Anopheles halophylus, a new species of the subgenus Nyssorhynchus (Diptera: Culicidae) from Brazil

Anopheles halophylus n.sp. is described from Central Brazil. It is distinguished from An. triannulatus (Neiva and Pinto) by morphological and morphometric characters, especially in the male genitalia, larva and egg. Illustrations of the male and female genitalia, egg, larva and pupa, and discussion of the status of names associated with An. triannulatus are provided.

Genetic diversity of Colombian sylvatic Trypanosoma cruzi isolates revealed by the ribosomal DNA

American trypanosomiasis is a common zoonosis in Colombia and Trypanosoma cruzi presents a wide distribution throughout the country. Although some studies based on enzyme electrophoresis profiles have described the population structure of the parasite, very few molecular analyses of genotipic markers have been conducted using Colombian strains. In this study, we amplified the non-transcribed spacer of the mini-gene by PCR, typing the isolates as T. cruzi I, T. cruzi zymodeme 3 or T. rangeli. In addition, the internal transcribed spacers of the ribosomal gene concomitant with the 5.8S rDNA were amplified and submitted to restriction fragment polymorphism analysis. The profiles were analyzed by a numerical methodology generating a phenetic dendrogram that shows heterogeneity among the T. cruzi isolates. This finding suggests a relationship between the complexity of the sylvatic transmission cycle in Colombia and the diversity of the sylvan parasites.

High prevalence anti-Trypanosoma cruzi antibodies, among blood donors in the State of Puebla, a non-endemic area of Mexico

Blood transfusion is the second most common transmission route of Chagas disease in many Latin American countries. In Mexico, the prevalence of Chagas disease and impact of transfusion of Trypanosoma cruzi-contaminated blood is not clear. We determined the seropositivity to T. cruzi in a representative random sample, of 2,140 blood donors (1,423 men and 647 women, aged 19-65 years), from a non-endemic state of almost 5 millions of inhabitants by the indirect hemagglutination (IHA) and enzyme linked immunosorbent assay (ELISA) tests using one autochthonous antigen from T. cruzi parasites, which were genetically characterized like TBAR/ME/1997/RyC-V1 (T. cruzi I) isolated from a Triatoma barberi specimen collected in the same locality. The seropositivity was up to 8.5% and 9% with IHA and ELISA tests, respectively, and up to 7.7% using both tests in common. We found high seroprevalence in a non-endemic area of Mexico, comparable to endemic countries where the disease occurs, e.g. Brazil (0.7%), Bolivia (13.7%) and Argentina (3.5%). The highest values observed in samples from urban areas, associated to continuous rural emigration and the absence of control in blood donors, suggest unsuspected high risk of transmission of T. cruzi, higher than those reported for infections by blood e.g. hepatitis (0.1%) and AIDS (0.1%) in the same region.

Biological characterization of a beta-galactosidase expressing clone of Trypanosoma cruzi CL strain

Clone CL B5 of Trypanosoma cruzi is a beta-galactosidase expressing organism that was genetically transfected to be used for in vitro pharmacological screening. Biological parameters were determined, evaluating growth kinetics of epimastigotes, metacyclogenesis, infectivity to mammalian cell lines, parasitemia kinetics in mice and sensibility to nifurtimox and benznidazole. Differences in relation to other strains and CL parental strain were found, the most important being the incapability to produce death to mice in spite of the high inoculum used. However, it possesses the required features to be used for in vitro drug screening. Data obtained demonstrate that heterogeneity of T. cruzi appears even among clones of the same strain, and that these differences found do not prevent the use of clone CL B5 for the purpose that was engineered.

