- Email: [email protected]
Phylogenetic reconstruction based on Cytochrome b (Cytb) gene sequences reveals distinct genotypes within Colombian Trypanosoma cruzi I populations
Acta Tropica 119 (2011) 61–65
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
Phylogenetic reconstruction based on Cytochrome b (Cytb) gene sequences reveals distinct genotypes within Colombian Trypanosoma cruzi I populations夽 Juan David Ramírez, María Clara Duque, Felipe Guhl ∗ Centro de Investigaciones en Microbiología y Parasitología Tropical (CIMPAT), Facultad de Ciencias, Universidad de los Andes, Bogotá Cra 1 No. 18A-20, Colombia
a r t i c l e
i n f o
Article history: Received 17 January 2011 Received in revised form 8 April 2011 Accepted 10 April 2011 Available online 16 April 2011 Keywords: Chagas disease DTU Trypanosoma cruzi Transmission cycles Cytb Maximum likelihood
a b s t r a c t Chagas disease caused by Trypanosoma cruzi comprises an important problem of public health in the Americas. This parasite has been recently divided into six Discrete Typing Units (DTUs) due to its high genetic diversity. We sequenced the Cytochorme b (Cytb) gene of 70 T. cruzi I Colombian clones finding four genotypes related to transmission cycles of Chagas disease in Colombia and also to specific hosts of T. cruzi. The genotypes herein described based on Cytb gene sequences are in accordance with those found using the mini-exon gene and reveals once again the enormous genetic diversity at sub-DTU level evidenced in T. cruzi I. © 2011 Elsevier B.V. All rights reserved.
Chagas disease is a complex zoonosis caused by the parasite Trypanosoma cruzi. This pathology currently affects 15 million people, and 21 million are at risk of acquiring the infection. In America, there are approximately 41,200 new cases and 12,500 deaths annually (WHO, 2007). The nomenclature of T. cruzi has recently been modified to reflect its high genetic variability. Six Discrete Typing Units (DTUs) have been proposed in T. cruzi (Zingales et al., 2009). Several studies have focused on elucidating the genetic variability within T. cruzi (Tc) II–VI, revealing the presence of hybrid groups as supported by the evidence of genetic recombination in vitro (Gaunt et al., 2003). Accordingly, TcII is considered to be an homogeneous group, TcIII and TcIV are considered to be hybrid groups created by potential recombination events between TcI and TcII, and TcV and TcVI are considered to be hybrid groups created by potential recombination events between TcIII/TcIV and TcII, though this last statement remains controversial and requires further research (Gaunt et al., 2003; Sturm and Campbell, 2009; Westenberger et al., 2005). Despite the fact that the nomenclature proposed in 1999 determined TcI to be an homogenous DTU (Anon, 1999), many studies have since reported the genetic variability of TcI using different molecular markers (Cura et al., 2010; Falla et al., 2009; Herrera et al., 2007, 2009; Llewellyn ˜ et al., 2009; Mejía-Jaramillo et al., 2009; Ocana-Mayorga et al.,
夽 GenBank NCBI accession numbers: HQ713679–HQ713748. ∗ Corresponding author. Tel.: +571 3324540. E-mail address: [email protected] (F. Guhl). 0001-706X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2011.04.009
2010; Salazar et al., 2006; Spotorno et al., 2008; Triana et al., 2006). Studies using a large number of TcI isolates have shown the high degree of genetic diversity within this group. In 2007, four genotypes were proposed (Ia–Id) in relation to the transmission cycles of Chagas disease in Colombia (Herrera et al., 2007, 2009). In 2010, these four genotypes were confirmed within the American continent, showing a defined geographical distribution, and one new genotype (Ie) was related to the domestic cycle in Chile and the sylvatic cycle in Bolivia (Cura et al., 2010). The microsatellite motif of the intergenic region of the mini-exon gene has been demonstrated to be highly polymorphic (Fernandes et al., 1998; O’Connor et al., 2007; Tomasini et al., 2010). However, new studies, based on other molecular markers, are required to propose suitable subdivisions within TcI, due to the recent description of the importance of TcI in the development of cardiomyopathies in Argentina (Burgos et al., 2010) and cardiac alterations in Colombia (Ramírez et al., 2010). In 2007, subgroups were reported within TcI isolates based on genetic polymorphisms in the Cytochrome b (Cytb) gene sequence (Spotorno et al., 2008). The objective of this study was to evaluate the genetic variability within TcI clones using the sequences of Cytb genes in Colombian clones and to determine whether any kind of concordance could be observed with the established genotypes proposed using other molecular markers. We used the Cytb gene sequences to examine the possible phylogenetic relationships between a well characterized set of TcI clones from different geographical regions of Colombia (Table 1). The strains were cloned and from five to ten clones were obtained
62
J.D. Ramírez et al. / Acta Tropica 119 (2011) 61–65
Table 1 Clones from the isolates analyzed using direct sequencing of Cytb gene that were genotyped as TcI based on the intergenic region of mini-exon gene and the divergent domain of the 24S␣ rDNA. International code
Abbreviated code
Host/vector
Geographical origin
Cycle
Mini-exon genotype
Cytochrome b genotype
