Blood meal sources of wild and domestic Triatoma infestans (Hemiptera: Reduviidae) in Bolivia: connectivity between cycles of transmission of Trypanosoma cruzi.

BACKGROUND: Chagas disease is a major public health problem in Latin America. Its etiologic agent, Trypanosoma cruzi, is mainly transmitted through the contaminated faeces of blood-sucking insects called triatomines. Triatoma infestans is the main vector in various countries in South America and recently, several foci of wild populations of this species have been described in Bolivia and other countries. These wild populations are suspected of affecting the success of insecticide control campaigns being carried out in South America. To assess the risk that these T. infestans populations pose to human health, it is helpful to determine blood meal sources. METHODS: In the present work, blood meals were identified in various Bolivian wild T. infestans populations and in three specific areas, in both wild and intra-peridomestic populations to assess the links between wild and domestic cycles of T. cruzi transmission. PCR-HDA and sequencing of Cytb gene were used to identify these blood meal sources. RESULTS AND DISCUSSION: Fourteen vertebrate species were identified as wild blood meal sources. Of those, the most prevalent species were two Andean endemic rodents, Octodontomys gliroides (36%) and Galea musteloides (30%), while humans were the third most prevalent source (18.7%). Of 163 blood meals from peridomestic areas, more than half were chickens, and the others were generally domestic animals or humans. Interestingly, blood from wild animals was identified in triatomines captured in the peridomestic and domestic environment, and blood from domestic animals was found in triatomines captured in the wild, revealing links between wild and domestic cycles of T. cruzi transmission. CONCLUSION: The current study suggests that wild T. infestans attack humans in the wild, but is also able to bite humans in domestic settings before going back to its natural environment. These results support the risk to human health posed by wild populations of T. infestans.

Putative panmixia in restricted populations of Trypanosoma cruzi isolated from wild Triatoma infestans in Bolivia.

Trypanosoma cruzi, the causative agent of Chagas disease, is subdivided into six discrete typing units (DTUs; TcI-TcVI) of which TcI is ubiquitous and genetically highly variable. While clonality is the dominant mode of propagation, recombinant events play a significant evolutive role. Recently, foci of wild Triatoma infestans have been described in Bolivia, mainly infected by TcI. Hence, for the first time, we evaluated the level of genetic exchange within TcI natural potentially panmictic populations (single DTU, host, area and sampling time). Seventy-nine TcI stocks from wild T. infestans, belonging to six populations were characterized at eight microsatellite loci. For each population, Hardy-Weinberg equilibrium (HWE), linkage disequilibrium (LD), and presence of repeated multilocus genotypes (MLG) were analyzed by using a total of seven statistics, to test the null hypothesis of panmixia (H0). For three populations, none of the seven statistics allowed to rejecting H0; for another one the low size did not allow us to conclude, and for the two others the tests have given contradictory results. Interestingly, apparent panmixia was only observed in very restricted areas, and was not observed when grouping populations distant of only two kilometers or more. Nevertheless it is worth stressing that for the statistic tests of "HWE", in order to minimize the type I error (i. e. incorrect rejection of a true H0), we used the Bonferroni correction (BC) known to considerably increase the type II error ( i. e. failure to reject a false H0). For the other tests (LD and MLG), we did not use BC and the risk of type II error in these cases was acceptable. Thus, these results should be considered as a good indicator of the existence of panmixia in wild environment but this must be confirmed on larger samples to reduce the risk of type II error.

Wild populations of Triatoma infestans are highly connected to intra-peridomestic conspecific populations in the Bolivian Andes.

