals followed institutional guidelines for the humane use of laboratory animals.

T. infestans of different instars were raised in an insectary at the Chagas Disease Multidisciplinary Research Laboratory. This colony, which was derived from wild bugs captured in the Central State of Goiás, Brazil, has been raised in captivity for the last 12 years. The insects are maintained in a closet within a tightly closed room, with three levels of barriers created to avoid dispersion. The insectary is kept at 28°C, 70% relative humidity, with a day/night cycle of 12 hours. T. infestans saliva spontaneously ejected from the insect’s proboscis upon contact with the researcher’s finger was immediately collected (2 μL) with a 20-μL pipette tip. Pools of saliva from 50 triatomines were stored at −80°C until use.

Immunization of chickens.

Loubel-Rouge chickens were immunized with triatomines’ crude saliva by two different routes. One group of 6 chickens was immunized by natural exposure in 9 different occasions, 3 weeks apart, each consisting of 80 adult T. infestans feeding upon chickens for 20 minutes. The other group of 6 chickens received 600 μg of saliva proteins injected subcutaneously at 15-day intervals. The first injection was made with the saliva in complete Freund’s adjuvant, and the second and third injections had the saliva in incomplete adjuvant. Each chicken received 6 subsequent immunizing injections of crude saliva alone. Because the Loubel-Rouge chickens raised in the open-pen were exposed to hematophagous insect bites, the congenic B12/B12 chickens raised in rooms inaccessible to insect bites were immunized; two chickens received the same dose of T. infestans saliva antigens with the frequency and routes described for outbred chickens.

Serum samples.

Venous blood (5 mL) was drawn from a wing vein of the chickens 1 week before the first immunization, 1 week after the third immunization, and thereafter at regular intervals of 30 days. The collected blood was allowed to coagulate at room temperature for 30 minutes. The serum samples were collected after centrifugation at 200g for 6 minutes. The serum was mixed with glycerol (1:1 v/v) and stored at −20°C.

ELISA assay.

This assay was performed as described.18 Briefly, 96-well plates sensitized with 50 μL of saliva antigen suspension (0.1 μg of protein per well) in 50 mM sodium carbonate buffer, pH 9.6, were incubated overnight at 4°C. The antigen excess was discarded, and plates were washed three times with PBS containing Tween 0.05% (v/v), pH 7.4. The plate wells were then blocked with 100 μL of a PBS-Tween solution to which was added defatted milk to a final 2% (w/v) concentration. After 1 hour of incubation at room temperature, plates receiving three additional washes were stored at −20°C until use.

Specific antibodies were detected in serum dilutions after incubation with saliva antigens in the wells for 2 hours at room temperature. At the end of incubation, the washes with PBS/Tween solution were repeated three times. Plates were air dried before the second antibody was added to the wells. It consisted of the addition of 50 μL of a 1:100 dilution of alkaline phosphatase-conjugated rabbit anti-chicken IgG antibody (Sigma). After incubation for 90 minutes at 37°C, the substrate p-nitrophenyl phosphate (Sigma) was added and optical densities (OD) were then read at 630 nm. Test and control sera assays were run in triplicate, and the OD results represent the means ± standard deviation.

Immunoblot.

A total 170 μg of crude saliva was dispensed in a well (5 × 0.8 × 0.1 cm, length × height × width) and proteins were resolved in a 10% SDS-PAGE. The protein bands were then transferred onto a nitrocellulose membrane, which was blocked and cut in strips 3 mm wide.18 Each strip was then separately incubated with control and with test serum samples (1:400 dilutions) for 90 minutes at room temperature. Next, each strip was incubated with a 1:1000 dilution of alkaline phosphatase-conjugated, rabbit anti-chicken IgG antibody (Sigma) for 90 minutes. The reaction was detected using the substrate nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma).

Assessment of prey’s immune status upon bug’s feeding pattern.

Fifty T. infestans fifth instars, which had been fasting for 20 days, were permitted to feed upon chickens that had never been in contact with triatomines. Two groups of three experimental birds were used. In one group, triatomines fed upon chickens for 15 minutes. In the second group, triatomines fed upon chickens for 40 minutes. The procedure with each experimental group of chickens was repeated every 3 weeks on nine occasions.