MHOM/CO/92/FCH/6 MHOM/CO/04/MG/15 MHOM/CO/92/JL/5 MHOM/CO/03/CG/6 MHOM/CO/05/JEM/2 MHOM/CO/01/DA/14 MHOM/CO/09/EH/1 MHOM/CO/07/EB/10 MHOM/CO/07/EM/12 MHOM/CO/YLY/14 MHOM/CO/07/DYR/15 MHOM/CO/FEC/15 MHOM/CO/SP/12 MHOM/CO/09/XCh/14 MHOM/CO/07/SEV/12 MHOM/CO/09/CACQ/14 MHOM/CO/09/LER/11 MHOM/CO/09/LJVP/7 MHOM/CO/10/SMA/8 MHOM/CO/09/LCV/3 MCAN/CO/00/H10/10 MCAN/CO/10/AACf1/2 ITr dimidiata/CO/Td3/10 ITr dimidiata/CO/Td11/7 ITr dimidiata/CO/Td/9 IRHO/CO/SN11/3 MDID/CO/00/Dm11/17 MDID/CO/D1/14 MDID/CO/00/Dm38/16 MDID/CO/7/6 MDID/CO/10/YDm1M/2 MDID/CO/10/YDm1B/3 MDID/CO/D16/10 MDID/CO/D18/10 MDID/CO/10/AADm1/1 MDID/CO/10/SLDm2/9 MDID/CO/10/SLDm1/9 MDID/CO/10/NDm1/7 XXX/CO/00/Cepa2/6 XXXX/CO/91/Gal61/16 XXXX/CO/SR2/7 MRAT/CO/10/NR1/10 MTAM/CO/10/YTT1/1 MALO/CO/10/YAS1/2 IRHO/CO/SN5/7 IRHO/CO/10/AAD6/7 IRHO/CO/10/YB1/2 IRHO/CO/10/YD1/5 IRHO/CO/10/AAC1/3 IRHO/CO/10/AAB3/2 IRHO/CO/10/AAA7/5 IRHO/CO/10/SLF5/10 IRHO/CO/10/SLA9/7 IRHO/CO/10/NA3/4 IRHO/CO/10/NB2/5 IRHO/CO/10/SLB3/2 IRHO/CO/10/SLD2/1 IRHO/CO/10/NC2/9 IRHO/CO/X380/11 IRHO/CO/X236/8 IRHO/CO/X1082/9 IRHO/CO/X1544/10 IRHO/CO/00/N5P14/7 IRHO/CO/00/Rp540/9 IRHO/CO/00/PAL/7 IEcuspidatus/CO/10/SLD1Ec/6 Itmaculata/CO/10/TmPA1/6 XXX/CO/00/Coy11/12 IRPAL/CO/00/Necoclí/6 ITr venosa/CO/04/TV/9
FcH cl6 MG cl 15 JL cl 5 CG cl 6 JEM cl 2 DA cl 14 EH cl 1 EEBB cl 10 EM cl 12 YLY cl 14 DYR c 15 FEC cl 15 SP cl 12 Xch cl 14 SEV cl12 CACQ cl 14 LER cl 11 LJVP cl 7 SMA cl 8 LCV cl3 H10 cl 10 AACf1 cl2 Td3 cl 10 Td11 cl 7 Td cl 9 SN11 cl 3 Dm11 cl 17 D1 cl 14 Dm38 cl 16 Dm7 cl 6 YDm1M cl 2 YDm1B cl 3 D16 cl 10 D18 cl 10 AADm1 cl 1 SLDm2 cl 9 SLDm1 cl 9 NDm1 cl 7 Cepa 2 cl 6 Gal61 cl 16 SR2 cl 7 NR1 cl 10 YTT1 cl 1 YAS1 cl 2 SN5 cl 7 AAD6 cl 7 YB1 cl 2 YD1 cl 5 AAC1 cl 3 AAB3 cl 2 AAA7 cl 5 SLF5 cl 10 SLA9 cl 7 NA3 cl 4 NB2 cl 5 SLB3 cl 2 SLD2 cl 1 NC2 cl 9 X380 cl 11 X236 cl 8 X1082 cl 9 X1544 cl 10 N5P14 cl 7 Rp540 cl 9 Palmas cl 7 SLD1Ec cl 6 TmPA1 cl 6 Coy11 cl12 Necocli cl 6 TV cl 2
Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Canis familiaris Canis familiaris Triatoma dimidiata Triatoma dimidiata Triatoma dimidiata Triatoma dimidiata Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Rodent Rodent Rodent Rattus rattus Tamandua tetradactyla Alouatta spp. Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Eratyrus cuspidatus Triatoma maculata Rhodnius colombiensis Rhodnius pallescens Triatoma venosa
Norte de Santander Arauca Arauca Caquetá Putumayo Boyacá Santander Boyacá Boyacá Arauca Boyacá Boyacá Casanare Santander Boyacá Santander Santander Santander Santander Santander Boyacá Casanare Boyacá Boyacá Boyacá Magdalena Tolima Tolima Tolima Tolima Casanare Casanare Tolima Tolima Casanare Casanare Casanare Casanare Casanare Sucre Casanare Casanare Casanare Casanare Magdalena Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Boyacá Boyacá Boyacá Boyacá Casanare Casanare Casanare Casanare Casanare Tolima Antioquia Boyacá
Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Domestic Domestic Domestic Domestic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic
Ib Ib Ib Ia Ia Ib Ib Ia Ia Ia Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Ic Ib Ib Ib Id Id Id Id Id Id Id Id
A A A A A A A A A A A A A A A A A A A A B B B B B B C C C C C C C C C C C C C C C C C C D D D D D D D D D D D D D D D D D D D D D D D C D D
J.D. Ramírez et al. / Acta Tropica 119 (2011) 61–65
63
Fig. 1. Phylogenetic tree obtained by the maximum composite likelihood method using a bootstrapping of 1000 iterations with the Kimura-2-parameter model, based on the GTR+G+I model.
by each strain. In this study we selected one clone per strain to develop the phylogenetic reconstruction. Seventy clones were previously characterized as TcI by amplification of the intergenic region of the mini-exon gene and the divergent domain of 24S␣ rDNA. We obtained 200 L aliquots of LIT-biphasic culture media from the exponential growth phase, which were submitted to DNA extraction using a QIamp kit (Quiagen). The DNA samples were amplified by PCR using the primers p18 (5 -GAC AGG ATT GAG AAG CGA GAG AG-3 ) and p20 (5 -CAA ACC TAT CAC AAA AAG CAT CTG-3 ), in a master mix reaction containing Buffer 1× (Corpogen, COL), 25 mM MgCl2 , 50 M of each primer, 5 U/L of Taq polymerase (Corpogen, COL), 10 M of dNTPs, 30 ng of DNA and sufficient water to obtain a final volume of 21 L. This mix was submitted to 35 cycles of amplification in a BIORAD thermocycler (Brisse et al., 2003). The products of amplification were visualized by electrophoresis in 2% agarose gels stained with ethidium bromide. The products of amplification were cloned and sequenced using the dideoxy terminal method in Macrogen (Korea). The sequences were edited in MEGA 5.0 (Tamura et al., 2007) and aligned with Clustal W 1.8. The sequence from the TcI isolate, Sylvio X10 (AJ130928), as reported in GenBank, was used as a control to edit the sequences. The sequences obtained were deposited in NCBI’s GenBank with the accession numbers HQ713679–HQ713748. Using the edited alignment, we detected 431 conserved sites, 86 variable sites and 49 parsimonious sites with a 0.06 coefficient of differentiation and an average evolutionary distance of 0.013 between the 70 DNA sequences. The sequence of the Cytb gene from T. c. marinkellei was used as an outgroup for the analyses developed. The alignment from the 71 sequences was tested using the AIC (Akaike Information Criterion) on MrModel Test 2.2, where the most suitable evolutive model was selected and GTR+G+I was considered. We developed a phylogenetic reconstruction using a
Maximum Composite Likelihood analysis (ML) with the RAxML 7.2.5 tool on the portal 2.0 from the CIPRES project (Cyberinfrastructure for Phylogenetic Research) (Miller et al., 2009), using the neighbour-joining method by the Kimura-2 parameter model with 1000 bootstrapping iterations to observe the robustness of the nodes. The results of the heuristic search were visualised on FigTree 4.4 (Fig. 1) and determined the presence of four welldifferentiated genotypes, validated with bootstrap values above 80% and −Ln = 1039 with GTR 4 rate. We used the alignment matrix in Nexus format to construct a network, using the median-joining analysis and the default parameters on Network 2.0 to observe the polymorphism differences between the four established genotypes (Fig. 2). The phylogenetic tree showed four genotypes with clear associations with the transmission cycles of Chagas disease in Colombia and also showed associations with specific