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    Hybridization patterns of xenodiagnosis samples from Mepraia spinolai (sp) and Triatoma infestans (i) and staining with ethidium bromide (EB) and Southern blot analyses with specific probes (TCI, TCII, TCV, and TCVI) for xenodiagnosis samples at A, time 0 (i.e., immediately after capture); B, one year later, C, two years later, and D, two and a half years later. Numbers 5 and 18 correspond to identification numbers for Octodon degus rodents.

  • 1.

    Zingales B, Andrade SG, Briones MR, Campbell DA, Chiari E, Fernandes O, Guhl F, Lages-Silva E, Macedo AM, Machado CR, Miles MA, Romanha AJ, Sturm NR, Tibayrenc M, Schijman AG, 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
  • 2.

    Tibayrenc M, 1998. Genetic epidemiology of parasitic protozoa and other infections agents: the need for an integrated approach. Int J Parasitol 28: 85104.

    • Search Google Scholar
    • Export Citation
  • 3.

    Coronado X, Zulantay I, Albrecht H, Rozas M, Apt W, Ortiz S, Rodriguez J, Sanchez G, Solari A, 2006. Variation in Trypanosoma cruzi clonal composition detected in blood patients and xenodiagnosis triatomines: implications in the molecular epidemiology of Chile. Am J Trop Med Hyg 74: 10081012.

    • Search Google Scholar
    • Export Citation
  • 4.

    Rozas M, Botto-Mahan C, Coronado X, Ortiz S, Cattan PE, Solari A, 2007. Coexistence of Trypanosoma cruzi genotypes in wild and peridomestic mammals in Chile. Am J Trop Med Hyg 77: 647653.

    • Search Google Scholar
    • Export Citation
  • 5.

    Coronado X, Rozas M, Botto-Maham C, Ortiz S, Cattan P, Solari A, 2009. Molecular epidemiology of Chagas disease in the wild transmission cycle: the evaluation in the sylvatic vector Mepraia spinolai from an endemic area of Chile. Am J Trop Med Hyg 81: 656659.

    • Search Google Scholar
    • Export Citation
  • 6.

    Dias E, 1940. Xenodiagnosticos seriados em caes infectados con amostras venezuelanas de Schizotrypanum cruzi. Bras Med 54: 859861.

  • 7.

    Schenone H, Contreras MC, Rojas A, 1991. Yielding of xenodiagnosis, according to the number of boxes used in 1,181 persons with chronic chagasic infection diagnosed with indirect hemagglutination reaction. Bol Chil Parasitol 46: 5861.

    • Search Google Scholar
    • Export Citation
  • 8.

    Westenberger SJ, Barnabé C, Campbell DA, Sturm NR, 2005. Two hybridization events define the population structure of Trypanosoma cruzi. Genetics 171: 527543.

    • Search Google Scholar
    • Export Citation
  • 9.

    Campos R, Acuña-Retamar M, Botto-Mahan C, Ortiz S, Cattan PE, Solari A, 2007. Susceptibility of Mepraia spinolai and Triatoma infestans to different Trypanosoma cruzi strains from naturally infected rodent hosts. Acta Trop 104: 2529.

    • Search Google Scholar
    • Export Citation
  • 10.

    Veas F, Breniere SF, Cuny G, Brengues C, Solari A, Tibayrenc M, 1991. General procedure to construct highly specific kDNA probes for clones of Trypanosoma cruzi for sensitive detection by polymerase chain reaction. Cell Mol Biol 37: 7384.

    • Search Google Scholar
    • Export Citation
  • 11.

    Lane JE, Ribeiro-Rodriguez R, Olivares-Villagomez D, Vnencak-Jones CL, McCurley T, Carter CE, 2003. Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction. Mem Inst Oswaldo Cruz 98: 373376.

    • Search Google Scholar
    • Export Citation
  • 12.

    de Lana M, da Silveira Pinto A, Barnabe C, Quesney V, Noel S, Tibayrenc M, 1998. Trypanosoma cruzi: compared vectorial transmissibility of three major clonal genotypes by Triatoma infestans. Exp Parasitol 90: 2025.