Influence of preying upon immune and non-immune chickens on the bug’s life cycle.

In these experiments, three groups of immune chickens were used. Group I consisted of 6 chickens fully and naturally immunized, preyed upon in five different occasions 1 month apart with the saliva ejected from glands of 50 adult T. infestans. Group II consisted of 6 chickens immunized with three subcutaneous injections of 600 μg of crude saliva proteins mixed with complete (first injection) and incomplete (second and third injections) Freund’s adjuvant. Each chicken received two additional injections of the same quantity of crude saliva in PBS, pH 7.4. In Group III, control non-immune chickens were preyed upon by triatomines. Assessment of fecundity, fertility, and period of time required for nymph development was carried out in three generations of T. infestans for a 42-month period. During this period of observation, each experimental group of triatomines was inspected, the insects in each nymph stage were counted, and rates of survival, fecundity, fertility, and time required for nymph development were recorded.

Immune status of chicken and feeding preference of T. infestans.

These experiments were conducted in a hut-like box measuring 60 × 62 × 84 cm, with safety features to prevent insect evasion similar to those described previously.19 The interior of a box was divided by a 2 × 2 cm iron mesh grate in two rooms that separated the chickens but allowed the insects free-transit (Figure 1). Hidden-away dwellings were slotted on the walls of each compartment such that the triatomines could rest after blood meals and not be reached by the chickens’ beaks. The immune and the non-immune, control chickens were each placed in one compartment. Then, fifth instars T. infestans were introduced into the box, 50 in each room. All night long, the triatomines had to avoid being devoured by the chickens; they could leave their lodgings just long enough to feed upon sleeping chickens and then return immediately to their hiding places. The next morning, we counted how many triatomines were hiding away in the immune and in the control, non-immune chickens’ compartments. In these experiments, we used chickens that had been immunized naturally and artificially, as described above. Each pair of chickens was used three times, one week apart.

Use of fluorochromes to determine source of chicken’s blood.

The results of preliminary experiments showing significant differences in the distribution of triatomines after feeding were confirmed with specific fluorochromes. This consisted of injection of 5.5 × 10−6 M of a red (PKH26) or a green (PKH67) fluorescent dye (Sigma) in a wing vein of immune and of non-immune chickens, respectively. Fluorescein-labeled chicken blood cells were placed in the hut-like box, and experiments were carried out and repeated as described. Each triatomine bug that had fed upon chicken’s blood then had its gut content microscopically examined in a glass smear under a fluorescent light microscope with emission filters of wavelength 567 and 502 nm, respectively, to detect red and green fluorescence-labeled cells.

Intradermal skin testing.

Delayed-type hypersensitivity to saliva antigens was detected by intradermal injection of 50 μg of crude saliva proteins diluted in 100 μL of 0.15 M NaCl. The injection sites were inspected at 24, 48, and 72 hours. The lesions were surgically biopsied, and tissues placed in Bouin fixative were embedded in paraffin. Five-micron-thick sections were stained by hematoxylin and eosin and examined microscopically.

Statistical analysis.

Student’s t test was used to data group analyses. The variance test of two values with repeat measures was used to analyze data obtained in the hut-box experiment. The P values ≤ 0.05 were considered statistically significant.

RESULTS

Influence of feeding frequency upon T. infestans probing time.

To determine the dynamics of T. infestans feeding upon birds, we used two groups of three chickens that were equally preyed upon by 50 fifth instar bugs. in Group I, triatomines were allowed to feed for 15 minutes, but in Group II they preyed upon chickens for 40 minutes. We used insects at fifth instars because they obtain their fills and engorge quickly, which made it easy to count the bugs. After set times, engorged triatomines in each experimental group were counted. Figure 2 illustrates results obtained when chickens were kept immobilized while triatomines retained in feeding boxes were brought into immediate contact with the skin region under the birds’ wings. It was seen that triatomines did not feed during the 5 min they were allowed to prey, although they inserted the proboscis in the chicken skin. However, if they were allowed to probe during the 40 minutes exposure, they were successful to obtain blood fills from immobilized chickens. Interestingly, after the third occasion that triatomines were exposed to chicken skin for 15 minutes, 50% of them became engorged with blood. It appears therefore that previous exposures to triatomine’s saliva resulted in shortening the probing time for feeding upon chickens.

