International Journal for Parasitology 39 (2009) 615–623 Contents lists available at ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara Trypanosoma cruzi in Brazilian Amazonia: Lineages TCI and TCIIa in wild primates, Rhodnius spp. and in humans with Chagas disease associated with oral transmission q Arlei Marcili a, Vera C. Valente b, Sebastião A. Valente b, Angela C.V. Junqueira c, Flávia Maia da Silva a, Ana Yecê das Neves Pinto b, Roberto D. Naiff d, Marta Campaner a, José R. Coura c, Erney P. Camargo a, Michael A. Miles e, Marta M.G. Teixeira a,* a Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900 São Paulo, SP, Brazil Laboratório de Doença de Chagas, Instituto Evandro Chagas, Belem, PA, Brazil c Laboratório de Doenças Parasitárias, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brazil d Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, Amazonas, Brazil e Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK b a r t i c l e i n f o Article history: Received 29 July 2008 Received in revised form 20 September 2008 Accepted 22 September 2008 Keywords: Trypanosoma cruzi lineages TCI and TCIIa Chagas disease Oral infection Non-human primates Amazonia ssrDNA Cytochrome b Evolution Phylogeny a b s t r a c t In this study, we provide phylogenetic and biogeographic evidence that the Trypanosoma cruzi lineages T. cruzi I (TCI) and T. cruzi IIa (TCIIa) circulate amongst non-human primates in Brazilian Amazonia, and are transmitted by Rhodnius species in overlapping arboreal transmission cycles, sporadically infecting humans. TCI presented higher prevalence rates, and no lineages other than TCI and TCIIa were found in this study in wild monkeys and Rhodnius from the Amazonian region. We characterised TCI and TCIIa from wild primates (16 TCI and five TCIIa), Rhodnius spp. (13 TCI and nine TCIIa), and humans with Chagas disease associated with oral transmission (14 TCI and five TCIIa) in Brazilian Amazonia. To our knowledge, TCIIa had not been associated with wild monkeys until now. Polymorphisms of ssrDNA, cytochrome b gene sequences and randomly amplified polymorphic DNA (RAPD) patterns clearly separated TCIIa from TCIIb-e and TCI lineages, and disclosed small intra-lineage polymorphisms amongst isolates from Amazonia. These data are important in understanding the complexity of the transmission cycles, genetic structure, and evolutionary history of T. cruzi populations circulating in Amazonia, and they contribute to both the unravelling of human infection routes and the pathological peculiarities of Chagas disease in this region. Ó 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction Trypanosoma cruzi occurs exclusively in the American continent where it has been parasitising sylvatic mammals for millions of years. American trypanosomiasis caused by T. cruzi, known as Chagas disease in humans, is considered to be one of the most important parasitic infections in Latin America (Miles et al., 2003; Coura, 2007). In regions endemic for Chagas disease, T. cruzi circulates between humans and domestic animals and is transmitted by domiciliated triatomine bugs. However, infection by T. cruzi is primarily a highly prevalent and widespread zoonosis that occurs from the southern half of the USA to the southernmost countries of South America, in a range of habitats that include the Amazonian rainforq Nucleotide sequences reported in this paper are available in the GenBank database under the Accession numbers listed in Table 1. * Corresponding author. Tel.: +55 11 30917268; fax: +55 11 30917417. E-mail address: [email protected] (M.M.G. Teixeira). est. In the sylvatic environment, hosts of T. cruzi encompass 180 species belonging to 25 mammalian families of virtually all-mammalian orders; Didelphimorpha, Xenarthra, Rodentia and Primata are the most frequently infected (Miles et al., 1979, 2003; Coura et al., 2002). Natural populations of T. cruzi are very heterogeneous in biological, immunological and molecular features (Miles et al., 2003; Coura, 2007). Based on multilocus enzyme electrophoresis (MLEE), T. cruzi populations were distributed in the following principal zymodemes: Z1; Z2; Z3 (Miles et al., 1978); Z3 with a Z1 ASAT character (Miles et al., 1981a); and Bolivian and Paraguayan ‘hybrid’ lineages (Tibayrenc and Miles, 1983; Chapman et al., 1984). By comparing randomly amplified polymorphic DNA (RAPD), ribosomal, mini-exon and cytochrome b gene markers, the divisions within T. cruzi were redesignated as TCI (Z1) and five lineages of TCII: TCIIa (Z3), TCIIb (Z2), TCIIc (Z3/Z1 ASAT), TCIId and TCIIe (the latter two are hybrids of TCIIb and TCIIc) (Souto et al., 1996; Anonymous, 1999; Brisse et al., 2000, 2001). The phylogenetic 0020-7519/$34.00 Ó 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2008.09.015 616 A. Marcili et al. / International Journal for Parasitology 39 (2009) 615–623 position of TCIIa and TCIIc in relation to other lineages varies according to the markers used (Brisse et al., 2001; Mendonça et al., 2002; Sturm et al., 2003; Westenberger et al., 2005, 2006; Freitas et al., 2006). Genetic distances to TCI and TCIIb led to the suggestion that TCIIa and TCIIc might, like TCIId and TCIIe, be hybrid lineages (Sturm et al., 2003; Westenberger et al., 2005, 2006) although this was not supported by the extent of heterozygosity, karyotyping, or analyses of microsatellites and the cytochrome oxidase II gene (Freitas et al., 2006; Pedroso et al., 2007). It has been hypothesized that the evolutionary history of T. cruzi might be correlated with a long-standing association with its vertebrate hosts. Early mammals of South America are the marsupials and the placentals of Xenarthra (armadillos, sloths and anteaters) from the end of the Cretaceous, 65 million years ago (mya), when this continent was separated from all other landmasses. It has been suggested that TCI evolved with marsupial didelphids and TCII in association with terrestrial mammals, such as armadillos (Miles et al., 1981a; Gaunt and Miles, 2000; Yeo et al., 2005), rather than forming recent associations with those ecological niches. A recent study supported this hypothesis, proposing at least three ancestral lineages (TCI, TCIIb and TCIIc) (Freitas et al., 2006). An alternative suggestion is that TCII entered South America from North America during the Pliocene (5 mya), or with primates and caviomorph rodents during the Oligocene (35 mya) (Briones et al., 1999; Kawashita et al., 2001). However, this is not consistent with the present distribution and hosts of TCII. Only TCIIa has been found in