Trypanosoma cruzi infection via oral route results in outbreaks or cases of acute Chagas disease (ACD) in different Brazilian regions and poses a novel epidemiological scenario. In the Espírito Santo state (southeastern Brazil), a fatal case of a patient with ACD led us to investigate the enzootic scenario to avoid the development of new cases. At the studied locality, Triatoma vitticeps exhibited high T. cruzi infection rates and frequently invaded residences. Sylvatic and domestic mammals in the Rio da Prata locality, where the ACD case occurred, and in four surrounding areas (Baia Nova, Buenos Aires, Santa Rita and Todos os Santos) were examined and underwent parasitological and serological tests. Triatomines were collected for a fecal material exam, culturing and mini-exon gene molecular characterization, followed by RFLP-PCR of H3/Alul. Paraffin-embedded cardiac tissue of a patient was washed with xylene to remove paraffin and DNA was extracted using the phenol-chloroform method. For genotype characterization, PCR was performed to amplify the 1f8, GPI and 18S rRNA genes. In the case of V7V8 SSU rRNA, the PCR products were molecularly cloned. PCR products were sequenced and compared to sequences in GenBank. Phylogenetic analysis using maximum likelihood method with 1000 bootstrap replicates was performed. None of the animals showed positive hemocultures. Three rodents and two dogs showed signs of infection, as inferred from borderline serological titers. T. vitticeps was the only triatomine species identified and showed T. cruzi infection by DTUs TcI and TcIV. The analysis of cardiac tissue DNA showed mixed infection by T. cruzi (DTUs I, II, III and IV) and Trypanosoma dionisii. Each case or outbreak of ACD should be analyzed as a particular epidemiological occurrence. The results indicated that mixed infections in humans may play a role in pathogenicity and may be more common than is currently recognized. Direct molecular characterization from biological samples is essential because this procedure avoids parasite selection. T. dionisii may under certain and unknown circumstances infect humans. The distribution of T. cruzi DTUS TcIII and TcIV in Brazilian biomes is broader than has been assumed to date.

Molecular phylogenetic studies have revealed the growing diversity of bat trypanosomes. Here, 14 isolates from blood samples of the vampire bat Desmodus rotundus (Phyllostomidae) from Rio de Janeiro, Southeast Brazil, were cultivated, and morphologically and molecularly characterized. All isolates represent a novel species named Trypanosoma madeirae n. sp. positioned in the Neobat lineage of the clade T. cruzi. The Neobat lineage also comprises closely related trypanosomes of clades Neotropic 1, 2 and 3 from diverse phyllostomid species. Trypanosomes of Neotropic 1, found in Trachops cirrhosus and Artibeus jamaicensis (phyllostomids), likely represent a different species or genotype closely related to T. madeirae. Consistent with its phylogenetic positioning, T. madeirae differs from Trypanosoma cruzi in morphology of both epimastigote and trypomastigote culture forms and does not infect Triatoma infestans. Similar to its closest relatives of Neobat lineage, T. madeirae was unable to develop within mammalian cells. To date, PCR-surveys on archived blood/liver samples unveiled T. madeirae exclusively in D. rotundus from Southern to Northern Brazil. The description of a new species of bat trypanosome associated with vampire bats increases the repertoire of trypanosomes infecting D. rotundus, currently comprised of Trypanosoma cruzi, T. cruzi marinkellei, Trypanosoma dionisii, Trypanosoma rangeli, Trypanosoma pessoai, and Trypanosoma madeirae.