Detection of immune responses against insect’s saliva antigens.

Specific antibodies to T. infestans saliva proteins were detected by ELISA in the serum of chickens that had been naturally or artificially immunized (Figure 3). The levels of serum antibodies in chickens that had been immunized with crude saliva were usually much higher than those observed in the control, non-immune chickens. Usually after the third immunization, differences in antibody titers were seen in many-fold serum dilutions (1:200), which were statistically significant (P < 0.01). Interestingly, antibody titers were higher in the serum of artificially immunized chickens as compared with those in the serum of naturally immunized chickens, and these differences were also statistically significant (P < 0.05). Figure 3 also shows that antibody titers increased threefold after the third immunizing injection and that after the sixth immunizing dose the titers increased at least fourfold as compared with those seen in non-immune, control birds. It was observed that levels of IgG antibodies anti-T. infestans saliva proteins were significantly higher (P < 0.05) in artificially immunized than in naturally immunized chickens (Figure 4). The differences in antibody titers in immune and in non-immune chickens were statistically significant (P < 0.01). The profiles of specific IgG antibodies anti-saliva antigens in the chickens remained stable after the ninth immunizing injection of triatomines’ saliva proteins. Immunoblot assays revealed that specific anti-saliva antiserum recognized 27 protein bands, with molecular masses ranging from 21 to 104 kDa (Figure 5), regardless of IgG antibodies deriving from artificial or natural immunizations. The bands formed by proteins with molecular masses of 100, 94, and 75 kDa were present in every immunized chicken’s serum tested. On the other hand, heterophile antibodies in the serum of some non-immune chickens revealed 10 bands in the triatomine’s saliva, with molecular masses 100 kDa and below.

Increasing capacity of T. infestans to blood feed upon immunized chickens.

In view of our previous observation (Figure 2) showing that the immune status of a chicken reduces the length of time required for triatomines engorgement, we conducted further experiments to find out whether this feeding preference would be a factor favoring prey selection by the hematophagous insect. In the wild, the triatomine’s search for blood in its environment requires the insect to move toward its prey. Therefore, we constructed a hut-like box where triatomines could move freely between compartments and select an animal to prey upon. In these experiments, 100 fifth instars triatomines were set free in a hut-box, and they could choose immune or non-immune chickens to prey upon in separate compartments. The results of these experiments are shown in Table 1. It was expected that triatomines would move between hut-box compartments overnight; surprisingly, 64.4 ± 4.7% of the set-free triatomines were found in the hiding places of the immune chicken room, whereas 35.6 ± 4.5% were present in the non-immune chicken compartment. Variance analysis of data generated by 12 repeat experiments revealed insects’ distribution did not occur by chance, because differences among those values are statistically highly significant (P < 0.001). Therefore, these results suggest that triatomines prefer to feed upon immune hosts showing specific antibodies against the salivary gland proteins.

To discard the possibility that an undisclosed factor would have interfered in the triatomines’ choice of a blood type, we then used fluorescent dyes to label immune and non-immune systems (Figure 6). The result of this experiment (Table 1) confirmed insects’ preference to feed upon immune chickens, with differences that were statistically significant (P < 0.001). Additionally, we conducted repeat mock experiment with two immune chickens in the box. After overnight exposure of naturally immunized chickens to the insects, we captured and counted engorged triatomines; the triatomines were present in both chicken rooms, and total counts were not statistically significant (a vs. b = 47 ± 3; N = 12, P > 0.05).

Influence of anti-saliva antibodies in triatomine’s life cycle.

These experiments were conducted with 24 selected adult male and female T. infestans that had reached molting in a single day. These triatomines were the founders (F0) used in experiments lasting for 3.5 years. Subsequently, 50 triatomines, which hatched within a period of 1 week, were chosen to compose generations F1, F2, and F3. The Group I experiment consisted of F0, F1, F2, and F3 triatomines, which were allowed to feed upon six immune chickens only after the sixth immunizing injection. The Group II experiments consisted of a total of 12 control chickens that had never been exposed to triatomines. Each control chicken was used only once. In Group I and II experiments, chickens were subjected to 50 triatomines. Figure 7A shows the comparative times of survival for adult T. infestans that were allowed to feed upon either immune chickens or upon non-immune chickens. These results showed that the immune status of a chicken does not influence a triatomine’s survival, for statistical analysis of the data showed no difference between test and control groups: F0, P = 0.69; F1, P = 042; F2, P = 0.12.

