Chronic myocardial damage in experimental T. cruzi infection of a new world primate, Cebus sp. monkey

Falasca, Carlos Alberto; Gili, Monica; Grana, Daniel; Gomez, Elena; Zoppi, Jorge; Mareso, Eduardo

doi:10.1590/S0036-46651990000300002

Abstracts

Eighteen Cebus apella monkeys, (juvenile and adult of both sexes) were inoculated five years ago, with three Trypanosoma cruzi strains (CA1, n = 10; Colombian, n=4 and Tulahuen, n=4), either by conjunctival or intraperitoneal route, once or repeatedly. Parasitological, hematological, serological, enzymatic, radiographic, electro and echocardiographic findings have been peviously published15 and they are similar to those observed in human pathology. The most frequent electrocardiographic alteration was right branch bundle block. Six animals, chosen at random, were sacrificed. Those sacrificed 20 to 25 months post-first inoculation showed focal accumuli of leukocytes with myocytolysis. Foci of diffuse interstitial fibrosis with mild infiltrate of leukocytes among fibers were observed in the animals sacrificed 36 to 47 months post-inoculation. No parasites were seen. The lesions were more prominent in the ventricular walls and the septum. The fact that the infiltrates were predominant in the animals sacrificed at a shorter time after first inoculation and that fibrosis was more severe in those sacrificed at a longer time suggests that there is a progression of the infiltrative lesions to fibrosis, with a leukocytic activity indicative of a chronic phase. These lesions are similar to those described in human chronic Chagas' disease. This would demonstrate that this model is useful in evaluating a progress in the knowledge of the pathogenesis which is still a controversial issue, immunology, immunogenesis and chemotherapeutic agents of the chronic and indeterminate phases of this disease.

Cebus monkey; Chagas' disease; Pathology

Dezoito macacos Cebus apella (jovens e adultos de ambos os sexos) foram inoculados há 5 anos atrás, com 3 cepas de T. cruzi (CA1, n = 10; Colombiana, n =4 e Tulahuen, n =4) seja por via conjuntival ou intraperitonial, uma única vez ou repetidamente. Os achados parasitológicos, hematológicos, sorológicos, enzimáticos, radiográficos, eletro e ecocardiográficos foram anteriormente publicados15 e são semelhantes àqueles vistos no homem. O achado eletrocardiográfico mais freqüente foi o bloqueio do ramo direito. Seis animais, escolhidos ao acaso, foram sacrificados. Aqueles sacrificados 20 a 25 meses após a primeira inoculação mostraram acúmulos focais de leucócitos com miocitólise. Focos de fibrose intersticial difusa com pequeno infiltrado de leucócitos entre as fibras foram observados em animais sacrificados 36 a 47 meses após a inoculação. Não foram encontrados parasitas. As lesões foram mais proeminentes nas paredes ventriculares e no septo. O achado de infiltrados predominantemente, nos animais sacrificados em tempo mais curto em relação à primeira inoculação e a fibrose mais severa naqueles sacrificados após um tempo maior sugere que existe uma progressão das lesões infiltrativas até a fibrose, com atividade leucocítica indicativa de fase crônica. Estas lesões são semelhantes àquelas descritas na doença de Chagas crônica humana. Este modelo, portanto, é útil na avaliação do progresso e conhecimento da patogênese da doença, assim como de sua imunologia, imunogenesis e da ação da quimioterapia, tanto na sua fase crônica como indeterminada.

ORIGINAL ARTICLES

Chronic myocardial damage in experimental T. cruzi infection of a new world primate, Cebus sp. monkey

Lesões miocárdicas crônicas na infecção experimental pelo T. cruzi no macaco (Cebus apella)

Carlos Alberto Falasca, M. D.; Monica Gili, M. D.; Daniel Grana, V. M.; Elena Gomez; Jorge Zoppi, M. D.; Eduardo Mareso, M. D.

^{Address for correspondence} Address for correspondence: Dr. Carlos A. Falasca Instituto Latinoamericano de Investigaciones Medicas Universidad del Salvador (ILAIMUS) Tucuman 1845 (1050) Buenos Aires, Argentina tel.: 42-2935/812-9846 Tlx.: 18660 UNIVDELSAL DELPHY AR

SUMMARY

Eighteen Cebus apella monkeys, (juvenile and adult of both sexes) were inoculated five years ago, with three Trypanosoma cruzi strains (CA1, n = 10; Colombian, n=4 and Tulahuen, n=4), either by conjunctival or intraperitoneal route, once or repeatedly. Parasitological, hematological, serological, enzymatic, radiographic, electro and echocardiographic findings have been peviously published¹⁵ and they are similar to those observed in human pathology. The most frequent electrocardiographic alteration was right branch bundle block.