Temperature influence on embryonic development of Anopheles albitarsis and Anopheles aquasalis

Temperature influence on the embryonic development of Anopheles aquasalis and An. albitarsis was investigated. At 26ºC, 75% and 60% of respectively An. aquasalis and An. albitarsis eggs hatched, with one peak of eclosion, between the 2nd and 3rd day after oviposition. At 20 ± 2ºC, around 66-70% of An. aquasalis eggs hatched, with one eclosion peak, on the 5th day. On the other hand, An. albitarsis eclosion at 21 ± 2ºC decreased to 10-22%, with two eclosion peaks, on the 4th-5th day and on the 9th-12th day. These data indicate a stronger temperature influence over An.albitarsis than over An. aquasalis embryos.

Systematic studies on Anopheles galvaoi Causey, Deane & Deane from the subgenus Nysssorhynchus blanchard (Diptera: Culicidae)

Anopheles galvaoi, a member of the subgenus Nyssorhynchus, is redescribed based on morphological characters of the adults male and female, fourth-instar larva and pupa. Female, male genitalia, larval and pupal stages are illustrated. Data about medical importance, bionomics, and distribution are given based on literature records. Adult female of An. galvaoi can be easily misidentified as An. benarrochi Gabaldón and An. aquasalis Curry. A few characters are indicated for identifying female and immatures of An. galvaoi. Phylogenetic relationships among An. galvaoi and six other species of the Oswaldoi Subgroup are estimated using COII mtDNA and ITS2 rDNA gene sequences. Lectotype of An. galvaoi, an adult female from Rio Branco, State of Acre, is invalidated.

Morphometric discrimination of females of five species of Anopheles of the subgenus Nyssorhynchus from Southern and Northwest Colombia

The most important vectors of human Plasmodium in the neotropics belong to the subgenus Nyssorhynchus. These species are generally sympatric in terms of their geographical distributions. Some are difficult to identify based solely on examination of adult females using the available morphological keys, in these cases examination of immature stages and male genitalia is required to make correct determinations. However, in epidemiological studies it is necessary to identify the species of adult females which are found near humans, i.e. in studies of malaria transmission or evaluation of control measures. The purpose of the present study was to evaluate the discrimination of adult females of different species of Nyssorhynchus isolated mainly from Southern Colombia (department of Putumayo), using morphometric analysis. Adult females were obtained after rearing larvae collected in natural breeding places and from the progeny of females collected on humans. The morphological characteristics of the immature stages allowed the identification of four species of the subgroup Oswaldoi from Southern Colombia: Anopheles rangeli Gabaldon, Cova Garcia & Lopez, An. oswaldoi (Peryassu), An. benarrochi Gabaldon, Cova Garcia & Lopez and An. triannulatus (Neiva & Pinto). The species An. nuneztovari (Gabaldon) from the Northwest of Colombia was included for comparison. Morphometric analysis allowed differentiation of the females of all species to a confidence level approaching 90% using principal components analysis of 10 wing and leg variables, followed by canonical variate analysis of the first four principal components. We conclude that morphometrics may represent a useful taxonomic tool for this group and that its use should be further studied.

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37°C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.

Should Trypanosoma cruzi be called "cruzi" complex? A review of the parasite diversity and the potential of selecting population after in Vitro culturing and mice infection

Morpho-biological diversity of Trypanosoma cruzi has been known since Chagas' first works in 1909. Several further studies confirmed the morphological differences among the parasite strains, which were isolated from different reservoirs and vectors, as well as from human beings. In the early sixties, antigenic differences were found in the parasite strains from various sources. These differences, coupled to the observation of regional variations of the disease, led to the proposal of the term cruzi complex to designate the taxon T. cruzi. Since then this protozoan has been typed in distinct biodemes, zymodemes and lineages which were consensually grouped into T. cruzi I, T. cruzi II and into non-grouped strains. T. cruzi genotypic characterization, initially carried out by schizodeme analysis and more recently by various other techniques, has shown a great diversity of the parasite strains. In fact, T. cruzi is formed by groups of heterogeneous sub-population, which present specific characteristics, including distinct histotropism. The interaction of the different infecting clones of the cruzi complex and the human host will determine the morbidity of the disease.

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37°C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.

A novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi

We report the molecular characterization of a novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi. Steady-state level of transcripts of this sequence family appeared to be developmentally regulated, since only in the replicative forms the parasite showed expression of related sequences with a major band around 3 kb. The presence of frame shifts or premature stop codons predicts that transcripts are not translated. The sequence family also contains truncated forms of retrotransposons elements that may become potential hot spots for retroelement insertion. Sequences homologous to this family are interspersed at many chromosomes including the subtelomeric regions.

In vitro evaluation of the activity of aromatic nitrocompounds against Trypanosoma cruzi

Fourteen compounds were evaluated for their activity against Trypanosoma cruzi blood stream forms at the concentration of 500 µg/ml. Six compounds were active and re-tested at lower concentrations.

Isoenzymatic variability among five Anopheles species belonging to the Nyssorhynchus and Anopheles subgenera of the Amazon region, Brazil

An isoenzymatic comparative analysis of the variability and genetics differentiation among Anopheles species was done in populations of An. (Nys.) intermedius and An. (Ano.) mattogrossensis of the Anopheles subgenus, and of An. darlingi, An. albitarsis and An. triannulatus of the Nyssorhynchus subgenus, with the aim of detecting differences between both subgenera and of estimating the degree of genetic intere specific divergence. Samples from Macapá, State of Amapá and Janauari Lake, near Manaus, State of Amazonas, were analyzed for eight isoenzymatic loci. Analysis revealed differences in the average number of alleles per locus (1.6-2.3) and heterozygosity (0.060-0.284). However, the proportion of polymorphic loci was the same for An. (Nys.) darlingi, An. (Nys.) triannulatus and An. (Ano.) mattogrossensis (50%), but differed for An. (Nys.) albitarsis (62.5%) and An. (Ano.) intermedius (25%). Only the IDH1 (P > 0.5) locus in all species studied was in Hardy-Weinberg equilibrium. The fixation index demonstrated elevated genetic structuring among species, based on values of Fst = 0.644 and genetic distance (0.344-0.989). Genetic difference was higher between An. (Nys.) triannulatus and An. (Ano.) intermedius (0.989) and smaller between An. (Nys.) albitarsis sensu lato and An. (Nys.) darlingi (0.344). The data show interspecific genetic divergence which differs from the phylogenetic hypothesis based on morphological characters.

Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39) were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana) and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6%) presented a mixed infection Leishmania complex species, 17 (58.6%) a mixed infection Leishmania-T. cruzi, and 4 (13.8%) a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%). The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages) dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V.) braziliensis, L. (L.) chagasi and L. (L.) mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V.) braziliensis-L. (L.) mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.

Trypanosoma cruzi-elicited CD8+ T cell-mediated myocarditis: chemokine receptors and adhesion molecules as potential therapeutic targets to control chronic inflammation?

In Chagas disease, during the acute phase, the establishment of inflammatory processes is crucial for Trypanosoma cruzi control in target tissues and for the establishment of host/parasite equilibrium. However, in about 30% of the patients, inflammation becomes progressive, resulting in chronic disease, mainly characterized by myocarditis. Although several hypothesis have been raised to explain the pathogenesis of chagasic myocardiopathy, including the persistence of the parasite and/or participation of autoimmune processes, the molecular mechanisms underlying the establishment of the inflammatory process leading to parasitism control but also contributing to the maintenance of T. cruzi-elicited chronic myocarditis remain unsolved. Trying to shed light on these questions, we have for several years been working with murine models for Chagas disease that reproduce the acute self-resolving meningoencephalitis, the encephalitis resulting of reactivation described in immunodeficient individuals, and several aspects of the acute and chronic myocarditis. In the present review, our results are summarized and discussed under the light of the current literature. Furthermore, rational therapeutic intervention strategies based on integrin-mediated adhesion and chemokine receptor-driven recruitment of leukocytes are proposed to control T. cruzi-elicited unbalanced inflammation.