• View in gallery

    TcI (A) and TcII (B) groups of Trypanosoma cruzi in epimastigote form stained with orcein. Note the difference in the protozoa nuclei: TcI presented the nucleus with positive heteropycnosis (A, arrow) and TcII presented the nucleus with negative heteropycnosis (B, arrow). Bar: 10 μm.

  • 1.

    Nouvellet P, Cucunubá ZM, Gourbière S, 2015. Ecology, evolution and control of Chagas disease: a century of neglected modelling and a promising future. Adv Parasitol 87: 135191.

    • Search Google Scholar
    • Export Citation
  • 2.

    World Health Organization, 2018. Chagas Disease (American Trypanosomiasis). Available at: http://www.who.int/chagas/en/. Accessed August 08, 2018.

    • Search Google Scholar
    • Export Citation
  • 3.

    Pan American Health Organization, 2017. Chagas in the Americas for the General. Available at: https://www.paho.org/hq/index.php?option=com_content&view=article&id=13566&Itemid=40721&lang=pt. Accessed August 08, 2018.

    • Search Google Scholar
    • Export Citation
  • 4.

    Angheben A, Boix L, Buonfrate D, Gobbi F, Bisoffi Z, Pupella S, Gandini G, Aprili G, 2015. Chagas disease and transfusion medicine: a perspective from non-endemic countries. Blood Transfus 13: 540550.

    • Search Google Scholar
    • Export Citation
  • 5.

    Howard EJ, Xiong X, Carlier Y, Sosa-Estani S, Buekens P, 2015. Frequency of the congenital transmission of Trypanosoma cruzi: a systematic review and meta-analysis. BJOG 121: 2233.

    • Search Google Scholar
    • Export Citation
  • 6.

    World Health Organization, 2017. Chagas Disease (American Trypanosomiasis). Available at: http://www.who.int/mediacentre/factsheets/fs340/en/. Accessed August 08, 2018.

    • Search Google Scholar
    • Export Citation
  • 7.

    Zingales B et al. 2012. The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect Genet Evol 12: 240253.

    • Search Google Scholar
    • Export Citation
  • 8.

    Marcili A, Lima L, Cavazzana M, Junqueira AC, Veludo HH, Maia da Silva F, Campaner M, Paiva F, Nunes VL, Teixeira MM, 2009. A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and histone H2B genes and genotyping based on ITS1 rDNA. Parasitology 136: 641655.

    • Search Google Scholar
    • Export Citation
  • 9.

    Zingales B et al. 2009. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz 104: 10511054.

    • Search Google Scholar
    • Export Citation
  • 10.

    Miles MA, Cedillos RA, Póvoa MM, Souza AA, Prata A, Macedo V, 1981. Do radically dissimilar Trypanosoma cruzi strains (zymodemes) cause Venezuelan and Brazilian forms of Chagas’ disease? Lancet 1: 13381340.

    • Search Google Scholar
    • Export Citation
  • 11.

    Freitas JM, Lages-Silva E, Crème E, Pena SD, Macedo AM, 2005. Real time PCR strategy for the identification of major lineages of Trypanosoma cruzi directly in chronically infected human tissues. Int J Parasitol 35: 411417.

    • Search Google Scholar
    • Export Citation
  • 12.

    Lages-Silva E, Ramírez LE, Pedrosa AL, Crema E, Cunha Galvão LM, Pena SD, Macedo AM, Chiari E, 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: 21672171.

    • Search Google Scholar
    • Export Citation
  • 13.

    Lima VS, Xavier SC, Maldonado IF, Roque AL, Vicente AC, Jansen AM, 2014. Expanding the knowledge of the geographic distribution of Trypanosoma cruzi TcII and TcV/TcVI genotypes in the Brazilian Amazon. PLoS One 9: e116137.

    • Search Google Scholar
    • Export Citation
  • 14.

    Silva LHP, Nussenzweig V, 1953. Sobre uma cepa de Trypanosoma cruzi altamente virulenta para o camundongo branco. Folia Clin Biol 20: 191203.

    • Search Google Scholar
    • Export Citation
  • 15.

    Pennington PM, Paiz C, Grajeda LM, Cordón-Rosales C, 2009. Concurrent detection of Trypanosoma cruzi lineages I and II in domestic Triatoma dimidiata from Guatemala. Am J Trop Med Hyg 80: 239241.

    • Search Google Scholar
    • Export Citation
  • 16.