Trypanosoma cruzi, the agent of Chagas disease in humans, has a vast reservoir of mammalian hosts in the Americas, and is classified into six genetic lineages, TcI-TcVI, with a possible seventh, TcBat. Elucidating enzootic cycles of the different lineages is important for understanding the ecology of this parasite, the emergence of new outbreaks of Chagas disease and for guiding control strategies. Direct lineage identification by genotyping is hampered by limitations of parasite isolation and culture. An indirect method is to identify lineage-specific serological reactions in infected individuals; here we describe its application with sylvatic Brazilian primates. Synthetic peptides representing lineage-specific epitopes of the T. cruzi surface protein TSSA were used in ELISA with sera from Atlantic Forest Leontopithecus chrysomelas (golden-headed lion tamarin), L. rosalia (golden lion tamarin), Amazonian Sapajus libidinosus (black-striped capuchin) and Alouatta belzebul (red-handed howler monkey). The epitope common to lineages TcII, TcV and TcVI was recognised by sera from 15 of 26 L. chrysomelas and 8 of 13 L. rosalia. For 12 of these serologically identified TcII infections, the identity of the lineage infection was confirmed by genotyping T. cruzi isolates. Of the TcII/TcV/TcVI positive sera 12 of the 15 L. chrysomelas and 2 of the 8 L. rosalia also reacted with the specific epitope restricted to TcV and TcVI. Sera from one of six S. libidinous recognised the TcIV/TcIII epitopes. This lineage-specific serological surveillance has verified that Atlantic Forest primates are reservoir hosts of at least TcII, and probably TcV and TcVI, commonly associated with severe Chagas disease in the southern cone region of South America. With appropriate reagents, this novel methodology is readily applicable to a wide range of mammal species and reservoir host discovery.

Trypanosomatids are diverse and can infect several host species, including small mammals (rodents and marsupials). Between 2012 and 2014, 91 small mammals were surveyed for trypanosomatid infection in the Estação Biológica FIOCRUZ Mata Atlântica (EFMA), an Atlantic Forest area in Rio de Janeiro that presents different levels of conserved and degraded areas. Blood, skin, liver, and spleen samples were submitted to parasitological, serological, and molecular assays to detect the infection and determine the taxonomic status of their parasites. Sixty-eight individuals (74.7%; n = 91) were infected by trypanosomatids, including fourteen mixed infected by different trypanosomatid parasites. These hosts were infected by: T. cruzi DTU TcI (n = 12), T. cruzi DTU TcIV (n = 2), T. janseni (n = 15), T. dionisii (n = 1), and T. rangeli A (n = 1) detected in blood or tissue cultures, in addition to T. cruzi DTU TcI (n = 9) and Leishmania sp. (n = 1) only by the molecular diagnosis. Serological diagnosis was positive in 38 (71.6%) individuals for T. cruzi, the same amount for Leishmania spp., and 23 (43.3%) individuals were mixed infected. These data indicate a remarkable richness of trypanosomatid species/genotypes infecting small mammals, even in a disturbed area with low mammal species diversity—as is the case of the EFMA—reinforcing the generalist aspect of these parasites.

This study describes the morphological, biochemical, and molecular differences among Trypanosoma dionisii isolates from hemocultures of hematophagous (Desmodus rotundus; n = 2) and insectivorous (Lonchorhina aurita; n = 1) bats from the Atlantic Rainforest of Rio de Janeiro, Brazil. Fusiform epimastigotes from the hematophagous isolates were elongated, whereas those of the insectivorous isolate were stumpy, reflected in statistically evident differences in the cell body and flagellum lengths. In the hemocultures, a higher percentage of trypomastigote forms (60%) was observed in the hematophagous bat isolates than that in the isolate from the insectivorous bat (4%), which demonstrated globular morphology. Three molecular DNA regions were analyzed: V7V8 (18S rDNA), glycosomal glyceraldehyde 3-phosphate dehydrogenase gene, and mitochondrial cytochrome b gene. The samples were also subjected to multilocus enzyme electrophoresis and random amplified polymorphic DNA analysis. All isolates were identified as T. dionisii by phylogenetic analysis. These sequences were clustered into two separate subgroups with high bootstrap values according to the feeding habits of the bats from which the parasites were isolated. However, other T. dionisii samples from bats with different feeding habits were found in the same branch. These results support the separation of the three isolates into two subgroups, demonstrating that different subpopulations of T. dionisii circulate among bats.