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.

We have focused on the role played by a carnivore, the coati (Nasua nasua), in the transmission cycle of Trypanosoma cruzi in the Brazilian Pantanal biome. We collected data during 2000/01 and 2005-07. Prevalence and pattern of T. cruzi infection were determined by serological tests and hemoculture. Isolates were characterized by miniexon molecular assay. Our results demonstrate that T. cruzi transmission cycle among coatis in the southern Pantanal seems to be well established, as we found high serum prevalences and high parasitemias throughout the two studied periods. Single infections by TCII (32.1%), TCI (28.0%) and Z3 (7.1%) were observed. Mixed infections by TCI/TCII (10.7%) and TCI/Z3 (3.6%) were also detected. Distinct genotypes of T. cruzi could be recovered during the 8 months follow-up of the same animals. As free-living coatis have high densities and inhabit all habitats, they may play an important role in the maintenance and dispersion of the main T. cruzi subpopulations. Considering that the Pantanal connects some of the major biomes of South America, it may be acting as a corridor for the spread of the main T. cruzi subpopulations. Our data give support that predator-prey links are important mechanisms for T. cruzi transmission and perpetuation in the wild.

Few studies have been conducted to investigate the role played by the order Chiroptera in the sylvatic transmission cycle of Trypanosoma cruzi or their putative association with the main genotypes of the parasite. Here, the purpose was to enlarge the knowledge of this issue, in this sense, 93 specimens of bats included in 4 families, respectively Molossidae, Noctilionidae, Phyllostomidae and Vespertilionidae collected in distinct regions of Brazil were submitted to fresh blood smears and hemocultures. No patent parasitemia was observed but positive hemocultures by T. cruzi were observed in 14% (13/93) of examined samples. The majority of the parasite isolates were obtained from Phyllostomus hastatus (80%) captured in one same buriti hollow palm tree in the Cerrado region. Multilocus enzyme electrophoresis (MLEE) analyses showed that the genetic distance among these isolates was 0.35, almost the same observed when all the isolates (excluding the reference strains) were analyzed (0.40). No correlation of zymodeme with bat genera, species or geographic region of its origin could be observed, moreover, correlation of zymodeme and genotype of the parasite was not strict. Ten out of 14 T. cruzi isolates obtained from bats corresponded to the TCII genotype. Chiropterans with TCI, TCII/TCIII mixed infection as well as Trypanosoma rangeli in single or mixed infections were observed. These results show that bats may harbor and are probably important maintainers of the main genotypes (TCI, TCII, TCIII/Z3) of T. cruzi. These results support the absence of an association of TCII with any mammal order and show that bats, mainly P. hastatus, may act as amplifier hosts of TCII subpopulations of T. cruzi.

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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.

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Philander frenata and Didelphis marsupialis harbor parasitism by Trypanosoma cruzi without developing any apparent disease and on the contrary to D. marsupialis, P. frenata maintains parasitism by T. cruzi II subpopulations. Here we compared the humoral immune response of the two didelphids naturally and experimentally infected with T. cruzi II group, employing SDS-PAGE/Western blot techniques and by an Indirect immunofluorescence assay. We also studied the histopathological pattern of naturally and experimentally infected P. frenata with T. cruzi. P. frenata sera recognized more antigens than D. marsupialis, and the recognition pattern did not show any change over the course of the follow up of both didelphid species. Polypeptides of 66 and 90kDa were the most prominent antigens recognized by both species in the soluble and enriched membrane fractions. P. frenata recognized intensely also a 45kDa antigen. Our findings indicate that: 1) there were no quantitative or qualitative differences in the patent or subpatent phases in the recognition pattern of P. frenata; 2) the significant differences in the recognition pattern of parasitic antigens by P. frenata and D. marsupialis sera suggest that they probably "learned" to live in harmony with T. cruzi by different strategies; 3) although P. frenata do not display apparent disease, tissular lesions tended to be more severe than has been described in D. marsupialis; and 4) Both didelphids probably acquired infection by T. cruzi after their evolutionary divergence.