Molecular typing of Trypanosoma cruzi isolates, United States

Studies have characterized Trypanosoma cruzi from parasite-endemic regions. With new human cases, increasing numbers of veterinary cases, and influx of potentially infected immigrants, understanding the ecology of this organism in the United States
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  Molecular Typing of Trypanosoma cruzi Isolates, United States Dawn M. Roellig,* Emily L. Brown,* Christian Barnabé,† Michel Tibayrenc,† Frank J. Steurer,‡ and Michael J. Yabsley* Studies have characterized Trypanosoma cruzi  from parasite-endemic regions. With new human cases, increas-ing numbers of veterinary cases, and in fl ux of potentially infected immigrants, understanding the ecology of this or-ganism in the United States is imperative. We used a clas-sic typing scheme to determine the lineage of 107 isolates from various hosts. I n Latin America, an estimated 10–12 million persons are infected with Trypanosoma cruzi , the etiologic agent of Chagas disease and a major contributor to heart disease within the region. Autochthonous human infections in the United States have been reported in 6 persons, with the most recent case reported from Louisiana ( 1 ). In addition, the parasite is euryxenous; it is able to infect a broad range of hosts, including domestic dogs, woodrats, raccoons, opossums, armadillos, and nonhuman primates.Associations between host species and parasite geno-type have been suggested and are important in understand-ing the domestic and sylvatic cycles of T  . cruzi  ( 2  –  4 ). Although studies conducted on US isolates suggest an as-sociation between T  . cruzi  genotype and host, these studies were limited because of low sample numbers, low host di-versity, and narrow geographic distribution ( 2 , 4  –  7  ). In the current investigation, we used the molecular typing scheme  proposed by Brisse et al. ( 8  ), in which isolates are delin-eated into 1 of the 6 lineages (types I and IIa–IIe) on the  basis of size polymorphisms of several PCR markers. We then expanded characterization of US isolates and show ad-ditional evidence for correlations between host speci fi city and genotype of T  . cruzi . The Study We analyzed 107 isolates of T  . cruzi  from multiple species of free-ranging and captive wildlife, domestic ani-mals, triatomine bug vectors, and humans who were au-tochthonously infected in the United States. Some isolates were obtained as liquid nitrogen–stored parasites from the Centers for Disease Control and Prevention (Atlanta, GA, USA), the Institut Pasteur (Paris, France), and the South-eastern Cooperative Wildlife Disease Study (Athens, GA, USA) and were established in axenic liver infusion tryptose medium as described ( 9 ). Additional isolates were obtained from wild-trapped animals in axenic liver infusion tryptose medium or canine macrophage cell culture as described ( 10 ). Isolated DNA was used as template for PCR ampli fi -cation of 3 gene targets, mini-exon, D7 divergent domain of 24S α  rRNA, and 18S rRNA, according to published methods ( 8  ). Locality data and results of molecular typ-ing of each isolate are shown in the online Appendix Table (available from All animals used in this study were cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee and under animal use protocol approved by the Institutional Animal Care and Use Com-mittee at the University of Georgia.Only 2 genotypes, T  . cruzi  I and T  . cruzi  IIa, were de-tected. Typical amplicon sizes of T  . cruzi  I and T  . cruzi  IIa isolates from the United States are shown in the Table. Atypical banding patterns and isolates that differ from the standard genotype from a particular host are also represent-ed. With the exception of human isolates, 1 primate isolate, and a few raccoon isolates, placental mammalian isolates, including those from raccoons, domestic dogs, ring-tailed lemurs, and skunks, were characterized as type IIa (online Appendix Table). All remaining isolates, including those from Virginia opossums (  Didelphis virginiana ), triatom-ine vectors, humans, and rhesus macaques from the United States, were identi fi ed as type I (online Appendix Table). Conclusions In contrast to studies conducted on South American isolates, for which 6 genotypes of T  . cruzi  have been iden-ti fi ed, only 2 genotypes (I and IIa) were identi fi ed in the current study. These data support results of investigations in Central America and Mexico in which a paucity of geno-types was found ( 14 , 15 ). Many investigations on T  . cruzi  evolutionary ecology have shown strict host–parasite spec-i fi city in regard to host species and parasite genotype ( 2  –  4 ), although exceptions have been observed. The presence of only 2 genotypes in the United States could be caused by a lack of introduction of other genotypes or a lower diver-sity of natural reservoir hosts for T  . cruzi  than in South America. A recent analysis of T  . cruzi  hosts in North and South America indicated that >48 host species representing 17 families were infected with >1 of the 6 strains ( 4 ). Only 6 of these hosts have established populations in the United States, and US isolates from these species were only char-acterized as types I or IIa ( 4 ).  