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Molecular Genotyping of Trypanosoma cruzi for Lineage Assignment and Population Genetics

  • Louisa A. Messenger
  • Matthew Yeo
  • Michael D. Lewis
  • Martin S. Llewellyn
  • Michael A. MilesEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1201)

Abstract

Trypanosoma cruzi, the etiological agent of Chagas disease, remains a major public health problem in Latin America. Infection with T. cruzi is lifelong and can lead to a spectrum of pathological sequelae ranging from subclinical to lethal cardiac and/or gastrointestinal complications. Isolates of T. cruzi can be assigned to six genetic lineages or discrete typing units (DTUs), which are broadly associated with disparate ecologies, transmission cycles, and geographical distributions. This extensive genetic diversity is also believed to contribute to the clinical variation observed among chagasic patients. Unravelling the population structure of T. cruzi is fundamental to understanding Chagas disease epidemiology, developing control strategies, and resolving the relationship between parasite genotype and clinical prognosis.

To date, no single, widely validated, genetic target allows unequivocal resolution to DTU-level. In this chapter we present standardized methods for strain DTU assignment using PCR-restriction fragment length polymorphism analysis (PCR-RFLP) and nuclear multilocus sequence typing (MLST). PCR-RFLPs have the advantages of simplicity and reproducibility, requiring limited expertise and few laboratory consumables. MLST data are more laborious to generate but more informative; DNA sequences are readily transferable between research groups and amenable to recombination detection and intra-lineage analyses. We also recommend a mitochondrial (maxicircle) MLST scheme and a panel of 28 microsatellite loci for higher resolution population genetics studies.

Due to the scarcity of T. cruzi in blood and tissue, all of these genotyping techniques have limited sensitivity when applied directly to clinical or biological specimens, particularly when targets are single (MLST) or low copy number (PCR-RFLPs). We therefore describe essential protocols to isolate parasites, derive biological clones, and extract T. cruzi genomic DNA from field and clinical samples.

Key words

Trypanosoma cruzi PCR Genotyping Phylogenetics Microsatellites MLST RFLP Mitochondria Sequencing 

Notes

Acknowledgments

Research detailed in this chapter was funded by support from the Wellcome Trust, the BBSRC and the European Union Seventh Framework Programme grant 223034 (“ChagasEpiNet”).

