Immunogenic salivary proteins of Triatoma infestans: development of a recombinant antigen for the detection of low-level infestation of triatomines.
ABSTRACT: 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.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.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.
Project description:BACKGROUND:Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas' disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas' disease. METHODS AND FINDINGS:The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas' disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion. CONCLUSIONS AND SIGNIFICANCE:Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas' disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms.
Project description:<h4>Background</h4>Salivary proteins of Triatoma infestans elicit humoral immune responses in their vertebrate hosts. These immune responses indicate exposure to triatomines and thus can be a useful epidemiological tool to estimate triatomine infestation. In the present study, we analyzed antibody responses of guinea pigs to salivary antigens of different developmental stages of four T. infestans strains originating from domestic and/or peridomestic habitats in Argentina, Bolivia, Chile and Peru. We aimed to identify developmental stage- and strain-specific salivary antigens as potential markers of T. infestans exposure.<h4>Methodology and principal findings</h4>In SDS-PAGE analysis of salivary proteins of T. infestans the banding pattern differed between developmental stages and strains of triatomines. Phenograms constructed from the salivary profiles separated nymphal instars, especially the 5th instar, from adults. To analyze the influence of stage- and strain-specific differences in T. infestans saliva on the antibody response of guinea pigs, twenty-one guinea pigs were exposed to 5th instar nymphs and/or adults of different T. infestans strains. Western blot analyses using sera of exposed guinea pigs revealed stage- and strain-specific variations in the humoral response of animals. In total, 27 and 17 different salivary proteins reacted with guinea pig sera using IgG and IgM antibodies, respectively. Despite all variations of recognized salivary antigens, an antigen of 35 kDa reacted with sera of almost all challenged guinea pigs.<h4>Conclusion</h4>Salivary antigens are increasingly considered as an epidemiological tool to measure exposure to hematophagous arthropods, but developmental stage- and strain-specific variations in the saliva composition and the respective differences of immunogenicity are often neglected. Thus, the development of a triatomine exposure marker for surveillance studies after triatomine control campaigns requires detailed investigations. Our study resulted in the identification of a potential antigen as useful marker of T. infestans exposure.
Project description:BACKGROUND:Chagas disease is caused by the parasite Trypanosoma cruzi and is transmitted through triatomines (Hemiptera: Reduviidae). In the last year, many studies of triatomine gut microbiota have outlined its potential role in modulating vector competence. However, little is known about the microbiota present in the salivary glands of triatomines. Bacterial composition of salivary glands in selected triatomine species was investigated, as well as environmental influences on the acquisition of bacterial communities. METHODOLOGY/PRINCIPAL FINDINGS:The diversity of the bacterial communities of 30 pairs of salivary glands of triatomines was studied by sequencing of the V1- V3 variable region of the 16S rRNA using the MiSeq platform (Illumina), and bacteria isolated from skin of three vertebrate hosts were identified based on 16S rRNA gene sequence analysis (targeting the V3-V5 region). In a comparative analysis of microbiota in the salivary glands of triatomine species, operational taxonomic units belonging to Arsenophonous appeared as dominant in Triatoma spp (74% of the total 16S coverage), while these units belonging to unclassified Enterobacteriaceae were dominant in the Rhodnius spp (57% of the total 16S coverage). Some intraspecific changes in the composition of the triatomine microbiota were observed, suggesting that some bacteria may have been acquired from the environment. CONCLUSIONS AND SIGNIFICANCE:Our study revealed the presence of a low-diversity microbiota associated to the salivary glands of the evaluated triatomines. The predominant bacteria genera are associated with triatomine genera and the bacteria can be acquired in the environment in which the insects reside. Further studies are necessary to determine the influence of bacterial communities on vector competence.
Project description:In the Gran Chaco region, control of Triatoma infestans has been limited by persistent domestic infestations despite the efforts of the Vector Control Services. In Paraguay, this region is the highest endemic area in the country, showing high levels of indoor and outdoor infestation. Although sylvatic T. infestans have been found in the Bolivian and Argentine Chaco, similar searches for sylvatic populations of this species in Paraguay had been unsuccessful over the last 20 years. Here we present a new approach to detecting sylvatic Triatominae, using a trained dog, which has successfully confirmed sylvatic populations of T. infestans and other triatomine species in Paraguay. A total of 22 specimens corresponding to dark morph forms of T. infestans were collected, and 14 were confirmed as T. infestans by the mitochondrial cytochrome B gene analysis. Through this analysis, one of which were previously reported and a second that was a new haplotype. Triatomines were captured from amongst vegetation such as dry branches and hollows trees of different species such Aspidosperma quebracho-blanco, Bulnesia sarmientoi and Stetsonia coryne. The colonies found have been small and without apparent infection with Trypanosoma cruzi. During the study, Triatoma sordida and Triatoma guasayana have also been found in ecotopes close to those of T. infestans.