North America (Brisse et al., 2001; Hall et al., 2007; Roellig et al., 2008). Furthermore, non-human primates appear to be naturally associated with TCI in both Atlantic and Amazonian forests (Lisboa et al., 2006; Maia da Silva et al., 2008). The discovery of free-ranging tamarins infected with TCII in a biological reserve of the Atlantic Forest must be carefully interpreted because some animals could be infected while in captivity before being re-introduced to the wild (Fernandes et al., 1999; Lisboa et al., 2004, 2006; Yeo et al., 2005). In addition, caviomorph rodents are naturally infected with TCI, except when living close to human dwellings and/or in regions endemic for Chagas disease (Herrera et al., 2005). Humans became accidental hosts of T. cruzi thousands of years ago, as evidenced by its presence in mummified human tissues (Aufderheide et al., 2004). The main sylvatic host of TCI is the opossum Didelphis marsupialis, which lives in both arboreal and terrestrial sylvatic and peridomestic ecotopes. Most isolates from humans, reservoirs and vectors from Amazonia belong to TCI with scarce TCIIa and TCIIc isolates. The sylvatic hosts of TCIIa are poorly known, despite records of both TCIIa/TCIIc associated with armadillos and marsupials (Monodelphis) in terrestrial ecotopes (Miles et al., 1981a; Póvoa et al., 1984; Gaunt and Miles, 2000; Yeo et al., 2005). There are no reports of TCIIb, TCIId and TCIIe lineages in this region, which are those that predominate in humans, domestic and peridomestic vectors in southern South America, whereas TCI occurs in sylvatic cycles and is only sporadically found in humans (Miles et al., 1981a; Coura et al., 2002; Fernandes et al., 1998, 2001; Teixeira et al., 2006). Thus, transmission dynamics of T. cruzi populations circulating in Amazonia are quite distinct. Although there is a sustained low-intensity transmission with sporadic autochthonous human cases, Chagas disease is not endemic to the Amazon region, probably because of the absence of species of triatomines adapted to human dwellings and the small human population (Miles et al., 1981b, 2003; Coura et al., 2002). In recent years, more than 400 acute cases have been reported, mostly from outbreaks that were probably due to oral transmission. The main transmission mechanisms of T. cruzi in this region are the sporadic invasion of domiciles by light-attracted triatomines from palms and ingestion of palm fruit juices contaminated with crushed triatomines in outbreaks of oral acute Chagas disease. Human settlements and deforestation in Amazonia could enhance the possibility of adaptation of sylvatic triatomines to human dwellings and the risk of Chagas disease transmission (Miles et al., 1981a; Valente et al., 1998, 1999; Coura et al., 2002; Albajar et al., 2003; Pinto et al., 2004; Xavier et al., 2006; Aguilar et al., 2007). Understanding the diversity of wild reservoirs and the dynamics of transmission cycles is important for evaluating the risk of emergence of distinct sylvatic lineages of T. cruzi as human pathogens. In this study, we characterised isolates from humans, wild primates and triatomines of the genus Rhodnius from Brazilian Amazonia. Besides genotyping by mini-exon and ribosomal markers, we inferred genetic relatedness between isolates from these hosts by comparing sequences of the variable V7–V8 region of ssrDNA and mitochondrial cytochrome b gene sequences. Intralineage genetic diversity was evaluated by ssrDNA sequence polymorphisms and by RAPD patterns. 2. Materials and methods 2.1. Study areas, capture and handling of wild mammals Field studies of T. cruzi-infected wild mammals were carried out in distantly separated locations of Brazilian Amazonia in the States of Rondônia and Acre (West), Amazonas (North), and Pará and Amapá (East) (Fig. 1; Table 1). The work with humans was approved by the Committee of Ethic of the Evandro Chagas Institute Fig. 1. Map of the Brazilian Amazon region showing the geographical origin and genotyping results of Trypanosoma cruzi isolates characterised in this study. (A) Geographical origin of T. cruzi isolates from humans (w), non-human primates (N), triatomines (d) and marsupials (j). (B) Genotyping patterns of T. cruzi isolates selected to illustrate genotypes found amongst isolates from these hosts. Reference strains: TCI (G), TCIIb (Y), TCIIa (JJ) and TCIIc (MT3869). Brazilian states: Amazonia (AM); Pará (PA); Amapá (AP); Rondônia (RO); and Acre (AC). 617 A. Marcili et al. / International Journal for Parasitology 39 (2009) 615–623 Table 1 Trypanosoma cruzi isolates used in this study; host and geographic origin, genotyping and sequences of ssrDNA and cytochrome b genes used for phylogenetic analysis. TryCCa Wild primates 11 337 338 463 1536 201 209 262 269 331 931 1173 1171, 1176 1229 1237 1236, 1290 1537, 538, 549 Isolateb Host Lineagec Geographic origin GenBank acession number ssrRNA Cyt b EU755215 EU755223 AY491762 EU755224 EU755251 EU755219 EU755220 AY491763 EU755221 EU755222 EU856376 EU856377 EU856378 EU856371 11225 Fuscicolis 15 Labiatus 17 2440 IM4817 AT-AEI M12127 AE-AAB AV-AAF AM-ANV AT-AEQ IM4828 IM4949/4935 IM5084 IM5046 IM5083/5053 IM4988/5028/5021 Aotus sp. Saguinus fuscicollis Saguinus labiatus Cebus albifrons Saguinus ustus Saimiri sciureus Aotus sp. Cebuella pygmaea Saguinus midas Cebus paella Saimiri sciureus Saguinus bicolor Saguinus bicolor Saguinus bicolor Saguinus bicolor Saguinus bicolor Belém Plácido de Castro Plácido de Castro Barcelos Manaus Marajó Island Belém Rio Branco Manaus Rio Branco Marajó Island Manaus Manaus Manaus Manaus Manaus PA/BR AC/BR AC/BR AM/BR AM/BR PA/BR PA/BR AC/BR AM/BR AC/BR PA/BR AM/BR AM/BR AM/BR AM/BR AM/BR TCIIa TCIIa TCIIa TCIIa TCIIa TCI TCI TCI TCI TCI TCI TCI TCI TCI TCI TCI CANIII JJ RBS 4766 EDS 3068 MBS 3659 IJR 3475 CSL 3665 M6241 cl6 Silvio X10 AM16 Cilene MNO CASS GSC DRS RBS 3081 MPR MPS 6823 OAD 7389 IMD 7390 MGS 7392 Tc15776 Tc5106 Tc15926 Y Peru Esmeraldo cl3 CBB cl3 MT3869 NRcl3 9280 cl1 Homo Homo Homo Homo sapiens sapiens sapiens sapiens Belém Barcelos Macapá Macapá PA/BR AM/BR AP/BR AP/BR Homo Homo Homo Homo Homo Homo Homo Homo Homo Homo sapiens sapiens sapiens sapiens sapiens sapiens sapiens sapiens sapiens sapiens Santarém Bragança Belém Belém Barcelos Barcelos Irituia Belém Ananindeua Macapá PA/BR PA/BR PA/BR PA/BR AM/BR AM/BR PA/BR PA/BR PA/BR AP/BR AJ009148 AY491761 EU755244 EU755246/ EU755249 EU755247 EU755248 Homo Homo Homo Homo Homo Homo Homo Homo Homo Homo Homo Homo Homo Homo Homo sapiens sapiens sapiens sapiens sapiens sapiens sapiens sapiens sapiens sapiens