    Rocha FL, Roque AL, Arrais RC, Santos JP, Lima VS, Xavier SC, Cordeir-Estrela P, D’Andrea PS, Jansen AM, 2013. Trypanosoma cruzi TcI and TcII transmission among wild carnivores, small mammals and dogs in a conservation unit and surrounding areas, Brazil. Parasitology 140: 160170.

    • Search Google Scholar
    • Export Citation
  • 17.

    Lisboa CV, Dietz J, Baker AJ, Russel NN, Jansen AM, 2000. Trypanosoma cruzi infection in Leontopithecus rosalia at the Reserva Biológica de Poco das Antas, Rio de Janeiro, Brazil. Mem Inst Oswaldo Cruz 95: 445452.

    • Search Google Scholar
    • Export Citation
  • 18.

    Funayama GK, Prado Junior JC, 1974. Estudo sobre os caracteres de una amostra boliviana do Trypanosoma cruzi. Rev Soc Bras Med Trop 8: 7581.

    • Search Google Scholar
    • Export Citation
  • 19.

    Ribeiro AR et al. 2018. Biological and molecular characterization of Trypanosoma cruzi strains from four states of Brazil. Am J Trop Med Hyg 98: 453463.

    • Search Google Scholar
    • Export Citation
  • 20.

    De Vaio ES, Grucci B, Castagnino AM, Franca ME, Martínez ME, 1985. Meiotic differences between three triatomine species (Heteroptera, Reduviidae). Genetica 67: 185191.

    • Search Google Scholar
    • Export Citation
  • 21.

    Alevi KCC, Mendonça PP, Pereira NP, Rosa JA, Azeredo-Oliveira MTV, 2012. Karyotype of Triatoma melanocephala Neiva and Pinto (1923). Does this species fit in the Brasiliensis subcomplex? Infect Genet Evol 12: 16521653.

    • Search Google Scholar
    • Export Citation
  • 22.

    Yasukawa K, Patel SM, Flash CA, Stager CE, Goodman JC, Woc-Colburn L, 2014. Trypanosoma cruzi meningoencephalitis in a patient with acquired immunodeficiency syndrome. Am J Trop Med Hyg 91: 8485.

    • Search Google Scholar
    • Export Citation
  • 23.

    Tomasini N, Diosque P, 2015. Evolution of: clarifying hybridisations, mitochondrial introgressions and phylogenetic relationships between major lineages. Mem Inst Oswaldo Cruz 110: 403413.

    • Search Google Scholar
    • Export Citation
  • 24.

    Kawashita SY, Sanson GFO, Fernandes O, Zingales B, Briones MRS, 2001. Maximum-likelihood divergence date estimates based on rRNA gene sequences suggest two scenarios of Trypanosoma cruzi intraspecific evolution. Mol Biol Evol 18: 22502259.

    • Search Google Scholar
    • Export Citation
  • 25.

    Machado CA, Ayala FJ, 2002. Sequence variation in the dihydrofolate reductasethymidylate synthase (DHFR-TS) and trypanothione reductase (TR) genes of Trypanosoma cruzi. Mol Biochem Parasitol 121: 3347.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

Cytotaxonomy of Trypanosoma cruzi (Chagas, 1909): Differentiation of T. cruzi I (TcI) and T. cruzi II (TcII) Genotypes Using Cytogenetic Markers

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  • 1 Laboratório de Biologia Celular, Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Júlio de Mesquita Filho”, IBILCE/UNESP, São José do Rio Preto, Brazil;
  • | 2 Departamento de Parasitologia, Instituto de Biologia, Universidade Estadual de Campinas, UNICAMP-Avenida Bertrand Russel, Campinas, Brazil;
  • | 3 Instituto de Biociências de Botucatu, Universidade Estadual Paulista “Júlio de Mesquita Filho”, IBB/UNESP, Botucatu, Brazil;
  • | 4 Laboratório de Parasitologia, Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista “Júlio de Mesquita Filho”, FCFAR/UNESP, Araraquara, São Paulo, Brazil

Chagas disease is a public health problem caused by the Trypanosoma cruzi, and the T. cruzi I (TcI) and T. cruzi II (TcII) groups are considered important genotypes from the clinical point of view. Currently, the groups need to be molecularly analyzed for their identification; thus, we cytogenetically analyzed these groups with the objective of developing more accessible techniques for the characterization of these parasites. TcI and TcII groups were differentiated by nucleus characterization with lacto-acetic orcein (TcI—nucleus with positive heteropycnosis and TcII—nucleus with negative heteropycnosis), emphasizing the importance of the application of this technique for epidemiological and clinical studies of Chagas disease.

