Regina Helena Riccioppo Mangia

Molecular epidemiology of American trypanosomiasis in Brazil based on dimorphisms of rRNA and mini-exon gene sequences

American trypanosomiasis is transmitted in nature via a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, or via a domestic cycle where the parasite comes into contact with humans through domiciliated triatomines. The pool of T. cruzi isolates consists of sub-populations presenting a broad genetic diversity. In contrast to the heterogeneity suggested by isoenzyme analysis, PCR amplification of sequences from the 24S alpha rRNA gene and from the non-transcribed spacer of the mini-exon gene indicated dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 157 T. cruzi isolates obtained from humans, triatomines and sylvatic mammalian reservoirs from 12 Brazilian states were analysed by the 24S alpha RNA and mini-exon typing approaches. The stocks were classified into the two proposed lineages and according to the domestic or sylvatic cycle of the parasite. Data presented provide evidence for a strong association of T. cruzi lineage 1 with the domestic cycle, while in the sylvatic cycle both lineages circulate equally. Molecular typing of human parasite isolates from three well-characterised endemic regions of Chagas disease (Minas Gerais, Paraiba and Piaui) and from Amazonas State, where T. cruzi is enzootic, suggests that in some endemic areas in Brazil there is a preferential linkage between both cycles mediated by lineage-1 stocks.

The complexity of the sylvatic cycle of Trypanosoma cruzi in Rio de Janeiro state (Brazil) revealed by the non-transcribed spacer of the mini-exon gene

American trypanosamiasis occurs in nature as a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, such as marsupials, rodents, armadillos and other animals. Due to difficulties in trying to isolate T. cruzi stocks from the sylvatic cycle, very few studies have been performed in order to understand the parasite infection in natural environments. Traditionally T. cruzi has been considered to be composed of a highly heterogeneous population of parasites. In contrast, the mini-exon and the 24S alpha rRNA gene loci have shown that T. cruzi stocks can be clustered in 2 major phylogenetic groups: lineage 1 and lineage 2. In this report, 68 recently isolated T. cruzi samples from the sylvatic cycle belonging to different geographical areas in Rio de Janeiro, Brazil, have been typed based on a variable spot in the non-transcribed spacer of the mini-exon gene. Eight isolates were from triatomines, 26 stocks were from golden-lion tamarins, 31 from opossums, 2 from rodents and 1 from a three-toed sloth. Thirty (44%-30/68) isolates were typed as lineage 1, while 36 (53%-36/68) isolates were typed as lineage 2. Two opossums presented mixed infection. Therefore, 3% (2/68) of the isolates were typed as lineage 1 + lineage 2. Using these geographical regions as models of sylvatic environments, it was observed that 96% of the Didelphis marsupialis were infected by lineage 2 isolates, while all 26 golden-lion tamarins were infected by lineage 1. The results show preferential association of the 2 lineages of T. cruzi with different hosts, composing the complexity of the sylvatic cycle.

Trypanosoma cruzi in the sylvatic environment: distinct transmission cycles involving two sympatric marsupials

Thirty-five specimens of Philander frenata and 36 Didelphis marsupialis were captured in the same Atlantic forest area of Brazil between 1992 and 1994. Haemocultures showed that 50% of P. frenata and 60% of D. marsupialis were infected with Trypansoma cruzi. Biological, biochemical and molecular characterization of the isolates suggested 2 distinct transmission cycles of T. cruzi occurred between these 2 sympatric didelphids. The T. cruzi isolates could be distinguished according to their association with each marsupial species. Biochemical characterization (multilocus enzyme electrophoresis) revealed 15 zymodemes; more variability was observed among the P. frenata isolates than among the isolates from D. marsupialis. The course of natural and experimental infection in D. marsupialis and P. frenata was different and suggested that D. marsupialis was more resistant to infection than P. frenata. In the studied area, P. frenata seems to be a more important reservoir of T. cruzi than D. marsupialis, since 40% of the characterized isolates from P. frenata belonged to the T. cruzi II group, which is associated with human infections.

Stable infection of primates with Trypanosoma cruzi I and II

In order to better comprehend the putative association between genotype Trypanosoma cruzi II and primates, an evaluation of the infection in free ranging primates and specimens born in captivity from different geographical areas, the Amazon and the Atlantic forest, was carried out. Seroprevalences of the T. cruzi infection among the primates was similar in both biomes (45.5% and 46%). The parasites were isolated from 8 and 4 different species of primates, respectively from the Amazon and Atlantic forest. Multi-locus enzyme electrophoresis (MLEE) typed the isolates from Amazon as zymodeme 1. Mini-exon gene analysis characterized all these isolates as T. cruzi I, the main genotype circulating in the region. In the Atlantic forest, primates infected with TCI and TCII, as well as a mixed infection (TCI and TCII), were detected. These findings prove that primates may maintain stable infections by both genotypes. Moreover, data show that T. cruzi can occur in a wide range of primate genera, independent of their social behaviour, niches or habitats. Considering the high seroprevalence and stability of T. cruzi infection among the primates, these animals play an important role in the maintenance of the parasite in nature.