sapiens sapiens sapiens sapiens sapiens Macapá Macapá Cachoeira do Arari Cachoeira do Arari Cachoeira do Arari Breves Barcarena Bagre AP/BR AP/BR PA/BR PA/BR PA/BR PA/BR PA/BR PA/BR SP/BR Peru BA/BR Chile AM/BR Chile Bolivia TCIIa TCIIa TCIIa TCIIa TCIIa TCIIa TCIIa TCIIa TCI TCI TCI TCI TCI TCI TCI TCI TCI TCI TCI TCI TCI TCI TCI TCI TCIIb TCIIb TCIIb TCIIb TCIIc TCIId TCIId RBX RBIII RBI/Rb777 Rb778 Rr351 Rr668 Rr661 Rr698 RBVI RBVII/Rb761/Rb776 Rr649/ Rr669 Rr651/ Rr675 MF6 PA 528 5306 IM5129 IM5112 Tehuentepec cl2 SC13 TU18cl2 CL14 MT3663 Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius Rhodnius TCIIa TCIIa TCIIa TCIIa TCIIa TCIIa TCIIa TCIIa TCI TCI TCI TCI TCI TCI TCI TCI EU755217 EU755218 EU856367 EU755232 EU856379 EU856369 EU856370 EU755238 EU755240/ EU755241 Humans 85 1434 144 1449 1441 1446 1339 29 743 821 823 835 971 1435 973 978 1396 1408 1454 1581 1593 1590 34 844 967 Triatomines 82 83 87, 777 778 351 668 661 698 77 78, 761, 776 649, 669 651, 675 1359 1403 1073 1166 1178 476 845 brethesi brethesi brethesi brethesi robustus robustus robustus robustus brethesi brethesi robustus robustus robustus robustus pictipes pictipes Triatoma sp. Rhodnius pallescens Triatoma infestans Triatoma infestans Panstrongylus geniculatus Carauari Barcelos Barcelos Barcelos Barcelos Monte Negro Monte Negro Monte Negro Monte Negro Barcelos Barcelos Monte Negro Monte Negro Cachoeira do Arari Santana Macapá Itacoatiara Manaus AM/BR AM/BR AM/BR AM/BR RO/BR RO/BR RO/BR RO/BR AM/BR AM/BR RO/BR RO/BR PA/BR AP/BR AP/BR AM/BR AM/BR México Colombia Bolívia RS/BR AM/BR TCI TCI TCIIb TCIIb TCIIc AF303659 EU856368 EU856380 AJ130933 AJ130928 EU755231 EU755233 EU755234/ EU755245 EU755235 EU755250 AF301912 X53917 AJ130931 AJ439722 AF303660 AF228685 AJ439725 EU856372 EU755226 EU755228 EU755216 EU856373 EU755225/ EU755227 EU755243 EU755236 EU755237/ EU755239 AJ130938 AJ130937 AJ130932 EU856375 AF288660 (continued on next page) 618 A. Marcili et al. / International Journal for Parasitology 39 (2009) 615–623 Table 1 (continued) TryCCa Didelphids 30 711 1334 712 131, 132 Isolateb Host SC43cl1 CL Brener Triatoma infestans Triatoma infestans G MS 2669 B 6020 Cuica cl1 MS 2682 IB74FB/IB74P Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Philander opossum Monodelphis brevicaudata Philander frenata Lineagec Geographic origin Barcelos Paraobebas Barcelos Ilha Bela GenBank acession number ssrRNA Cyt b Bolívia RS/BR TCIId TCIIe AF232214 AJ439721 AJ130935 AM/BR AM/BR PA/BR SP/BR AM/BR SP/BR TCI TCI TCI TCI TCIIc TCIIc AF239981 EU755229 EU755242 EU755230 AJ439719 EU856374 BR, Brazil, Brazilian states; PA, Pará; AC, Acre; AM, Amazonas; AP, Amapá; SP, São Paulo; BA, Bahia; RO, Rondônia; RS, Rio Grande do Sul. a TryCC, code number of the isolates/strains cryopreserved in the trypanosomatid culture collection (TCC), Department of Parasitology, University of São Paulo, São Paulo, Brazil. b Original codes of isolates. c Genotyping using the method developed by Fernandes et al. (2001). (IEC), Belém, Pará State. Animals were field-captured, identified and manipulated for blood sample collection according to permits from IBAMA (Instituto Brasileiro do Meio Ambiente) during research projects conducted by the primatologists Dr. Carmem Brigido (Center of Primatology, Belém), Dr. Cibele Bonvicino (INCA, Rio de Janeiro) and Dr. Marcelo Gordo (INPA, Manaus). 2.2. Isolation and culture of T. cruzi from wild mammals, humans and triatomines For T. cruzi isolation and culturing, blood samples from wild mammals and humans were inoculated into vacutainer tubes containing a biphasic medium consisting of 15% rabbit red blood cells mixed with 4% Blood Agar Base overlaid with liquid liver infusion tryptose (LIT) medium with 10% FBS, incubated at 25–28 °C, and expanded in LIT medium as previously described (Maia da Silva et al., 2007). Triatomines were collected from palm trees in sylvatic and peridomestic environments, and identified by morphology and molecular taxonomy as previously described (Maia da Silva et al., 2007) (Table 1). Field-collected triatomines were dissected, their intestinal contents were examined by phase microscopy, and samples positive for trypanosomes were inoculated into the same medium used for hemocultures. Isolates used in this study are cryopreserved in liquid nitrogen in the trypanosomatid culture collection (TCC) of the Department of Parasitology, University of São Paulo. Isolates from Pará and Amapá are also preserved in the Culture Collection of the Evandro Chagas Institute, Brazil. More than 300 isolates from wild mammals, triatomines and humans were previously genotyped using the PCR developed by Fernandes et al. (2001) (data not shown) in order to separate all the isolates of TCIIa, as well as some of the TCI isolates from the same hosts found to be infected by TCIIa, for further characterisation in this study. 2.4. PCR amplification, sequencing and data analysis of ssrDNA and Cyt b sequences DNA of T. cruzi isolates from humans, non-human primates and triatomines were used as templates for amplification of a 900 bp DNA fragment containing a partial ssrDNA sequence (V7–V8 variable region) using primers 609F and 706R and standardised PCR reactions as described previously (Maia da Silva et al., 2004a). A 500 bp DNA fragment of the cytochrome b (Cyt b) gene was amplified using primers described previously (Brisse et al., 2003). Amplification products were automatically sequenced using the same primers employed from PCR amplification. Alignments of new sequences with corresponding sequences of reference T. cruzi isolates from GenBank (Table 1) were made using ClustalW and then were manually refined and used to construct dendrograms using parsimony analysis (bootstrap analysis done with 100 replicates) and similarity matrix as previously described (Maia da Silva et al., 2004a). The alignments used in this study are available from the authors upon request and can be obtained via the EMBLALIGN database via SRS at http://srs.ebi.ac.uk under accession numbers: ALIGN-001278 and ALIGN-001279, respectively, for ssrDNA and Cyt b. 2.5. RAPD fingerprinting For primer selection and standardization of RAPD assays, we initially tested 10 decameric primers to amplify DNA from T. cruzi isolates of all phylogenetic lineages. Then, for analysis of all TCIIa and selected TCI isolates from the Amazonian region we employed three primers that yielded the most discriminating RAPD patterns: 650 (AGTATGCAGC), 625 (CCGCTGGAGC) and 672 (TACCGTGGCG). Amplifications were performed as previously described (Maia da Silva et al., 2004b). The amplification products were separated on 2.0% agarose gel and stained with ethidium bromide. 