Trypanosoma cruzi high infectivity in vitro is related to cardiac lesions during long-term infection in Beagledogs

Trypanosoma cruzi is a hemoflagelate parasite associated with heart dysfunctions causing serious problems in Central and South America. Beagle dogs develop the symptoms of Chagas disease in humans, and could be an important experimental model for better understanding the immunopathogenic mechanisms involved in the chagasic infection. In the present study we investigated the relation among biological factors inherent to the parasite (trypomastigote polymorphism and in vitro infectivity) and immunoglobulin production, inflammation, and fibrosis in the heart of Beagle dogs infected with either T. cruzi Y or Berenice-78 strains. In vitro infectivity of Vero cells as well as the extension of cardiac lesions in infected Beagle was higher for Y strain when compared to Berenice-78 strain. These data suggested that in vitro infectivity assays may correlate with pathogenicity in vivo. In fact, animals infected with Y strain, which shows prevalence of slender forms and high infectivity in vitro, presented cardiomegaly, inflammation, and fibrosis in heart area. Concerning the immunoglobulin production, no statistically significant difference was observed for IgA, IgM or IgG levels among T. cruzi infected animals. However, IgA together IgM levels have shown to be a good marker for the acute phase of Chagas disease.

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.

Cladistic analysis of the subgenus Anopheles (Anopheles) Meigen (Diptera: Culicidae) based on morphological characters

In the present study, we used morphological characters to estimate phylogenetic relationships among members of the subgenus Anopheles Meigen. Phylogenetic analyses were carried out for 36 species of Anopheles (Anopheles). An. (Stethomyia) kompi Edwards, An. (Lophopodomyia) gilesi (Peryassú), Bironella hollandi Taylor, An. (Nyssorhynchus) oswaldoi (Peryassú) and An. (Cellia) maculatus Theobald were employed as outgroups. One hundred one characters of the external morphology of the adult male, adult female, fourth-instar larva, and pupa were scored and analyzed under the parsimony criterion in PAUP. Phylogenetic relationships among the series and several species informal groups of Anopheles (Anopheles) were hypothesized. The results suggest that Anopheles (Anopheles) is monophyletic. Additionally, most species groups included in the analysis were demonstrated to be monophyletic.

Population genetic structure of the major malaria vector Anopheles darlingi (Diptera: Culicidae) from the Brazilian Amazon, using microsatellite markers

The population genetic structure of Anopheles darlingi, the major human malaria vector in the Neotropics, was examined using seven microsatellite loci from nine localities in central and western Amazonian Brazil. High levels of genetic variability were detected (5-25 alleles per locus; H E = 0.519-0.949). There was deviation from Hardy-Weinberg Equilibrium for 59.79% of the tests due to heterozygote deficits, while the analysis of linkage disequilibrium was significant for only two of 189 (1.05%) tests, most likely caused by null alleles. Genetic differentiation (F ST = 0.001-0.095; Nm = 4.7-363.8) indicates that gene flow is extensive among locations < 152 km apart (with two exceptions) and reduced, but not absent, at a larger geographic scale. Genetic and geographic distances were significantly correlated (R² = 0.893, P < 0.0002), supporting the isolation by distance (IBD) model. The overall estimate of Ne was 202.4 individuals under the linkage disequilibrium model, and 8 under the heterozygote excess model. Analysis of molecular variance showed that nearly all variation (~ 94%) was within sample locations. The UPGMA phenogram clustered the samples geographically, with one branch including 5/6 of the state of Amazonas localities and the other branch the Acre, Rondônia, and remaining Amazonas localities. Taken together, these data suggest little genetic structure for An. darlingi from central and western Amazonian Brazil. These findings also imply that the IBD model explains nearly all of the differentiation detected. In practical terms, populations of An. darlingi at distances < 152 km should respond similarly to vector control measures, because of high gene flow.

Systematic notes on Anopheles Meigen (Diptera: Culicidae) species in the state of Amapá, Brazil

Identification of Anopheles nuneztovari Gabaldón and An. goeldii Rozeboom and Gabaldón based on the male genitalia traits is discussed. An. goeldii is in the synonymy of An. nuneztovari, however, characters of the aedeagus of male genitalia distinguish both species. We hypothesize that An. goeldii may be a valid species, however, further studies using molecular characters, especially ITS2 rDNA sequences will be necessary to elucidate the taxonomic status of the species. An. konderi Galvão and Damasceno and An. forattinii Wilkerson and Sallum are registered for the first time in the state of Amapá.