Next, we observed the influence of feeding habits upon the triatomines’ fertility. It consisted of counting eggs laid down by triatomines that obtained their fills from immune or from non-immune chickens. Figure 7B shows that differences among totals of eggs laid down by F0, F1, and F2 were not statistically significant: F0, P = 0.26; F1, P = 0.42; and F2, P = 0.12. To demonstrate whether feeding upon immune or non-immune chickens would interfere with fecundity, we counted nymphs after hatching from eggs laid by triatomines in each experimental group. Figure 7C shows these results have no differences with statistical significance: F0, P = 0.33; F1, P = 0.16; F2, P = 0.10. We then assessed whether a feeding pattern would interfere with the length of time required for nymph development. Usually, triatomines that fed upon immune birds belonging to F1 and F2 progeny reached adult stage 40 days before those that had fed upon non-immune birds (Figure 7D). These differences were statistically significant: F1, P < 0.01; F2, P < 0.05.

Furthermore, we observed the triatomines of generation F3 until 280 days after hatching; by that length of time, we found that 34 triatomines in the group that had fed upon immune birds reached adult stage while just 4 triatomines in the group that had fed upon the non-immune chickens did. These data suggest that the habit of feeding upon immune chicken blood,

Skin hypersensitivity.

We also investigated skin sensitivity to saliva proteins. First, we challenged Loubel-Rouge immune and control chickens with 60 μg of saliva proteins in 100 μL of saline solution. An indurate skin lesion was usually formed at the site of antigen injection, and it reached its maximal intensity 72 hours thereafter. Skin biopsy of the site of antigen injection showed very severe inflammatory infiltrates in the dermis of immune chickens. However, inflammatory infiltrates were also present in the dermis of control chickens, albeit to a lesser intensity. In view of Loubel-Rouge chickens being raised in open-air pens, they were subjected to bites of hematophagous insects, mainly Culex quinquefasciatus.18 This observation explains the inflammatory infiltrates and the presence of heterophile antibodies in the serum of non-immune, control chickens. Therefore, we decided to investigate the delayed-type hypersensitivity in chickens raised indoors.20 The results of these experiments are shown in Figure 8A and B. At the site of saliva antigen injection in the skin of non-immune chickens, no reaction was observed after 72 hours, and the epidermis showed normal histologic features. In marked contrast, an indurate skin lesion appeared at the site of saliva antigen injection in immune chickens, which increased progressively in 24–48 hours and reached its maximal size, showing prominent borders and a necrotic central area by 72 hours. Biopsy histology from a lesion border showed severe inflammatory infiltrates, edema, and congestion (Figure 8B).

DISCUSSION

In this study, we have shown that T. infestans appears to prefer an immune prey to feed upon. This observation was consistently reproduced in birds naturally immunized by repeated triatomines bites as well as in birds that were immunized by intradermal injections of the insects’ saliva antigens. The skin hypersensitivity lesion consisting of inflammation and blood congestion has suggested that triatomines cannulate wide-open blood vessels that facilitate feeding upon immune chickens. Previous work had shown that mice exposed to Phlebotomus papatasi bites developed strong delayed-type hypersensitivity, and this response was associated with an increase of blood flow and a reduction in probing time.9

The labeling of blood mononuclear cells of immune and non-immune chickens with fluorescent dyes, which was obtained by injection of minute amounts of fluorochromes, allowed confirmation of a this triatomine preference to feed upon immune chickens. This preference was consistently observed, regardless of birds having been subjected to saliva antigen injections or to triatomines’ blood feeding. In a series of repeated experiments, triatomines that had been equally distributed in both compartments of a hut-like box were captured consistently in the immune chicken dwelling, where they hid and rested after obtaining their fills. These results appear to have a broad epidemiologic significance as for spreading off the enzootics, because an immunologic status of vertebrate host populations appears to favor the predator and main transmitter of the parasite agent of Chagas disease.