2.3. Molecular diagnosis and genotyping of T. cruzi isolates 3. Results Cultured trypanosomes were processed for DNA extraction using the traditional phenol/chloroform method. All new isolates of T. cruzi were identified using a T. cruzi-specific PCR assay based on ribosomal sequences that are able to distinguish T. cruzi and Trypanosoma rangeli (Souto et al., 1999). Genotyping of T. cruzi isolates was done using a PCR assay based 24Sa-lsrRNA (Souto et al., 1996) and mini-exon sequences (Fernandes et al., 2001). Reference strains/isolates of major T. cruzi lineages were used as controls: TCI (G and Silvio X10), TCIIa (CANIII and JJ), TCIIb (Y and Peru), TCIIc (MT3663, MT3869) and TCIId (NRcl3 and SC43cl1). 3.1. Genotyping of T. cruzi isolates from wild primates, humans and triatomines from Brazilian Amazonia Eleven new isolates of T. cruzi from nine species of Amazonian wild primates of three families were characterised in this study: Callitrichidae (Saguinus midas, Saguinus fuscicollis, Saguinus labiatus and Saguinus ustus); Aotidae (Aotus sp.); and Cebidae (Cebuella pygmaea, Saimiri sciureus and Cebus albifrons). In addition to these new isolates, we included 10 isolates from Saguinus bicolor (Maia da A. Marcili et al. / International Journal for Parasitology 39 (2009) 615–623 619 Silva et al., 2008). Moreover, 19 new isolates from humans with acute Chagas disease living in the States of Pará and Amapá obtained by hemocultures or xenodiagnosis, and 17 new isolates from Rhodnius spp. were selected for this study (Fig. 1A; Table 1). All new T. cruzi isolates were genotyped by PCR based on ribosomal and mini-exon genes (Souto et al., 1996; Fernandes et al., 2001) and assigned to lineages TCI or TCIIa/c (Z3). Despite distinguishing the lineages TCI, TCIIb and TCIIa/c (Z3), these methods do not reliably distinguish between TCIIa and TCIIc/d/ e as shown in this study using mini-exon-derived PCR (Fig. 1B). Sixteen of the 17 new TCIIa/c isolates were further identified as TCIIa by sequencing the variable V7–V8 region of ssrDNA, as described in the following section. New isolates from wild primates were assigned to TCIIa (five isolates) or TCI (six isolates) and compared with sequences determined in this study for three out 10 isolates from S. bicolor previously genotyped as TCI (Maia da Silva et al., 2008) (Table 1). We characterised 19 new isolates from humans, of which 14 were assigned to TCI and five to TCIIa. In addition, two human isolates each from TCI and TCIIa had been described previously (Miles et al., 1981a; Fernandes et al., 2001). Moreover, in this study we included 17 new isolates from Rhodnius robustus (II and IV genetic populations), Rhodnius brethesi and Rhodnius pictipes that were collected in palm trees (Table 1), and five additional TCIIa isolates previously genotyped from R. brethesi (Fernandes et al., 2001). While TCI was detected in the three species of Rhodnius examined, TCIIa was isolated only from R. robustus and R. brethesi. Analyses of trypanosomes directly from guts of Rhodnius spp. revealed mixed TCI and TCIIa infections, in addition to T. rangeli (data not shown), which suggest lineage selection during isolation in culture and, thus, hampers any association between species of Rhodnius and T. cruzi lineages. 3.2. Analyses of polymorphism and genetic relatedness amongst Amazonian TCI and TCIIa from humans, non-human primates and triatomines using ssrDNA and cytochrome b sequences To evaluate polymorphisms within Amazonian TCI and TCIIa isolates from humans, non-human primates and triatomines as well as to analyse the degree of genetic relatedness amongst these isolates and those of other lineages, we compared sequences of V7–V8 ssrDNA and Cyt b genes aligned with those from reference strains of T. cruzi lineages from GenBank (Table 1). Isolates selected from this study were from Pará, Amapá, Amazonas, Acre and Rondônia. ssrDNA sequences of 13 isolates from monkeys, 11 from humans, 13 from Rhodnius spp. and four from D. marsupialis were determined in this study. Cytochrome b sequences were determined for six isolates from monkeys, two from humans, four from Rhodnius spp., one from Panstrongylus geniculatus and one from Monodelphis brevicaudata (Table 1). In the dendrogram constructed using ssrDNA sequences, most isolates from monkeys, humans and vectors from Amazonia nested in a complex clade harbouring exclusively TCI isolates, which also included TCI reference strains (G and Silvio X10) from D. marsupialis and humans. TCI isolates revealed more heterogeneous ssrDNA sequences (98.5%) than TCIIa (99.8%). All isolates from monkeys, humans and Rhodnius sp. assigned to TCIIa clustered together with CANIII (the prototype strain of TCIIa) in an assemblage comprising exclusively isolates from Amazonia. The dendrogram based on 49 ssrDNA sequences evidenced four major and well-supported clades: TCI, TCIIa, TCIIb and TCIIc/TCIId. TCIIa diverged 5.2% from TCI, 4.4% from TCIIb, 2.8% from TCIIc (represented in this study by reference-isolates from humans (MT3869), P. geniculatus (MT3663) and by one new isolate from M. brevicaudata in Amazonia), and 2.5% from reference strains SC43cl1 and NRd3 of TCIId (Fig. 2A). Fig. 2. Dendrograms inferred based on (A) V7–V8 ssrDNA sequences (804 characters, 68 parsimony informative) of 49 Trypanosoma cruzi isolates, and (B) cytochrome b sequences (490 characters, 72 parsimony informative) of 25 isolates. Both analyses included isolates from humans (w) and non-human primates (N), triatomines (d) and marsupials (j). Reference strains of the distinct lineages were used for comparative purposes (Table 1). The numbers at the nodes correspond to parsimony percentage bootstrap values derived from 100 replicates. Analysis of 25 Cyt b sequences corroborated the general pattern and degrees of genetic relatedness amongst T. cruzi isolates revealed by ssrDNA. This analysis supported high similarity within TCIIa (99.8%) and TCI (99.6%) and large genetic distances separated TCIIa from TCIIb (11%) and TCI (7.0%), whereas small divergences separated TCIIa from TCIIc (1.2%) and TCIId/e (0.2%) (Fig. 2B). The affinity of TCIIa and TCIIc with TCIId/e on the basis of Cyt b sequences accords with the previous analysis of mitochondrial sequences by Machado and Ayala (2001), in which TCIIa, TCIIc, TCIId/e clustered into their single mitochondrial clade B; 620 A. Marcili et al. / International Journal for Parasitology 39 (2009) 615–623 Fig. 3. Agarose gels (2%) stained