hosts. Genotype A was associated with clones isolated from humans, with a bootstrap of 91% related to the domestic cycle of transmission, and was concordant with the TcIa and TcIb genotypes, based on the intergenic region of the mini-exon gene. Genotype B was associated with clones isolated from Triatoma dimidiata, captured in the peridomicile, and Canis familiaris, with a bootstrap of 87% related to the peridomiciliary transmission cycle and concordant with the TcIb genotype, as determined by the mini-exon gene. Genotype C was associated with sylvatic/synanthropic reservoirs, such as Didelphis marsupialis, Rattus rattus, Tamandua tetradactyla and Alouatta seniculus, with a bootstrap of 83%, and genotype D was associated with sylvatic triatomines, such as Rhodnius prolixus, Triatoma maculata, Triatoma venosa, Eratyrus cuspidatus, Rhodnius pallescens and R. colombiensis, with a bootstrap of 83%, which are concordant with the TcId genotype, as determined by the mini-exon gene. These results corroborate the previous studies conducted using the intergenic region of the mini-exon gene, in which five
64
J.D. Ramírez et al. / Acta Tropica 119 (2011) 61–65
Fig. 2. Genotype network determined using Network 2.0 based on the median-joining analysis using the default parameters.
genotypes have been previously established in relation to the transmission cycles of Chagas disease in America Cura et al., 2010; Herrera et al., 2007, 2009). Likewise, the studies developed in Chile, using the Cytb gene in TcI isolates, reported the presence of two phylogenetically robust subgroups associated with human infection and domestic vectors from Chile, Brazil and Colombia, and a second subgroup mainly related to caviomorph reservoirs, showing the importance of this reservoir in the evolution of trypanosomes (Spotorno et al., 2008). Our results are congruent with the initial descriptions from Chile: the subgroup isolated from domestic vectors could be considered to be genotype A, as reported in this study, and likewise, the subgroup from the caviomorph reservoirs could be considered to be genotype C but a phylogenetic reconstruction using these sequences must be addressed to confirm this premise. Previous reports, using microsatellite markers on a significant number of TcI isolates, showed genotypes linked to human infection, which could be associated with genotype A, reported in this study (Llewellyn et al., 2009). Studies developed in Brazil with TcI, TcII, TcIII and TcIV isolates, using the Cytb gene sequences, showed a differentiation regarding the ecological ecotopes of the reservoirs and also unravelled a close relatedness between TcI and TcIII/TcIV isolates (Marcili et al., 2009). These results show the great utility of this molecular marker in elucidating the genetic diversity of T. cruzi DTUs and suggesting the relationships between the variants of the parasite and their respective hosts, which is in accordance with the proposed genotypes in this report, where the evidence indicates that certain genotypes are related to specific hosts. The purpose of evaluating the genetic diversity of T. cruzi must be related to the clinical forms of Chagas disease, where studies have reported the impact of specific TcI genotypes on the symptoms of patients with chronic cardiomyopathy (Burgos et al., 2010; Ramírez et al., 2010). Also, this genetic variability shows the evolutive processes that the parasite has undergone, showing a possible coevolution with its hosts, clearly reflected in the presence of genotype C (sylvatic/synanthropic reservoirs) and genotype D (sylvatic triatomines), as determined by Cytb, the subgroup 2, reported by Spotorno et al. (2008) and the genotype TcId, reported by Herrera et al. (2009) and Cura et al. (2010). It is important to mention that our findings show a differentiation of R. prolixus and the other species of sylvatic vectors, with a bootstrap of 83%, from six
clones from sylvatic triatomines, such as Triatoma maculata, Triatoma venosa, E. cuspidatus, R. pallescens and R. colombiensis. This can be related to the adaptation of the parasite to different vectors, reflecting a form of coevolution. The results of the genotype network allowed us to corroborate the usefulness of Cytb in associating specific T. cruzi genotypes with specific hosts. This is shown by the fact that genotype C showed two types of frequencies: one that includes clones isolated from D. marsupialis and R. rattus and another that includes clones isolates from Tamandua tetradactyla and A. seniculus and a clone isolated from a D. marsupialis with congenital infection, revealing specific nucleotide changes related to certain hosts (Fig. 2). In conclusion, we report, for the first time, distinct genotypes within Colombian T. cruzi I clones isolated from different geographical regions and hosts, based on polymorphisms of the Cytb gene, validated with high bootstrap values and developed in clones instead of strains. Our results corroborate the genetic diversity that can be found within T. cruzi I and are concordant with the previously reported genotypes based on the mini-exon gene (Cura et al., 2010; Herrera et al., 2009) and the subgroups reported using Cytb in Chile (Spotorno et al., 2008). It is important to continue studies using this molecular marker in different American countries in order to unravel the processes and evolutionary drivers of TcI and its implications in the transmission, and the different clinical forms, of Chagas disease. Acknowledgments Financial support was provided by the Project Chagas EpiNet from The European Union Seventh Framework Programme, contract No. 223034. We thank the research fund of the faculty of sciences from the Universidad de los Andes and the group of parasitology from the Instituto Nacional de Salud in Colombia for providing some human isolates. References Anon, 1999. Recommendations from a satellite meeting. Mem. Inst. Oswaldo Cruz 94, 429–432. Brisse, S., Henriksson, J., Barnabe, C., Douzery, E.J.P., Berkyens, D., Serrano, M., De Carvalho, R., Buck, G.A., Dujardin, J.C., Tibayrenc, M., 2003. Evidence for genetic
