Distribution of sibling species of Anopheles culicifacies s.l. and Anopheles fluviatilis s.l. and their vectorial capacity in eight different malaria endemic districts of Orissa, India

The study was undertaken in eight endemic districts of Orissa, India, to find the members of the species complexes of Anopheles culicifacies and Anopheles fluviatilis and their distribution patterns. The study area included six forested districts (Keonjhar, Angul, Dhenkanal, Ganjam, Nayagarh and Khurda) and two non-forested coastal districts (Puri and Jagatsingpur) studied over a period of two years (June 2007-May 2009). An. culicifacies A, B, C and D and An. fluviatilis S and T sibling species were reported. The prevalence of An. culicifacies A ranged from 4.2-8.41%, B from 54.96-76.92%, C from 23.08-33.62% and D from 1.85-5.94% (D was reported for the first time in Orissa, except for occurrences in the Khurda and Nayagarh districts). The anthropophilic indices (AI) were 3.2-4.8%, 0.5-1.7%, 0.7-1.37% and 0.91-1.35% for A, B, C and D, respectively, whereas the sporozoite rates (SR) were 0.49-0.54%, 0%, 0.28-0.37% and 0.41-0.46% for A, B, C and D, respectively. An. fluviatilis showed a similarly varied distribution pattern in which S was predominant (84.3% overall); its AI and SR values ranged from 60.7-90.4% and 1.2-2.32%, respectively. The study observed that the co-existence of potential vector sibling species of An. culicifacies (A, C and D) and An. fluviatilis S (> 50%) was responsible for the high endemicity of malaria in forested districts such as Dhenkanal, Keonjhar, Angul, Ganjam, Nayagarh and Khurda (> 5% slide positivity rate). Thus, the epidemiological scenario for malaria is dependent on the distribution of the vector sibling species and their vectorial capacity.

Confirmation of Anopheles (Anopheles) calderoni Wilkerson, 1991 (Diptera: Culicidae) in Colombia and Ecuador through molecular and morphological correlation with topotypic material

The morphologically similar taxa Anopheles calderoni, Anopheles punctimacula, Anopheles malefactor and Anopheles guarao are commonly misidentified. Isofamilies collected in Valle de Cauca, Colombia, showed morphological characters most similar to An. calderoni, a species which has never previously been reported in Colombia. Although discontinuity of the postsubcostal pale spots on the costa (C) and first radial (R1) wing veins is purportedly diagnostic for An. calderoni, the degree of overlap of the distal postsubcostal spot on C and R1 were variable in Colombian specimens (0.003-0.024). In addition, in 98.2% of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7% of specimens examined. Correlation of DNA sequences of the second internal transcribed spacer and mtDNA cytochrome c oxidase subunit I gene (COI) barcodes (658 bp of the COI gene) generated from Colombian progeny material and wild-caught mosquitoes from Ecuador with those from the Peruvian type series of An. calderoni confirmed new country records. DNA barcodes generated for the closely related taxa, An. malefactor and An. punctimacula are also presented for the first time. Examination of museum specimens at the University of the Valle, Colombia, revealed the presence of An. calderoni in inland localities across Colombia and at elevations up to 1113 m.

A new mtDNA COI gene lineage closely related to Anopheles janconnae of the Albitarsis complex in the Caribbean region of Colombia

An understanding of the taxonomic status and vector distribution of anophelines is crucial in controlling malaria. Previous phylogenetic analyses have supported the description of six species of the Neotropical malaria vector Anopheles (Nyssorhynchus) albitarsis s.l. (Diptera: Culicidae): An. albitarsis, Anopheles deaneorum, Anopheles marajoara, Anopheles oryzalimnetes, Anopheles janconnae and An. albitarsis F. To evaluate the taxonomic status of An. albitarsis s.l. mosquitoes collected in various localities in the Colombian Caribbean region, specimens were analyzed using the complete mitochondrial DNA cytochrome oxidase I (COI) gene, the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) region and partial nuclear DNA white gene sequences. Phylogenetic analyses of the COI gene sequences detected a new lineage closely related to An. janconnae in the Caribbean region of Colombia and determined its position relative to the other members of the complex. However, the ITS2 and white gene sequences lacked sufficient resolution to support a new lineage closely related to An. janconnae or the An. janconnae clade. The possible involvement of this new lineage in malaria transmission in Colombia remains unknown, but its phylogenetic closeness to An. janconnae, which has been implicated in local malaria transmission in Brazil, is intriguing.