Results stemming from immunization experiments suggest that prospects for immunoprophylaxis against T. cruzi infections are rather feeble. These results are consistent with previous attempts to prevent T. cruzi invasion or against its settlement in the host’s body.21 In this respect, many researchers have dedicated themselves to the study of insect’s saliva proteins, aiming at the means to curtail blood feeding and transmission of T. cruzi forms contaminating triatomines’ bite wound. Unfortunately, results of experiments reported here have failed to show single evidence in favor of a saliva-based vaccine development. Contrastingly, immune hosts facilitate triatomines obtaining fills of blood, and nymphs hatched from a colony fed on immune chickens reached adult life usually earlier than those feeding upon non-immune chickens. These results are consistent with others in the literature.2224

Explanation of our data and those in the literature requires remembering that arthropods date from the Phanerozoic, superior Davonian initiated 400 million years ago (mya). Since early times, insects have widely spread to every ecosystem. An early fossil of a hematophagous insect dates from the Jurassic, 150 mya, time enough for the emergence of the 20 different Orders already known.25 Therefore, we believe that if specific antibodies against T. infestans saliva had imposed any sort of barrier against its survival and reproduction, this hematophagous species would not have survived this long. A convergent evolution, resulting in redundancy of saliva proteins with a major role played in insects’ feeding ability,10,11,26 appears to explain the existing reactions of prey against different insect bites, not necessarily limited to species that were the source of immunizing saliva antigens.27,28

Some insects have developed mechanisms for eluding the host’s immune responses. For example, male and female mite Rhipicephalus appendiculatus are used to preying at the same time, together. This synergism has been considered advantageous because there is an Ig-binding protein in the male saliva, and, therefore, a smaller amount of Ig is supposedly ingested by the female.7 We believe that this mechanism may not be functional in T. infestans because we did not find a single evidence of any Ig impeding triatomine feeding. Therefore, a possible role played by the insect-secreted Ig superfamily in neutralizing the prey’s immune factors requires further investigation.29

Immunoblot analysis of saliva proteins with antibodies raised in chickens revealed a unique 100-kDa band that was present in each immune and in some non-immune chicken sera. Interestingly, a protein with similar molecular mass was observed in sera of mice subjected to T. infestans bites.30 It could be suggested that such proteins could be candidate targets for producing a recombinant vaccine against insect bites. Unfortunately, data discussed here and previously have described redundancy of proteins with anti-coagulant, anti-platelet aggregation, and anti-vasodilator activities in saliva of hematophagous insects, which could present a powerful barrier difficult to overcome with current biotechnologies; therefore, this appears to be a puzzle deserving further attention in forthcoming research studies.31,32

Table 1

T. infestans prefers to feed upon immune chickens

Triatomines captured (%)
Repeat experiment
Exp.Chicken statusNumber123
Note: Experiment A, unlabeled chickens; B, experiment fluorescence-labeled chicken blood leukocytes. Chickens were placed in separate compartments and exposed to 100 triatomines overnight. On the next day, triatomines hiding in immune or in nonimmune chicken compartments were counted: chickens 1, 2, 9, and 10 were immunized with saliva antigen injections; chickens 3, 4, 11, and 12 were immunized by triatomines’ bites. Chickens 5–8 and 13–16 are control, non-immune chickens (a vs, b, P < 0.05; c vs. d, P < 0.05).
AImmune1656763
2657470
3606762
4616455(a) 64.4 ± 4.7
Non-immune5353337
6342630
7403338
8393645(b) 35.6 ± 4.5
BImmune9786770
10778062
11736777
12847478(c) 74 ± 6
Non-immune13223330
14232038
15273323
16162622(d) 26 ± 6
Figure 1.
Figure 1.

Top view of hut box, lid open. Double-headed arrow shows the position of the wire grate separating chickens’ rooms. The 1.5-cm wire grates are also seen on the floor. Triatomines set free in the box can move from one room to another and hide safely in dwellings inaccessible to chicken beaks.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.893

Figure 2.
Figure 2.

Natural immunization by repeated exposure of chickens to T. infestans bites shortens the insect’s probing time. A 40-min exposure was required for 90% of the insects to obtain their fill from non-immune chickens; however, a 15-min exposure was required for 50% of the insects to obtain their fill and to become engorged with blood from chickens that had undergone natural immunization with saliva injected through the insects’ bites.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.893

Figure 3.
Figure 3.