Trypanosoma cruzi ribosomal protein S4: characterization of its coding locus, analysis of transcripts, and antigenicity of the protein

Two allelic genomic fragments containing ribosomal protein S4 encoding genes (rpS4) from Trypanosoma cruzi (CL-Brener strain) were isolated and characterized. One allele comprises two complete tandem repeats of a sequence encoding an rpS4 gene. In the other, only one rpS4 gene is found. Sequence comparison to the accessed data in the genome project database reveals that our two-copy allele corresponds to a variant haplotype. However, the deduced aminoacid sequence of all the gene copies is identical. The rpS4 transcripts processing sites were determined by comparison of genomic sequences with published cDNA data. The obtained sequence data demonstrates that rpS4 genes are expressed in epimastigotes, amastigotes, and trypomastigotes. A recombinant version of rpS4 was found to be an antigenic: it was recognized by 62.5% of the individuals with positive serology for T. cruzi and by 93.3% of patients with proven chronic chagasic disease.

Experimental transmission of the parasitic flagellates Trypanosoma cruzi and Trypanosoma rangeli between triatomine bugs or mice and captive neotropical bats

Trypanosoma cruzi and Trypanosoma rangeli-like trypanosomes have been found in a variety of neotropical bat species. In this study, bats (Artibeus lituratus, Carollia perspicillata, Desmodus rotundus, Glossophaga soricina, Molossus molossus, Phyllostomus hastatus) were maintained under controlled conditions, and experiments were conducted to determine how they might become infected naturally with trypanosomes. All bats were first screened for existing infections by hemoculture and the examination of blood smears, and only apparently uninfected animals were then used in the experiments. Proof was obtained that the triatomine bug Rhodnius prolixus would readily feed upon some of the bats, and two species became infected after being bitten by bugs infected with T. rangeli. Some bats also became infected by ingesting R. prolixus carrying T. cruzi, or following subcutaneous or intragastic inoculation with fecal suspensions of R. prolixus containing T. cruzi. P. hastatus became infected after ingesting mice carrying T. cruzi. All of the bats studied inhabit roosts that may be occupied by triatomine bugs and, with the exception of D. rotundus, all also feed to at least some extent upon insects. These findings provide further evidence of how bats may play significant roles in the epidemiology of T. cruzi and T. rangeli in the New World tropics.

Transferrin uptake may occur through detergent-resistant membrane domains at the cytopharynx of Trypanosoma cruzi epimastigote forms

Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.

Population structure of the malaria vector Anopheles darlingi in Rondônia, Brazilian Amazon, based on mitochondrial DNA

Anopheles darlingi is the most important Brazilian malaria vector, with a widespread distribution in the Amazon forest. Effective strategies for vector control could be better developed through knowledge of its genetic structure and gene flow among populations, to assess the vector diversity and competence in transmitting Plasmodium. The aim of this study was to assess the genetic diversity of An. darlingi collected at four locations in Porto Velho, by sequencing a fragment of the ND4 mitochondrial gene. From 218 individual mosquitoes, we obtained 20 different haplotypes with a diversity index of 0.756, equivalent to that found in other neotropical anophelines. The analysis did not demonstrate significant population structure. However, haplotype diversity within some populations seems to be over-represented, suggesting the presence of sub-populations, but the presence of highly represented haplotypes complicates this analysis. There was no clear correlation among genetic and geographical distance and there were differences in relation to seasonality, which is important for malarial epidemiology.

Systematics of Anopheles lanei Galvão and Amaral, a species of the subgenus Nyssorhynchus Blanchard (Diptera: Culicidae)

Anopheles (Nyssorhynchus) lanei Galvão and Amaral is here redescribed using morphological characteristics of adult, male and female, fourth instar larva and pupa. The larva, pupa, and male genitalia are illustrated. Diagnostic morphological characters of adults, male genitalia, fourth instar larva and pupa are provided to distinguish An. lanei from other species of the Argyritarsis section. Species distribution data are based on the published literature records and bionomics data are based on both literature records and field data.

Diverse population dynamics of three Anopheles species belonging to the Triannulatus Complex (Diptera: Culicidae)

The Triannulatus Complex of Anopheles (Nyssorhynchus) consists of at least three sibling species, namely Anopheles triannulatus s.s., Anopheles halophylus and a third undescribed member herein referred to as An. triannulatus "C". Sympatric anophelines belonging to species complexes, even though closely related, may exploit different environments such as larval habitats. In this paper we hypothesize that rainfall and seasonal flooding would distinctly influence the availability of larval habitats and consequently the seasonal population dynamics of sympatric members of the Triannulatus Complex. A reflection of this is distinct seasonal biting frequencies exhibited by three members of the Triannulatus Complex at a site in Central Brazil. Population dynamics seem to be influenced by the water level in the local rivers, although biting frequency of all three species was negatively influenced by rainfall. An. triannulatus s.s. was more abundant following the end of the rainy season, but notably 30 to 60 days after flooding. On the other hand, An. halophylus and An. triannulatus C peaked during the middle of the dry season, when water impoundments have no inflow, are somewhat reduced in size and the water becomes brackish. Differences in population dynamics were greater between An. triannulatus s.s. and An. halophylus and An. triannulatus C than between An. halophylus and An. triannulatus C. This might reflect differences in larval habitat exploitation and therefore spatial segregation among these members of the complex.