J.D. Ramírez et al. / Acta Tropica 119 (2011) 61–65 exchange and hybridization in Trypanosoma cruzi based on nucleotide sequences and molecular karyotype. Infect. Genet. Evol. 2, 173–183. Burgos, J.M., Diez, M., Vigliano, C., Bisio, M., Risso, M., Duffy, T., Cura, C., Brusses, B., Favaloro, L., Leguizamon, S., Lucero, R.H., Laguens, R., Levin, M.J., Favaloro, R., Schijman, A.G., 2010. Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation. Clin. Infect. Dis. 51, 485–495. Cura, C.I., Mejia-Jaramillo, A.M., Duffy, T., Burgos, J.M., Rodriguero, M., Cardinal, M.V., Kjos, S., Gurgel-Goncalves, R., Blanchet, D., De Pablos, L.M., Tomasini, N., da Silva, A., Russomando, G., Cuba, C.A., Aznar, C., Abate, T., Levin, M.J., Osuna, A., Gurtler, R., Diosque, P., Solari, A., Triana-Chavez, O., Schijman, A.G., 2010. Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced leader genes. Int. J. Parasitol. 40, 1599–1607. Falla, A., Herrera, C., Fajardo, A., Montilla, M., Vallejo, G.A., Guhl, F., 2009. Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans. Acta Trop. 110, 15–21. 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. Gaunt, M.W., Yeo, M., Frame, I.A., Stothard, J.R., Carrasco, H.J., Taylor, M.C., Mena, S.S., Veazey, P., Miles, G.A., Acosta, N., de Arias, A.R., Miles, M.A., 2003. Mechanisms of genetic exchange in American trypanosomes. Nature 421, 936–939. Herrera, C., Bargues, M.D., Fajardo, A., Montilla, M., Triana, O., Vallejo, G.A., Guhl, F., 2007. Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia. Infect. Genet. Evol. 7, 535–539. Herrera, C., Guhl, F., Falla, A., Fajardo, A., Montilla, M., Vallejo, G.A., Bargues, M.D., 2009. Genetic variability and phylogenetic relationships within Trypanosoma cruzi I isolated in Colombia based on miniexon gene sequences. J. Parasitol. Res., doi:10.1155/2009/897364. Llewellyn, M.S., Miles, M.A., Carrasco, H.J., Lewis, M.D., Yeo, M., Vargas, J., Torrico, F., Diosque, P., Valente, V., Valente, S.A., Gaunt, M.W., 2009. Genome-scale multilocus microsatellite typing of Trypanosoma cruzi discrete typing unit I reveals phylogeographic structure and specific genotypes linked to human infection. PLoS Pathog. 5, e1000410. Marcili, A., Lima, L., Valente, V.C., Valente, S.A., Batista, J.S., Junqueira, A.C.V., Souza, A.I., da Rosa, J.A., Campaner, M., Lewis, M.D., Llewellyn, M.S., Miles, M.A., Teixeira, M.G., 2009. Comparative phylogeography of Trypanosoma cruzi TCIIc: new hosts, association with terrestrial ecotopes and spatial clustering. Infect. Genet. Evol. 9, 1265–1274. Mejía-Jaramillo, A.M., Arboleda-Sánchez, S., Rodríguez, I.B., Cura, C., Salazar, A., Del Mazo, J., Triana-Chávez, O., Schijman, A.G., 2009. Geographical clustering of Trypanosoma cruzi I groups from Colombia revealed by low-stringency single specific primer-PCR of the intergenic regions of spliced-leader genes. Parasitol. Res. 104, 399–410.
65
Miller, M., Holder, M., Vos, R., Midford, P., Liebowitz, T., Chan, L., Hoover, P., Warnow, T., 2009. The CIPRES portals. CIPRES, 2009-08-04. URL: http://www.phylo.org/sub sections/portal. (Archivado por WebCite.(r) en http://www.webcitation.org/5imQlJeQa). ˜ Ocana-Mayorga, S., Llewellyn, M.S., Costales, J.A., Miles, M.A., Grijalva, M.J., 2010. Sex, subdivision, and domestic dispersal of Trypanosoma cruzi lineage I in Southern Ecuador. PLoS Negl. Trop. Dis. 4, e915, doi:10.1371/journal.pntd.0000915. O’Connor, O., Bosseno, M.F., Barnabé, C., Douzery, E.J.P., Breniére, S.F., 2007. Genetic clustering of Trypanosoma cruzi I lineage evidenced by intergenic miniexon sequencing. Infect. Genet. Evol. 7, 587–593. Ramírez, J.D., Guhl, F., Rendón, L.M., Rosas, F., Marin-Neto, J.A., Morillo, C., 2010. Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic Chagasic patients. PLoS Negl. Trop. Dis 4, e899, doi:10.1371/journal.pntd.0000899. Salazar, A., Schijman, A.G., Triana, O., 2006. High variability of Colombian Trypanosoma cruzi lineage I stocks as revealed by low-stringency single primer-PCR minicircle signatures. Acta Trop. 100, 110–118. Spotorno, A.E., Córdova, L., Solari, A., 2008. Differentiation of Trypanosoma cruzi I subgroups through characterization of cytochrome b gene sequences. Infect. Genet. Evol. 8, 898–900. Sturm, N., Campbell, D.A., 2009. Alternative lifestyles: the population structure of Trypanosoma cruzi. Acta Trop. 33, 205–214. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596– 1599. Tomasini, N., Lauthier, J.J., Monje Rumi, M.M., Ragone, P.G., Alberti, A., Brandan, C.P., Cura, C.I., Schijman, A.G., Barnabé, C., Tibayrenc, M., Basombrio, M.A., Falla, A., Herrera, C., Guhl, F., Diosque, P., 2010. Interest and limitations of spliced leader intergenic region sequences for analyzing Trypanosoma cruzi I phylogenetic diversity in the Argentinean Chaco. Infect. Genet. Evol. 11, 300– 307. Triana, O., Ortiz, S., Dujardin, J.C., Solari, A., 2006. Trypanosoma cruzi; variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis. Exp. Parasitol. 113, 62–66. Westenberger, S.J., Barnabe, C., Campbell, D.A., Sturm, N.R., 2005. Two hybridization events define the population structure of Trypanosoma cruzi genetics. Genetics 171, 527–543. World Health Organization (WHO) Special Programme for Research and Training in Tropical Diseases (TDR), 2007. Report of Scientific Group in Chagas disease, Buenos Aires, Argentina, April 17–20, 2005, Update July 2007. Zingales, B., Andrade, S.G., Briones, M.R.S., Campbell, D.A., Chiari, E., Fernandes, O., Guhl, F., Lages-Silva, E., Macedo, A.M., Machado, C.R., Miles, M.A., Romanha, A.J., Sturm, N.R., Tibayrenc, M., Schijman, A.G., 2009. A new consensus for Trypanosoma cruzi intraespecific nomenclature: second revision meeting recommends TcI to TcVI. Mem. Inst. Oswaldo Cruz 104, 1051–1054.