Effect of immunization by repeated exposures to insect bites or by injections of crude saliva antigens in chickens. IgG antibody titers in 1:200 serum dilutions were detected by ELISA assays (see Materials and Methods). After the third immunizing procedure, antibodies in the serum of chickens that received saliva injections were approximately twofold higher than in the serum of chickens that had been immunized through insect bites. Each point represents the mean ± SD of six repeated experiments.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.893

Figure 4.
Figure 4.

Profiles of IgG anti-saliva antibody titers detected by ELISA in immune and in non-immune, control chickens. At serum dilutions from 1:100 to 1:1600, the immune chickens, regardless of the route of immunization, showed significantly higher specific IgG antibodies titers (P < 0.001) than the control, non-immune birds. The bars indicate the mean ± SD of six repeated experiments.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.893

Figure 5.
Figure 5.

Detection of T. infestans saliva protein bands with chicken serum IgG. Lanes 1 and 5, bands formed with sera from chickens immunized with crude saliva antigens injected in Loubel-Rouge and in congenic B12/B12 chickens, respectively; lanes 2 and 4, bands formed with heterophile antibodies from a non-immune Loubel-Rouge serum (note the 100-kDa band present in each lane); lane 3, bands formed with serum antibodies of a Loubel-Rouge chicken immunized by exposure to T. infestans bites; lane 6, control non-immune B12/B12 chicken serum.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.893

Figure 6.
Figure 6.

Blood source detection. The immune chicken had fluorescein-labeled (apple green) blood leukocytes and the non-immune chicken had rhodamine-labeled (red) leukocytes. Leukocytes (A) in the immune chicken’s blood smears fluoresce under light stimuli (B), and leukocytes from the gut (C) of an insect that fed upon that chicken also stain apple-green (D). Similarly, leukocytes in the non-immune chicken’s blood smears (E) stain red (F), and leukocytes in the gut (G) of an insect that fed upon that chicken also stain red (H). This method allowed identification of the chicken that T. infestans had preyed upon. This figure appears in color at www.ajtmh.org. which accelerated growth of F1 and F2 progeny, appeared evident in the next generation.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.893

Figure 7.
Figure 7.

Immune status influence upon T. infestans life cycle. (A) Survival ratios of triatomines feeding upon immune and non-immune chickens were similar in the three generations observed. (B) Fertility ratios observed in the three generations were not statistically significant (see text). (C) Fecundity measured by the ratios of eggs to hatching nymphs was also similar. (D) Length of time required for nymph (F1 and F2) development was 40 days shorter when the bugs fed upon the immune chicken as compared with the time required for development and molting of nymphs fed upon non-immune chickens (F1, P < 0.01; F2, P < 0.05).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.893

Figure 8.
Figure 8.

Effect of a chicken immune status toward elicitation of delayed-type skin reactivity against T. infestans saliva proteins. (A, B) Congenic chicken normal skin 72 h after injection of 60 μg of saliva in 100 μL of 0.15 M saline solution. (C) Indurate skin lesion with central necrotic area is seen in an immune congenic chicken challenged with the same dose of saliva antigen. (D) Intense inflammatory infiltrates and dilated blood vessels in the dermis (arrows). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 5; 10.4269/ajtmh.2006.75.893

*

Address correspondence to Antonio R. L. Teixeira, Chagas Disease Multidisciplinary Research Laboratory, Faculty of Medicine, University of Brasília, P.O. Box 04536, 70919-970, Brasília, DF, Brazil. E-mail: ateixeir@unb.br

Authors’ addresses: Mariana M. Hecht, Ana Carolina Bussacos, Silene P. Lozzi, Jaime M. Santana, and Antonio R.L. Teixeira, Chagas Disease Multidisciplinary Research Laboratory, Faculty of Medicine, University of Brasília, P.O. Box 04536, 70919-970, Brasília, Federal District, Brazil.

Acknowledgments: The authors thank Meire Maria de Lima and José Marcos Ribeiro for exchanging ideas and criticism and Dr. Eduardo Freitas da Silva for statistical analysis.

Financial support: Support was provided by FINEP-Financiadora de Estudos e Projetos, and CNPq (Conselho Nacional de Pesquisas), Brazil.

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