Phylogeny of snake trypanosomes inferred by SSU rDNA sequences, their possible transmission by phlebotomines, and taxonomic appraisal by molecular, cross-infection and morphological analysis.

Blood examination by microhaematocrit and haemoculture of 459 snakes belonging to 37 species revealed 2.4% trypanosome prevalence in species of Viperidae (Crotalus durissus and Bothrops jararaca) and Colubridae (Pseudoboa nigra). Trypanosome cultures from C. durissus and P. nigra were behaviourally and morphologically indistinguishable. In addition, the growth and morphological features of a trypanosome from the sand fly Viannamyia tuberculata were similar to those of snake isolates. Cross-infection experiments revealed a lack of host restriction, as snakes of 3 species were infected with the trypanosome from C. durissus. Phylogeny based on ribosomal sequences revealed that snake trypanosomes clustered together with the sand fly trypanosome, forming a new phylogenetic lineage within Trypanosoma closest to a clade of lizard trypanosomes transmitted by sand flies. The clade of trypanosomes from snakes and lizards suggests an association between the evolutionary histories of these trypanosomes and their squamate hosts. Moreover, data strongly indicated that these trypanosomes are transmitted by sand flies. The flaws of the current taxonomy of snake trypanosomes are discussed, and the need for molecular parameters to be adopted is emphasized. To our knowledge, this is the first molecular phylogenetic study of snake trypanosomes.

Characterization and classification of leishmanial parasites from humans, wild mammals, and sand flies in the Amazon region of Brazil.

Ninety-four leishmanial isolates from the Brazilian Amazon Region (Amapá, Amazonas, Pará, and Rondônia) were identified and classified using specific monoclonal antibodies and an indirect radioimmunoassay (serodeme analysis); eighty-two were also characterized by enzyme electrophoresis (zymodeme analysis), the results of which were subjected to a numerical phenetic analysis. Six isolates from humans (3), Didelphis marsupialis (1), Lutzomyia olmeca nociva (1), and Lu, reducta (1) showed reactivity patterns and isoenzyme profiles similar to those obtained with the Leishmania amazonensis reference strains, and were identified as this species. Eighty-six stocks were classified as members of the L. braziliensis complex; of these, 61 were L. guyanensis or variants, which presented three serodeme subtypes, but whose isoenzyme profiles were all similar to the reference strain. A total of 15 isolates were distinguished as L. braziliensis or variants and were classified into five serodeme subtypes. The isolate from Psychodopugus davisi appeared, from the numerical analysis, to be a distinct parasite species. Ten isolates showed reactivity patterns and isoenzyme profiles similar to those obtained with the L. naiffi reference strain. A parasite isolated from Ps. claustrei appeared to be different from all reference strains by both techniques, and was classified as probably being a new species. The importance of these results with respect to the taxonomic status of the New World Leishmania, and their implications for both clinical and epidemiologic data are discussed.

Biochemical characterization of some species of Trypansoma (Schizotrypanum) from bats (Microchiroptera).

Trypanosoma (Schizotryanum) species isolated from bats (Microchirotera) in Europe and Latin America were examined by determining the buoyant densities of their nuclear and kinetoplastic deoxyribonucleic acid (DNA) and the electrophoretic patterns of six isoenzymes. By these criteria they were separated into three distinct groups -- two from Europe (T. dionisii and T. vespertilionis) and one from America. T. dionisii was also separable by its morphology in vitro. Geographical location and DNA buoyant densities suggested that the American stocks were more closely related to T. cruzi than to the European species, though they differed from it marginally in kinetoplastic DNA density and in being noninfective to mice. Similar stocks studied by other workers have been shown to differ from T. cruzi also in reduced infectivity to, and lack of natural association with, Triatominae, and in antigenic composition. It is therefore proposed that trypanosomes of the subgenus Schizotryanum occuring naturally in Microchiroptera and differing from T. cruzi sensu stricto as outlined above should be treated as a distinct subspecies, T. cruzi marinkellei ssp. nov. T. cruzi sensu stricto thus becomes the nominate subspecies T. cruzi cruze Chagas 1909.