Emerging Infectious Diseases • • Vol. 14, No. 7, July 2008 1123 *University of Georgia, Athens, Georgia, USA; †Institut de Recher-che pour le Developpement, Montpellier, France; and ‡Centers for Disease Control and Prevention, Atlanta, Georgia, USADOI: 10.3201/eid1407.080175  Our data for US isolates correspond with those of pre-vious studies in which  Didelphis  spp. are reservoirs for type I T  . cruzi  ( 4 ); no infections with type II parasites were observed. The Virginia opossum (and its ancestors), which is the only marsupial present in the United States (it mi-grated from South America ≈ 4.5 million years ago), is a  possible host for T  . cruzi  I. This evidence suggests that T  . cruzi  was not recently introduced into North America or the United States ( 5 ). Additionally, suf  fi cient time may have  passed for random and rare genetic exchange events to oc-cur independent of those found in South American isolates ( 13 ), enabling the lineage to infect atypical reservoirs (i.e., raccoons) in North America.The second major natural reservoir of T  . cruzi  in the United States is the raccoon. In general, the nonprimate  placental mammals in our study were infected with type IIa, a strain that is commonly found in sylvatic cycles in the Southern Cone of South America. Our data con fi rm previ-ous typing of US isolates by multilocus enzyme electro- phoresis or random ampli fi ed polymorphic DNA analysis ( 5 ), in which 11 raccoons from Georgia were characterized as zymodeme 3 (equivalent to IIa). Although raccoons are  predominately infected with T  . cruzi  IIa, 4 known excep-tions include 3 isolates from Georgia and Florida in the current study and 1 raccoon from Louisiana from a previ-ous study ( 5 ).These data are in contrast to typing data for Virginia opossum isolates, which have all found T  . cruzi  I. This fi nding suggests that opossums primarily maintain  persistent infections with T  . cruzi  I.All characterized human isolates from autochthonous US cases of infection with T  .  cruzi  are T  . cruzi  I. This genotype is predominantly responsible for Chagas disease north of the Amazon Basin and is part of the domiciliary cycle of the parasite. Our fi ndings correspond with data from Mexico where T  . cruzi  I is the predominate strain de-tected in humans ( 14 ). It would be useful to differentiate  biologic characteristics and polymorphisms by using addi-tional gene targets in human type I isolates and compare them with those in opossum, triatomine vectors, and rhe-sus macaque isolates from the United States. Additionally, comparing these US isolates and Mexican reference strains with those from South America may indicate why type I typically infects humans in North America and multiple strains are found in humans in South America.Our results provide additional evidence that T  . cruzi  has distinct genotypes that preferentially infect 1 host spe-cies or a group of hosts. Humans and marsupials are typi-cally infected with type I T  . cruzi , but raccoons, skunks, domestic dogs, and prosimians are typically infected with type IIa. Although we only detected T  . cruzi  I in triatomid  bugs, other studies have detected T  . cruzi  IIa in triatomids from the United States ( 5 ). The mechanism is unknown by which persistent infections with a particular genotype of T  . cruzi  develop in certain hosts. Further analysis of iso-lates from an increased host diversity and geographic range should be pursued. Determining basic infection dynamics of reservoir hosts experimentally infected with various T  . cruzi  genotypes may provide additional insight into the host–parasite dichotomy.  