with ethidium bromide showing randomly amplified polymorphic DNA patterns of selected Amazonian isolates from humans, non-human primates and triatomines illustrative of the high inter-lineage polymorphism, almost identical profiles shared by TCIIa isolates, and small polymorphism within TCI, generated using primers 625 (A) and 672 (B). based on nuclear sequences TCIIa formed their fourth clade D, with their clades B (TCIIc) and C (TCIIb) each encompassing separate ‘parental’ haplotypes of the hybrid lineages TCIId/e. The relevant divergence between TCIIa and TCIIc demonstrated here with analyses of two loci corroborated a previous study of an additional nine loci (Westenberger et al., 2006). Data from the present study did not provide evidence that TCIIa have hybrid characteristics like TCIId/e. Moreover, the same TCIIa isolates from humans (JJ) and from R. brethesi employed in this study were tightly clustered together in a previous analysis of homologous chromosomes (Pedroso et al., 2007). Altogether, TCIIa data suggest that if they are hybrids, they originated from hybridization followed by an extensive genome homogenisation as proposed by Westenberger et al. (2005). 3.3. Intra-lineage polymorphism analysis determined by RAPD patterns For a sensitive evaluation of intra-lineage polymorphism, we compared RAPD patterns generated for three selected primers using DNA of isolates from human and non-human primates and from Rhodnius spp. RAPD profiles from all isolates assigned to TCIIa and selected isolates of TCI from the Amazonian region were compared with patterns generated for reference-isolates of other lineages. Two primers (625 and 672) generated RAPD profiles that allowed lineage differentiation, whereas isolates within the same lineages shared more similar patterns. Almost identical patterns were shared by all TCIIa isolates, and profiles of TCI isolates revealed small polymorphisms as exemplified with selected isolates (Fig. 3). 4. Discussion Natural cycles of T. cruzi transmission are abundant and complex in Amazonia, where a remarkable diversity of sylvatic mammals and vectors infected by distinct T. cruzi lineages circulate in separate cycles according to ecotopes and particular niches. How- ever, the sylvatic hosts and transmission cycles of the lineages are not yet entirely resolved (Miles et al., 1981a; Gaunt and Miles, 2000; Coura et al., 2002). Data from this study corroborate previous studies showing that TCI, TCIIc and TCIIa are the only T. cruzi lineages so far reported in Brazilian Amazonia (Miles et al., 1978, 1981a; Póvoa et al., 1984). TCI has a vast geographical distribution from North to South America, predominating from the Amazonian region northwards, where domestic and re-invading sylvatic triatomine species sustain transmission of Chagas disease (Miles et al., 1981b; Añez et al., 2004; Samudio et al., 2007; Fitzpatrick et al., 2008). Previous studies revealed that TCI is the most common lineage in Brazilian Amazonia, where it has been described in several arboreal mammal species, especially in Didelphis, less frequently in terrestrial mammals and, sporadically, in peridomestic mammals and humans (Miles et al., 1981a; Póvoa et al., 1984; Valente et al., 1998; Fernandes et al., 2001; Coura et al., 2002). Despite a lack of field evidence, TCIIc and TCIIa have been considered as having similar terrestrial transmission cycles. In Amazonia and elsewhere, TCIIc has been sporadically found in humans whereas it has been commonly isolated from armadillos, especially Dasypus novemcinctus, and from a few other wild terrestrial mammals such as the marsupial Monodelphis. The transmission cycle of TCIIc has been well studied. This lineage has a widespread distribution from Northeastern Brazil to Paraguay and Argentina in southern South America. To date, TCIIc has only been isolated from triatomine species that are considered to be predominantly terrestrial, P. geniculatus, Triatoma infestans and Triatoma rubrovaria (Barrett et al., 1980; Miles et al., 1981a; Barnabé et al., 2001; Yeo et al., 2005; Cardinal et al., 2008; Martins et al., 2008). The natural mammalian reservoirs of TCIIa in the Amazon Basin are not conclusively known. Despite previous records in Dasypus, Monodelphis and Panstrongylus from zymodeme analyses (Miles et al., 1981a; Póvoa et al., 1984; Gaunt and Miles, 2000; Yeo et al., 2005), and although human isolates CANI-IV were all identified by MLEE, only two human isolates (CANIII and JJ) were confirmed as TCIIa using distinct molecular markers. Moreover, A. Marcili et al. / International Journal for Parasitology 39 (2009) 615–623 vectors of this lineage have also been poorly characterised, except R. brethesi, which is restricted to Northern Amazonia (Fernandes et al., 1998, 2001; Coura et al., 2002; Mendonça et al., 2002; Pedroso et al., 2007), and possibly P. geniculatus (Miles et al., 1981a; Gaunt and Miles, 2000), which has a widespread distribution. Here, we have shown that TCIIa is a well-supported and very homogeneous lineage clearly separated from TCIIc and from all other lineages according to both ribosomal and cytochrome b gene analyses. We also demonstrated that TCIIa is common in wild monkeys, R. robustus and R. brethesi in Brazilian Amazonia, circulating in an arboreal transmission cycle distinct from the terrestrial cycle usually attributed to TCIIc. In this study, we confirmed TCI as being the most common lineage that infects wild primates in Amazonia (76%), which is in agreement with our previous work restricted to S. bicolor (Maia da Silva et al., 2008). Five out of 21 isolates from wild primates belonged to TCIIa, a lineage, until now, not associated with monkeys although two isolates of wild primates from Bolivia and Venezuela were previously assigned to TCIIa (Westenberger et al., 2006). We believe this is the first study that describes genotyping, RAPD, ssrDNA and cytochrome b gene polymorphism analyses of TCIIa and TCI isolates from wild primates and from acute cases of Chagas disease associated with oral infection. Results generated by analyses of all these molecular markers showed that human isolates were very similar to isolates from wild primates and Rhodnius spp. assigned to the same lineages, indicating that they are transmitted by the same vectors in the Amazonian region. Before this study, TCIIa had only been reported in five Amazonian cases of human Chagas disease, four being simultaneous acute cases (CANI-IV) in a single household and one being a chronic case (JJ) (Miles et al., 1978, 1981a; Lainson et al., 1979; Fernandes