    • Search Google Scholar
    • Export Citation
  • 13.

    Vago AR, Andrade LO, Leite AA, d`Avila Reis D, Macedo AM, Adad SJ, Tostes S Jr, Moreira MC, Filho GB, Pena SD, 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: 18051809.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Temporal Fluctuation of Infection with Different Trypanosoma cruzi Genotypes in the Wild Rodent Octodon degus

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  • Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, e Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile

We identified and followed-up for two years Octodon degus rodents infected with Trypanosoma cruzi genotypes by using xenodiagnosis with two vector species (Mepraia spinolai and Triatoma infestans), polymerase chain reaction DNA-based detection of insect dejections, Southern blot analysis, and minicircle hybridization with genotype-specific probes. Results show temporal fluctuations of infection with four parasite lineages (TCI, TCII, TCV, and TCVI) in one co-infected O. degus. Results are discussed in the context of parasitemia level and infection control in mammal hosts.

Chagas disease is a vector-borne zoonosis caused by the protozoa Trypanosoma cruzi. This taxon had been described as composed of two lineages (TCI and TCII) and five subgroups (IIa–IIe), but a recent study reported six lineages or discrete typing units (DTUs) (T. cruzi I–VI).1 These lineages are defined as sets of stocks that are genetically more related to each other than to any other stock and are identifiable by common genetic molecular and immunologic markers.2

Trypanosoma cruzi populations circulate in nature in multiple T. cruzi genotypes that coexist in different hosts, including Octodon degus rodents.35 After a short acute or primary infection, the mammal host sustains subclinical infections, which are microscopically undetectable in peripheral blood during the undetermined and chronic phases. Conversely, parasitemia in those phases is detected only by polymerase chain reaction (PCR). The classic parasitologic diagnostic method for Chagas disease xenodiagnosis, which can amplify T. cruzi after feeding on infected hosts.6 Although xenodiagnosis is specific, it lacks sensitivity and is limited to high levels of parasitemia.7 The epidemiology of Chagas disease and clinical symptoms are associated with the infective T. cruzi genotypes.8 Therefore, would be useful to know the dynamics of these genotypes.

In the present study, we assess the occurrence of temporal fluctuations of T. cruzi DTUs in peripheral blood of two naturally infected wild reservoir specimens of O. degus by using a combination of two diagnosis methods: 1) xenodiagnosis with domestic and sylvatic vectors (Triatoma infectans and Mepraia spinolai), respectively, and 2) PCR DNA-based detection specific for minicircles and hybridization analyses with T. cruzi genotype-specific probes.

Ten nymphs (stages II and III) of each vector species were allowed to feed simultaneously on anesthetized O. degus rodents for 30 minutes or until engorgement on the rodent (mean ± SD weight of ingested blood = 0.2 ± 0.05 mg). After 30 days, feces and intestinal contents of the triatomines were observed under a light microscope. The minimal theoretical parasitemia detected under these conditions is approximately 5 parasites/mL (1 parasite/0.2 mL). However, because several but not all insects (2–5) were parasite positive by visual examination, the estimated parasitemia would be > 10–25 parasites/mL.

After microscopic inspection, the intestinal contents of each vector species pool was collected and PCR was performed as reported.9 Amplicons were subjected to electrophoresis on an agarose gel and transferred to nylon membranes. Copies of these membranes were hybridized separately with each probe under high stringency conditions.3 Construction of genotype-specific probes was performed as described.10 Different T. cruzi clones were used as templates to generate DNA probes to determine parasite genotypes. The probes were P32-labeled.4

A total of 35 O. degus were captured at the field and analyzed. Overall, only two O. degus showed infection with both vector species and six were positive only for M. spinolai.9 The two O. degus samples positive for both vector species were subjected to serial xenodiagnosis to determine the genotype of the T. cruzi population circulating at different times: time 0, one year, two years, and two and a half years. Results for O. degus sample 5, which was infected with a one genotype (TCI), are shown in Figure 1. This result was confirmed with both vector species at different times. Results obtained with O. degus sample 8 showed mixed infection with DTUs TCI, TCII, and TCVI at time zero for M. spinolai, but only TCII for T. infestans. However, one year later, both vectors showed mixed infections with lineages TCI and TCV. After two years, both vectors contained only genotype TCII. After two and a half years, vectors were still infected with TCII.