Triatoma infestans, the major vector of Chagas disease south of the Amazon in South America, has a large distribution of wild populations, contrary to what has previously been stated. These populations have been suspected of being the source of reinfestation of human habitats and could impede the full success of vector control campaigns. This study examined gene flow between intra-peridomestic populations and wild populations collected in the surround areas in three Andean localities in Bolivia. The populations were defined according to temporal, ecological, and spatial criteria. After DNA extraction from the legs of each insect, the samples were analyzed using seven microsatellite markers. First, the analysis of molecular variance (AMOVA) detected an absence of differentiation between wild and intra-peridomestic populations, although strong structuring was observed between the populations within each environment. Then for some populations, the Bayesian method of assignment to inferred populations showed very similar assignment patterns of the members of wild or intra-peridomestic populations in each locality. Finally, the detection of the first-generation migrants within the different populations provided evidence of insect displacement from the wild to the intra-peridomestic environment. This result indicates that, after control campaigns in the Andes, controlling this new paradigm of vector transmission risk stemming from the invasion of human habitats by wild populations of T. infestans requires long-term maintenance of public monitoring to keep the risk at a minimal level. Since wild populations of T. infestans have also been detected elsewhere in Argentina, Paraguay, and Chile, there is an urgent need to take these populations into account in future monitoring of Chagas disease transmission.

Risk of transmission of Trypanosoma cruzi by wild Triatoma infestans (Hemiptera: Reduviidae) in Bolivia supported by the detection of human blood meals.

We analyzed the food sources of Bolivian wild Triatoma infestans (the main vector of Chagas disease in this country), to assess the role of these populations in the epidemiological context of Chagas disease. Ninety-eight blood meals were identified by heteroduplex assay and sequencing. Most of them were from wild mammals but surprisingly 27 were from humans. This brings to light the occurrence of human-vector contacts at risk of Trypanosoma cruzi transmission in the wild environment by highly infected insects.

Combination of cytochrome b heteroduplex-assay and sequencing for identification of triatomine blood meals.

The identification of blood meals in vectors contributes greatly to the understanding of interactions between vectors, microorganisms and hosts. The aim of the current work was to complement the validation of cytochrome b (Cytb) heteroduplex assay (HDA) previously described, and to add the sequencing of the Cytb gene of some samples for the identification of blood meals in triatomines. Experimental feedings of reared triatomines helped to clarify the sensitivity of the HDA. Moreover, the sequencing coupled with the HDA, allowed the assessment of the technique's taxonomic level of discrimination. The primers used to produce DNA fragments of Cytb genes for HDA had a very high sensitivity for vertebrate DNAs, rather similar for mammals, birds and reptiles. However, the formation of heteroduplex depended on blood meal's quality rather than its quantity; a correlation was observed between blood meals' color and the positivity of HDA. HDA electrophoresis profiles were reproducible, and allowed the discrimination of blood origins at the species level. However, in some cases, intraspecific variability of Cytb gene generated different HDA profiles. The HDA based on comparison of electrophoresis profiles is a very useful tool for screening large samples to determine blood origins; the subsequent sequencing of PCR products of Cytb corresponding to different HDA profiles allowed the identification of species whatever the biotope in which the vectors were captured.

Population genetic structure of Meccus longipennis (Hemiptera, Reduviidae, Triatominae), vector of Chagas disease in West Mexico.

The originally wild species of the Meccus complex are important vectors of Chagas disease in Mexico. In West Mexico, Meccus longipennis plays an important epidemiological role. To understand the genetic structure of the domestic and wild populations of this species, a preliminary study with five polymorphic microsatellite loci was conducted. The population genetics analysis showed high structuring between peridomestic biotopes, with breeding subunits detected in a single peridomestic structure. In the wild environment, two genetic patterns were observed according to the biotope, possible breeding subunits in large rocky formations and a larger panmictic unit in agropastoral areas, suggesting considerable dispersal of bugs in this biotope. Moreover, the discovery of two foci of wild populations at the edge of Guadalajara city raises the question of new urban areas where the phenomenon of bug incursions into households could constitute a risk of transmission of Chagas disease.

Trypanosoma cruzi discrete typing units (DTUs): microsatellite loci and population genetics of DTUs TcV and TcI in Bolivia and Peru.

