Signaling pathways that regulate Trypanosoma cruzi infection and immune response.

Current understanding of key cellular pathways, which are activated by the interaction between T. cruzi and host immunity, is crucial for controlling T. cruzi infection and also for limiting the development of the immunopathological symptoms of Chagas´ disease. Here, we focus on recent advances in the knowledge of modulation of innate receptors such as TLRs and NLRs, especially NLRP3, by T. cruzi in different cells of the immune system. On the other hand, the modulation of macrophage activation may be instrumental in allowing parasite persistence and long-term host survival. In this sense, we discuss the importance of the metabolism of two amino acids: L-arginine and tryptophan, and evaluate the role of iNOS, arginase and IDO enzymes in the regulation of innate and adaptive immune response during this infection; and, finally, we also discuss how T. cruzi exploits the AhR, mTOR and Wnt signaling pathways to promote their intracellular replication in macrophages, thus evading the host's immune response.

The limpet transcription factors of Triatoma infestans regulate the response to fungal infection and modulate the expression pattern of defensin genes.

As part of the innate humoral response to microbial attack, insects activate the expression of antimicrobial peptides (AMP). Understanding the regulatory mechanisms of this response in the Chagas disease vector Triatoma infestans is important since biological control strategies against pyrethroid-resistant insect populations were recently addressed by using the entomopathogenic fungus Beauveria bassiana. By bioinformatics, gene expression, and silencing techniques in T. infestans nymphs, we achieved sequence and functional characterization of two variants of the limpet transcription factor (Tilimpet) and studied their role as regulators of the AMP expression, particularly defensins, in fungus-infected insects. We found that Tilimpet variants may act differentially since they have divergent sequences and different relative expression ratios, suggesting that Tilimpet-2 could be the main regulator of the higher expressed defensins and Tilimpet-1 might play a complementary or more general role. Also, the six defensins (Tidef-1 to Tidef-6) exhibited different expression levels in fungus-infected nymphs, consistent with their phylogenetic clustering. This study aims to contribute to a better understanding of T. infestans immune response in which limpet is involved, after challenge by B. bassiana infection.

Identification and characterization of the causative triatomine bugs of anaphylactic shock in Zhanjiang, China.

BACKGROUND: Two health concerns primarily related to triatomine bugs are transmission of Trypanosoma cruzi through infective feces, and allergic reactions induced by triatomine bites. In the Southwestern United States, reduviid bugs bites commonly cause insect allergy. In South China, four cases of anaphylactic shock have been reported after this bite exposure. To further classify the species of these bugs and confirm the sensitization of the triatomine saliva, we caught triatomine bugs from the region where the bites occurred and performed phylogenetic and immunohistochemical (IHC) analysis. METHODS: Triatomine bugs were collected in Donghai Island of Zhanjiang City in South China. The genomic DNA was extracted from three legs of the bugs. The fragments of mitochondrial 16S rRNA, cytochrome c oxidase subunit I (COI) gene and nuclear ribosomal 18S and 28S rRNA genes were obtained by PCR and sequenced. A phylogenetic tree was constructed based on the sequence of 16S rRNA gene using a maximum likelihood method with MEGA 7.0 software. Trypanosomal specific fragments and vertebrate COI genes were amplified from the fecal DNA to detect the infection of trypanosomes and analyze the blood feeding patterns, respectively. Paraffin-embedded sections were then prepared from adult triatomines and sent for IHC staining. RESULTS: We collected two adult triatomine bugs in Donghai Island. Morphological and molecular analyses indicated that the triatomines were Triatoma rubrofasciata. No fragments of T. cruzi or other trypanosomes were detected from the fecal DNA. Mitochondrial gene segments of Homo sapiens and Mus musculus were successfully amplified. The allergens which induced specific IgE antibodies in human serum were localized in the triatomine saliva by IHC assay. CONCLUSIONS: The two triatomine bugs from Donghai Island were T. rubrofasciata. They had bitten humans and mice. Their saliva should contain the allergens related to the allergic symptoms and even anaphylactic shock of exposed residents. Great consideration should be given to this triatomine bugs due to their considerable distribution and potential threat to public health in South China.

Comparative genomics analysis of triatomines reveals common first line and inducible immunity-related genes and the absence of Imd canonical components among hemimetabolous arthropods.