Chagas disease is caused by the protozoan Trypanosoma cruzi (Chagas, 1909) and represents a serious public health problem.1 It is estimated that eight million people worldwide are infected by T. cruzi2 and about 70 million people are living in areas with contamination risk.3

Transmission of T. cruzi can occur, for example, through organ transplantation of an infected donor, laboratory accidents, or ingestion of contaminated food or liquids (sugarcane, acai, and raw meat). In addition, it is important to note that the transmission can be also congenital (vertically between mother and child) or can happen through blood transfusion. The latter two forms lead to infections in urban and non-endemic areas and are targeted by control measures in an attempt to reduce disease.4,5 However, despite nonvector forms, the main mechanism of transmission of T. cruzi is through triatomines.6

There are seven discrete typing units (DTUs) for T. cruzi, classified based on different molecular markers,7 namely, T. cruzi I (TcI), T. cruzi II (TcII), T. cruzi III (Tc III), T. cruzi IV (Tc IV), T. cruzi V (Tc V), T. cruzi VI (Tc VI), and T. cruzi VII (Tc VII).8,9 TcI and TcII groups are important DTUs from the clinical point of view because TcI is associated with chronic cases of cardiomyopathy and severe meningoencephalitis and TcII is related to chronic forms of Chagas disease that cause cardiac and digestive manifestations.7,1012

TcI has a wide geographic distribution in the American continent (South, Central, and North America)7 and has been reported as the most frequently isolated of all mammalian taxa throughout the geographic range of the parasite, in a wide variety of biomes and habitats.7,13 TcII has a more restricted distribution [Southern Cone and North (sporadic)]7 that, although initially was mainly associated with human infection,14 more recently has been isolated from a wide range of mammalian species in various biomes.7,13,15,16 However, when compared with the number of TcI reservoirs, it has been proposed that this DTU occurs in more focal cycles.17

Methods that are currently used to determine T. cruzi DTUs require isolation and molecular analysis for their identification7; therefore, it is important to develop more accessible techniques for the identification of DTUs. Based on this priority, the present work aimed to cytogenetically analyze the TcI and TcII groups of T. cruzi.

As a representative of the TcI group, we used the Bolivia strain, isolated from feces from specimens of Triatoma infestans (Klug, 1834) captured in Vitichi, Bolivia.18 As a representative of TcII, we used the Y strain, isolated from a human case.14 The maintenance of populations of T. cruzi may be related to intrinsic characteristics of the parasite, such as its infection ability.19 To minimize this potential confounding factor, the strains used in the study were cryopreserved at −80° and then grown in LIT medium.

TcI and TcII strains were cultured in LIT medium in the epimastigote form of the parasite, and slides were prepared with 100 μL of culture spread. After drying, the material was fixed with methanol. Subsequently, the slides were stained by lacto-acetic orcein,20 with modifications described by Alevi et al.21 and analyzed using the Jenaval light microscope (Zeiss), coupled with a digital camera and an image analyzer system AxioVision LE 4.8 (Copyright ©2006-2009 Carl Zeiss Imaging Solutions Gmb H). The images were magnified by a factor of 1,000.

Cytogenetic analyses allowed us to differentiate TcI and TcII groups of T. cruzi by characterization of the protozoan nucleus because TcI presented as a nucleus with positive heteropycnosis (Figure 1A, arrow), whereas TcII showed the nucleus with negative heteropycnosis (Figure 1B, arrow).

Figure 1.
Figure 1.

TcI (A) and TcII (B) groups of Trypanosoma cruzi in epimastigote form stained with orcein. Note the difference in the protozoa nuclei: TcI presented the nucleus with positive heteropycnosis (A, arrow) and TcII presented the nucleus with negative heteropycnosis (B, arrow). Bar: 10 μm.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 3; 10.4269/ajtmh.18-0650

Currently, T. cruzi DTUs cannot be differentiated by classic analyses with Giemsa, which only allow evaluation of the presence/absence of this protozoan in biological material samples,22 and molecular analyses are necessary for the classification of DTUs.7 Based on current findings, the cytogenetic characterization by orcein may help in entoepidemiological studies, allowing investigators to evaluate if the triatomines are infected by T. cruzi and, above all, the type of DTU that is present in these vectors. Furthermore, it may also help in the clinical diagnosis of Chagas disease.

Although T. cruzi DTUs share a common ancestry, TcI and TcII are distant from the phylogenetic point of view.8,9,23 The dates of divergence date between TcI and TcII have been estimated between 88 and 37 million years ago (based on small subunit rDNA)24 or between 16 and 3 million years ago (based on dihydrofolate reductase-thymidylate synthase and trypanothione reductase genes).25 These estimates support the nuclear divergences observed for the analyzed strains.