In vitro effects of citral on Trypanosoma cruzi metacyclogenesis

Citral, the main constituent of lemongrass (Cymbopogon citratus) essential oil, was added to Trypanosoma cruzi cultures grown in TAU3AAG medium to observe the effect on the epimastigote-to-trypomastigote differentiation process (metacyclogenesis). Our results showed that citral (20 μg/mL) did not affect epimastigote viability or inhibit the differentiation process. Concentrations higher than 60 μg/mL, however, led to 100% cell death (both epimastigote and trypomastigote forms). Although epimastigotes incubated with 30 μg/mL citral were viable and able to adhere to the substrate, we observed around 50% inhibition in metacyclogenesis, with a calculated concentration that inhibited metacyclogenesis by 50% after 24 h (IC50/24 h) of about 31 μg/mL. Treatment with 30 μg/mL citral did not hinder epimastigote multiplication because epimastigote growth resumed when treated cells were transferred to a drug-free liver infusion tryptose culture medium. Metacyclogenesis was almost totally abolished at 40 μg/mL after 24 h of incubation. Furthermore, the metacyclic trypomastigotes obtained in vitro were similarly susceptible to citral, with an IC50/24 h, concentration that killed 50% of the cells after 24 h, of about 24.5 μg/mL. Therefore, citral appears to be a good candidate as an inhibitory drug for further studies analyzing the T. cruzi metacyclogenesis process.

Developmental and reproductive patterns of Triatoma brasiliensis infected with Trypanosoma cruzi under laboratory conditions

The aim of this work was to study the interaction between Trypanosoma cruzi-1 and Triatoma brasiliensis. A group of 1st instar nymphs was initially fed on T. cruzi-infected mice and a control group was fed on uninfected mice. From the second feeding onwards, both groups were otherwise fed on non-infected mice. The resulting adults were grouped in pairs: infected male/uninfected female, uninfected male/infected female, infected male and female and uninfected male/uninfected female. The infection affected only the 1st instar nymphs, which took significantly more time to reach the 2nd instar than uninfected nymphs. The differences in the molting time between the infected and uninfected nymphs from the 2nd to the 5th instars were not statistically significant. Both groups presented similar rates of nymphal mortality and reproductive performance was not significantly affected by infection in any of the treatments.

Vaccination with Trypanosoma rangeli modulates the profiles of immunoglobulins and IL-6 at local and systemic levels in the early phase of Trypanosoma cruzi experimental infection

In America, there are two species of Trypanosoma that can infect humans: Trypanosoma cruzi, which is responsible for Chagas disease and Trypanosoma rangeli, which is not pathogenic. We have developed a model of vaccination in mice with T. rangeli epimastigotes that protects against T. cruzi infection. The goal of this work was to study the pattern of specific immunoglobulins in the peritoneum (the site of infection) and in the sera of mice immunized with T. rangeli before and after challenge with T. cruzi. Additionally, we studied the effects triggered by antigen-antibodies binding and the levels of key cytokines involved in the humoral response, such as IL-4, IL-5 and IL-6. The immunization triggered the production of antibodies reactive with T. cruzi in peritoneal fluid (PF) and in serum, mainly IgG1 and, to a lesser magnitude, IgG2. Only immunized mice developed specific IgG3 antibodies in their peritoneal cavities. Antibodies were able to bind to the surface of the parasites and agglutinate them. Among the cytokines studied, IL-6 was elevated in PF during early infection, with higher levels in non-immunized-infected mice. The results indicate that T. rangeli vaccination against T. cruzi infection triggers a high production of specific IgG isotypes in PF and sera before infection and modulates the levels of IL-6 in PF in the early periods of infection.

Characterization of TcSTI-1, a homologue of stress-induced protein-1, in Trypanosoma cruzi

The life cycle of the protozoan Trypanosoma cruzi exposes it to several environmental stresses in its invertebrate and vertebrate hosts. Stress conditions are involved in parasite differentiation, but little is known about the stress response proteins involved. We report here the first characterization of stress-induced protein-1 (STI-1) in T. cruzi (TcSTI-1). This co-chaperone is produced in response to stress and mediates the formation of a complex between the stress proteins HSP70 and HSP90 in other organisms. Despite the similarity of TcSTI-1 to STI-1 proteins in other organisms, its expression profile in response to various stress conditions, such as heat shock, acidic pH or nutrient starvation, is quite different. Neither polysomal mRNA nor protein levels changed in exponentially growing epimastigotes cultured under any of the stress conditions studied. Increased levels of TcSTI-1 were observed in epimastigotes subjected to nutritional stress in the late growth phase. Co-immunoprecipitation assays revealed an association between TcSTI-1 and TcHSP70 in T. cruzi epimastigotes. Immunolocalization demonstrated that TcSTI-1 was distributed throughout the cytoplasm and there was some colocalization of TcSTI-1 and TcHSP70 around the nucleus. Thus, TcSTI-1 associates with TcHSP70 and TcSTI-1 expression is induced when the parasites are subjected to stress conditions during specific growth phase.