BACKGROUND: Insects operate complex humoral and cellular immune strategies to fend against invading microorganisms. The majority of these have been characterized in Drosophila and other dipterans. Information on hemipterans, including Triatominae vectors of Chagas disease remains incomplete and fractionated. RESULTS: We identified putative immune-related homologs of three Triatominae vectors of Chagas disease, Triatoma pallidipennis, T. dimidiata and T. infestans (TTTs), using comparative transcriptomics based on established immune response gene references, in conjunction with the predicted proteomes of Rhodnius prolixus, Cimex lecticularis and Acyrthosiphon pisum hemimetabolous. We present a compressive description of the humoral and cellular innate immune components of these TTTs and extend the immune information of other related hemipterans. Key homologs of the constitutive and induced immunity genes were identified in all the studied hemipterans. CONCLUSIONS: Our results in the TTTs extend previous observations in other hemipterans lacking several components of the Imd signaling pathway. Comparison with other hexapods, using published data, revealed that the absence of various Imd canonical components is common in several hemimetabolous species.

Feces from wild Triatoma dimidiata induces local inflammation and specific immune response in a murine model

In endemic regions for Triatoma dimidiata the vector for Chagas disease, subjects can be in contact with insect`s feces several times through a lifetime. The triatomine's digestive tract is colonized by diverse but few dominant genera of microorganisms. The immune responses to microbiota feces are poorly known in mammal hosts. The goal of this paper is to describe the local inflammation at the port of inoculation and the humoral immune response in a murine model mimicking natural contamination of feces from wild Triatoma dimidiata and its identification of bacterial community. Feces from twenty T. dimidiata insects captured in peridomestic and domestic ecotopes were used for bacteria isolation and phenotypic identification. Five microliters of whole feces or bacteria isolated colonies were used for intradermal inoculation of mice for detection of humoral immune response and local inflammation at the inoculation site. The bacterial community identified corresponded to Kytococcus, Brevibacillus, Kocuria, Chryseobacterium, Pantoe, Proteus, Burkholderia, Acinetobacter and Stapylococcus. The local inflammation at the inoculation site was dominated by neutrophils infiltration, and specific seric IgG immune response was recognized against whole feces as well as Burkholderia, Acinetobacter and Staphylococcus isolates. In conclusion, feces from T. dimidiata were colonized by few culturable microorganism genera that are able to induce local inflammation and IgG immune response in a murine model.

Relationships between altitude, triatomine (Triatoma dimidiata) immune response and virulence of Trypanosoma cruzi, the causal agent of Chagas' disease.

Little is known about how the virulence of a human pathogen varies in the environment it shares with its vector. This study focused on whether the virulence of Trypanosoma cruzi (Trypanosomatida: Trypanosomatidae), the causal agent of Chagas' disease, is related to altitude. Accordingly, Triatoma dimidiata (Hemiptera: Reduviidae) specimens were collected at three different altitudes (300, 700 and 1400 m a.s.l.) in Chiapas, Mexico. The parasite was then isolated to infect uninfected T. dimidiata from the same altitudes, as well as female CD-1 mice. The response variables were phenoloxidase (PO) activity, a key insect immune response, parasitaemia in mice, and amastigote numbers in the heart, oesophagus, gastrocnemius and brain of the rodents. The highest levels of PO activity, parasitaemia and amastigotes were found for Tryp. cruzi isolates sourced from 700 m a.s.l., particularly in the mouse brain. A polymerase chain reaction-based analysis indicated that all Tryp. cruzi isolates belonged to a Tryp. cruzi I lineage. Thus, Tryp. cruzi from 700 m a.s.l. may be more dangerous than sources at other altitudes. At this altitude, T. dimidiata is more common, apparently because the conditions are more beneficial to its development. Control strategies should focus activity at altitudes around 700 m a.s.l., at least in relation to the region of the present study sites.