Trypanosoma cruzi, the agent of Chagas disease, is usually subdivided into six discrete typing units (DTUs), TcI to TcVI, among which TcI and TcV are most common in human infections in Bolivia. Multilocus microsatellite typing (MLMT) was selected to further explore the structure of the natural populations belonging to these DTUs. The analysis showed that microsatellite clustering does not fully match the six DTUs, but it is relevant for the within DTUs analyses. Population genetics analysis was conducted on 11 relevant subsamples of stocks from Bolivia and Peru, belonging to TcI (6) and TcV (5), defined by four criterions: DTU, vector species, geographic origin, and date of isolation. Most TcV strains presented the same multilocus genotype over all subsamples with the puzzling characteristic that five loci were heterozygous and the other five homozygous. In TcI, four clusters were defined according to the vector species. Most of them appeared in agreement with clonal propagation (stocks isolated from Triatoma infestans and Triatoma sordida), while a few highly homozygous stocks (e.g. those isolated from Rhodnius stali) suggested that scarce sex events can occur. The poor role played by spatio-temporal factors in describing the observed genetic diversity suggested that ecology, in particular as regard to host played a significant role. These results highlight the extreme heterogeneity of T. cruzi and suggest that further population genetics surveys will need to target the most possible precise spatio-temporal and ecological scales.

New insights on the Chagas disease main vector Triatoma infestans (Reduviidae, Triatominae) brought by the genetic analysis of Bolivian sylvatic populations.

Triatoma infestans is the main and most widespread vector of Chagas disease in South America. For the first time, a large sample of sylvatic populations of T. infestans was analyzed by ITS-2 and mtCytB sequencing. ITS-2 showed a low level of polymorphism but revealed a dichotomy between the Andean and non-Andean sylvatic populations. On the contrary, mtCytB sequences showed a high polymorphism (19 haplotypes determined by 35 variable sites) revealing a strong structuring between most of the sylvatic populations and possible ancient isolation and bottleneck in the Northern Andes. The dichotomy Andean vs. non-Andean populations was not observed with this marker. Moreover, mtCytB haplotype genealogies showed that the non-Andean haplotypes would have derived from the Andean ones, supporting somewhat an Andean origin of the species. Nevertheless, a non-Andean origin could not be discarded because a remarkable genetic diversity was found in the non-Andean sample. The comparison of the sylvatic haplotypes with the domestic ones from GenBank suggested multiple events of T. infestans domestication in Andean and non-Andean areas, instead of a major and unique domestication event in the Bolivian Andes, as previously proposed.

Community participation and domiciliary occurrence of infected Meccus longipennis in two Mexican villages in Jalisco state.

The entomological features of Chagas disease in two western Mexican villages were analyzed through triatomines collection by the inhabitants and active research in the peridomicile. The inhabitant collections have the following comparable characteristics: 1) Meccus longipennis was the dominant species (> 91%), 2) around 43% of the insects were collected indoors, 3) about 70% of triatomines were adults, 4) cumulated rates of infestation of the dwellings reached 40-50%, 5) the triatomine infection rate by Trypanosoma cruzi was > 50%, and 6) the indoor triatomines frequently feed on humans (range 38.5-56.2%). However, the collection was twice as abundant in the first village and the peridomicile infestation, evaluated by the active collection, reached up to 60% and only 4.9% in the other village. Furthermore, females predominated in the first village, whereas males in the other. The current results allow discussing the course of action to prevent Chagas disease in this region.

First report of widespread wild populations of Triatoma infestans (Reduviidae, Triatominae) in the valleys of La Paz, Bolivia.

Wild populations of Triatoma infestans, the main vector of Chagas disease in the Southern Cone countries, may be involved in reinfestation of human dwellings, limiting the success of vector-control campaigns in Bolivia. Knowledge of the distribution of these populations remains incomplete. We report here the detection of T. infestans wild populations in large areas in the department of La Paz, Bolivia. Among 18 sylvatic areas investigated, 17 were positive with T. infestans specimens. The infection rate of captured T. infestans with Trypanosoma cruzi was 85.7% in adult specimens. These results expand the geographical distribution of wild populations of T. infestans; it may be distributed throughout the Inter-Andean Dry Forest eco-region of Bolivia. The current information allows us to propose the hypothesis that a sylvatic origin of the reinfestation is located in the valleys of La Paz.

