Results
By indirect immunofluorescence test for anti-T. cruzi antibodies in human blood collected on filter paper[30], 212 (0.83%) of persons tested had specific antibodies for T. cruzi infections (Figure 3). Positive serologic results in young populations indicate recent transmission and acute infection. Forty-six children ≤10 years of age (0.18% of the total study population) were antibody-positive for T. cruzi and were considered acutely infected.
Figure 3. Human population growth and acute T. cruzi infections in the Amazonian county Paço do Lumiar[19,20,21,22,23,24,25,26,27,28]. In A, population density increased approximately fourfold in the last three decades. In B, autoctonous acute T. cruzi infections increased fourfold in the same timespan (comparing percentages of seropositivity in 41- to 50-year-olds vs. 11- to 20-year-olds), affecting younger age groups.
Our results, which show seroprevalence of T. cruzi infections in the absence of hematophagous bugs or their vestiges (excreta and molted skins) in houses, prompted us to search for triatomines in the ecosystem where the population was infected or continues to be at risk. The strategy for capture of triatomines consisted in surveillance of houses by residents (household members captured bugs in the house and placed them in plastic containers) or in dissection of palm trees in backyards of houses[16,45,46,47,48,49]. This householder-assisted surveillance and capture method yielded 52 triatomine bugs (36 R. pictipes and 16 R. neglectus). Triatomine excreta and molted skins in these houses were neither reported by inhabitants nor detected by field workers. Adult triatomines were captured in houses only during the rainy season. We also captured 133 triatomines in 23 palm trees cut down in backyards in five villages. Careful dissection of these trees allowed detection of different developmental stages of triatomines in clefts of palm frond-sheets (Figure 4, Tables 2-4).
Figure 4. Palm tree (Attalaea phalerata) frond-sheet microsystem and cycle of transmission of Trypanosoma cruzi. This natural dwelling of triatomine species and top predators (Didelphis marsupialis, circle) consists of clefts formed by the insertion of frond sheets into the stipe of babassu palm.
Remains of animal species (e.g., nests, hair, feathers) on which triatomine bugs prey were identified in palm trees in backyards in five villages. Twice when a tree was cut down, adult opossums (Didelphis, Figure 4) ran out of the fronds into the forest. Nests of marsupials and birds were easily detected on dissection of palm fronds and crowns. In addition to opossums and birds, we identified molds and captured and identified different species of various taxa of invertebrate and vertebrate animals[50,51,52,53,54,55,56,57,58,59,60] in the 23 palm trees (Table 5, Figure 5). Molds were found in stipes, fronds, and crowns, and insects in roots, stipes, inflorescence, fruits, fronds, crowns, and leaves. The clefts formed by frond sheets were particularly rich in Amphibia, Arachnida, and Hemiptera. Triatomines were detected at the bottom of clefts where marsupials built their nests. Bird nests were found in the fronds and crowns where abundant species of insects were available for predation.
Figure 5. The informal economy: man carrying a babassu palm frond to be sold as soft thatch. A single palm tree enclosing five trophic levels associated with the Rhodnius pictipes (Hemiptera: Reduviidae) transmission of T. cruzi in the babassu microsystem.
Scarcity of blood flagellates in marsupials precluded detection by direct microscopy. However, the metacyclic flagellates recovered by xenodiagnosis were subinoculated in weanling mice. Two weeks after injection, trypomastigote forms of the parasitic protozoan morphologically indistinguishable from T. cruzi were detected in blood of the mice. To define and further characterize these isolates, we used phenotypic and genotypic molecular characterizations. In the first group, antibodies in sera of chronic Chagas disease patients reacted indistinctly with antigenic determinants in the surface of T. cruzi Berenice and with isolates Dm1, Dm2, and Dm3 from D. marsupialis and with Rp1 from R. pictipes.
Genotypic kDNA and nuclear DNA (nDNA) markers were used to genetically characterize these wild flagellate protozoan isolates. PCR amplification of template DNA from each of these T. cruzi isolates showed that kDNA primers S35/36[35,36,37] amplified cruzi, a laboratory standard for virulent T. cruzi. In addition, when we used PCR with mini-exon intergenic spacer primers TC/TC1/TC2[38] and rDNA primers D71/72[39,40,41], amplification resulted in the same bands, using template DNA from wild T. cruzi Dm 28c, Dm1, Dm2, Dm3, and Rp1 and from Berenice T. cruzi isolated from a patient with acute Chagas disease (Figure 6). These molecular features allowed classification of wild isolates of T. cruzi as phylogenetic type I, while Berenice was T. cruzi II[11,38,39,40,41]. These results were further confirmed by in situ hybridization of wild T. cruzi with a biotinilated 198-bp sequence derived from Berenice template DNA (Figure 7), which was amplified with specific nDNA primers Tcz1/2[37].
Figure 6. Genotypic characterization of wild-type flagellates by PCR amplification with rDNA and mini-exon specific primers derived from Trypanosoma cruzi. A, template DNAs amplified with mini-exon intergenic spacer primers (38): Blank, negative control; Tcb, archetypic type II T. cruzi Berenice; Rp1, Dm1, Dm2 and Dm3, flagellates isolated from Rhodnius pictipes and from Didelphis marsupialis; Dm28, standard type I, sylvatic T. cruzi isolate. B, same template DNAs amplified with rDNA primers (39-41). Tcb yielded typical 300-bp band of type II lineage, whereas Rp1, Dm1, Dm2, and Dm3 and Dm28 yielded a 350-bp band of type I T. cruzi lineage, with mini-exon spacer primers. In addition, Tcb yielded typical a 125-bp band of type II, whereas the sylvatic T. cruzi isolates yielded a 110-bp band of type I, with rDNA primers. These findings confirm sylvatic Rp1, Dm1, Dm2, and Dm3 as T. cruzi.
Figure 7. In situ hybridization of Rhodnius pictipes-derived flagellates with an archetypic Berenice Trypanosoma cruzi-specific probe. The byotinylated 198-bp sequence amplified with Tcz1/2 nuclear DNA probe[37] was revealed with fluorescein-conjugated streptavidin. This genotypic marker, representing 12.5% of total parasitic nuclear DNA, also confirms this sylvatic isolate as T. cruzi.
Emerging Infectious Diseases. 2001;7(1) © 2001 Centers for Disease Control and Prevention (CDC)
Cite this: Emerging Chagas Disease: Trophic Network and Cycle of Transmission of Trypanosoma cruzi from Palm Trees in the Amazon - Medscape - Feb 01, 2001.
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