Pre-exposure to faeces or saliva of Triatoma dimidiata decreases parasitemia in mice challenged with Trypanosoma cruzi: a description of the inflammatory reaction at the inoculation site

Under natural conditions, Trypanosoma cruzi infection is transmitted to mammals when faeces contaminated with metacyclic trypomastigotes gain access through skin lesions, mucosa or bite wounds. Natural infection of bugs with T. cruzi can vary greatly from less than 1% up to 70%, depending on triatomine species: in the case of Triatoma dimidiata, the percentage of infection is around 30%. In this work uses biological fluids (saliva and faeces) from Triatoma dimidiata to inoculate experimental animals once or multiple times, before inoculation with faeces contaminated with metacyclic trypomastigotes discrete type unit Ia (TcI). The site of infection was analyzed for histological changes based on hematoxile-eosine technique and toluide blue stain for mast cells. Inoculation with saliva led to the recruitment of eosinophils and mononuclear cells at the inoculation site, whereas inoculation with faeces led to the recruitment of neutrophils. Mice inoculated multiple times exhibited a strong inflammatory reaction from the first hour. Mono- or multi-exposure to T. dimidiata fluids before inoculation with metacyclic trypomastigotes helped to control the level of parasitemia. Previous contact with saliva or faeces of T. dimidiata reduces parasitemia in T. cruzi I -infected mice.

Assessing gene expression during pathogenesis: Use of qRT-PCR to follow toxin production in the entomopathogenic fungus Beauveria bassiana during infection and immune response of the insect host Triatoma infestans.

Entomopathogenic fungi secrete toxic secondary metabolites during the invasion of the insect hemocoel as part of the infection process. Although these compounds have been frequently mentioned as virulence factors, the roles of many of them remain poorly understood, including the question of whether they are expressed during the infection process. A major hurdle to this issue remains the low sensitivity of biochemical detection techniques (e.g., HPLC) within the complex samples that may contain trace quantities of fungal molecules inside the insect. In this study, quantitative reverse transcription real-time PCR (qRT-PCR) was used to measure the transcript levels within the insect fungal pathogen Beauveria bassiana, that encode for the synthetase enzymes of the secondary metabolites tenellin (BbtenS), beauvericin (BbbeaS) and bassianolide (BbbslS) during the infection of Triatoma infestans, a Chagas disease insect vector. Absolute quantification was performed at different time periods after insect treatment with various concentrations of propagules, either by immersing the insects in conidial suspensions or by injecting them with blastospores. Both BbtenS and BbbeaS were highly expressed in conidia-treated insects at days 3 and 12 post-treatment. In blastospore-injected insects, BbtenS and BbbeaS expression peaked at 24h post-injection and were also highly expressed in insect cadavers. The levels of BbbslS transcripts were much lower in all conditions tested. The expression patterns of insect genes encoding proteins that belong to the T. infestans humoral immune system were also evaluated with the same technique. This qPCR-based methodology can contribute to decifering the dynamics of entomopathogenic fungal infection at the molecular level.

An insight into the sialotranscriptome of Triatoma rubida (Hemiptera: Heteroptera).

The kissing bug Triatoma rubida (Uhler, 1894) is found in southwestern United States and parts of Mexico where it is found infected with Trypanosoma cruzi, invades human dwellings and causes allergies from their bites. Although the protein salivary composition of several triatomine species is known, not a single salivary protein sequence is known from T. rubida. Furthermore, the salivary diversity of related hematophagous arthropods is very large probably because of the immune pressure from their hosts. Here we report the sialotranscriptome analysis of T. rubida based on the assembly of 1,820 high-quality expressed sequence tags, 51% of which code for putative secreted peptides, including lipocalins, members of the antigen five family, apyrase, hemolysin, and trialysin families. Interestingly, T. rubida lipocalins are at best 40% identical in primary sequence to those of T. protracta, a kissing bug that overlaps its range with T. rubida, indicating the diversity of the salivary lipocalins among species of the same hematophagous genus. We additionally found several expressed sequence tags coding for proteins of clear Trypanosoma spp. origin. This work contributes to the future development of markers of human and pet exposure to T. rubida and to the possible development of desensitization therapies. Supp. Data 1 and 2 (online only) of the transcriptome and deducted protein sequences can be obtained from http://exon.niaid.nih.gov/transcriptome/Trubida/Triru-S1-web.xlsx and http://exon.niaid.nih.gov/transcriptome/Trubida/Triru-S2-web.xlsx.

Anti-triatomine saliva immunoassays for the evaluation of impregnated netting trials against Chagas disease transmission.