Trypanosoma cruzi: parasite persistence in tissues in chronic chagasic Brazilian patients

Chagas disease in the chronic phase may develop into cardiac and/or digestive forms. The pathogenesis of the disease is not yet clear and studies have been carried out to elucidate the role of parasite persistence in affected organs. The aim of this study was to detect and quantify Trypanosoma cruzi in paraffin-embedded tissue samples from chronic patients using NPCR (nested polymerase chain reaction) and QPCR (quantitative polymerase chain reaction) methods. These results were correlated to anatomopathological alterations in the heart and gastrointestinal tract (GIT). Of the 23 patients studied, 18 presented the cardiac form and five presented the cardiodigestive form of Chagas disease. DNA samples were randomly isolated from formalin-fixed paraffin-embedded sections of heart and GIT tissue of 23 necropsies and were analyzed through NPCR amplification. T. cruzi DNA was detected by NPCR in 48/56 (85.7%) heart and 35/42 (83.3%) GIT samples from patients with the cardiac form. For patients with the cardiodigestive form, NPCR was positive in 12/14 (85.7%) heart and in 14/14 (100%) GIT samples. QPCR, with an efficiency of 97.6%, was performed in 13 samples (11 from cardiac and 2 from cardiodigestive form) identified previously as positive by NPCR. The number of T. cruzi copies was compared to heart weight and no statistical significance was observed. Additionally, we compared the number of copies in different tissues (both heart and GIT) in six samples from the cardiac form and two samples from the cardiodigestive form. The parasite load observed was proportionally higher in heart tissues from patients with the cardiac form. These results show that the presence of the parasite in tissues is essential to Chagas disease pathogenesis.

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0% oocyst rates were obtained, in comparison to the 86.67-100% oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0% sporozoite rates were obtained, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09%, 6.67% and 11.76% sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67% and 64.29% sporozoite rates were obtained, respectively, in comparison to 90% sporozoite rates recovered from An. cracens.

Studying nanotoxic effects of CdTe quantum dots in Trypanosoma cruzi

Semiconductor nanoparticles, such as quantum dots (QDs), were used to carry out experiments in vivo and ex vivo with Trypanosoma cruzi. However, questions have been raised regarding the nanotoxicity of QDs in living cells, microorganisms, tissues and whole animals. The objective of this paper was to conduct a QD nanotoxicity study on living T. cruzi protozoa using analytical methods. This was accomplished using in vitro experiments to test the interference of the QDs on parasite development, morphology and viability. Our results show that after 72 h, a 200 μM cadmium telluride (CdTe) QD solution induced important morphological alterations in T. cruzi, such as DNA damage, plasma membrane blebbing and mitochondrial swelling. Flow cytometry assays showed no damage to the plasma membrane when incubated with 200 μM CdTe QDs for up to 72 h (propidium iodide cells), giving no evidence of classical necrosis. Parasites incubated with 2 μM CdTe QDs still proliferated after seven days. In summary, a low concentration of CdTe QDs (2 μM) is optimal for bioimaging, whereas a high concentration (200 μM CdTe) could be toxic to cells. Taken together, our data indicate that 2 μM QD can be used for the successful long-term study of the parasite-vector interaction in real time.

Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

Trypanosoma cruzi, a protozoan parasite that causes Chagas disease, exhibits unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes, RNA editing and trans-splicing. In the absence of mechanism controlling transcription initiation, organized subsets of T. cruzi genes must be post-transcriptionally co-regulated in response to extracellular signals. The mechanisms that regulate stage-specific gene expression in this parasite have become much clearer through sequencing its whole genome as well as performing various proteomic and microarray analyses, which have demonstrated that at least half of the T. cruzi genes are differentially regulated during its life cycle. In this review, we attempt to highlight the recent advances in characterising cis and trans-acting elements in the T. cruzi genome that are involved in its post-transcriptional regulatory machinery.