Wild ecotopes and food habits of Triatoma longipennis infected by Trypanosoma cruzi lineages I and II in Mexico.

The control of wild triatomine populations that can invade dwellings is a major challenge for Chagas disease control in Mexico, but a better knowledge of the biology of these populations is required to develop appropriate control methods. We describe a new terrestrial ecotope of Triatoma longipennis, a principal vector in the occidental part of Mexico, in addition to its previously identified niche in rock pile boundary walls. Analysis of feeding hosts in the two ecotopes showed that this species is able to diversify its food sources outside of the principal hosts, Dasypus novemcinctus and Procyon lotor, and to disperse in search of new meals. Moreover, T. longipennis are strongly infected not only by the Trypanosoma cruzi I lineage found in the domestic cycle, but also by T. cruzi lineage II. The impact of T. cruzi II on human infection remains to be determined.

Conocimiento epidemiológico y situación actual de la enfermedad de Chagas en el estado de Jalisco, México / Epidemiologic knowledge and current situation of Chagas disease in the state of Jalisco, Mexico

La enfermedad de Chagas en el estado de Jalisco, México, apareció por primera vez en 1967, aunque su conocimiento ha seguido un proceso lento. Entre los años de 1967 y 2006 se describió la enfermedad en sus formas agudas y crónicas; se identificaron las especies de vectores y se aisló el parásito Trypanosoma cruzi, que luego se caracterizó en el plano genético. La magnitud de la infección en el hombre se determinó con estudios serológicos en diversas poblaciones, así como en donadores de sangre. En la actualización presente del conocimiento de la enfermedad en el estado de Jalisco se mostró la necesidad de incrementar las investigaciones sobre la epidemiología de la enfermedad de Chagas, así como los estudios clínicos para determinar la salud de los individuos y las poblaciones.

Chagas disease in the state of Jalisco, Mexico was described for the first time in 1967; however, knowledge on the disease remains in a slow process. Between 1967 and 2006, the disease was described in its acute and chronic forms. The vector species have been identified, and the parasite Trypanosoma cruzi has been isolated and genetically characterized. Also, the magnitude of the infection in humans has been determined through serological studies of different populations as well as of blood donors. The up-to-dateness of knowledge of the disease in the state of Jalisco, unveils a necessity of increased research on the epidemiology of Chagas disease as well as on clinical studies to assess the health of individuals and the populations.

[Epidemiologic knowledge and current situation of Chagas disease in the state of Jalisco, Mexico]. / Conocimiento epidemiológico y situación actual de la enfermedad de Chagas en el estado de Jalisco, México.

Chagas disease in the state of Jalisco, Mexico was described for the first time in 1967; however, knowledge on the disease remains in a slow process. Between 1967 and 2006, the disease was described in its acute and chronic forms. The vector species have been identified, and the parasite Trypanosoma cruzi has been isolated and genetically characterized. Also, the magnitude of the infection in humans has been determined through serological studies of different populations as well as of blood donors. The up-to-dateness of knowledge of the disease in the state of Jalisco, unveils a necessity of increased research on the epidemiology of Chagas disease as well as on clinical studies to assess the health of individuals and the populations.

Exploring the FL-160-CRP gene family through sequence variability of the complement regulatory protein (CRP) expressed by the trypomastigote stage of Trypanosoma cruzi.