Insecticide-impregnated nets can kill triatomine bugs, but it remains unclear whether they can protect against Chagas disease transmission. In a field trial in Quequeña, Peru, sentinel guinea pigs placed in intervention enclosures covered by deltamethrin-treated nets showed significantly lower antibody responses to saliva of Triatoma infestans compared with animals placed in pre-existing control enclosures. Our results strongly suggest that insecticide-treated nets prevent triatomine bites and can thereby protect against infection with Trypanosoma cruzi. Anti-salivary immunoassays are powerful new tools to evaluate intervention strategies against Chagas disease.

Trypanosoma cruzi infection modulates the expression of Triatoma brasiliensis def1 in the midgut.

Antimicrobial peptides are an essential component of the insect immune system. One of the most ubiquitous is defensin, which has been identified in all examined insect orders. Triatoma brasiliensis (Heteroptera, Triatominae), the main Trypanosoma cruzi vector in semi-arid regions of north-eastern Brazil, expresses def1, a defensin encoding gene, predominantly in the anterior region (cardia and stomach) of the midgut. In the present study, we compared the transcript abundance of T. brasiliensis def1 in the anterior (stomach) and posterior midgut (small intestine) regions of naïve bugs with those infected with a familiar T. cruzi isolate. In the stomach, only slight differences between the two insect groups were visible, whereas in the small intestine wide differences (up to 9.6-fold) between infected and noninfected bugs become apparent. The highly increased def1 transcript abundance at 20 days after the infective blood meal might be a response to the T. cruzi infection, suggesting a potential function of intestinal defensin in the parasite population control.

IgM-antibody responses of chickens to salivary antigens of Triatoma infestans as early biomarkers for low-level infestation of triatomines.

The recombinant form of a highly immunogenic 14.6 kDa protein in Triatoma infestans saliva (rTiSP14.6) is a potential epidemiological marker for the detection of triatomine bug populations using IgG responses in peridomestic chickens. However, the persistence of the IgG response prevents it being of value for several months in areas where triatomine control programmes have been implemented. In this investigation, IgM-antibody reactions to crude salivary antigens or rTiSP14.6 decayed rapidly after exposure of chickens and were measurable for only 18 days after a single challenge with T. infestans. In serial exposure experiments, chickens from low and high exposure groups showed no significant differences in anti-saliva and anti-rTiSP14.6 IgM-antibody titres. Highly immunogenic salivary antigens of 12 and 14 kDa were recognised by all chicken sera. Sera from peridomestic chickens from sites of known T. infestans infestation in Bolivia also recognised these two antigens and no differences in the IgM responses of sera from chickens from low and high infestation households were detected. IgM responses were specific to infested households and could not be detected in sera from non-infested households. Cross-reactivity studies showed that at least four other triatomine species share the 14.6 kDa salivary antigen. No IgM responses were detected against salivary proteins of mosquitoes and sandflies. Thus, we believe that rTiSP14.6 represents a promising epidemiological marker for the detection of low numbers of triatomines in peridomestic habitats, and the comparison of IgM and IgG responses can be used to detect re-infestation soon after insecticide-based control programmes.

Immunogenic salivary proteins of Triatoma infestans: development of a recombinant antigen for the detection of low-level infestation of triatomines.

BACKGROUND: Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The most effective vector, Triatoma infestans, has been controlled successfully in much of Latin America using insecticide spraying. Though rarely undertaken, surveillance programs are necessary in order to identify new infestations and estimate the intensity of triatomine bug infestations in domestic and peridomestic habitats. Since hosts exposed to triatomines develop immune responses to salivary antigens, these responses can be evaluated for their usefulness as epidemiological markers to detect infestations of T. infestans. METHODOLOGY/PRINCIPAL FINDINGS: T. infestans salivary proteins were separated by 2D-gel electrophoresis and tested for their immunogenicity by Western blotting using sera from chickens and guinea pigs experimentally exposed to T. infestans. From five highly immunogenic protein spots, eight salivary proteins were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) and comparison to the protein sequences of the National Center for Biotechnology Information (NCBI) database and expressed sequence tags of a unidirectionally cloned salivary gland cDNA library from T. infestans combined with the NCBI yeast protein sub-database. The 14.6 kDa salivary protein [gi|149689094] was produced as recombinant protein (rTiSP14.6) in a mammalian cell expression system and recognized by all animal sera. The specificity of rTiSP14.6 was confirmed by the lack of reactivity to anti-mosquito and anti-sand fly saliva antibodies. However, rTiSP14.6 was recognized by sera from chickens exposed to four other triatomine species, Triatoma brasiliensis, T. sordida, Rhodnius prolixus, and Panstrongylus megistus and by sera of chickens from an endemic area of T. infestans and Chagas disease in Bolivia. CONCLUSIONS/SIGNIFICANCE: The recombinant rTiSP14.6 is a suitable and promising epidemiological marker for detecting the presence of small numbers of different species of triatomines and could be developed for use as a new tool in surveillance programs, especially to corroborate vector elimination in Chagas disease vector control campaigns.