Six animals, chosen at random, were sacrificed. Those sacrificed 20 to 25 months post-first inoculation showed focal accumuli of leukocytes with myocytolysis. Foci of diffuse interstitial fibrosis with mild infiltrate of leukocytes among fibers were observed in the animals sacrificed 36 to 47 months post-inoculation. No parasites were seen. The lesions were more prominent in the ventricular walls and the septum. The fact that the infiltrates were predominant in the animals sacrificed at a shorter time after first inoculation and that fibrosis was more severe in those sacrificed at a longer time suggests that there is a progression of the infiltrative lesions to fibrosis, with a leukocytic activity indicative of a chronic phase.

These lesions are similar to those described in human chronic Chagas' disease. This would demonstrate that this model is useful in evaluating a progress in the knowledge of the pathogenesis which is still a controversial issue, immunology, immunogenesis and chemotherapeutic agents of the chronic and indeterminate phases of this disease.

Key words: Cebus monkey; Chagas' disease; Pathology.

RESUMO

Dezoito macacos Cebus apella (jovens e adultos de ambos os sexos) foram inoculados há 5 anos atrás, com 3 cepas de T. cruzi (CA1, n = 10; Colombiana, n =4 e Tulahuen, n =4) seja por via conjuntival ou intraperitonial, uma única vez ou repetidamente. Os achados parasitológicos, hematológicos, sorológicos, enzimáticos, radiográficos, eletro e ecocardiográficos foram anteriormente publicados¹⁵ e são semelhantes àqueles vistos no homem. O achado eletrocardiográfico mais freqüente foi o bloqueio do ramo direito.

Seis animais, escolhidos ao acaso, foram sacrificados. Aqueles sacrificados 20 a 25 meses após a primeira inoculação mostraram acúmulos focais de leucócitos com miocitólise. Focos de fibrose intersticial difusa com pequeno infiltrado de leucócitos entre as fibras foram observados em animais sacrificados 36 a 47 meses após a inoculação. Não foram encontrados parasitas. As lesões foram mais proeminentes nas paredes ventriculares e no septo. O achado de infiltrados predominantemente, nos animais sacrificados em tempo mais curto em relação à primeira inoculação e a fibrose mais severa naqueles sacrificados após um tempo maior sugere que existe uma progressão das lesões infiltrativas até a fibrose, com atividade leucocítica indicativa de fase crônica.

Estas lesões são semelhantes àquelas descritas na doença de Chagas crônica humana. Este modelo, portanto, é útil na avaliação do progresso e conhecimento da patogênese da doença, assim como de sua imunologia, imunogenesis e da ação da quimioterapia, tanto na sua fase crônica como indeterminada.

Full text available only in PDF format.

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ACKNOWLEDGMENT

The authors wish to thank Prof. Dr. Zilton Andrade, Director Centro de Pesquisas Gonçalo Moniz, Brazil, for reviewing the manuscript and helpful criticism and the technical assistance of Ms. Nery Rolon and Mrs. Claudia Chiesa.

Recebido para publicação em 4/9/1989.

This investigation received support from the UNDP/WORLD Bank/WHO Special Programme for Research and Training in Tropical Disease (ID 790122), and by the Universidad del Salvador, Buenos Aires, Argentina.