References

  1. 1.
    Rassi A Jr, Rassi A, Marin-Neto JA (2010) Chagas disease. Lancet 375:1388–1402CrossRefPubMedGoogle Scholar
  2. 2.
    Prata A (2001) Clinical and epidemiological aspects of Chagas disease. Lancet Infect Dis 1:92–100CrossRefPubMedGoogle Scholar
  3. 3.
    Miles MA, Cedillos RA, Póvoa MM et al (1981) Do radically dissimilar Trypanosoma cruzi strains (zymodemes) cause Venezuelan and Brazilian forms of Chagas disease? Lancet 1:1338–1340CrossRefPubMedGoogle Scholar
  4. 4.
    Campbell DA, Westenberger SJ, Sturm NR (2004) The determinants of Chagas disease: connecting parasite and host genetics. Curr Mol Med 4:549–562CrossRefPubMedGoogle Scholar
  5. 5.
    Macedo AM, Machado CR, Oliveira RP et al (2004) Trypanosoma cruzi: genetic structure of populations and relevance of genetic variability to the pathogenesis of Chagas disease. Mem Inst Oswaldo Cruz 99:1–12CrossRefPubMedGoogle Scholar
  6. 6.
    Fernandes O, Souto R, Castro J et al (1998) Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences. Am J Trop Med Hyg 58:807–811PubMedGoogle Scholar
  7. 7.
    Souto RP, Fernandes O, Macedo AM et al (1996) DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Mol Biochem Parasitol 83:141–152CrossRefPubMedGoogle Scholar
  8. 8.
    Hamilton PB, Lewis MD, Cruickshank C et al (2011) Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding. Infect Genet Evol 11:44–51CrossRefPubMedGoogle Scholar
  9. 9.
    Oliveira RP, Broude NE, Macedo AM et al (1998) Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites. Proc Natl Acad Sci U S A 95:3776–3780CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Llewellyn MS, Miles MA, Carrasco HJ et al (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:e1000410CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Ocaña-Mayorga S, Llewellyn MS, Costales JA et al (2010) Sex, subdivision, and domestic dispersal of Trypanosoma cruzi lineage I in Southern Ecuador. PLoS Negl Trop Dis 4:e915CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Burgos JM, Altcheh J, Bisio M et al (2007) Direct molecular profiling of minicircle signatures and lineages of Trypanosoma cruzi bloodstream populations causing congenital Chagas disease. Int J Parasitol 37:1319–1327CrossRefPubMedGoogle Scholar
  13. 13.
    Telleria J, Lafay B, Virreira M et al (2006) Trypanosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles. Exp Parasitol 114:279–288CrossRefPubMedGoogle Scholar
  14. 14.
    Lages-Silva E, Ramírez LE, Pedrosa AL et al (2006) Variability of kinetoplast DNA gene signatures of Trypanosoma cruzi II strains from patients with different clinical forms of Chagas disease in Brazil. J Clin Microbiol 44:2167–2171CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Zingales B, Miles MA, Campbell DA et al (2012) The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect Gent Evol 12:240–253CrossRefGoogle Scholar
  16. 16.
    Tibayrenc M (1998) Genetic epidemiology of parasitic protozoa and other infectious agents: the need for an integrated approach. Int J Parasitol 28:85–104CrossRefPubMedGoogle Scholar
  17. 17.
    Miles MA, Llewellyn MS, Lewis MD et al (2009) The molecular epidemiology and phylogeography of Trypanosoma cruzi and parallel research on Leishmania: looking back and to the future. Parasitology 136:1509–1528CrossRefPubMedGoogle Scholar
  18. 18.
    Zingales B, Andrade SG, Briones MR et al (2009) A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz 104:1051–1054CrossRefPubMedGoogle Scholar
  19. 19.
    Lewis MD, Llewellyn MS, Yeo M et al (2011) Recent, independent and anthropogenic origins of Trypanosoma cruzi hybrids. PLoS Negl Trop Dis 4:e1363CrossRefGoogle Scholar
  20. 20.
    Yeo M, Mauricio IL, Messenger LA et al (2011) Multilocus sequence typing (MLST) for lineage assignment and high resolution diversity studies in Trypanosoma cruzi. PLoS Negl Trop Dis 5:e1049CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Brisse S, Henriksson J, Barnabé C (2003) Evidence for genetic exchange and hybridization in Trypanosoma cruzi based on nucleotide sequences and molecular karyotype. Infect Genet Evol 2:173–183CrossRefPubMedGoogle Scholar