Antibody responses of domestic animals to salivary antigens of Triatomainfestans as biomarkers for low-level infestation of triatomines.

Hematophagous arthropods such as Triatoma infestans, the vector of Trypanosoma cruzi, elicit host-immune responses during feeding. Characterization of antibody responses to salivary antigens offers the potential to develop immunologically based monitoring techniques for exposure to re-emergent triatomine bug populations in peridomestic animals. IgG-antibody responses to the salivary antigens of T.infestans have been detected in chickens as soon as 2 days after the first exposure to five adult bugs. Chickens and guinea pigs regularly exposed to this number of triatomines showed a significantly lower anti-saliva antibody titre than animals exposed to 25 adults and fifth instars of four different T.infestans strains originating from Bolivia and from Northern Chile. Highly immunogenic salivary antigens of 14 and 21kDa were recognised by all chicken sera and of 79kDa by all guinea pig sera. Cross-reactivity studies using saliva or salivary gland extracts from different hematophagous species, e.g. different triatomines, bed bugs, mosquitoes, sand flies and ticks, as well as chicken sera exposed to triatomines and mosquitoes, demonstrated that the 14 and 21kDa salivary antigens were only found in triatomines. Sera from peridomestic chickens and guinea pigs in sites of known T.infestans challenge in Bolivia also recognised the 14 and 21kDa antigens. These represent promising epidemiological markers for the detection of small numbers of feeding bugs and hence may be a new tool for vector surveillance in Chagas disease control programs.

An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas' disease.

Triatoma infestans is a hemiptera, vector of Chagas' disease that feeds exclusively on vertebrate blood in all life stages. Hematophagous insects' salivary glands (SG) produce potent pharmacological compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its SG was randomly sequenced. Also, salivary proteins were submitted to two-dimensional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1534 (SG) cDNA sequences, 645 of which coded for proteins of a putative secretory nature. Most salivary proteins described as lipocalins matched peptide sequences obtained from proteomic results.

Sugar epitopes as potential universal disease transmission blocking targets.

One promising method to prevent vector-borne diseases is through the use of transmission blocking vaccines (TBVs). However, developing several anti-pathogen TBVs may be impractical. In this study, we have identified a conserved candidate carbohydrate target in the midguts of several Arthropod vectors. A screen of the novel GlycoChip glycan array found that the anti-carbohydrate malaria transmission blocking monoclonal antibody (MG96) preferentially recognized D-mannose (alpha) and the type II lactosamine disaccharide. The specificity for D-mannose was confirmed by competition ELISA using alpha-methyl mannoside as inhibitor. Con A, which identifies terminal mannose residues, did not inhibit MG96 reactivity with mosquito midgut lysates, suggesting that Con A has differential recognition of this monosaccharide. However, the jack bean lectin, Jacalin, which recognizes D-mannose (alpha), d-galactose (alpha/beta) and the T antigen, not only displays a similar banding profile to that recognized by MG96 on immunoblot but was also shown to effectively inhibit MG96. Wheat-germ agglutinin, which recognizes N-acetyllactosamine units, only partially inhibited MG96 reactivity. This highlights the contribution of both glycan moieties to the MG96 epitope or glycotope. Enzyme deglycosylation results suggest that MG96 recognizes a mannose alpha1-6 substitution on an O-linked oligosaccharide. Taken together, the data suggest that MG96 recognizes a discontinuous glycotope composed of Manalpha1-6 proximal to Galbeta1-4GlcNAc-alpha-O-R glycans on arthropod vector midguts. As such, these glycotopes may represent potential transmission blocking vaccine targets for a wide range of vector-borne pathogens.

Proteinase and gelatinolytic properties of a Triatoma infestans extract.