Prevalence of parasitemia and seroreactivity to Trypanosoma cruzi in a rural population of Northeast Brazil.

Age-specific prevalence rates of parasitemia and seroreactivity to Trypanosoma cruzi were determined in a rural area endemic for Chagas' disease in Northeast Brazil. Parasitemia was detected by blood cultures and xenodiagnosis, and serum antibodies to the parasite were measured by the complement fixation (CF) and indirect immunofluorescence (IFA) tests. Of the 116 persons examined, 39 (33.7%) had antibodies and 23 (19.8%) had parasitemia. Ninety-six percent of parasitemic individuals were seropositive and 56% of seropositive individuals were parasitemic. The percentage of seropositive individuals with detectable parasitemia declined with age; all seropositive children in the 1- to 4-year age group and two-thirds of seropositive persons 5-19 years old had parasitemia while only one-third of seropositive adults above 19 years had parasitemia. CF and IFA tests were equally sensitive in detecting persons with parasitemia. Xenodiagnosis was more sensitive than culture for detecting parasitemia, but the two methods together were more sensitive than either method alone. Using the age-dependent relationship of parasitemia to seropositivity determined in this study, the prevalence rate of T. cruzi parasitemia was estimated in a much larger adjacent population in which seropositivity rates and the demographic structure were already known.

Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences.

Traditional molecular and biochemical methods, such as schizodeme analysis, karyotyping, DNA fingerprinting, and enzyme electrophoretic profiles, have shown a large variability among Trypanosoma cruzi isolates. In contrast to those results, polymerase chain reaction (PCR) amplification of sequences from the 24S alpha ribosomal RNA gene and from the mini-exon gene nontranscribed spacer indicated a dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 86 T. cruzi field stocks (68 isolated from humans with defined presentations of Chagas' disease and 18 from triatomines) derived from four Brazilian geographic areas were typed by the PCR assay based on the DNA sequences of the mini-exon and 24S alpha rRNA genes. These stocks were ordered into the two major T. cruzi lineages. Lineage 1 was associated mainly with human isolates and lineage 2 with the sylvatic cycle of the parasite.

An outbreak of acute Chagas's disease in the São Francisco Valley region of Bahia, Brazil: triatomine vectors and animal reservoirs of Trypanosoma cruzi.

Following reports of an unusually high incidence of acute Chagas's disease and the appearance of large numbers of Triatoma infestans in the southwestern region of the State of Bahia, triatomine bugs (Hemiptera: Reduviidae) and domestic animals in one of the affected communities were surveyed and examined for infection with Trypanosoma cruzi. Triatoma infestans was prevalent in houses and was also found in peridomestic habitats. T. sordida and T. pseudomaculata occupied peridomestic and sylvatic habitats and T. brasiliensis was found only among rocks far from houses. Panstrongylus megistus, formerly present in the region, was not found. Trypanosoma cruzi was detected in 19.5% of Triatoma infestans, 11.5% of T. sordida, 19% of dogs, 29% of cats and 100% of rats examined. A disproportionate number of early instar bugs were infected with Trypanosoma cruzi, suggesting that a rapid increase in the rate of transmission had recently occurred. The history of the domestic triatomine fauna of the region since 1912 is reviewed, and it is proposed that the relatively recent arrival of Triatoma infestans initiated a domestic cycle linked to peridomestic and sylvatic cycles of Trypanosoma cruzi transmission. Increased human mobility, the use of DDT for malaria control, and drought conditions are considered as factors which might have contributed to the outbreak of human infection.

Epidemiological aspects of three Trypanosoma cruzi zymodemes in Bahia State, Brazil.