et al., 1998, 2001). However, more than 70 isolates from oral outbreaks of Chagas disease have been genotyped as TCI, corroborating its predominance not only in sylvatic mammals but also in humans living in Amazonia (Valente et al., unpublished data). In Amazonia where most cases of autochthonous Chagas disease are associated with TCI, as confirmed in this study, clinical manifestations ranged from sudden fever, myalgia, dyspnea and signs of heart failure, and included a growing number of fatal cases (Pinto et al., 2004; Xavier et al., 2006; Aguilar et al., 2007). These findings, along with several cases of acute and chronic myocardiopathy in countries where TCI is the only or the most prevalent lineage infecting humans, such as Mexico, Panama, Venezuela and Colombia, indicate that this lineage may be responsible for severe disease despite the absence of megasyndromes (Miles et al., 1981b, 2003; Añez et al., 2004; Ruíz-Sánchez et al., 2005; Samudio et al., 2007). Humans from whom new isolates of TCI and TCIIa characterised in this study were obtained showed variable symptoms of acute disease as described previously for other human cases from Amazonia (Pinto et al., 2004). A few cases of symptomatic chronic infections were reported in Amazonia without lineage identification (Albajar et al., 2003; Xavier et al., 2006; Aguilar et al., 2007). It remains to be clarified if distinct clinical forms of Chagas disease can be correlated to specific T. cruzi lineages, transmission routes and/or host genetics (Coura, 2007). In wild Amazonian monkeys, T. cruzi infection rates ranged from 10.3% to 46% by parasitological or serological methods, respectively (Ziccardi and Lourenço-de-Oliveira, 1997; Lisboa et al., 2006; Maia da Silva et al., 2008). Monkeys, apart from their importance in the epizootiology, develop pathological manifestations typical of acute and chronic Chagas disease (Miles et al., 1979; Monteiro et al., 2006). The impact of T. cruzi infections in the preservation of wild primates has not been investigated. How wild primates become infected is not known, but ingestion of infected triatomines appears to be the most important route of T. cruzi 621 infection in the enzootic transmission cycles (Maia da Silva et al., 2008). Together, molecular and ecogeographical analyses strongly suggested that very similar isolates of either TCI or TCIIa circulate amongst wild primates and are transmitted by R. robustus, R. pictipes and R. brethesi in Brazilian Amazonia. Although palms are the specific ecotopes of a majority of Rhodnius spp., vector-ecotope association can vary. Even those species of Panstrongylus preferring burrows, such as P. geniculatus, can sporadically be found in palms, nests and tree cavities in Amazonia (Miles et al., 1981b; Gaunt and Miles, 2000). The limited data about T. cruzi genotypes in wild reservoirs and triatomines in Amazonia are insufficient to rule out other arboreal or even terrestrial mammals and vectors as natural hosts of TCIIa. Besides overlapping arboreal cycles in Amazonia, TCI and TCIIa also share ecotopes, hosts and vectors with T. rangeli (Maia da Silva et al., 2007). Despite sharing vectors and ecotopes with TCI, TCIIa was not found in more than 50 isolates from D. marsupialis that were captured during this study in the same places of Amazonia (data not shown) and were all assigned to TCI corroborating a strong association of this lineage with Didelphis (Yeo et al., 2005). Taken together, the data suggest that sympatric T. cruzi lineages of Amazonia circulate in independent transmission cycles determined by their preferential mammalian hosts and by the specific ecotopes of their vertebrate and invertebrate hosts. Nevertheless, finding that the same lineages of T. cruzi infect mammals of distinct orders in sylvatic transmission cycles confirms that lineage association with mammals is far from absolute (Yeo et al., 2005; O’Connor et al., 2007). Interestingly, T. cruzi isolates from lemurs, racoons and domestic dogs from USA were assigned to TCIIa (Hall et al., 2007; Roellig et al., 2008). However, zymodeme and RAPD patterns suggested that TCIIa from Amazonia and North America are not identical lineages (Barnabé et al., 2001; Brisse et al., 2003; Marcili et al., unpublished data). The capacity of T. cruzi to infect multiple mammalian hosts, including human and non-human primates, and to circulate in sylvatic, peridomestic and domestic reservoirs and ecotopes are risk factors for its emergence as an important human pathogen in Amazonia. Unravelling the dynamics of T. cruzi populations and their complex multi-host communities, which vary according to biomes, ecotopes and specific niches, is very important in understanding the structure and evolutionary history of T. cruzi populations. Results from this study are helpful in revealing the complexity and dynamics of transmission cycles of T. cruzi that circulate in the Amazon region. In addition, these results could help clarify peculiarities of Chagas disease associated with oral infection in Amazonia. Acknowledgements We are grateful to several colleagues for their continuing collaboration, and especially indebted to technical assistants F S. Gomes, A. Freitas, R.N. Almeida, R.B. Nascimento and J.M. Nascimento from the Instituto Evandro Chagas (IEC) for their inestimable help in fieldwork and collaboration in the isolation of trypanosomes. We thank Martin Llewellyn and Michael Lewis (LSHTM, UK) for helpful discussions. This work was supported by grants from the Brazilian agencies CNPq (UNIVERSAL) and FAPESP (PRONEX) to M.M.G.T. and from IEC and ECLAT to S.A.V. Maia da Silva, F. is a postdoctoral fellow sponsored by CAPES (PRODOC– PROTAX), and Marcili, A. and Junqueira A.C.V. were recipients of scholarships from CNPq. References Aguilar, H.M., Abad-Franch, F., Dias, J.C., Junqueira, A.C., Coura, J.R., 2007. Chagas disease in the Amazon region. Mem. Inst. Oswaldo Cruz 30, 47–56. 622 A. Marcili et al. / International Journal for Parasitology 39 (2009) 615–623 Albajar, P., Laredo, S.V., Terrazas, M.B., Coura, J.R., 2003. Miocardiopatia dilatada em pacientes com infecção chagásica crônica. Relato de dois casos fatais autóctones do Rio Negro, Estado do Amazonas. Rev. Soc. Bras. Med. Trop. 36, 401–407. Añez, N., Crisante, G., Rojas, A., 2004. Update on Chagas disease in Venezuela – a review. Mem. Inst. Oswaldo Cruz 99, 781–787. Anonymous, 1999. Recommendations from a satellite meeting. Mem. Inst. Oswaldo Cruz 94, 429–432. Aufderheide, A.C., Salo, W., Madden, M., Streitz, J., Buikstra, J., Guhl, F., Arriaza, B., Renier, C., Wittmers Jr., L.E., Fornaciari, G., Allison, M., 2004. A 9, 000-year record of Chagas’ disease. Proc. Natl. Acad. Sci. U S A 17, 2034–2039. Barnabé, C., Yaeger, R., Pung, O., Tibayrenc, M., 2001. Trypanosoma cruzi: a considerable phylogenetic divergence indicates that the agent of Chagas disease is indigenous to the native fauna of the United States. Exp. Parasitol. 