Carbon dioxide baited trap catches do not correlate with human landing collections of Anopheles aquasalis in Suriname

Three types of carbon dioxide-baited traps, i.e., the Centers for Disease Control Miniature Light Trap without light, the BioGents (BG) Sentinel Mosquito Trap (BG-Sentinel) and the Mosquito Magnet® Liberty Plus were compared with human landing collections in their efficiency in collecting Anopheles (Nyssorhynchus) aquasalis mosquitoes. Of 13,549 total mosquitoes collected, 1,019 (7.52%) were An. aquasalis. Large numbers of Culex spp were also collected, in particular with the (BG-Sentinel). The majority of An. aquasalis (83.8%) were collected by the human landing collection (HLC). None of the trap catches correlated with HLC in the number of An. aquasalis captured over time. The high efficiency of the HLC method indicates that this malaria vector was anthropophilic at this site, especially as carbon dioxide was insufficiently attractive as stand-alone bait. Traps using carbon dioxide in combination with human odorants may provide better results.

A high corticosterone/DHEA-s ratio in young rats infected with Trypanosoma cruzi is associated with increased susceptibility

We have previously established that young male rats are more susceptible to the effects of Trypanosoma cruzi infection than adult rats. To explore underlying age-associated differences in disease outcome, we simultaneously assessed hormone levels and cytokine release throughout the acute infection period in young and adult rats infected with T. cruzi. Young rats were inoculated with 1 x 10(6) and adult rats with 7 x 10(6) blood trypomastigotes, according to their relative body weight. At zero, seven, 14, 21 and 28 days after infection, blood was collected for the determination of gonadal and adrenal hormones, tumor necrosis factor α (TNF-α), interleukin (IL)-10 and specific IgM and IgG subtypes. Young animals displayed significantly higher parasitaemia values and an endocrine pattern that was characterised by elevated values in corticosterone (CT) and the CT/dehydroepiandrosterone-sulfate ratio, which favours immunosuppression and susceptibility. In contrast, adult male rats were able to restrict the parasite burden, which likely resulted from increased IgG antibody synthesis and oestradiol levels. Adult rats also showed a reduced TNF-α/IL-10 ratio and less tissue damage. We conclude that young animals exhibited increased vulnerability to T. cruzi infection compared with adults and this is associated with an unsuitable immunoendocrine milieu.

Distribution and infection of triatomines (Hemiptera: Reduviidae) by Trypanosoma cruzi in the state of Michoacán, Mexico

An entomological study of triatomine species was carried out to assess their prevalence in 10 localities of the state of Michoacán, Mexico. Entomological indices were calculated to estimate the risk for vector-borne transmission of Trypanosoma cruzi to the human population in this area. Four triatomine species (Triatoma barberi, Triatoma dimidiata, Meccus pallidipennis and Meccus longipennis) were collected from the study area. This is the first report of M. longipennis and T. dimidiata in Michoacán. M. pallidipennis was significantly (p < 0.05) more abundant than any of the other species collected in the study area. Infection indices were greater than 50% for each of the four collected triatomine species. Significantly more triatomines were collected from intradomiciliary areas than from peridomiciliary or sylvatic areas. Infestation, crowding and density indices were low, whereas colonisation indices were high in five localities. The current vectorial conditions in the study area require continuous entomological and serological surveillance to diminish the risk of T. cruzi transmission to human populations.

Comparison of the efficacy of long-lasting insecticidal nets PermaNet® 2.0 and Olyset® against Anopheles albimanus under laboratory conditions

Insecticide-treated nets provide a reduction in human-vector contact through physical barrier, mortality and/or repellent effects that protect both users and non-users, thereby protecting the wider community from vector-borne diseases like malaria. Long-lasting insecticide-treated nets (LLINs) are the best alternative. This study evaluated the bioefficacy of LLINs PermaNet® 2.0 and Olyset® under laboratory conditions with Anopheles albimanus. The laboratory strain was evaluated for insecticide susceptibility with selected insecticides used for malarial control. Regeneration time and wash resistance were evaluated with the standard bioassay cone technique following WHO guidelines. Heat assistance was used for Olyset® nets; the nets were exposed to four different temperatures to speed the regeneration process. The regeneration study of PermaNet® 2.0 showed that efficacy was fully recovered by 24 h after one and three washes and wash resistance persisted for 15 washes. Regeneration of Olyset® nets was not observed for nets washed three times, even with the different temperature exposures for up to seven days. Thus, for Olyset® the wash resistance evaluation could not proceed. Differences in response between the two LLINs may be associated with differences in manufacturing procedures and species response to the evaluated LLINs. PermaNet® 2.0 showed higher and continuous efficacy against An. albimanus.