Distinct patterns of Trypanosoma cruzi infection in Leontopithecus rosalia in distinct Atlantic Coastal Rainforest fragments in Rio de Janeiro – Brazil

Previous studies on infection of Trypanosoma cruzi in the Poço das Antas Biological Reserve population of wild free-ranging Leontopithecus rosalia have shown the presence of genotype T. cruzi II, associated in Brazil with human disease. Herein, this study has been extended, the infection being evaluated in L. rosalia of 3 different tamarin populations, inhabiting distinct forest areas located in the same Atlantic Coastal Rainforest. Edentata, Marsupialia, Rodentia and Chiroptera were examined exclusively in the Poço das Antas Biological Reserve. Excluding Chiroptera, T. cruzi infection was found in all orders. Biochemical and molecular characterization demonstrated that golden lion tamarins maintained stable infections by T. cruzi II. The isolates from the other mammals corresponded to T. cruzi I, suggesting independent transmission cycles occurring among the sylvatic mammals inside Poço das Antas Biological Reserve. Significant differences in the infection patterns presented by the 3 populations of wild and captive-born golden lion tamarins were noticed. In Poço das Antas a considerably higher number of positive haemocultures from tamarins with positive serological titres was observed in comparison to those obtained from other areas. The implications for conservation and public health of an active sylvatic cycle in the Atlantic Coastal Rainforest of Rio de Janeiro are discussed.

Trypanosoma cruzi: Correlations of Biological Aspects of the Life Cycle in Mice and Triatomines

The infection pattern in Swiss mice and Triatomine bugs (Rhodnius neglectus) of eleven clones and the original stock of a Trypanosoma cruzi isolate, derived from a naturally infected Didelphis marsupialis, were biochemically and biologically characterized. The clones and the original isolate were in the same zymodeme (Z1) except that two clones were found to be in zymodeme 2 when tested with G6PDH. Although infective, neither the original isolate nor the clones were highly virulent for the mice and lesions were only observed in mice infected with the original stock and one of the clones (F8). All clones and the original isolate infected bugs well while only the original isolate and clones E2 and F3 yielded high metacyclogenesis rates. An observed correlation between absence of lesions in the mammal host and high metacyclogenesis rates in the invertebrate host suggest a evolutionary trade off i.e. a fitness increase in one trait which is accompanied by a fitness reduction in a different one. Our results suggest that in a species as heterogeneous as T. cruzi, a cooperation effect among the subpopulations should be considered.

The Occurrence of Rhodnius prolixus Stal, 1859, Naturally Infected by Trypanosoma cruzi in the State of Rio de Janeiro, Brazil (Hemiptera, Reduviidae, Triatominae)

Rhodnius prolixus, one of the most importantvector of Chagas’ disease in Venezuela, was ba-sically considered in this country as related to thedomiciliar transmission cycle (O Cova-Garcia etal. 1959 Publ Div Malariol 11: 209-253). In thesylvatic environment it was observed that this spe-cies was mainly adapted to palm tree crowns andnests of Mycteria americana . R. prolixus is a veryeclectic species feeding on marsupials, rats, birdsand reptilians. Moreover, it is agreed that the ad-aptation of R. prolixus to human dwellings was aconsequence of human colonization of the naturalfoci of Trypanosoma cruzi (F Pifano1973 ArchVenezol Med Trop Parasitol Med 5: 3-29).During a search for triatomids performed in anAtlantic rain forest area in Teresopolis, State ofRio de Janeiro, we collected a total of 23 speci-mens (2 adults and 21 nymphs) of R. prolixus fromwhich 13 were infected by T. cruzi . Eleven nymphswere found in the axils of Pteridophyta leaves; 2adults and 4 nymphs in leafages, probably opos-sum nests and 6 nymphs were collected on the trunkof palm-tree. Several eggs could be found in theaxils of 1 Pterydophyta (Fig. 1). The studied area, Granja Florestal, Teresopolis,can be characterized as a secondary rain forest withpoor human dwellings on the forests borders. Thelocal population live basically on small agricul-ture and hunting. Weekly searches were performedbetween September and March (1994-95) and in-cluded palm-trees, bracts of pteridophyta, bird andmammal nests, leafages and bromeliaceae. The collected insects were maintained in glassflasks, fed through a membrane (ES Garcia et al.1975 Rev Brasil Biol 35 : 207-210) and the nymphswere allowed to moult. Seven isolates of T. cruziwere obtained through inoculation of swiss micewith the feces of the infected bugs. Axenic me-dium derived metacyclic forms (10