By means of the cytogenetic characterization of the parasite nucleus, the lacto-acetic orcein stain was shown to be an efficient technique for the differentiation of T. cruzi belonging to the TcI and TcII DTUs. These results may complement epidemiological studies and help in the clinical diagnosis of Chagas disease.

Acknowledgments:

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Process number 2017/05015-7) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

REFERENCES

  • 1.

    Nouvellet P, Cucunubá ZM, Gourbière S, 2015. Ecology, evolution and control of Chagas disease: a century of neglected modelling and a promising future. Adv Parasitol 87: 135191.

    • Search Google Scholar
    • Export Citation
  • 2.

    World Health Organization, 2018. Chagas Disease (American Trypanosomiasis). Available at: http://www.who.int/chagas/en/. Accessed August 08, 2018.

    • Search Google Scholar
    • Export Citation
  • 3.

    Pan American Health Organization, 2017. Chagas in the Americas for the General. Available at: https://www.paho.org/hq/index.php?option=com_content&view=article&id=13566&Itemid=40721&lang=pt. Accessed August 08, 2018.

    • Search Google Scholar
    • Export Citation
  • 4.

    Angheben A, Boix L, Buonfrate D, Gobbi F, Bisoffi Z, Pupella S, Gandini G, Aprili G, 2015. Chagas disease and transfusion medicine: a perspective from non-endemic countries. Blood Transfus 13: 540550.

    • Search Google Scholar
    • Export Citation
  • 5.

    Howard EJ, Xiong X, Carlier Y, Sosa-Estani S, Buekens P, 2015. Frequency of the congenital transmission of Trypanosoma cruzi: a systematic review and meta-analysis. BJOG 121: 2233.

    • Search Google Scholar
    • Export Citation
  • 6.

    World Health Organization, 2017. Chagas Disease (American Trypanosomiasis). Available at: http://www.who.int/mediacentre/factsheets/fs340/en/. Accessed August 08, 2018.

    • Search Google Scholar
    • Export Citation
  • 7.

    Zingales B et al. 2012. The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect Genet Evol 12: 240253.

    • Search Google Scholar
    • Export Citation
  • 8.

    Marcili A, Lima L, Cavazzana M, Junqueira AC, Veludo HH, Maia da Silva F, Campaner M, Paiva F, Nunes VL, Teixeira MM, 2009. A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and histone H2B genes and genotyping based on ITS1 rDNA. Parasitology 136: 641655.

    • Search Google Scholar
    • Export Citation
  • 9.

    Zingales B et al. 2009. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz 104: 10511054.

    • Search Google Scholar
    • Export Citation
  • 10.

    Miles MA, Cedillos RA, Póvoa MM, Souza AA, Prata A, Macedo V, 1981. Do radically dissimilar Trypanosoma cruzi strains (zymodemes) cause Venezuelan and Brazilian forms of Chagas’ disease? Lancet 1: 13381340.

    • Search Google Scholar
    • Export Citation
  • 11.

    Freitas JM, Lages-Silva E, Crème E, Pena SD, Macedo AM, 2005. Real time PCR strategy for the identification of major lineages of Trypanosoma cruzi directly in chronically infected human tissues. Int J Parasitol 35: 411417.

    • Search Google Scholar
    • Export Citation
  • 12.

    Lages-Silva E, Ramírez LE, Pedrosa AL, Crema E, Cunha Galvão LM, Pena SD, Macedo AM, Chiari E, 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: 21672171.

    • Search Google Scholar
    • Export Citation
  • 13.

    Lima VS, Xavier SC, Maldonado IF, Roque AL, Vicente AC, Jansen AM, 2014. Expanding the knowledge of the geographic distribution of Trypanosoma cruzi TcII and TcV/TcVI genotypes in the Brazilian Amazon. PLoS One 9: e116137.

    • Search Google Scholar
    • Export Citation
  • 14.

    Silva LHP, Nussenzweig V, 1953. Sobre uma cepa de Trypanosoma cruzi altamente virulenta para o camundongo branco. Folia Clin Biol 20: 191203.

    • Search Google Scholar
    • Export Citation
  • 15.

    Pennington PM, Paiz C, Grajeda LM, Cordón-Rosales C, 2009. Concurrent detection of Trypanosoma cruzi lineages I and II in domestic Triatoma dimidiata from Guatemala. Am J Trop Med Hyg 80: 239241.

    • Search Google Scholar
    • Export Citation
  • 16.