Culture forms of 104 stocks of Trypanosoma cruzi isolated in different regions of the State of Bahia were compared by electrophoresis of six enzymes. The three distinct combinations of isoenzyme patterns seen were designated ZI, Z2 and Z3. In an area of endemic Chagas's disease in eastern Bahia, T. cruzi Z1 was associated with sylvatic mammals and sylvatic triatomines, whereas T. cruzi Z2 was associated with a separate domestic cycleof transmission. T. cruzi Z1 was also found in sylvatic triatomines from other parts of the State. In contrast, in an area of the São Francisco Valley region of western Bahia, both T. cruzi Z1 and Z2 were isolated from man, domestic animals, and peridomestic rats. T. cruzi Z3 was isolated from an armadillo and from Panstrongylus geniculatus, a triatomine commonly found in armadillo burrows. Both T. cruzi Z1 and Z2 appeared to be pathogenic in man: T. cruzi Z1 was isolated from patients with acute Chagas's disease and from a single patient with chronic cardiac manifestations. T. cruzi Z2 was isolated from some asymptomatic individuals but was also associated with acute disease and chronic cardiac and digestive syndromes.

New records of Histoplasma capsulatum from wild animals in the Brazilian Amazon.

Twenty-eight isolates of Histoplasma capsulatum were obtained from eight species of forest mammals from the States of Amazonas, Pará and Rondônia in the Amazon Region of Brazil. Primary isolates were obtained by inoculating triturated liver and spleen tissue intradermally and intraperitoneally in hamsters. Mycological diagnosis in hamsters presenting lesions was confirmed by histopathology and culture on Sabouraud dextrose-agar. Infected hamsters developed signs of disease within two to nine months; all had disseminated visceral lesions and most also had skin lesions at the sites of inoculation. None of the hamsters inoculated with skin macerates of the original hosts developed histoplasmosis, and histopathological examination of the viscera of the wild hosts failed to reveal H. capsulatum. Prevalence of infection was considerably higher in females than in males both for the opossum Didelphis marsupialis and for total wild animals (479) examined. It is proposed that canopy-dwelling mammals may acquire the infection from conidia borne on convective currents in hollow trees with openings at ground-level.

[Wild vectors of Chagas' disease in an urban area of Manaus (AM): flying activity during dry and rainy seasons]. / Vetores selváticos de doença de Chagas na área urbana de Manaus (AM): atividade de vôo nas estações secas e chuvosas.

Adults of Rhodnius prolixus, R. pictipes and Panstrongylus geniculatus found in houses in Manaus have high rates of Trypanosoma cruzi infection. Rhodnius spp. were found throughout the year, but males of P. geniculatus were significantly more frequent in the dry season.

[Human populations attacked by wild Triatominae in the Amazonas: a new form of transmission of Chagas disease?]. / Ataque de populações humanas por triatomíneos silvestres no Amazonas: uma nova forma de transmissao da infecção chagásica?

An "attack" of wild triatomines (Rhodnius brethesi) to piaçaca workers (Leopoldinia piaçaba) is confirmed in the locality of Acuquaia, at Padauari river, affluent of Rio Negro in the municipality of Barcelos, State of Amazonas, Brazil. A serological prevalence of 12.5% for T. cruzi antibodies in human population, in the city of Barcelos, has already been described in a previous paper. A strong association between the serological positivity and the population contact with wild triatomines, known in the area as "piaçava's lice", was verified.

Distribution of sandflies (Diptera: Phlebotominae) in forest remnants and adjacent matrix habitats in Brazilian Amazonia.

We studied the distribution of sandflies (Diptera: Phlebotominae)--insect vectors of several diseases, including leishmaniasis--at the interface between primary forest and cattle pasture and between primary forest and secondary forest (< 15 yr old) in Southern Brazilian Amazonia. Sandflies were collected by using a combination of light traps and traps having vertebrates as baits. Strong differences in abundance and species richness were found between primary forests and pastures. Very few sandfly species were found in the pastures, and those that were found generally occurred at lower densities when compared to the adjacent forest. At least one species (Lutzomyia lainsoni), however, can become extremely abundant in pastures, possibly depending on the presence of cattle and water bodies. Differences between primary forests and secondary forests were not so strong, although the latter usually had fewer species and lower population abundances. No species were exclusively found in pastures or secondary forests; the species present in these two habitats were a subset of those found in primary forests. The distance to the edge did not affect the abundance, richness and composition of sandfly species in primary forests. The abundance and richness of sandflies, however, was greater in forest edges facing pastures than those facing secondary forests. This pattern could not be explained by an influx of species and individuals from the adjacent pasture, suggesting the existence of in situ differences between the different types of forest edges studied.