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
Phylogenetic reconstruction based on Cytochrome b (Cytb) gene sequences reveals distinct genotypes within Colombian Trypanosoma cruzi I populations夽 Juan David Ramírez, María Clara Duque, Felipe Guhl ∗ Centro de Investigaciones en Microbiología y Parasitología Tropical (CIMPAT), Facultad de Ciencias, Universidad de los Andes, Bogotá Cra 1 No. 18A-20, Colombia
a r t i c l e
i n f o
Article history: Received 17 January 2011 Received in revised form 8 April 2011 Accepted 10 April 2011 Available online 16 April 2011 Keywords: Chagas disease DTU Trypanosoma cruzi Transmission cycles Cytb Maximum likelihood
a b s t r a c t Chagas disease caused by Trypanosoma cruzi comprises an important problem of public health in the Americas. This parasite has been recently divided into six Discrete Typing Units (DTUs) due to its high genetic diversity. We sequenced the Cytochorme b (Cytb) gene of 70 T. cruzi I Colombian clones finding four genotypes related to transmission cycles of Chagas disease in Colombia and also to specific hosts of T. cruzi. The genotypes herein described based on Cytb gene sequences are in accordance with those found using the mini-exon gene and reveals once again the enormous genetic diversity at sub-DTU level evidenced in T. cruzi I. © 2011 Elsevier B.V. All rights reserved.
Chagas disease is a complex zoonosis caused by the parasite Trypanosoma cruzi. This pathology currently affects 15 million people, and 21 million are at risk of acquiring the infection. In America, there are approximately 41,200 new cases and 12,500 deaths annually (WHO, 2007). The nomenclature of T. cruzi has recently been modified to reflect its high genetic variability. Six Discrete Typing Units (DTUs) have been proposed in T. cruzi (Zingales et al., 2009). Several studies have focused on elucidating the genetic variability within T. cruzi (Tc) II–VI, revealing the presence of hybrid groups as supported by the evidence of genetic recombination in vitro (Gaunt et al., 2003). Accordingly, TcII is considered to be an homogeneous group, TcIII and TcIV are considered to be hybrid groups created by potential recombination events between TcI and TcII, and TcV and TcVI are considered to be hybrid groups created by potential recombination events between TcIII/TcIV and TcII, though this last statement remains controversial and requires further research (Gaunt et al., 2003; Sturm and Campbell, 2009; Westenberger et al., 2005). Despite the fact that the nomenclature proposed in 1999 determined TcI to be an homogenous DTU (Anon, 1999), many studies have since reported the genetic variability of TcI using different molecular markers (Cura et al., 2010; Falla et al., 2009; Herrera et al., 2007, 2009; Llewellyn ˜ et al., 2009; Mejía-Jaramillo et al., 2009; Ocana-Mayorga et al.,
夽 GenBank NCBI accession numbers: HQ713679–HQ713748. ∗ Corresponding author. Tel.: +571 3324540. E-mail address: [email protected] (F. Guhl). 0001-706X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2011.04.009
2010; Salazar et al., 2006; Spotorno et al., 2008; Triana et al., 2006). Studies using a large number of TcI isolates have shown the high degree of genetic diversity within this group. In 2007, four genotypes were proposed (Ia–Id) in relation to the transmission cycles of Chagas disease in Colombia (Herrera et al., 2007, 2009). In 2010, these four genotypes were confirmed within the American continent, showing a defined geographical distribution, and one new genotype (Ie) was related to the domestic cycle in Chile and the sylvatic cycle in Bolivia (Cura et al., 2010). The microsatellite motif of the intergenic region of the mini-exon gene has been demonstrated to be highly polymorphic (Fernandes et al., 1998; O’Connor et al., 2007; Tomasini et al., 2010). However, new studies, based on other molecular markers, are required to propose suitable subdivisions within TcI, due to the recent description of the importance of TcI in the development of cardiomyopathies in Argentina (Burgos et al., 2010) and cardiac alterations in Colombia (Ramírez et al., 2010). In 2007, subgroups were reported within TcI isolates based on genetic polymorphisms in the Cytochrome b (Cytb) gene sequence (Spotorno et al., 2008). The objective of this study was to evaluate the genetic variability within TcI clones using the sequences of Cytb genes in Colombian clones and to determine whether any kind of concordance could be observed with the established genotypes proposed using other molecular markers. We used the Cytb gene sequences to examine the possible phylogenetic relationships between a well characterized set of TcI clones from different geographical regions of Colombia (Table 1). The strains were cloned and from five to ten clones were obtained
62
J.D. Ramírez et al. / Acta Tropica 119 (2011) 61–65
Table 1 Clones from the isolates analyzed using direct sequencing of Cytb gene that were genotyped as TcI based on the intergenic region of mini-exon gene and the divergent domain of the 24S␣ rDNA. International code
Abbreviated code
Host/vector
Geographical origin
Cycle
Mini-exon genotype
Cytochrome b genotype
MHOM/CO/92/FCH/6 MHOM/CO/04/MG/15 MHOM/CO/92/JL/5 MHOM/CO/03/CG/6 MHOM/CO/05/JEM/2 MHOM/CO/01/DA/14 MHOM/CO/09/EH/1 MHOM/CO/07/EB/10 MHOM/CO/07/EM/12 MHOM/CO/YLY/14 MHOM/CO/07/DYR/15 MHOM/CO/FEC/15 MHOM/CO/SP/12 MHOM/CO/09/XCh/14 MHOM/CO/07/SEV/12 MHOM/CO/09/CACQ/14 MHOM/CO/09/LER/11 MHOM/CO/09/LJVP/7 MHOM/CO/10/SMA/8 MHOM/CO/09/LCV/3 MCAN/CO/00/H10/10 MCAN/CO/10/AACf1/2 ITr dimidiata/CO/Td3/10 ITr dimidiata/CO/Td11/7 ITr dimidiata/CO/Td/9 IRHO/CO/SN11/3 MDID/CO/00/Dm11/17 MDID/CO/D1/14 MDID/CO/00/Dm38/16 MDID/CO/7/6 MDID/CO/10/YDm1M/2 MDID/CO/10/YDm1B/3 MDID/CO/D16/10 MDID/CO/D18/10 MDID/CO/10/AADm1/1 MDID/CO/10/SLDm2/9 MDID/CO/10/SLDm1/9 MDID/CO/10/NDm1/7 XXX/CO/00/Cepa2/6 XXXX/CO/91/Gal61/16 XXXX/CO/SR2/7 MRAT/CO/10/NR1/10 MTAM/CO/10/YTT1/1 MALO/CO/10/YAS1/2 IRHO/CO/SN5/7 IRHO/CO/10/AAD6/7 IRHO/CO/10/YB1/2 IRHO/CO/10/YD1/5 IRHO/CO/10/AAC1/3 IRHO/CO/10/AAB3/2 IRHO/CO/10/AAA7/5 IRHO/CO/10/SLF5/10 IRHO/CO/10/SLA9/7 IRHO/CO/10/NA3/4 IRHO/CO/10/NB2/5 IRHO/CO/10/SLB3/2 IRHO/CO/10/SLD2/1 IRHO/CO/10/NC2/9 IRHO/CO/X380/11 IRHO/CO/X236/8 IRHO/CO/X1082/9 IRHO/CO/X1544/10 IRHO/CO/00/N5P14/7 IRHO/CO/00/Rp540/9 IRHO/CO/00/PAL/7 IEcuspidatus/CO/10/SLD1Ec/6 Itmaculata/CO/10/TmPA1/6 XXX/CO/00/Coy11/12 IRPAL/CO/00/Necoclí/6 ITr venosa/CO/04/TV/9