Acknowledgments We thank B. Wilcox, B. Hanson, and D. Kavanaugh for fi eld assistance; C. Paddock for providing 1 isolate used in the study; and P. Dorn for providing blood for isolation of 1 isolate.This study was supported by grant R15 AI067304 from the  National Institutes of Health, National Institute of Allergy and In-fectious Diseases.Ms Roellig is a doctoral student in infectious diseases at the University of Georgia. Her research interests are vector-borne zoonotic diseases, including Chagas disease in wildlife and tick- borne rickettsial pathogens. References  1. Dorn PL, Perniciaro L, Yabsley MJ, Roellig DM, Balsamo G, Diaz J, et al. Autochthonous transmission of Trypanosoma cruzi , Louisiana. Emerg Infect Dis. 2007;13:605–7. 2. Clark CG, Pung OJ. Host speci fi city of ribosomal DNA variation in sylvatic Trypanosoma cruzi  from North America. Mol Biochem Parasitol. 1994;66:175–9. DOI: 10.1016/0166-6851(94)90052-3 DISPATCHES 1124 Emerging Infectious Diseases • • Vol. 14, No. 7, July 2008 Table. Approximate amplicon sizes of gene targets and lineage determination in Trypanosoma cruzi  Strain Mini-exon, bp 24S   rRNA, bp 18S rRNA, bp Lineage FL Opo 15*   350 110 175 I GA Rac 103*   None 120 155 IIa FL Rac 5*   400 120 155 IIa 93053103R cl3   350 110 175 I FL Rac 13 350 110, 120 155, 175 I/IIa†   FL Rac 46 400 110, 120 155 I/IIa†   Griffin Dog 350 110, 120 155 I/IIa†   Monk RH89–40 None 110 155 I/IIa†   *Denotes isolates used as representative banding patterns seen for classic lineage typing. †Because of atypical banding patterns, a clear definition of an isolate as type I vs. type IIa could not be obtained.  Molecular Typing of T . cruzi  Isolates  3. Briones MR, Souto RP, Stolf BS, Zingales B. The evolution of two Trypanosoma cruzi  subgroups inferred from rRNA genes can be cor-related with the interchange of American mammalian faunas in the Cenozoic and has implications to pathogenicity and host speci fi city. Mol Biochem Parasitol. 1999;104:219–32. DOI: 10.1016/S0166-6851(99)00155-3 4. Yeo M, Acost N, Llewellyn M, Sánchez H, Adamson S, Miles GA, et al. 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. 2005;35:225–33. DOI: 10.1016/j.ijpara.2004.10.024 5. Barnabé C, Yaeger R, Pung O, Tibayrenc M. 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. 2001;99:73–9. DOI: 10.1006/expr.2001.4651 6. Miles MA, Souza A, Povoa M, Shaw JJ, Lainson E, Toye PJ. Isozy-mic heterogeneity of Trypanosoma cruzi  in the fi rst autochtho-nous patients with Chagas’ disease in Amazonian Brazil. Nature. 1978;272:819–21. DOI: 10.1038/272819a0 7. Yabsley MJ, Noblet GP. Biological and molecular characterization of a raccoon isolate of Trypanosoma cruzi  from South Carolina. J Parasitol. 2002;88:1273–6. 8. Brisse S, Verhoef J, Tibayrenc M. Characterisation of large and small subunit rRNA and mini-exon genes further support the distinction of six Trypanosoma cruzi  lineages. Int J Parasitol. 2001;31:1218–26. DOI: 10.1016/S0020-7519(01)00238-7 9. Castellani O, Ribeiro LV, Fernandes JF. Differentiation of Trypano-soma cruzi  in culture. J Protozool. 1967;14:447–51.10. Yabsley MJ, Norton TM, Powell MR, Davidson WR. Molecular and serologic evidence of tick-borne ehrlichiae in three species of le-murs from St. Catherine’s Island, Georgia, USA. J Zoo Wildl Med. 2004;35:503–9.11. de Freitas JM, Augusto-Pinto L, Pimenta JR, Bastos-Rodrigues L, Goncalves VF, Teixeira SM, et al. Ancestral genomes, sex and the population structure of Trypanosoma cruzi.  PLoS Pathog. 2006;2:e24. DOI: 10.1371/journal.ppat.002002412. Brisse S, Barnabé C, Tibayrenc M. Trypanosoma cruzi  clonal di-versity: identi fi cation of discrete phylogenetic lineages by random ampli fi ed polymorphic DNA and multilocus enzyme electrophoresis analysis. Int J Parasitol. 2000;30:35–44. DOI: 10.1016/S0020-7519-(99)00168-X13. Machado CA, Ayala FJ. Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi.  Proc Natl Acad Sci U S A. 2001;98:7396–401. DOI: 10.1073/ pnas.12118719814. Bosseno M-F, Bernabé C, Gastélum EM, Kasten FL, Ramsey J, Espinoza B, et al. Predominance of Trypanosoma cruzi  lineage I in Mexico. J Clin Microbiol. 2002;40:627–32. DOI: 10.1128/JCM.40.2.627-632.200215. Iwagami M, Higo H, Miura S, Yanagi T, Tada I, Kano S, et al. Mo-lecular phylogeny of Trypanosoma cruzi  from Central America (Guatemala) and a comparison with South American strains. Parasi-tol Res. 2007;102:129–34. DOI: 10.1007/s00436-007-0739-9Address for correspondence: Dawn M. Roellig, Southeastern Cooperative Wildlife Disease Study, Department of Population Health, 589 DW Brooks Dr, Wildlife Health Bldg, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA; email:  Emerging Infectious Diseases • • Vol. 14, No. 7, July 2008 1125 All material published in Emerging Infectious Diseases is in the  public domain and may be used and reprinted without special  permission; proper citation, however, is required. Search past Issues
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