The complement regulatory protein (CRP) of Trypanosoma cruzi is a surface glycoprotein which confers to the infectious trypomastigote forms a protection against the lytic activity of the host complement. CRP belongs to the large family of the trans-sialidase-like proteins and its sequence is highly similar to those of the flagellar FL-160 and chronic exoantigen proteins, encoded by a multigene family. To further define the gene family encoding the CRP, we investigated the protein diversity among several strains of T. cruzi through the sequencing of trypomastigote transcripts, and used a phylogenetic analysis based on the multiple alignment of these proteins with the top scoring sequences detected by a database sequence homology search. Intrastrain variations in CRP sequences revealed the existence of several copies per strain. The interstrain variability of CRP was consistent with the genetic subdivisions of T. cruzi into lineages and discrete typing units. The phylogenetic analysis based on a 227 amino acid alignment of CRP sequences with the 200 putative proteins retrieved from the protein databases (including the sequences from the T. cruzi genome project) revealed that the CRP sequences clustered with the FL-160 proteins into a monophyletic group characterized by the presence of the 12 amino acid mimicry epitope that mimics nervous tissues. The phylogeny did not differentiate between the CRP and the FL-160 proteins. The identification of this group of CRP-like proteins and the high sequence similarity observed within it open up new prospects for the exploration of the localization, structure and function of these proteins and a better understanding of their involvement in key aspects of host-parasite interactions, such as the resistance to the complement. This work provides also information for the T. cruzi genome annotation of the trans-sialidase-like putative proteins.

Sequence diversity and differential expression of Tc52 immuno-regulatory protein in Trypanosoma cruzi: potential implications in the biological variability of strains.

Trypanosoma cruzi is highly heterogeneous in terms of genetics and biological properties. To explore the diversity of T. cruzi, we focused our study on the T. cruzi Tc52 protein playing a critical immunosuppressive role during infection. Sequence variability and expression levels of this virulence factor were analysed in various strains. Among the 40 amino acid substitutions detected in the Tc52 coding sequences, three substitutions may have an impact on protein activity or function, as two are localized in sites involved in the glutathione binding and the third is present in the region bearing immunomodulatory function. This sequence variability was consistent with the genetic subdivisions of T. cruzi. Moreover, we observed that the level of Tc52 transcripts and proteins varied between the different strains, but we did not find a significant correlation between Tc52 expression and the phylogeny of the parasite. Thus, both diversity in the sequences and differences in the expression levels of Tc52 protein may be involved in the biological variability of T. cruzi, especially in virulence and immunosuppression properties of T. cruzi strains.

Genetic clustering of Trypanosoma cruzi I lineage evidenced by intergenic miniexon gene sequencing.

American trypanosomiasis or Chagas disease is endemic in Latin America and caused by the flagellate Trypanosoma cruzi, which exhibits broad genetic variation. In various areas, the transmission of Chagas disease is ensured by sylvatic vectors, mainly carrying the evolutionary lineage I of T. cruzi. Despite its epidemiological importance, this lineage is poorly studied. Here, we investigated the genetic variability and the phylogenetic relationships within T. cruzi I using sequences of the non-transcribed spacer of miniexon genes. The variability was firstly analysed between 10 repeats of spacer-miniexon genes in two strains of T. cruzi I and in the CL Brener strain, showing lower intra-strain variability than inter-strain. Furthermore, the phylogenetic analysis of 19 T. cruzi I strains (49 copies in total) clusters the copies into at least three groups. Two evolutionary phenomena can be proposed to explain the partition of the strains: (i) an association between strains and Didelphis sp. hosts and (ii) geographical clustering between the North American and South American strains. Thereby, the miniexon gene is an attractive marker to establish the phylogeny of lineage I and explore relationships between T. cruzi and mammal hosts.

Peridomicilary habitat and risk factors for Triatoma infestation in a rural community of the Mexican occident.

An examination of peridomestic area organization and triatomine collection in an endemic village for Chagas disease (Jalisco State) identified the habitat of Triatoma longipennis (dominant species) and the risk factors of peridomestic infestation. In 100 visited peridomestic areas, 369 structures (permanent, temporary, and natural) were submitted to active manual research of triatomines. Storage shelters had a higher infestation of T. longipennis than piles of brick and tile; baked clay material had higher degrees of infestation than others. The secondary species Triatoma barberi shares a wide range of peridomicilary habitats with T. longipennis. Peridomestic area infestation risks (evaluated with multivariate logistic regression analysis) are number of closed storage shelters, number of brick and tile piles, number of houses per peridomestic areas, and distance of peridomicile from natural environment. Because both species present great adaptability to different artificial habitats, strategies of control must involved improving the overall management of peridomestic areas to prevent stable colonization.