Trypanosomes of the subgenus Megatrypanum from armadillos (Xenarthra:Dasypodidae).

A new species of trypanosome, Trypanosoma (Megatrypanum) peba, is described from the peripheral blood of the armadillo Euphractus sexcinctus setosus from Bahia State, Brazil. Ten out of 29 specimens of the armadillo Dasypus novemcinctus from Pará State were found to have trypanosomes, including epimastigote forms, in impression smears of subcutaneous lymph nodes. The trypanosomes from D. novemcinctus are illustrated and were identified as belonging to the subgenus Megatrypanum on the basis of their general appearance, although they failed to multiply in blood-agar culture medium and no bloodstream forms were seen. This is the first published record of trypanosomes of this subgenus from armadillos and the first demonstration of epimastigote trypanosomes in the mammalian host other than in the bloodstream, or in the anal glands of opossums.

Lutzomyia derelicta (Diptera: Psychodidae) a singular new phlebotomine sand fly from an Inselberg in Northeastern Amazonia.

Lutzomyia derelicta n. sp. is described from specimens collected in an isolated xeric habitat in the rainforest in the north of the State of Pará, Brazil. The new species lacks the posterior bulge in the dorsal wall of the cibarium characteristic of the New World genus Lutzomyia, and the armature of the male genitalia is of the pattern found elsewhere only in the Old World species of Sergentomyia. L. derelicta is phenetically intermediate between the known species of Lutzomyia and Sergentomyia, and cannot readily be placed in any existing subgenus or species group of either genus.

Epidemiological and nosological aspects of Leishmania naiffi Lainson & Shaw, 1989.

Leishmania naiffi was isolated from 10 out of 64 armadillos (Dasypus novemcinctus) examined in Amazonas, Pará and Rondônia States in the Brazilian Amazon Region. The isolates were obtained in culture from samples of liver (3), spleen (3), lymph nodes (2), skin (1) and blood (1) from the infected animals. Heavy infections with the same parasite were detected for the first time in Psychodopygus squamiventris, a common man-biting phlebotomine, in Amazonas and Pará. A new case of cutaneous leishmaniasis caused by L. naiffi is described from the Manaus area, making a total of three known cases of human infection by this parasite.

Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron.

Trypanosoma cruzi is currently classified into 2 major phylogenetic lineages, T. cruzi I and II, that correlate with the formerly described zymodeme 1 and 2, respectively. Another isoenzymic group (zymodeme 3-Z3) was also described. In this study, we analysed the genetic diversity among Z3 isolates of the Brazilian Amazon by restriction fragment length polymorphism of the intergenic transcribed spacers (ITSs) of the ribosomal RNA cistron and the size of the divergent domain D7 of the 24Salpha rRNA gene. DNAs from 12 T. cruzi Z3 isolates obtained from humans (2), Panstrongylus geniculatus (1), and Rhodnius brethesi (9) were submitted to PCR amplification of the ITSs plus the 5.8S rDNA. The PCR products were digested with 4 distinct endonucleases and the profiles analysed by a numerical methodology. The phenetic dendrogram revealed a clear dichotomy in the Z3 group, defining 2 groups that were named Z3-A and Z3-B. Dimorphism was also found in the band sizes of the amplified D7 divergent domain of the 24Salpha rDNA, which showed a perfect correlation with the ITSs clustering. The organization of the ribosomal cistron was investigated by Southern blotting and shown to be conserved in the genome of the 2 Z3 groups. This study shows that the rDNA cistron allows the definition of 2 distinct subclusters in Z3 isolates.