FcH cl6 MG cl 15 JL cl 5 CG cl 6 JEM cl 2 DA cl 14 EH cl 1 EEBB cl 10 EM cl 12 YLY cl 14 DYR c 15 FEC cl 15 SP cl 12 Xch cl 14 SEV cl12 CACQ cl 14 LER cl 11 LJVP cl 7 SMA cl 8 LCV cl3 H10 cl 10 AACf1 cl2 Td3 cl 10 Td11 cl 7 Td cl 9 SN11 cl 3 Dm11 cl 17 D1 cl 14 Dm38 cl 16 Dm7 cl 6 YDm1M cl 2 YDm1B cl 3 D16 cl 10 D18 cl 10 AADm1 cl 1 SLDm2 cl 9 SLDm1 cl 9 NDm1 cl 7 Cepa 2 cl 6 Gal61 cl 16 SR2 cl 7 NR1 cl 10 YTT1 cl 1 YAS1 cl 2 SN5 cl 7 AAD6 cl 7 YB1 cl 2 YD1 cl 5 AAC1 cl 3 AAB3 cl 2 AAA7 cl 5 SLF5 cl 10 SLA9 cl 7 NA3 cl 4 NB2 cl 5 SLB3 cl 2 SLD2 cl 1 NC2 cl 9 X380 cl 11 X236 cl 8 X1082 cl 9 X1544 cl 10 N5P14 cl 7 Rp540 cl 9 Palmas cl 7 SLD1Ec cl 6 TmPA1 cl 6 Coy11 cl12 Necocli cl 6 TV cl 2
Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Canis familiaris Canis familiaris Triatoma dimidiata Triatoma dimidiata Triatoma dimidiata Triatoma dimidiata Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Didelphis marsupialis Rodent Rodent Rodent Rattus rattus Tamandua tetradactyla Alouatta spp. Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Rhodnius prolixus Eratyrus cuspidatus Triatoma maculata Rhodnius colombiensis Rhodnius pallescens Triatoma venosa
Norte de Santander Arauca Arauca Caquetá Putumayo Boyacá Santander Boyacá Boyacá Arauca Boyacá Boyacá Casanare Santander Boyacá Santander Santander Santander Santander Santander Boyacá Casanare Boyacá Boyacá Boyacá Magdalena Tolima Tolima Tolima Tolima Casanare Casanare Tolima Tolima Casanare Casanare Casanare Casanare Casanare Sucre Casanare Casanare Casanare Casanare Magdalena Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Casanare Boyacá Boyacá Boyacá Boyacá Casanare Casanare Casanare Casanare Casanare Tolima Antioquia Boyacá
Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Synanthropic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Domestic Domestic Domestic Domestic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic
Ib Ib Ib Ia Ia Ib Ib Ia Ia Ia Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Ib Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Id Ic Ib Ib Ib Id Id Id Id Id Id Id Id
A A A A A A A A A A A A A A A A A A A A B B B B B B C C C C C C C C C C C C C C C C C C D D D D D D D D D D D D D D D D D D D D D D D C D D
J.D. Ramírez et al. / Acta Tropica 119 (2011) 61–65
63
Fig. 1. Phylogenetic tree obtained by the maximum composite likelihood method using a bootstrapping of 1000 iterations with the Kimura-2-parameter model, based on the GTR+G+I model.
by each strain. In this study we selected one clone per strain to develop the phylogenetic reconstruction. Seventy clones were previously characterized as TcI by amplification of the intergenic region of the mini-exon gene and the divergent domain of 24S␣ rDNA. We obtained 200 L aliquots of LIT-biphasic culture media from the exponential growth phase, which were submitted to DNA extraction using a QIamp kit (Quiagen). The DNA samples were amplified by PCR using the primers p18 (5 -GAC AGG ATT GAG AAG CGA GAG AG-3 ) and p20 (5 -CAA ACC TAT CAC AAA AAG CAT CTG-3 ), in a master mix reaction containing Buffer 1× (Corpogen, COL), 25 mM MgCl2 , 50 M of each primer, 5 U/L of Taq polymerase (Corpogen, COL), 10 M of dNTPs, 30 ng of DNA and sufficient water to obtain a final volume of 21 L. This mix was submitted to 35 cycles of amplification in a BIORAD thermocycler (Brisse et al., 2003). The products of amplification were visualized by electrophoresis in 2% agarose gels stained with ethidium bromide. The products of amplification were cloned and sequenced using the dideoxy terminal method in Macrogen (Korea). The sequences were edited in MEGA 5.0 (Tamura et al., 2007) and aligned with Clustal W 1.8. The sequence from the TcI isolate, Sylvio X10 (AJ130928), as reported in GenBank, was used as a control to edit the sequences. The sequences obtained were deposited in NCBI’s GenBank with the accession numbers HQ713679–HQ713748. Using the edited alignment, we detected 431 conserved sites, 86 variable sites and 49 parsimonious sites with a 0.06 coefficient of differentiation and an average evolutionary distance of 0.013 between the 70 DNA sequences. The sequence of the Cytb gene from T. c. marinkellei was used as an outgroup for the analyses developed. The alignment from the 71 sequences was tested using the AIC (Akaike Information Criterion) on MrModel Test 2.2, where the most suitable evolutive model was selected and GTR+G+I was considered. We developed a phylogenetic reconstruction using a
Maximum Composite Likelihood analysis (ML) with the RAxML 7.2.5 tool on the portal 2.0 from the CIPRES project (Cyberinfrastructure for Phylogenetic Research) (Miller et al., 2009), using the neighbour-joining method by the Kimura-2 parameter model with 1000 bootstrapping iterations to observe the robustness of the nodes. The results of the heuristic search were visualised on FigTree 4.4 (Fig. 1) and determined the presence of four welldifferentiated genotypes, validated with bootstrap values above 80% and −Ln = 1039 with GTR 4 rate. We used the alignment matrix in Nexus format to construct a network, using the median-joining analysis and the default parameters on Network 2.0 to observe the polymorphism differences between the four established genotypes (Fig. 2). The phylogenetic tree showed four genotypes with clear associations with the transmission cycles of Chagas disease in Colombia and also showed associations with specific hosts. Genotype A was associated with clones isolated from humans, with a bootstrap of 91% related to the domestic cycle of transmission, and was concordant with the TcIa and TcIb genotypes, based on the intergenic region of the mini-exon gene. Genotype B was associated with clones isolated from Triatoma dimidiata, captured in the peridomicile, and Canis familiaris, with a bootstrap of 87% related to the peridomiciliary transmission cycle and concordant with the TcIb genotype, as determined by the mini-exon gene. Genotype C was associated with sylvatic/synanthropic reservoirs, such as Didelphis marsupialis, Rattus rattus, Tamandua tetradactyla and Alouatta seniculus, with a bootstrap of 83%, and genotype D was associated with sylvatic triatomines, such as Rhodnius prolixus, Triatoma maculata, Triatoma venosa, Eratyrus cuspidatus, Rhodnius pallescens and R. colombiensis, with a bootstrap of 83%, which are concordant with the TcId genotype, as determined by the mini-exon gene. These results corroborate the previous studies conducted using the intergenic region of the mini-exon gene, in which five
64
J.D. Ramírez et al. / Acta Tropica 119 (2011) 61–65
Fig. 2. Genotype network determined using Network 2.0 based on the median-joining analysis using the default parameters.