99, 73–79. Barrett, T.V., Hoff, R.H., Mott, K.E., Miles, M.A., Godfrey, D.G., Teixeira, R., Almeida de Souza, J.A., Sherlock, I.A., 1980. Epidemiological aspects of three Trypanosoma cruzi zymodemes in Bahia State Brazil. Trans. R. Soc. Trop. Med. Hyg. 74, 84–90. Briones, M.R., Souto, R.P., Stolf, B.S., Zingales, B., 1999. The evolution of two Trypanosoma cruzi subgroups inferred from rRNA genes can be correlated with the interchange of American mammalian faunas in the Cenozoic and has implications to pathogenicity and host specificity. Mol. Biochem. Parasitol. 30, 219–232. Brisse, S., Henriksson, J., Barnabé, C., Douzery, E.J., Berkvens, D., Serrano, M., De Carvalho, M.R., Buck, G.A., Dujardin, J.C., Tibayrenc, M., 2003. Evidence for genetic exchange and hybridization in Trypanosoma cruzi based on nucleotide sequences and molecular karyotype. Infect. Genet. Evol. 2, 173–183. Brisse, S., Verhoef, J., Tibayrenc, M., 2001. Characterisation of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages. Int. J. Parasitol. 31, 1218–1226. Brisse, S., Barnabe, C., Tibayrenc, M., 2000. Identification of six Trypanosoma cruzi phylogenetic lineages by random amplified polymorphic DNA and multilocus enzyme electrophoresis. Int. J. Parasitol. 30, 35–44. Cardinal, M.V., Lauricella, M.A., Ceballos, L.A., Lanati, L., Marcet, P.L., Levin, M.J., Kitron, U., Gürtler, R.E., Schijman, A.G., 2008. Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina. Int. J. Parasitol. 38, 1533–1543. Chapman, M.D., Baggaley, R.C., Godfrey-Fausset, P.F., Malpas, T.J., White, G., Canese, J., Miles, M.A., 1984. Trypanosoma cruzi from the Paraguayan Chaco: isoenzyme profiles of strains isolated at Makthlawaiya. J. Protozool. 31, 482–486. Coura, J.R., 2007. Chagas disease: What is know and what is needed – A background article. Mem. Inst. Oswaldo Cruz 102, 113–122. Coura, J.R., Junqueira, A.C., Fernandes, O., Valente, S.A., Miles, M.A., 2002. Emerging Chagas disease in Amazonian Brazil. Trends Parasitol. 18, 171–176. Fernandes, O., Mangia, R.H., Lisboa, C.V., Pinho, A.P., Morel, C.M., Zingales, B., Campbell, D., Jansen, A.M., 1999. The complexity of sylvatic cycle of Trypanosoma cruzi in the Rio de Janeiro Sate (Brazil) revealed by the nontranscribed spacer of the mini-exon gene. Parasitology 118, 161–1666. Fernandes, O., Santos, S.S., Cupolillo, E., Mendonça, B., Derre, R., Junqueira, A.C.V., Santos, L.C., Sturm, N.R., Naiff, R.D., Barret, T.V., Campbell, D.A., Coura, J.R., 2001. A mini-exon multiplex polymerase chain reaction to distinguish the major groups of Trypanosoma cruzi and T. Rangeli in the Brazilian Amazon. Trans. R. Soc. Trop. Med. Hyg. 95, 97–99. Fernandes, O., Sturm, N.R., Derré, R., Campbell, D.A., 1998. The mini-exon gene: a genetic marker for zymodeme III of Trypanosoma cruzi. Mol. Biochem. Parasitol. 95, 129–133. Fitzpatrick, S., Feliciangeli, M.D., Sanchez-Martin, M.J., Monteiro, F.A., Miles, M.A., 2008. Molecular genetics reveal that silvatic Rhodnius prolixus do colonise rural houses. PLoS Negl. Trop. Dis. 2, e210. Freitas, J.M., Augusto-Pinto, L., Pimenta, J.R., Bastos-Rodrigues, L., Gonçalves, V.F., Teixeira, S.M.R., Chiari, E., Junqueira, A.C.V., Fernandes, O., Macedo, C.R., Pena, S.D.J., 2006. Ancestral genomes, sex, and the population structure of Trypanosoma cruzi. PLoS Pathoge. 2, e24. Gaunt, M., Miles, M., 2000. The ecotopes and evolution of triatomine bugs (triatominae) and their associated trypanosomes. Mem. Inst. Oswaldo Cruz 95, 557–565. Hall, C.A., Polizzi, C., Yabsley, M.J., Norton, T.M., 2007. Trypanosoma cruzi prevalence and epidemiologic trends in lemurs on St. Catherines Island, Georgia. J. Parasitol. 93, 93–96. Herrera, L., D’Andrea, P.S., Xavier, S.C., Mangia, R.H., Fernandes, O., Jansen, A.M., 2005. Trypanosoma cruzi infection in wild mammals of the National Park ‘Serra da Capivara’ and its surroundings (Piaui, Brazil), an area endemic for Chagas disease. Trans. R. Soc. Trop. Med. Hyg. 99, 379–388. Kawashita, S.Y., Sanson, G.F., Fernandes, O., Zingales, B., Briones, M.R., 2001. Maximum-likelihood divergence date estimates based on rRNA gene sequences suggest two scenarios of Trypanosoma cruzi intraspecific evolution. Mol. Biol. Evol. 18, 2250–2259. Lainson, R., Shaw, J.J., Fraiha, H., Miles, M.A., Draper, C.C., 1979. Chaga’s disease in the Amazon Basin: 1. Trypanosoma cruzi infections in silvatic mammals, triatomine bugs and man in the State of Pará, north Brazil. Trans. R. Soc. Trop. Med. Hyg. 73, 193–204. Lisboa, C.V., Mangia, R.H., Luz, S.L., Kluczkovski Jr., A., Ferreira, L.F., Ribeiro, C.T., Fernandes, O., Jansen, A.M., 2006. Stable infection of primates with Trypanosoma cruzi I and II. Parasitology 133, 603–611. Lisboa, C.V., Mangia, R.H., De Lima, N.R., Martins, A., Dietz, J., Baker, A.J., RamonMiranda, C.R., Ferreira, L.F., Fernandes, O., Jansen, A.M., 2004. Distinct patterns of Trypanosoma cruzi infection in Leontopithecus rosalia in distinct Atlantic coastal rainforest fragments in Rio de Janeiro–Brazil. Parasitology 129, 703– 711. Machado, C.A., Ayala, F.J., 2001. Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi. Proc. Natl. Acad. Sci. U S A 19, 7396–7401. Maia da Silva, F., Naiff, R.D., Marcili, A., Gordo, M., D’Affonseca Neto, J.A., Naiff, M.F., Franco, A.M.R., Campaner, M., Valente, V., Valente, A.S., Camargo, E.P., Teixeira, M.M.G., Miles, M., 2008. Infection rates and genotypes of Trypanosoma rangeli and Trypanosoma cruzi infecting free-ranging Saguinus bicolor (Callitrichidae), a critically endangered primate of the Amazon rainforest. Acta Trop. 107, 168–173. Maia Da Silva, F., Junqueira, A.C., Campaner, M., Rodrigues, A.C., Crisante, G., Ramirez, L.E., Caballero, Z.C., Monteiro, F.A., Coura, J.R., Añez, N., Teixeira, M.M.G., 2007. Comparative phylogeography of Trypanosoma rangeli and Rhodnius (Hemiptera: Reduviidae) supports a long coexistence of parasite lineages and their sympatric vectors. Mol. Ecol. 16, 3361–3373. Maia da Silva, F., Noyes, H., Campaner, M., Junqueira, A.C., Coura, J.R., Añez, N., Shaw, J.J., Stevens, J.R., Teixeira, M.M.G., 2004a. Phylogeny, taxonomy and grouping of Trypanosoma rangeli isolates from man, triatomines and sylvatic mammals from widespread geographical origin based on SSU and ITS ribosomal sequences. Parasitology 129, 549–561. Maia da Silva, F., Rodrigues, A.C., Campaner, M., Takata, C.S.A., Brigido, M.C., Junqueira, A.C.V., Coura, J.R., Takeda, G.F., Shaw, J.J., Teixeira, M.M.G., 2004b. Randomly amplified polymorphic DNA analysis of Trypanosoma rangeli and allied species from human, monkeys and other sylvatic mammals of the Brazilian Amazon disclosed a new group and a species–specific marker. Parasitology 128, 283–294. Martins, L.P.A., Marcili, A., Castanho, R.E.P., Therezo, A.L.S., Oliveira, J.C.P., Suzuki, R.B., Teixeira, M.M.G., Rosa, J.A., Sperança, M.A., 2008. Rural Triatoma rubrovaria from southern Brazil harbors Trypanosoma cruzi of lineage IIc. Am. J. Trop. Med. Hyg. 79, 427–434. Mendonça, M.B., Nehme, N.S., Santos, S.S., Cupolillo, E., Vargas, N., Junqueira, A., Naiff, R.D., Barrett, T.V., Coura, J.R., Zingales, B., Fernandes, O., 2002. Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron. Parasitology 124, 177–184. Miles, M.A., Feliciangeli, M.D., de Arias, A.R., 2003. American trypanosomiasis (Chagas’ disease) and the role of molecular epidemiology in guiding control strategies. Brit. Med. J. 28, 1444–1448. Miles, M.A., Povoa, M.M., de Souza, A.A., Lainson, R., Shaw, J.J., Ketteridge, D.S., 1981a. Chaga’s disease in the Amazon Basin: II. The distribution of Trypanosoma cruzi zymodemes 1 and 3 in Pará State, north Brazil. Trans. R. Soc. Trop. Med. Hyg. 75, 667–674. Miles, M.A., Cedillos, R.A., Povoa, M.M., de Souza, A.A., Prata, A., Macedo, V., 1981b. Do radically dissimilar Trypanosoma cruzi strains (zymodemes) cause Venezuelan and Brazilian forms of Chagas’ disease? Lancet 20, 1338–1340. Miles, M.A., Marsden, P.D., Pettitt, L.E., Draper, C.C., Watson, S., Seah, S.K., Hutt, M.S., Fowler, J.M., 1979. Experimental Trypanosoma cruzi infection in rhesus monkeys 111. Electrocardiographic and histopathological findings. Trans. R. Soc. Trop. Med. Hyg. 73, 528–532. Miles, M.A., Souza, A., Povoa, M., Shaw, J.J., Lainson, R., Toye, P.J., 1978. Isozymic heterogeneity of Trypanosoma cruzi in the first autochthonous patients with Chagas’ disease in Amazonian Brazil. Nature 27, 819–821. Monteiro, R.V., Baldez, J., Dietz, J., Baker, A., Lisboa, C.V., Jansen, A.M., 2006. Clinical, biochemical, and electrocardiographic aspects of Trypanosoma cruzi infection in free-ranging golden lion tamarins (Leontopithecus rosalia). J. Med. Primatol. 35, 48–55. O’Connor, O., Bosseno, M.F., Barnabé, C., Douzery, E.J., Brenière, S.F., 2007. Genetic clustering of Trypanosoma cruzi I lineage evidenced by intergenic miniexon gene sequencing. Infect. Genet. Evol. 7, 587–593. Pedroso, A., Cupolillo, E., Zingales, B., 2007. Trypanosoma cruzi: exploring the nuclear genome of zymodeme 3 stocks by chromosome size polymorphism. Exp. Parasitol. 116, 71–76. Pinto, A.Y., Valente, S.A., Valente, V. da C., 2004. Emerging acute Chagas disease in Amazonian Brazil: case reports with serious cardiac involvement. Braz. J. Infect. Dis. 8, 454–460. Póvoa, M.M., de Souza, A.A., Naiff, R.D., Arias, J.R., Naiff, M.F., Biancardi, C.B., Miles, M.A., 1984. Chagas’ disease in the Amazon basin IV. Host records of Trypanosoma cruzi zymodemes in the states of Amazonas and Rondonia, Brazil. Ann. Trop. Med. Parasitol. 78, 479–487. Roellig, D.M., Brown, E.L., Barnabé, C., Tibayrenc, M., Steurer, F.J., Yabsley, M.J., 2008. Molecular Typing of Trypanosoma cruzi Isolates, United States. Emerg. Infect. Dis. 14, 1123–1125. Ruíz-Sánchez, R., León, M.P., Matta, V., Reyes, P.A., López, R., Jay, D., Monteón, V.M., 2005. Trypanosoma cruzi isolates from Mexican and Guatemalan acute and chronic chagasic cardiopathy patients belong to Trypanosoma cruzi I. Mem. Inst. Oswaldo Cruz 100, 281–283. Samudio, F., Ortega-Barría, E., Saldaña, A., Calzada, J., 2007. Predominance of Trypanosoma cruzi I among Panamanian sylvatic isolates. Acta Trop. 101, 178–181. Souto, R.P., Vargas, N., Zingales, B., 1999. Trypanosoma rangeli: discrimination from Trypanosoma cruzi based on a variable domain from the large subunit ribosomal RNA gene. Exp. Parasitol. 91, 306–314. Souto, R.P., Fernandes, O., Macedo, A.M., Campbell, D.A., Zingales, B., 1996. DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Mol. Biochem. Parasitol. 83, 141–152. Sturm, N.R., Vargas, N.S., Westenberger, S.J., Zingales, B., Campbell, D.A., 2003. Evidence for multiple hybrid groups in Trypanosoma cruzi. Int. J. Parasitol. 33, 269–279. A. Marcili et al. / International Journal for Parasitology 39 (2009) 615–623 Teixeira, M.M.G., Maia da Silva, F., Marcili, A., Umezawa, E.S., Shikanai-Yasuda, M.A., Cunha-Neto, E., Kalil, J., Stolf, N., Stolf, A.M.S., 2006. Trypanosoma cruzi lineage I in endomyocardial biopsy from a north-eastern Brazilian patient at end-stage chronic Chagasic cardiomyopathy. Trop. Med. Int. Health 11, 294–298. Tibayrenc, M., Miles, M.A., 1983. A genetic comparison between Brazilian and Bolivian zymodemes of Trypanosoma cruzi. Trans. R. Soc. Trop. Med. Hyg. 77, 76–83. Valente, S.A.S., Valente, V.C., Fraiha Neto, H., 1999. Considerations on the epidemiology of Chagas Disease in the Brazilian Amazon. Mem. Inst. Oswaldo Cruz 94, 395–398. Valente, V.C., Valente, S.A., Noireau, F., Carrasco, H.J., Miles, M.A., 1998. Chagas disease in the Amazon Basin: association of Panstrongylus geniculatus (Hemiptera: Reduviidae) with domestic pigs. J. Med. Entomol. 35, 99–103. Xavier, S.S., Sousa, A.S., Viñas, P.A., Junqueira, A.C., Bóia, M.N., Coura, J.R., 2006. Chronic chagasic cardiopathy in the Rio Negro, Amazon State. Report of three 623 new autochthonous cases confirmed by serology, clinical examination, chest Xrays, electro and echocardiography. Rev. Soc. Bras. Med. Trop. 39, 211–216. Ziccardi, M., Lourenço-de-Oliveira, R., 1997. The infection rates of trypanosomes in squirrel monkeys at two sites in the Brazilian Amazon. Mem. Inst. Oswaldo Cruz 92, 465–470. Yeo, M., Acosta, N., Llewellyn, M., Sánchez, H., Adamson, S., Miles, G.A., López, E., González, N., Patterson, J.S., Gaunt, M.W., de Arias, A.R., Miles, M.A., 2005. Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids. Int. J. Parasitol. 35, 225–233. Westenberger, S.J., Barnabé, C., Campbell, D.A., Sturn, N.R., 2005. Two hybridization events define the population structure of Trypanosoma cruzi. Genetics 171, 527–543. Westenberger, S.J., Sturm, N.R., Campbell, D.A., 2006. Trypanosoma cruzi 5S rRNA arrays define five groups and indicate the geographic origins of an ancestor of the heterozygous hybrids. Int. J. Parasitol. 36, 337–346.
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