Project description:Triatomines are hematophagous insects that transmit Trypanosoma cruzi, the etiological agent of Chagas disease. This neglected tropical disease represents a global health issue as it is spreading worldwide. The saliva of Triatominae contains miscellaneous proteins crucial for blood feeding acquisition, counteracting host's hemostasis while performing vasodilatory, anti-platelet and anti-coagulant activities, besides modulating inflammation and immune responses. Since a set of biological processes are mediated by protein complexes, here, the sialocomplexomes (salivary protein complexes) of five species of Triatominae were studied to explore the protein-protein interaction networks. Salivary multiprotein complexes from Triatoma infestans, Triatoma dimidiata, Dipetalogaster maxima, Rhodnius prolixus, and Rhodnius neglectus were investigated by Blue-Native- polyacrylamide gel electrophoresis coupled with liquid chromatography tandem mass spectrometry. More than 70 protein groups, uncovering the landscape of the Triatominae salivary interactome, were revealed. Triabin, actin, thioredoxin peroxidase and an uncharacterized protein were identified in sialocomplexes of the five species, while hexamerin, heat shock protein and histone were identified in sialocomplexes of four species. Salivary proteins related to triatomine immunity as well as those required during blood feeding process such as apyrases, antigen 5, procalins, and nitrophorins compose different complexes. Furthermore, unique proteins for each triatomine species were revealed. This study represents the first Triatominae sialocomplexome reference to date and shows that the approach used is a reliable tool for the analysis of Triatominae salivary proteins assembled into complexes.
Project description:The dataset in this report is related to the research article with the title: "Salivary gland transcripts of the kissing bug, Panstrongylus chinai, a vector of Chagas disease" (Kato et al., 2017) . Lipocalin family proteins were identified as the dominant component in P. chinai saliva, and phylogenetic analysis of the salivary lipocalins resulted in the formation of five major clades. For further characterization, each clade of P. chinai lipocalin was s alignment and phylogenetic analyses together with homologous triatomine lipocalins; pallidipin 2, an inhibitor of collagen-induced platelet aggregation identified from saliva of Triatoma pallidipennis (clade I), pallidipin-like salivary lipocalin from Triatoma dimidiata (clade II), salivary lipocalin from T. dimidiata (clade III), triatin-like salivary lipocalin identified in the saliva of T. dimidiata (clade IV), and lipocalin-like TiLipo37 from Triatoma infestans (clade V).
Project description:Chagas disease is a trypanosomiasis whose causative agent is the protozoan parasite Trypanosoma cruzi, which is transmitted to humans by hematophagous insects known as triatomines and affects a large proportion of South America. The digestive tract of the insect vectors in which T. cruzi develops constitutes a dynamic environment that affects the development of the parasite. Thus, we set out to investigate the chemical composition of the triatomine intestinal tract through a metabolomics approach. We performed Direct Infusion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry on fecal samples of three triatomine species (Rhodnius prolixus, Triatoma infestans, Panstrongylus megistus) fed with rabbit blood. We then identified groups of metabolites whose frequencies were either uniform in all species or enriched in each of them. By querying the Human Metabolome Database, we obtained putative identities of the metabolites of interest. We found that a core group of metabolites with uniform frequencies in all species represented approximately 80% of the molecules detected, whereas the other 20% varied among triatomine species. The uniform core was composed of metabolites of various categories, including fatty acids, steroids, glycerolipids, nucleotides, sugars, and others. Nevertheless, the metabolic fingerprint of triatomine feces differs depending on the species considered. The variable core was mainly composed of prenol lipids, amino acids, glycerolipids, steroids, phenols, fatty acids and derivatives, benzoic acid and derivatives, flavonoids, glycerophospholipids, benzopyrans, and quinolines. Triatomine feces constitute a rich and varied chemical medium whose constituents are likely to affect T. cruzi development and infectivity. The complexity of the fecal metabolome of triatomines suggests that it may affect triatomine vector competence for specific T. cruzi strains. Knowledge of the chemical environment of T. cruzi in its invertebrate host is likely to generate new ways to understand the factors influencing parasite proliferation as well as methods to control Chagas disease.