An extract of Triatoma infestans has previously been demonstrated to produce specific IgG and IgE both in animals and in atopic humans with rhinitis/asthma as well as hypersensitivity pneumonitis in guinea pigs aerosolized with T. infestans. We attempted to determine whether the antigen or antigens responsible belonged to the protease group, as occurs with other allergens such as house dust mites and cockroaches. To do this, T. infestans was studied by SDS-PAGE, Western blots and gelatinolysis with and without the use of specific protease inhibitors such as E-64, TLCK, TPCK, PMSF, leupeptin, o-phenanthrolene and pepstatin-A. These assays revealed serine-like proteolytic and gelatinolytic activities. The presence of 10 to 12 bands of between 14 and 100 kDa was detected. The proteolytic activity pattern of T. infestans was greatest at pH 8.5 and gelatinolytic activity was highly sensitive to PMSF, suggesting that this enzyme could be characterized as a serine protease. Western blots revealed that two bands of 17 and 58 kDA reacted with the sera of atopic humans with respiratory diseases and anti-IgE. However, whether these bands correlated with allergenicity is unclear since the presence of several proteins in each of these bands does not rule out the possibility that this correlation could exist, especially because cross-reactions with antigens from the cockroach Periplaneta americana and its specific antiserum in animals and atopic humans have been demonstrated. The role of proteases in the etiopathogenesis of perennial rhinitis and bronchial asthma in inhabitants of the area of Argentina infested by T. infestans requires further investigation.

Allergic reactions to Triatoma bites.

BACKGROUND: Triatoma bugs are best known in the medical community as vectors of trypanosomiasis (Chagas disease). However, bites of Triatoma bugs are a cause of local cutaneous reactions and anaphylaxis, mainly in the western and southwestern United States. The reactions typically occur at night during sleep, and the bite may not be recognized. There is continuing public interest in medical complications of bites of these bugs, although the scope of the problem remains undefined. OBJECTIVE: To review the relevant medical literature, identify present knowledge, and determine future research goals for allergy to Triatoma. DATA SOURCES: Computerized databases were used to search the medical literature for articles in the English language on Triatoma bites, allergy and entomology, and Chagas disease. STUDY SELECTION: Almost all identified articles on Triatoma allergy were used. Only selected articles on Triatoma bites and entomology were pertinent to the objectives. Articles on Chagas disease were limited to cases in the United States. RESULTS: Bites of Triatoma bugs have been known to cause anaphylaxis for more than a century. These insects inhabit a large area of the United States, but to date most reports of allergic reactions to their bites have originated in the West and Southwest. The reactions typically occur at night during sleep following a bite on uncovered skin and may be unrecognized. Procalin has been identified as the major salivary allergen of Triatoma protracta and was recently cloned and expressed through recombinant technique. Allergenic reactivity has been demonstrated to salivary gland extracts of 2 species. The extracts of these 2 species have not shown immunologic cross-reactivity. Immunotherapy using a salivary gland extract appeared to be beneficial in a small number of patients; however, no commercial testing or treatment allergen is available. CONCLUSIONS: Triatoma bites appear to be an important cause of anaphylaxis, especially in the western and southwestern United States. Because exposure to these insects often occurs during sleep, the incidence of allergic reactions to them is unclear. An epidemiologic study should be performed to determine the incidence, prevalence, and range of allergic responses to the bites of these insects. The lack of commercial antigen limits diagnostic and treatment capabilities. The development of an allergen under the Orphan Drug Act should be encouraged.

Human IgG1 and IgG4: the main antibodies against Triatoma infestans (Hemiptera: Reduviidae) salivary gland proteins.

The Triatoma infestans salivary gland proteins (TSGP) can induce local and systemic hypersensitivity reactions in humans. IgG antibodies against TSGP were present in higher levels in sera of Chagas disease patients, and in individuals living in triatomine-infested areas than in controls living in triatomine-free areas. TSGP-specific IgG1 was found in sera of Chagas patients, and of individuals living in triatomine-infested rural areas, and uniquely specific IgG4 was present in sera of Chagas patients living in triatomine-infested areas, reactive against TSGP. Unique specificities were not detected in sera of individuals reacting against the ubiquitous mosquito Culex quinquifasciatus saliva proteins (CSGP). In conclusion, IgG1 reactive against TSGP is the main antibody present in individuals living in the triatomine-infested study areas. Also, IgG4 is found in the sera of insect-transmitted Chagas disease patients living in study areas.