Detection of Chagas infections using Trypanosoma evansi crude antigen demonstrates high cross-reactions with Trypanosoma cruzi.

Antigenic similarities between salivarian trypanosomes are known for a long time, but similarities between salivarian and stercorarian trypanosomes have been very little investigated. Phylogenetically, these genus and species appear to be far. However, in a preliminary work we had shown strong reactions of chagasic human sera using T. evansi antigens in Western-blotting and ELISA. In the current work an ELISA test using T. evansi crude antigens was probed with one hundred and two sera of chagasic Bolivian patients previously diagnosed which presented different pathologies. The sensitivity of the ELISA T. evansi was 92.6% similar to that of ELISA T. cruzi. The specificity evaluated using 20 sera of patients infected by Leishmania sp. reaches a comparable value of that obtained with the T. cruzi immunofluorescent assay. Finally, the sensitivity and the specificity of the ELISA T. evansi were not really different from conventional serology of Chagas. In spite of their taxonomic position in various sections and their old divergence, these observations prove a strong antigenic community between T. cruzi and T. evansi. Consequently, the common antigens which remain to be characterized, could be an alternative source of antigen for the detection of antibodies against T. cruzi. Given that T. evansi seems to have strong antigenic communities with the majority of the pathogenic current trypanosomoses of mammals, it is very attractive to identify and characterize these highly conserved antigens which could be suitable targets to develop tools for diagnosis, prophylaxy and chemotherapy against several human and animal trypanosomoses.

Peridomestic colonization of Triatoma longipennis (Hemiptera, Reduviidae) and Triatoma barberi (Hemiptera, Reduviidae) in a rural community with active transmission of Trypanosoma cruzi in jalisco state, Mexico.

Infestation of peridomiciles is likely a major risk factor for Chagas disease transmission in Jalisco state, Mexico. An entomological and serological survey of a typical village was conducted between July and September 2003. The peridomestic areas of 100 dwellings were visited and triatomines were searched manually in 369 potential sites. A total of 1821 Triatoma longipennis (93.2%) or Triatoma barberi was captured. Both species frequently occurred in sympatry. The infestation index was 60% for T. longipennis and 16% for T. barberi. T. longipennis occurred throughout the village. Colonization indices were high for T. longipennis (93%) and T. barberi (75%), suggesting that both species have adapted to peridomestic habitats. The bug population size was larger for T. longipennis than for T. barberi. Five very large colonies of T. longipennis were recorded whereas only 1 or 2 bugs were observed in 38% of the positive sites, which suggests intense dispersal activity. Both species exhibited high infection prevalence with Trypanosoma cruzi (46%). Only T. cruzi lineage I was detected. Human seroprevalence was 1.8%. This study serves as an entomological overview of peridomiciliar triatomine colonization in a Mexican village and highlights the current risk of Chagas disease transmission.

Polymerase chain reaction detection and serologic follow-up after treatment with benznidazole in Bolivian children infected with a natural mixture of Trypanosoma cruzi I and II.

Thirty-five Bolivian children (5-10 years of age) seropositive for infection with T. cruzi underwent specific chemotherapy with benznidazole. Before treatment, 57.1% had a positive parasitologic diagnosis. Some patients presented an early conversion by polymerase chain reaction of blood samples, while others were still positive four and seven months after the end of the treatment, which indicated an absence of parasite clearance. Strain typing showed that most patients were infected by a mixture of clones I and II of T. cruzi. Serologic conversion in conventional tests and antibodies to shed acute-phase antigen were observed in two and four patients, respectively. For the other patients, the average rate of antibody decay was half the initial rate. The parasitologic and serologic data indicated that chemotherapy acts throughout the course of infection in a long-lasting process in which the decrease of specific antibody production is related to the reduction of the live parasite load.