Project description:Triatomine bugs (Hemiptera: Reduviidae) are vectors of the flagellate Trypanosoma cruzi, the causative agent of Chagas disease. The study of triatomine gut microbiota has gained relevance in the last years due to its possible role in vector competence and prospective use in control strategies. The objective of this study is to examine changes in the gut microbiota composition of triatomines in response to a T. cruzi-infected blood meal and identifying key factors determining those changes.We sampled colony-reared individuals from six triatomine vectors (Panstrongylus megistus, Rhodnius prolixus, Triatoma brasiliensis, T. infestans, T. juazeirensis and T. sherlocki) comparing experimentally T. cruzi strain 0354-challenged and non-challenged insects. The microbiota of gut and gonad tissues was characterized using high throughput sequencing of region V3-V4 of bacterial 16S rRNA gene. The triatomine microbiota had a low intra-individual diversity, and a high inter-individual variation within the same host species. Arsenophonous appeared as the dominant triatomine bacterial symbiont in our study (59% of the total 16S coverage), but there were significant differences in the distribution of bacterial genera among vectors. In Rhodnius prolixus the dominant symbiont was Pectobacterium.Trypanosoma cruzi-challenge significantly affects microbiota composition, with challenged vectors harbouring a significantly more diverse bacterial community, both in the gut and the gonads. Our results show that blood-feeding with T. cruzi epimastigotes strongly affects microbiota composition in a species-specific manner. We suggest that triatomine-adapted enterobacteria such as Arsenophonus could be used as stable vectors for genetic transformation of triatomine bugs and control of Chagas disease.
Project description:BACKGROUND:Rapid reinfestation of insecticide-treated dwellings hamper the sustained elimination of Triatoma infestans, the main vector of Chagas disease in the Gran Chaco region. We conducted a seven-year longitudinal study including community-wide spraying with pyrethroid insecticides combined with periodic vector surveillance to investigate the house reinfestation process in connection with baseline pyrethroid resistance, housing quality and household mobility in a rural section of Pampa del Indio mainly inhabited by deprived indigenous people (Qom). METHODOLOGY/PRINCIPAL FINDINGS:Despite evidence of moderate pyrethroid resistance in local T. infestans populations, house infestation dropped from 31.9% at baseline to 0.7% at 10 months post-spraying (MPS), with no triatomine found at 59 and 78 MPS. Household-based surveillance corroborated the rare occurrence of T. infestans and the house invasion of other four triatomine species. The annual rates of loss of initially occupied houses and of household mobility were high (4.6-8.0%). Housing improvements did not translate into a significant reduction of mud-walled houses and refuges for triatomines because most households kept the former dwelling or built new ones with mud walls. CONCLUSIONS/SIGNIFICANCE:Our results refute the assumption that vector control actions performed in marginalized communities of the Gran Chaco are doomed to fail. The larger-than-expected impacts of the intervention program were likely associated with the combined effects of high-coverage, professional insecticide spraying followed by systematic vector surveillance-and-response, broad geographic coverage creating a buffer zone, frequent housing replacement and residential mobility. The dynamical interactions among housing quality, mobility and insecticide-based control largely affect the chances of vector elimination.
Project description:Integrated vector management strategies depend on local eco-bio-social conditions, community participation, political will and inter-sectorial partnership. Previously identified risk factors for persistent Triatoma dimidiata infestation include the presence of rodents and chickens, tiled roofs, dirt floors, partial wall plastering and dog density.A community-based intervention was developed and implemented based on cyclical stakeholder and situational analyses. Intervention implementation and evaluation combined participatory action research and cluster randomized pre-test post-test experimental designs. The intervention included modified insecticide application, education regarding Chagas disease and risk factors, and participatory rodent control.At final evaluation there was no significant difference in post-test triatomine infestation between intervention and control, keeping pre-test rodent and triatomine infestations constant. Knowledge levels regarding Chagas disease and prevention practices including rodent control, chicken management and health service access increased significantly only in intervention communities. The odds of nymph infection and rat infestation were 8.3 and 1.9-fold higher in control compared to intervention communities, respectively.Vector control measures without reservoir control are insufficient to reduce transmission risk in areas with persistent triatomine infestation. This integrated vector management program can complement house improvement initiatives by prioritizing households with risk factors such as tiled roofs. Requirement for active participation and multi-sectorial coordination poses implementation challenges.