Importance of TNF-alpha in the course of acute infection with Trypanosoma cruzi: influence of its inhibition by pentoxifylline treatment

Infection of C3H/He mice with the Peruvian strain of Trypanosoma cruzi (Biodeme type I, Z2b), a macrophagotropic strain, determined severe parasitism of macrophages, necrosis of the spleen, and high host mortality. In the present study, pentoxifylline (PTX), an inhibitor of TNF-alpha was investigated on its action upon splenic necrosis, parasitemia and host survival. Immunohistochemical data suggested the importance of this cytokine in parasite destruction and decreasing of parasitemia, although paradoxically contributing to the high mortality of infected mice. Necrotic lesions involving several organs, specially the heart, in acute Chagas disease, are important aggravating factors, increasing cardiac morbidity. Advantage of inhibiting TNF-alpha action was herein investigated. Infected mice were divided into two groups: untreated (n = 24), and PTX treated mice (n = 25). PTX was administered in two daily doses of 30 mg/kg/bw, by intraperitoneal route. Normal controls either treated with PTX or saline were also included. Histopathology of the spleen and in situ immunolabeling of TNF-alpha, using anti-TNF-alpha monoclonal antibody, were performed. Necrotic areas were evaluated by morphometry. Mice treated with PTX showed a significant decrease of necrotic areas and diminution of TNF-alpha expression in spleen tissue, suggesting that PTX treatment could control TNF-alpha effects, and thus be used as an adjuvant in the treatment of acute Chagas' disease.

Morphological analysis of three populations of Anopheles (Nyssorhynchus) nuneztovari Gabaldón (Diptera: Culicidae) from Colombia

Based on the results of comparative analyses of 1,039 specimens of several progenies of Anopheles nuneztovarifrom three localities in Colombia, eight costal wing spot patterns were observed. Patterns I and III were the most frequent: 77.96% and 11.36%, respectively. Using the diagnostic characters ratio of the length of the basal dark area of hind tarsomere II/length of hind tarsomere II, ratio of the length of the humeral pale spot/length of the pre-humeral dark spot, and the ratio of the length of the subcostal pale spot/length of the distal sector dark spot (DS-III2/Ta-III2, HP/PHD, SCP/DSD) approximately 5% of the adult females were misidentified as a species of Nyssorhynchus, different from An. nuneztovari. Approximately 5% of the specimens showed DS-III2/Ta-III2 ratio less than 0.25 (range 0.21 - 0.24), and among them 3.34% shared a HP/PHD ratio less than 1.50. Consequently, 1.52% of An. nuneztovari individuals can be misidentified as Anopheles oswaldoi. In those specimens with the DS-III2/Ta-III2 ratios higher than 0.25, 34.45% displayed SCP/DSD values greater than 0.50 and of these, 3.65% displayed HP/PHD values greater than 1.8. This combination of characters could lead one to misidentify samples of An. nuneztovari as Anopheles rangeli. Similarly, 2.43% of the females could be identified erroneously as either Anopheles aquasalis or Anopheles benarrochi. Individuals with a HP/PHD ratio greater than 2.0, could be misidentified as Anopheles trinkae, Anopheles strodei or Anopheles evansae. A distinct combination of diagnostic characters for An. nuneztovari from Colombia is proposed.

Purification and biochemical characterization of four iron superoxide dismutases in Trypanosoma cruzi

Four superoxide dismutase (SOD) activities (SOD I, II, III, and IV) have been characterized in the epimastigote form of Trypanosoma cruzi. The total extract was subjected to two successive ammonium sulphate additions between 35 and 85%, and the resulting fraction was purified using two continuous chromatography processes (ion exchange and filtration). Enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing SODs. The molecular masses of the different SODs were 20 kDa (SOD I), 60 kDa (SOD II), 50 kDa (SOD III) and 25 kDa (SOD IV), whereas the isoelectric points were 6.9, 6.8, 5.2 and 3.8, respectively. Subcellular location and digitonin experiments have shown that these SODs are mainly cytosolic, with small amounts in the low-mass organelles (SOD II and SOD I) and the mitochondrion (SOD III), where these enzymes play an important role in minimizing oxidative damage.