1. AMORIN, D. Cardiopatia chagasica. Modelos experimentales. Arq. bras. Cardiol., 42: 243, 1984.
2. ANDRADE, Z.A. & ANDRADE, S.G. Patologia, In: BRENER, Z. & ANDRADE, Z.A. Trypanosoma cruzi e doença de Chagas, Rio de Janeiro, Guanabara Koogan, 1979. p. 199-248.
3. ANDRADE, Z.A. Mechanisms of myocardial damage in Trypanosoma cruzi infection. In: Cytopathology of parasitic disease. London, Pitman, 1983. p. 214-233. (Ciba Foundation Symposium, 99).
4. BOLOMO, N.J.; MILEI, J.; COSSIO, P.M.; SEGURA, E.; LAGUENS, R.; FERNANDEZ, L. & ARANA, R. Experimental Chagas' disease in South American non human primate (Cebus sp. monkey). Medicina (B. Aires), 40: 667-672, 1980.
5. BOLOMO, N.J.; MILEI, J.; COSSIO, P.; NAGLE, C.; ARANA, R.; DEL PRADO, C.E. & SEGURA, E.L. Infección cronica por Trypanosoma cruzi en el Cebus sp. adulto. Medicina (B. Aires), 41: 678-679, 1981.
6. BRENER, Z. Pathogenesis and immunopathology of chronic Chagas' disease. Mem. Inst. Oswaldo Cruz, 82 (Suppl. 1): 205-213, 1987.
7. BRENER, Z. Progress recents dans le domaine de la maladie de Chagas. Bull. Org. mond. Santé, 60: 845-856, 1982.
8. BRUMPT, E.O. O xenodiagnóstico. Aplicaçăo do diagnóstico de algumas infecçőes parasitárias em particular trypanosomose de Chagas. An. paul. Med. Cirurg., 3(5): 97-117, 1914.
9. CAPRIS, T.A. & FERNANDEZ MOORES, A.J. Alteraciones electrocardiográficas en la cardiopatia chagasica cronica. Estudio de 174 enfermos. Rev. argent. Cardiol., 34: 200-204, 1967.
10. CHAGAS, C. Nova tripanosomiaze humana: estudos sobre a morfolojia e o ciclo evolutivo do Schizotrypanum cruzi, n.gen.n.sp. ajente etiológico de nova entidade morbida do homem. Mem. inst. Oswaldo Cruz, 1: 159-219, 1909.
11. COSSIO, P.M.; DIEZ, C.; LAGUENS, R.P. & ARANA, R.M. Inmunopatologia de la enfermedad de Chagas. Hechos y perspectivas. Medicina (B. Aires), 40 (Suppl. 1): 222, 1980.
12. COSSIO, P.M.; LAGUENS, R.P.; DIEZ, C.; SZARFMAN, A.; SEGAL, A. & ARANA, R.M. Chagasic cardiopathy: antibodies reacting with plasma membrane of striated muscle and endothelial cells. Circulation, 50: 1252-1259, 1974.
13. DORLAND, J.D. Infection in monkeys with strains of Trypanosoma cruzi isolated in the United States. Publ. Hlth. Rep., 158: 1006-1010, 1943.
14. EIGUCHIDE PALMERO, K.; CARBONETTO, Ch.; MALCHIODI, E.L.; MARGNI, A. & FALASCA, C.A. Humoral and cellular parameters of the immune system of Cebus apella monkeys. Cross reactivity between monkey and human immunoglobulins. Vet. Immunol. Immunopath., 19: 341-349, 1988.
15. FALASCA, C.A.; GRANA, D.; BUCCOLO, J.; GILI, M.; MERLO, A.; ZOPPI, J. & MARESO, E. Susceptibility of the Cebus apella monkey to different strains of T. cruzi after single or repeated inoculation. Bull. Pan Amer. Hlth. Org., 20: 117-137, 1986.
16. FIGUEIREDO, F.; MARIN-NETO, J.A. & ROSSI, M.A. The evolution of experimental Trypanosoma cruzi cardiomyopathy in rabbits: further parasitological, morphological and functional studies. Int. J. Cardiol., 10: 277-290, 1986.
17. GILI, M.; GRANA, D.; MARESO, E.; GARCILAZO, E. & FALASCA, C.A. Electrocardiograma normal y patológico, en el mono Cebus apella, un modelo experimental de cardiopatia chagasica cronica. Rev. argent. Cardiol., 55(4), 1987.
18. GILI, M.; GRANA, D.; GARCILAZO, E. & FALASCA, C.A. Alteraciones electro y ecocardiograficas (Modo M) en un modelo experimental crônico de la enfermedad de Chagas, el primate Cebus apella. Medicina (B. Aires), 46: 553, 1986.