genotypes have been previously established in relation to the transmission cycles of Chagas disease in America Cura et al., 2010; Herrera et al., 2007, 2009). Likewise, the studies developed in Chile, using the Cytb gene in TcI isolates, reported the presence of two phylogenetically robust subgroups associated with human infection and domestic vectors from Chile, Brazil and Colombia, and a second subgroup mainly related to caviomorph reservoirs, showing the importance of this reservoir in the evolution of trypanosomes (Spotorno et al., 2008). Our results are congruent with the initial descriptions from Chile: the subgroup isolated from domestic vectors could be considered to be genotype A, as reported in this study, and likewise, the subgroup from the caviomorph reservoirs could be considered to be genotype C but a phylogenetic reconstruction using these sequences must be addressed to confirm this premise. Previous reports, using microsatellite markers on a significant number of TcI isolates, showed genotypes linked to human infection, which could be associated with genotype A, reported in this study (Llewellyn et al., 2009). Studies developed in Brazil with TcI, TcII, TcIII and TcIV isolates, using the Cytb gene sequences, showed a differentiation regarding the ecological ecotopes of the reservoirs and also unravelled a close relatedness between TcI and TcIII/TcIV isolates (Marcili et al., 2009). These results show the great utility of this molecular marker in elucidating the genetic diversity of T. cruzi DTUs and suggesting the relationships between the variants of the parasite and their respective hosts, which is in accordance with the proposed genotypes in this report, where the evidence indicates that certain genotypes are related to specific hosts. The purpose of evaluating the genetic diversity of T. cruzi must be related to the clinical forms of Chagas disease, where studies have reported the impact of specific TcI genotypes on the symptoms of patients with chronic cardiomyopathy (Burgos et al., 2010; Ramírez et al., 2010). Also, this genetic variability shows the evolutive processes that the parasite has undergone, showing a possible coevolution with its hosts, clearly reflected in the presence of genotype C (sylvatic/synanthropic reservoirs) and genotype D (sylvatic triatomines), as determined by Cytb, the subgroup 2, reported by Spotorno et al. (2008) and the genotype TcId, reported by Herrera et al. (2009) and Cura et al. (2010). It is important to mention that our findings show a differentiation of R. prolixus and the other species of sylvatic vectors, with a bootstrap of 83%, from six
clones from sylvatic triatomines, such as Triatoma maculata, Triatoma venosa, E. cuspidatus, R. pallescens and R. colombiensis. This can be related to the adaptation of the parasite to different vectors, reflecting a form of coevolution. The results of the genotype network allowed us to corroborate the usefulness of Cytb in associating specific T. cruzi genotypes with specific hosts. This is shown by the fact that genotype C showed two types of frequencies: one that includes clones isolated from D. marsupialis and R. rattus and another that includes clones isolates from Tamandua tetradactyla and A. seniculus and a clone isolated from a D. marsupialis with congenital infection, revealing specific nucleotide changes related to certain hosts (Fig. 2). In conclusion, we report, for the first time, distinct genotypes within Colombian T. cruzi I clones isolated from different geographical regions and hosts, based on polymorphisms of the Cytb gene, validated with high bootstrap values and developed in clones instead of strains. Our results corroborate the genetic diversity that can be found within T. cruzi I and are concordant with the previously reported genotypes based on the mini-exon gene (Cura et al., 2010; Herrera et al., 2009) and the subgroups reported using Cytb in Chile (Spotorno et al., 2008). It is important to continue studies using this molecular marker in different American countries in order to unravel the processes and evolutionary drivers of TcI and its implications in the transmission, and the different clinical forms, of Chagas disease. Acknowledgments Financial support was provided by the Project Chagas EpiNet from The European Union Seventh Framework Programme, contract No. 223034. We thank the research fund of the faculty of sciences from the Universidad de los Andes and the group of parasitology from the Instituto Nacional de Salud in Colombia for providing some human isolates. References Anon, 1999. Recommendations from a satellite meeting. Mem. Inst. Oswaldo Cruz 94, 429–432. Brisse, S., Henriksson, J., Barnabe, C., Douzery, E.J.P., Berkyens, D., Serrano, M., De Carvalho, R., Buck, G.A., Dujardin, J.C., Tibayrenc, M., 2003. Evidence for genetic