    Rocha FL, Roque AL, Arrais RC, Santos JP, Lima VS, Xavier SC, Cordeir-Estrela P, D’Andrea PS, Jansen AM, 2013. Trypanosoma cruzi TcI and TcII transmission among wild carnivores, small mammals and dogs in a conservation unit and surrounding areas, Brazil. Parasitology 140: 160170.

    • Search Google Scholar
    • Export Citation
  • 17.

    Lisboa CV, Dietz J, Baker AJ, Russel NN, Jansen AM, 2000. Trypanosoma cruzi infection in Leontopithecus rosalia at the Reserva Biológica de Poco das Antas, Rio de Janeiro, Brazil. Mem Inst Oswaldo Cruz 95: 445452.

    • Search Google Scholar
    • Export Citation
  • 18.

    Funayama GK, Prado Junior JC, 1974. Estudo sobre os caracteres de una amostra boliviana do Trypanosoma cruzi. Rev Soc Bras Med Trop 8: 7581.

    • Search Google Scholar
    • Export Citation
  • 19.

    Ribeiro AR et al. 2018. Biological and molecular characterization of Trypanosoma cruzi strains from four states of Brazil. Am J Trop Med Hyg 98: 453463.

    • Search Google Scholar
    • Export Citation
  • 20.

    De Vaio ES, Grucci B, Castagnino AM, Franca ME, Martínez ME, 1985. Meiotic differences between three triatomine species (Heteroptera, Reduviidae). Genetica 67: 185191.

    • Search Google Scholar
    • Export Citation
  • 21.

    Alevi KCC, Mendonça PP, Pereira NP, Rosa JA, Azeredo-Oliveira MTV, 2012. Karyotype of Triatoma melanocephala Neiva and Pinto (1923). Does this species fit in the Brasiliensis subcomplex? Infect Genet Evol 12: 16521653.

    • Search Google Scholar
    • Export Citation
  • 22.

    Yasukawa K, Patel SM, Flash CA, Stager CE, Goodman JC, Woc-Colburn L, 2014. Trypanosoma cruzi meningoencephalitis in a patient with acquired immunodeficiency syndrome. Am J Trop Med Hyg 91: 8485.

    • Search Google Scholar
    • Export Citation
  • 23.

    Tomasini N, Diosque P, 2015. Evolution of: clarifying hybridisations, mitochondrial introgressions and phylogenetic relationships between major lineages. Mem Inst Oswaldo Cruz 110: 403413.

    • Search Google Scholar
    • Export Citation
  • 24.

    Kawashita SY, Sanson GFO, Fernandes O, Zingales B, Briones MRS, 2001. Maximum-likelihood divergence date estimates based on rRNA gene sequences suggest two scenarios of Trypanosoma cruzi intraspecific evolution. Mol Biol Evol 18: 22502259.

    • Search Google Scholar
    • Export Citation
  • 25.

    Machado CA, Ayala FJ, 2002. Sequence variation in the dihydrofolate reductasethymidylate synthase (DHFR-TS) and trypanothione reductase (TR) genes of Trypanosoma cruzi. Mol Biochem Parasitol 121: 3347.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Ana Beatriz Bortolozo de Oliveira, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Júlio de Mesquita Filho”, IBILCE/UNESP, Rua Cristóvão Colombo 2265, São José do Rio Preto, Brazil. E-mail: anabbortolozo@gmail.com

Authors’ addresses: Ana Beatriz Bortolozo de Oliveira, Fernanda Fernandez Madeira, and Maria Tercília Vilela de Azeredo-Oliveira, Laboratório de Biologia Celular, Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Júlio de Mesquita Filho”, IBILCE/UNESP, São José do Rio Preto, Brazil, E-mails: anabbortolozo@gmail.com, fernanda.bio56@hotmail.com, and tercilia@ibilce.unesp.br. Aline Rimoldi Ribeiro, Departamento de Parasitologia, Instituto de Biologia, Universidade Estadual de Campinas, UNICAMP-Avenida Bertrand Russel, Campinas, Brazil, E-mail: line2rimoldi@gmail.com. Natália Regina Cesaretto, Instituto de Biociências de Botucatu, Universidade Estadual Paulista “Júlio de Mesquita Filho”, IBB/UNESP, Botucatu, Brazil, E-mail: nrcesaretto@gmail.com. João Aristeu da Rosa and Kaio Cesar Chaboli Alevi, Laboratório de Parasitologia, Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista “Júlio de Mesquita Filho”, FCFAR/UNESP, Araraquara, Brazil, E-mails: joaoaristeu@gmail.com and kaiochaboli@hotmail.com.

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