  22. 22.
    Machado CA, Ayala FJ (2001) Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi. Proc Natl Acad Sci U S A 98:7396–7401CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    De Freitas JM, Augusto-Pinto L, Pimenta JR et al (2006) Ancestral genomes, sex, and the population structure of Trypanosoma cruzi. PLoS Pathog 2:e24CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Westenberger SJ, Barnabé C, Campbell DA et al (2005) Two hybridization events define the population structure of Trypanosoma cruzi. Genetics 171:527–543CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Sturm NR, Campbell DA (2010) Alternative lifestyles: the population structure of Trypanosoma cruzi. Acta Trop 115:35–43CrossRefPubMedGoogle Scholar
  26. 26.
    Añez N, Crisante G, Da Silva FM et al (2004) Predominance of lineage I among Trypanosoma cruzi isolates from Venezuelan patients with different clinical profiles of acute Chagas disease. Trop Med Int Health 9:1319–1326CrossRefPubMedGoogle Scholar
  27. 27.
    Ramirez JD, Guhl F, Rendón LM et al (2010) Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic Chagasic patients. PLoS Negl Trop Dis 4:e899CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Barnabé C, Brisse S, Tibayrenc M (2000) Population structure and genetic typing of Trypanosoma cruzi, the agent of Chagas disease: a multilocus enzyme electrophoresis approach. Parasitology 120:513–526CrossRefPubMedGoogle Scholar
  29. 29.
    Roellig DM, Brown EL, Barnabé C et al (2008) Molecular typing of Trypanosoma cruzi isolates, United States. Emerg Infect Dis 14:1123–1125CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Gaunt M, Miles M (2000) The ecotopes and evolution of triatomine bugs (Triatominae) and their associated trypanosomes. Mem Inst Oswaldo Cruz 95:557–565CrossRefPubMedGoogle Scholar
  31. 31.
    Marcili A, Lima L, Valente VC et al (2009) Comparative phylogeography of Trypanosoma cruzi TcIIc: new hosts, association with terrestrial ecotopes and spatial clustering. Infect Genet Evol 9:1265–1274CrossRefPubMedGoogle Scholar
  32. 32.
    Yeo M, Acosta N, Llewellyn M et al (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 35:225–233CrossRefPubMedGoogle Scholar
  33. 33.
    Llewellyn MS, Lewis MD, Acosta N (2009) Trypanosoma cruzi IIc: phylogenetic and phylogeographic insights from sequence and microsatellite analysis and potential impact on emergent Chagas disease. PLoS Negl Trop Dis 3:e510CrossRefPubMedCentralPubMedGoogle Scholar
  34. 34.
    Marcili A, Valente VC, Valente SA et al (2009) Trypansoma cruzi in Brazilian Amazonia: lineages TCI and TCIIa in wild primates, Rhodnius spp. and in humans with Chagas disease associated with oral transmission. Int J Parasitol 39:615–623CrossRefPubMedGoogle Scholar
  35. 35.
    Valente SA, Valente VC, Neves Pinto AY et al (2009) Analysis of an acute Chagas disease outbreak in the Brazilian Amazon: human cases, triatomines, reservoir mammals and parasites. Trans R Soc Trop Med Hyg 103:291–297CrossRefPubMedGoogle Scholar
  36. 36.
    Vagos AR, Andrade LO, Leite AA et al (2000) Genetic characterization of Trypanosoma cruzi directly from tissues of patients with chronic Chagas disease: differential distribution of genetic types into diverse organs. Am J Pathol 156:1805–1809CrossRefGoogle Scholar
  37. 37.
    Burgos JM, Begher S, Silva HM et al (2008) Molecular identification of Trypanosoma cruzi I tropism for central nervous system in Chagas reactivation due to AIDS. Am J Trop Med Hyg 78:294–297PubMedGoogle Scholar
  38. 38.
    Llewellyn MS, Rivett-Carnac JB, Fitzpatrick S et al (2011) Extraordinary Trypanosoma cruzi diversity within single mammalian reservoir hosts implies and mechanism of diversifying selection. Int J Parasitol 41:609–614CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Bosseno MF, Telleria J, Vargas F et al (1996) Trypanosoma cruzi: study of the distribution of two widespread clonal genotypes in Bolivian Triatoma infestans vectors shows a high frequency of mixed infections. Exp Parasitol 83:275–282CrossRefPubMedGoogle Scholar