19. HELD, J.R. Appropiate animal models. Ann. N. Y. Acad. Sci., 406: 13-19, 1983.
20. JORG, M.E. Tripanosomiasis cruzi; anarquia angiotopografica por descapilarization mesenquimorreactiva, co-factor patogenico de la miocardiopatia crônica. Pren. méd. argent., 61: 94-106, 1974.
21. KÖBERLE, F. Pathologic anatomy of enteromegaly in Chagas' disease. In: MEETING BOCKUS ALUMNI INTERNATIONAL SOCIETY OF GASTROENTEROLOGY, 2., Rio de Janeiro, 1960. Proceedings. Rio de Janeiro, 1961. p. 92-103.
22. LAGUENS, R.P.; MECKERT, P.C. & GELPI, R.J. Chronic Chagas' disease in the mouse. I. Electrocardiographic and morphological patterns of the cardiopathy. Medicina (B. Aires), 41: 35-30, 1981.
23. MARSDEN, P. & HAGSTROM, J.W.C. Experimental Trypanosoma cruzi infection in beagle puppies. The effect of variations in the dose and source of infecting trypanosomes and the route of inoculations of the course of the infection. Trans. roy. Soc. trop. Med. Hyg., 62: 816-824, 1968.
24. MARSDEN, P.; SEAH, S.K.K.; DRAPER, C.C.; PETTITT, L.E.; MILES, M.A. & VOLLER, A. Experimental T. cruzi infections in Rhesus monkeys II. The early chronic phase. Trans. roy Soc. trop. Med. Hyg., 70: 247-251, 1976.
25. MOLINA, H.A.; MILEI, J.; RIMOLDI, M.T.; GONZALEZ CAPPA, S.M. & STORINO, R.A. - Histopathology of the heart conducting system in experimental Chagas disease in mice. Trans. roy. Soc. trop. Med. Hyg., 82: 241-246, 1988.
26. OLSEN, E.G.J. Examination of the heart. In: OLSEN, E.G.J. The pathology of the heart. 2 nd. ed. London, Macmillan Press, 1980. p. 5-10.
27. PAN AMERICAN HEALTH ORGANIZATION. Report of a study group on Chagas' disease. Washington, 1970 (Scientific Publication No. 195).
28. REVELLI, S.S.; AMERIO, N.; MORENO, H.S.; VALENTI, J.L.; BALBARREY, H. & MORINI, J.C. Enfermedad de Chagas cronica en la rata. Características serologicas, electrocardiograficas e histopatologicas. Medicina (B. Aires), 40 (Suppl. 1): 69-76, 1980.
29. ROSEMBAUM, M.B. & ALVAREZ, A. The electrocardiogram in chronic chagasic myocarditis. Amer. Heart J., 50: 492-527, 1955.
30. STEFANINI, M.; DE MARTINO, C. & ZAMBONI, L. Fixation of ejeculated spermatozoa for electron microscopy. Nature, 216: 173, 1976.
31. STROUT, R.G. A method for concentrating hemoflagellates. J. Parasit., 48: 100, 1962.
32. TEIXEIRA, A.R.L.; TEIXEIRA, L. & SANTOS-BUSH, C.A. The immunology of experimental Chagas' disease. IV. The production of lesions in rabbits similar to those of chronic Chagas' disease in man. Amer. J. Path, 80: 163-177, 1975.
33. TEIXEIRA, A.R.L.; FIGUEIREDO, F.; REZENDE, F.J. & MACEDO, V. Chagas' disease: a clinical, parasitological, immunological and pathological study in rabbits. Amer. J. trop. Med. Hyg., 32: 258-272, 1983.
34. TORRES, C.M. Miocitolise e fibrose do miocárdio na doença de Chagas. Mem. Inst. Oswaldo Cruz, 58: 161-182, 1960.
35. TORRES, C.M. & TAVARES, B.M. Miocardite no macaco Cebus após inoculaçőes repetidas com Schizotrypanum cruzi. Mem. Inst. Oswaldo Cruz, 5: 85-152, 1958.
36. VIANNA, G. Contribuiçăo para o estudo da anatomia patológica da moléstia de Carlos Chagas. Mem. Inst. Oswaldo Cruz, 3: 276-294, 1911.
37. VOLLER, A.; DRAPER, C.C.; BIDWELL, D.F. & BARTLETT, A. Microplate enzyme-Linked immunosorbent assay for Chagas' disease. Lancet, 1: 426-427, 1975.
38. WESSLER, S. Introduction: What is a model? Animal models of thrombosis and hemorrhagic disease. Bethesda. National Institutes of Health, 1976.