J.D. Ramírez et al. / Acta Tropica 119 (2011) 61–65 exchange and hybridization in Trypanosoma cruzi based on nucleotide sequences and molecular karyotype. Infect. Genet. Evol. 2, 173–183. Burgos, J.M., Diez, M., Vigliano, C., Bisio, M., Risso, M., Duffy, T., Cura, C., Brusses, B., Favaloro, L., Leguizamon, S., Lucero, R.H., Laguens, R., Levin, M.J., Favaloro, R., Schijman, A.G., 2010. Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation. Clin. Infect. Dis. 51, 485–495. Cura, C.I., Mejia-Jaramillo, A.M., Duffy, T., Burgos, J.M., Rodriguero, M., Cardinal, M.V., Kjos, S., Gurgel-Goncalves, R., Blanchet, D., De Pablos, L.M., Tomasini, N., da Silva, A., Russomando, G., Cuba, C.A., Aznar, C., Abate, T., Levin, M.J., Osuna, A., Gurtler, R., Diosque, P., Solari, A., Triana-Chavez, O., Schijman, A.G., 2010. Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced leader genes. Int. J. Parasitol. 40, 1599–1607. Falla, A., Herrera, C., Fajardo, A., Montilla, M., Vallejo, G.A., Guhl, F., 2009. Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans. Acta Trop. 110, 15–21. 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. Gaunt, M.W., Yeo, M., Frame, I.A., Stothard, J.R., Carrasco, H.J., Taylor, M.C., Mena, S.S., Veazey, P., Miles, G.A., Acosta, N., de Arias, A.R., Miles, M.A., 2003. Mechanisms of genetic exchange in American trypanosomes. Nature 421, 936–939. Herrera, C., Bargues, M.D., Fajardo, A., Montilla, M., Triana, O., Vallejo, G.A., Guhl, F., 2007. Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia. Infect. Genet. Evol. 7, 535–539. Herrera, C., Guhl, F., Falla, A., Fajardo, A., Montilla, M., Vallejo, G.A., Bargues, M.D., 2009. Genetic variability and phylogenetic relationships within Trypanosoma cruzi I isolated in Colombia based on miniexon gene sequences. J. Parasitol. Res., doi:10.1155/2009/897364. Llewellyn, M.S., Miles, M.A., Carrasco, H.J., Lewis, M.D., Yeo, M., Vargas, J., Torrico, F., Diosque, P., Valente, V., Valente, S.A., Gaunt, M.W., 2009. Genome-scale multilocus microsatellite typing of Trypanosoma cruzi discrete typing unit I reveals phylogeographic structure and specific genotypes linked to human infection. PLoS Pathog. 5, e1000410. Marcili, A., Lima, L., Valente, V.C., Valente, S.A., Batista, J.S., Junqueira, A.C.V., Souza, A.I., da Rosa, J.A., Campaner, M., Lewis, M.D., Llewellyn, M.S., Miles, M.A., Teixeira, M.G., 2009. Comparative phylogeography of Trypanosoma cruzi TCIIc: new hosts, association with terrestrial ecotopes and spatial clustering. Infect. Genet. Evol. 9, 1265–1274. Mejía-Jaramillo, A.M., Arboleda-Sánchez, S., Rodríguez, I.B., Cura, C., Salazar, A., Del Mazo, J., Triana-Chávez, O., Schijman, A.G., 2009. Geographical clustering of Trypanosoma cruzi I groups from Colombia revealed by low-stringency single specific primer-PCR of the intergenic regions of spliced-leader genes. Parasitol. Res. 104, 399–410.
65
Miller, M., Holder, M., Vos, R., Midford, P., Liebowitz, T., Chan, L., Hoover, P., Warnow, T., 2009. The CIPRES portals. CIPRES, 2009-08-04. URL: http://www.phylo.org/sub sections/portal. (Archivado por WebCite.(r) en http://www.webcitation.org/5imQlJeQa). ˜ Ocana-Mayorga, S., Llewellyn, M.S., Costales, J.A., Miles, M.A., Grijalva, M.J., 2010. Sex, subdivision, and domestic dispersal of Trypanosoma cruzi lineage I in Southern Ecuador. PLoS Negl. Trop. Dis. 4, e915, doi:10.1371/journal.pntd.0000915. O’Connor, O., Bosseno, M.F., Barnabé, C., Douzery, E.J.P., Breniére, S.F., 2007. Genetic clustering of Trypanosoma cruzi I lineage evidenced by intergenic miniexon sequencing. Infect. Genet. Evol. 7, 587–593. Ramírez, J.D., Guhl, F., Rendón, L.M., Rosas, F., Marin-Neto, J.A., Morillo, C., 2010. Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic Chagasic patients. PLoS Negl. Trop. Dis 4, e899, doi:10.1371/journal.pntd.0000899. Salazar, A., Schijman, A.G., Triana, O., 2006. High variability of Colombian Trypanosoma cruzi lineage I stocks as revealed by low-stringency single primer-PCR minicircle signatures. Acta Trop. 100, 110–118. Spotorno, A.E., Córdova, L., Solari, A., 2008. Differentiation of Trypanosoma cruzi I subgroups through characterization of cytochrome b gene sequences. Infect. Genet. Evol. 8, 898–900. Sturm, N., Campbell, D.A., 2009. Alternative lifestyles: the population structure of Trypanosoma cruzi. Acta Trop. 33, 205–214. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596– 1599. Tomasini, N., Lauthier, J.J., Monje Rumi, M.M., Ragone, P.G., Alberti, A., Brandan, C.P., Cura, C.I., Schijman, A.G., Barnabé, C., Tibayrenc, M., Basombrio, M.A., Falla, A., Herrera, C., Guhl, F., Diosque, P., 2010. Interest and limitations of spliced leader intergenic region sequences for analyzing Trypanosoma cruzi I phylogenetic diversity in the Argentinean Chaco. Infect. Genet. Evol. 11, 300– 307. Triana, O., Ortiz, S., Dujardin, J.C., Solari, A., 2006. Trypanosoma cruzi; variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis. Exp. Parasitol. 113, 62–66. Westenberger, S.J., Barnabe, C., Campbell, D.A., Sturm, N.R., 2005. Two hybridization events define the population structure of Trypanosoma cruzi genetics. Genetics 171, 527–543. World Health Organization (WHO) Special Programme for Research and Training in Tropical Diseases (TDR), 2007. Report of Scientific Group in Chagas disease, Buenos Aires, Argentina, April 17–20, 2005, Update July 2007. Zingales, B., Andrade, S.G., Briones, M.R.S., Campbell, D.A., Chiari, E., Fernandes, O., Guhl, F., Lages-Silva, E., Macedo, A.M., Machado, C.R., Miles, M.A., Romanha, A.J., Sturm, N.R., Tibayrenc, M., Schijman, A.G., 2009. A new consensus for Trypanosoma cruzi intraespecific nomenclature: second revision meeting recommends TcI to TcVI. Mem. Inst. Oswaldo Cruz 104, 1051–1054.