Figure 1.
Figure 1.

Hybridization patterns of xenodiagnosis samples from Mepraia spinolai (sp) and Triatoma infestans (i) and staining with ethidium bromide (EB) and Southern blot analyses with specific probes (TCI, TCII, TCV, and TCVI) for xenodiagnosis samples at A, time 0 (i.e., immediately after capture); B, one year later, C, two years later, and D, two and a half years later. Numbers 5 and 18 correspond to identification numbers for Octodon degus rodents.

Citation: The American Society of Tropical Medicine and Hygiene 83, 2; 10.4269/ajtmh.2010.09-0797

Trypanosoma cruzi colonizes several tissues and evades the immune response by a concomitant low parasitemia level not detectable by several diagnosis methods.11 Parasites circulate as mixed infections. This finding is common for T. cruzi because several mammals and vectors are infected with more than one T. cruzi genotype,4,5 which results in recombination and hybrid genotypes.8

We report that infection of rodents can show temporal fluctuations with different T. cruzi genotypes, which is probably the result of fluctuation of relative proportions of parasite loads of different genotypes in peripheral blood. We detected infections in this O. degus with at least three of the four T. cruzi genotypes during the complete follow-up (xenodiagnosis at time 0). Two genotypes (TCII and TCVI) disappeared, and another one (TCV) appeared one year later. During the second year, only one genotype (TCII) was detected and maintained. A different scenario was detected for O. degus sample 5, which showed infection with only TCI during the entire sampling period.

In this study, we preferentially detected genotype TCII in both vector species. This genotype was likely circulating at high parasitemia levels in O. degus sample 18 because experimental infections in T. infestans with different T. cruzi DTUs indicated that genotype TCII is transmitted at a low rate; genotype TCI is transmitted at a high rate.12 Our results for T. cruzi genotypes in these two animals are consistent with local prevalence in the study area.4 Recent studies of T. cruzi genotypes circulating in the wild vector in this disease-endemic area showed that TCI and TCII are the most prevalent genotypes.5

We suggest that both rodent species showed moderate or high levels of parasitemia. We used xenodiagnosis with two triatomine species because insect vectors amplify T. cruzi in the midgut, which enables easy detection. Our results indicate fluctuation in specific genotype infections in a T. cruzi-infected sylvatic rodent.

The temporal fluctuation of the four T. cruzi genotypes could be explained by at least two hyptheses that are not mutually exclusive. First, colonization of different tissues with T. cruzi described in patients and experimentally infected animals with organ damage11,13 releases T. cruzi into the vascular system; these parasites then colonize other tissues. Second, infection is controlled by the immune system. Both processes might reach an equilibrium and explain the low parasitemia levels observed in immunocompetent patients in the chronic phase of Chagas disease. Future parasitologic studies of molecular pathogenesis may be necessary to understand the mechanisms underlying infection control in naturally infected hosts.

  • 1.

    Zingales B, Andrade SG, Briones MR, Campbell DA, Chiari E, Fernandes O, Guhl F, Lages-Silva E, Macedo AM, Machado CR, Miles MA, Romanha AJ, Sturm NR, Tibayrenc M, Schijman AG, 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
  • 2.

    Tibayrenc M, 1998. Genetic epidemiology of parasitic protozoa and other infections agents: the need for an integrated approach. Int J Parasitol 28: 85104.

    • Search Google Scholar
    • Export Citation
  • 3.

    Coronado X, Zulantay I, Albrecht H, Rozas M, Apt W, Ortiz S, Rodriguez J, Sanchez G, Solari A, 2006. Variation in Trypanosoma cruzi clonal composition detected in blood patients and xenodiagnosis triatomines: implications in the molecular epidemiology of Chile. Am J Trop Med Hyg 74: 10081012.

    • Search Google Scholar
    • Export Citation
  • 4.