  40. 40.
    Cardinal MV, Lauricella MA, Ceballos LA et al (2008) Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina. Int J Parasitol 38:1533–1543CrossRefPubMedCentralPubMedGoogle Scholar
  41. 41.
    Yeo M, Lewis MD, Carrasco HJ et al (2007) Resolution of multiclonal infections of Trypansoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium. Int J Parasitol 37:111–120CrossRefPubMedGoogle Scholar
  42. 42.
    Herrera L, D’Andrea PS, Xavier SC et al (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 99:379–388CrossRefPubMedGoogle Scholar
  43. 43.
    Macedo AM, Pimenta JR, Aguiar RS et al (2001) Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi. Mem Inst Oswaldo Cruz 96:407–413CrossRefPubMedGoogle Scholar
  44. 44.
    Ramirez JD, Guhl F, Messenger LA et al (2012) Contemporary cryptic sexuality in Trypanosoma cruzi. Mol Ecol 21:4216–4226CrossRefPubMedGoogle Scholar
  45. 45.
    Rougeron V, De Meeûs T, Hide M et al (2009) Extreme inbreeding in Leishmania braziliensis. Proc Natl Acad Sci U S A 106:10224–10229CrossRefPubMedCentralPubMedGoogle Scholar
  46. 46.
    Miles MA (1993) Culturing and biological cloning of Trypanosoma cruzi. In: Hyde JE (ed) Protocols in molecular parasitology, vol 21. Springer, London, pp 15–28CrossRefGoogle Scholar
  47. 47.
    Lewis MD, Ma J, Yeo M et al (2009) Genotyping of Trypanosoma cruzi: systematic selection of assays allowing rapid and accurate discrimination of all known lineages. Am J Trop Med Hyg 81:1041–1049CrossRefPubMedCentralPubMedGoogle Scholar
  48. 48.
    D’Avila DA, Macedo AM, Valadares HM et al (2009) Probing population dynamics of Trypanosoma cruzi during progression of the chronic phase in chagasic patients. J Clin Microbiol 47:1718–1725CrossRefPubMedCentralPubMedGoogle Scholar
  49. 49.
    Burgos JM, Diez M, Vigliano C (2010) Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease and reactivation after heart transplantation. Clin Infect Dis 51:485–495CrossRefPubMedGoogle Scholar
  50. 50.
    Van der Auwera G., Maes I., Lewis M.D. et al. (2012) Standardized method for direct determination of Trypanosoma cruzi discrete typing units. Trans R Soc Trop Med Hyg submittedGoogle Scholar
  51. 51.
    Lauthier JL, Tomasini N, Barnabé C et al (2012) Candidate targets for Multilocus Sequence Typing of Trypanosoma cruzi: validation using parasite stocks from the Chaco Region and a set of reference strains. Infect Genet Evol 12:350–358CrossRefPubMedGoogle Scholar
  52. 52.
    Diosque P, Tomasini N, Lauthier JJ et al (2014) Optimized multilocus sequence typing scheme (MLST) for Trypanosoma cruzi. PLoS Negl Trop Dis (in press.Google Scholar
  53. 53.
    Andersson B (2011) The Trypanosoma cruzi genome; conserved core genes and extremely variable surface molecule families. Res Microbiol 162:619–625CrossRefPubMedGoogle Scholar
  54. 54.
    Ellegren H (2000) Microsatellite mutations in the germline: implications for evolutionary inference. Trends Genet 16:551–558CrossRefPubMedGoogle Scholar
  55. 55.
    Hoffman J, Amos W (2005) Microsatellite genotyping errors: detection approaches, common sources and consequences for paternal exclusion. Mol Ecol 14:599–612CrossRefPubMedGoogle Scholar
  56. 56.
    Messenger LA, Llewellyn MS, Bhattacharyya T et al (2012) Multiple mitochondrial introgression events and heteroplasmy in Trypanosoma cruzi revealed by maxicircle MLST and next generation sequencing. PLoS Negl Trop Dis 6:e1584CrossRefPubMedCentralPubMedGoogle Scholar
  57. 57.
    Herwaldt BL (2001) Laboratory-acquired parasitic infections from accidental exposures. Clin Microbiol Rev 14:659–688CrossRefPubMedCentralPubMedGoogle Scholar
  58. 58.
    Hall TA (1999) Bioedit: a user-friendly biological sequence alignment edit and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  59. 59.