    Rozas M, Botto-Mahan C, Coronado X, Ortiz S, Cattan PE, Solari A, 2007. Coexistence of Trypanosoma cruzi genotypes in wild and peridomestic mammals in Chile. Am J Trop Med Hyg 77: 647653.

    • Search Google Scholar
    • Export Citation
  • 5.

    Coronado X, Rozas M, Botto-Maham C, Ortiz S, Cattan P, Solari A, 2009. Molecular epidemiology of Chagas disease in the wild transmission cycle: the evaluation in the sylvatic vector Mepraia spinolai from an endemic area of Chile. Am J Trop Med Hyg 81: 656659.

    • Search Google Scholar
    • Export Citation
  • 6.

    Dias E, 1940. Xenodiagnosticos seriados em caes infectados con amostras venezuelanas de Schizotrypanum cruzi. Bras Med 54: 859861.

  • 7.

    Schenone H, Contreras MC, Rojas A, 1991. Yielding of xenodiagnosis, according to the number of boxes used in 1,181 persons with chronic chagasic infection diagnosed with indirect hemagglutination reaction. Bol Chil Parasitol 46: 5861.

    • Search Google Scholar
    • Export Citation
  • 8.

    Westenberger SJ, Barnabé C, Campbell DA, Sturm NR, 2005. Two hybridization events define the population structure of Trypanosoma cruzi. Genetics 171: 527543.

    • Search Google Scholar
    • Export Citation
  • 9.

    Campos R, Acuña-Retamar M, Botto-Mahan C, Ortiz S, Cattan PE, Solari A, 2007. Susceptibility of Mepraia spinolai and Triatoma infestans to different Trypanosoma cruzi strains from naturally infected rodent hosts. Acta Trop 104: 2529.

    • Search Google Scholar
    • Export Citation
  • 10.

    Veas F, Breniere SF, Cuny G, Brengues C, Solari A, Tibayrenc M, 1991. General procedure to construct highly specific kDNA probes for clones of Trypanosoma cruzi for sensitive detection by polymerase chain reaction. Cell Mol Biol 37: 7384.

    • Search Google Scholar
    • Export Citation
  • 11.

    Lane JE, Ribeiro-Rodriguez R, Olivares-Villagomez D, Vnencak-Jones CL, McCurley T, Carter CE, 2003. Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction. Mem Inst Oswaldo Cruz 98: 373376.

    • Search Google Scholar
    • Export Citation
  • 12.

    de Lana M, da Silveira Pinto A, Barnabe C, Quesney V, Noel S, Tibayrenc M, 1998. Trypanosoma cruzi: compared vectorial transmissibility of three major clonal genotypes by Triatoma infestans. Exp Parasitol 90: 2025.

    • Search Google Scholar
    • Export Citation
  • 13.

    Vago AR, Andrade LO, Leite AA, d`Avila Reis D, Macedo AM, Adad SJ, Tostes S Jr, Moreira MC, Filho GB, Pena SD, 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: 18051809.

    • Search Google Scholar
    • Export Citation

Author Notes

*Address correspondence to Aldo Solari, Program of Cellular and Molecular Biology, Faculty of Medicine, University of Chile, Casilla 70086, Santiago 7, Chile. E-mail: asolari@machi.med.uchile.cl

Financial support: This study was supported by Fondo Nacional de Desarrollo Científico y Tecnológico 1085154 (A. Solari), Comisión Nacional de Investigación Científica y Tecnológica–Programa Bicentenario de Ciencia y Tecnología PSD66, and Fondo Nacional de Desarrollo Científico y Tecnológico 11090086 (C. Botto-Mahan).

Disclosure: The authors have no conflicts of interest in relation to this work.

Authors' addresses: Ricardo Campos, Sylvia Ortiz, Ximena Coronado, and Aldo Solari, Program of Cellular and Molecular Biology, Faculty of Medicine, University of Chile, Casilla, Santiago, Chile, E-mail: asolari@machi.med.uchile.cl. Carezza Botto-Mahan and Sylvia Ortiz, Department of Ecological Sciences, Faculty of Sciences, University of Chile, Casilla, Santiago, Chile, E-mail: cbotto@uchile.cl.

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