    Huson DH, Bryant D (2006) Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 23:254–267CrossRefPubMedGoogle Scholar
  60. 60.
    Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256CrossRefPubMedGoogle Scholar
  61. 61.
    Tamura K, Peterson D, Peterson N et al (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefPubMedCentralPubMedGoogle Scholar
  62. 62.
    Guindon S, Dufayard JF, Lefort V et al (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321CrossRefPubMedGoogle Scholar
  63. 63.
    Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574CrossRefPubMedGoogle Scholar
  64. 64.
    Minch E, Ruiz-Linares A, Goldstein D et al (1997) MICROSAT v1.5d: a computer programme for calculating various statistics on microsatellite allele data. Department of Genetics, Stanford University, Stanford, CAGoogle Scholar
  65. 65.
    Felsenstein J (1989) PHYLIP – phylogeny inference package (version 3.2). Cladistics 5:164–166Google Scholar
  66. 66.
    Park SDE (2001) Trypanotolerance in West African cattle and the population genetic effects of selection. Ph.D. thesis, University of DublinGoogle Scholar
  67. 67.
    Goudet J (1995) FSTAT (version 1.2): a computer program to calculate F-statistics. J Hered 86:485–486Google Scholar
  68. 68.
    Excoffier L, Schneider S (2005) Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol Bioinform Online 1:47–50PubMedCentralGoogle Scholar
  69. 69.
    Agapow PM, Burt A (2001) Indices of multilocus linkage disequilibrium. Mol Ecol Notes 1:101–102CrossRefGoogle Scholar
  70. 70.
    Peakall R, Smouse P (2006) GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295CrossRefGoogle Scholar
  71. 71.
    Liu N, Zhao H (2006) A non-parametric approach to population structure inference using multilocus genotypes. Hum Genomics 2:353–364CrossRefPubMedCentralPubMedGoogle Scholar
  72. 72.
    Jombart T, Devillard S, Balloux F (2010) Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Genet 11:94CrossRefPubMedCentralPubMedGoogle Scholar
  73. 73.
    Brisse S, Verhoef J, Tibayrenc M (2001) Characterisation of large small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages. Int J Parasitol 31:1218–1226CrossRefPubMedGoogle Scholar
  74. 74.
    Sturm NR, Vargas NS, Westenberger SJ et al (2003) Evidence for multiple hybrid groups in Trypanosoma cruzi. Int J Parasitol 33:269–279CrossRefPubMedGoogle Scholar
  75. 75.
    Gaunt MW, Yeo M, Frame IA et al (2003) Mechanism of genetic exchange in American trypanosomes. Nature 421:936–939CrossRefPubMedGoogle Scholar
  76. 76.
    Franzén O, Ochaya S, Sherwood E et al (2011) Shotgun sequencing analysis of Trypanosoma cruzi I Sylvio X10/1 and comparison with T. cruzi VI CL Brener. PLoS Negl Trop Dis 5:e984CrossRefPubMedCentralPubMedGoogle Scholar
  77. 77.
    Kawashita SY, Sanson GFO, Fernandes O et al (2001) Maximum-likelihood divergence date estimates based on rRNA gene sequences suggests two scenarios of Trypanosoma cruzi intraspecific evolution. Mol Biol Evol 18:2250–2259CrossRefPubMedGoogle Scholar
  78. 78.
    Jombart T (2008) adegenet: a R package for the multivariate analysis of genetic markers. Bioinformatics 24:1403–1405CrossRefPubMedGoogle Scholar
  79. 79.
    Weathery DB, Boehlke C, Tarleton RL (2009) Chromosome level assembly of the hybrid Trypanosoma cruzi genome. BMC Genomics 10:255CrossRefGoogle Scholar
  80. 80.
    Tomasini N, Lauthier JJ, Llewellyn MS et al. (2013) MLSTest: novel software for multi-locus sequence data analysis in eukaryotic organisms. Infect Genet Evol 20:188–196Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Louisa A. Messenger
    • 1
  • Matthew Yeo
    • 2
  • Michael D. Lewis
    • 2
  • Martin S. Llewellyn
    • 3
  • Michael A. Miles
    • 3
    Email author
  1. 1.London School of Hygiene and Tropical MedicineLondonUK
  2. 2.London School of Hygiene and Tropical MedicineLondonUK
  3. 3.London School of Hygiene and Tropical MedicineLondonUK

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