Address for correspondence:

Dr. Carlos A. Falasca

Instituto Latinoamericano de Investigaciones Medicas Universidad del Salvador (ILAIMUS)

Tucuman 1845 (1050)

Buenos Aires, Argentina

tel.: 42-2935/812-9846 Tlx.: 18660 UNIVDELSAL DELPHY AR

Publication Dates

Publication in this collection
13 Sept 2006
Date of issue
June 1990

History

Received
04 Sept 1989

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. AMORIN, D. Cardiopatia chagasica. Modelos experimentales. Arq. bras. Cardiol., 42: 243, 1984.

[2] 2. ANDRADE, Z.A. & ANDRADE, S.G. Patologia, In: BRENER, Z. & ANDRADE, Z.A. Trypanosoma cruzi e doença de Chagas, Rio de Janeiro, Guanabara Koogan, 1979. p. 199-248.

[3] 3. ANDRADE, Z.A. Mechanisms of myocardial damage in Trypanosoma cruzi infection. In: Cytopathology of parasitic disease. London, Pitman, 1983. p. 214-233. (Ciba Foundation Symposium, 99).

[4] 4. BOLOMO, N.J.; MILEI, J.; COSSIO, P.M.; SEGURA, E.; LAGUENS, R.; FERNANDEZ, L. & ARANA, R. Experimental Chagas' disease in South American non human primate (Cebus sp. monkey). Medicina (B. Aires), 40: 667-672, 1980.

[5] 5. BOLOMO, N.J.; MILEI, J.; COSSIO, P.; NAGLE, C.; ARANA, R.; DEL PRADO, C.E. & SEGURA, E.L. Infección cronica por Trypanosoma cruzi en el Cebus sp. adulto. Medicina (B. Aires), 41: 678-679, 1981.

[6] 6. BRENER, Z. Pathogenesis and immunopathology of chronic Chagas' disease. Mem. Inst. Oswaldo Cruz, 82 (Suppl. 1): 205-213, 1987.

[7] 7. BRENER, Z. Progress recents dans le domaine de la maladie de Chagas. Bull. Org. mond. Santé, 60: 845-856, 1982.

[8] 8. BRUMPT, E.O. O xenodiagnóstico. Aplicaçăo do diagnóstico de algumas infecçőes parasitárias em particular trypanosomose de Chagas. An. paul. Med. Cirurg., 3(5): 97-117, 1914.

[9] 9. CAPRIS, T.A. & FERNANDEZ MOORES, A.J. Alteraciones electrocardiográficas en la cardiopatia chagasica cronica. Estudio de 174 enfermos. Rev. argent. Cardiol., 34: 200-204, 1967.

[10] 10. CHAGAS, C. Nova tripanosomiaze humana: estudos sobre a morfolojia e o ciclo evolutivo do Schizotrypanum cruzi, n.gen.n.sp. ajente etiológico de nova entidade morbida do homem. Mem. inst. Oswaldo Cruz, 1: 159-219, 1909.

[11] 11. COSSIO, P.M.; DIEZ, C.; LAGUENS, R.P. & ARANA, R.M. Inmunopatologia de la enfermedad de Chagas. Hechos y perspectivas. Medicina (B. Aires), 40 (Suppl. 1): 222, 1980.

[12] 12. COSSIO, P.M.; LAGUENS, R.P.; DIEZ, C.; SZARFMAN, A.; SEGAL, A. & ARANA, R.M. Chagasic cardiopathy: antibodies reacting with plasma membrane of striated muscle and endothelial cells. Circulation, 50: 1252-1259, 1974.

[13] 13. DORLAND, J.D. Infection in monkeys with strains of Trypanosoma cruzi isolated in the United States. Publ. Hlth. Rep., 158: 1006-1010, 1943.

[14] 14. EIGUCHIDE PALMERO, K.; CARBONETTO, Ch.; MALCHIODI, E.L.; MARGNI, A. & FALASCA, C.A. Humoral and cellular parameters of the immune system of Cebus apella monkeys. Cross reactivity between monkey and human immunoglobulins. Vet. Immunol. Immunopath., 19: 341-349, 1988.

[15] 15. FALASCA, C.A.; GRANA, D.; BUCCOLO, J.; GILI, M.; MERLO, A.; ZOPPI, J. & MARESO, E. Susceptibility of the Cebus apella monkey to different strains of T. cruzi after single or repeated inoculation. Bull. Pan Amer. Hlth. Org., 20: 117-137, 1986.

[16] 16. FIGUEIREDO, F.; MARIN-NETO, J.A. & ROSSI, M.A. The evolution of experimental Trypanosoma cruzi cardiomyopathy in rabbits: further parasitological, morphological and functional studies. Int. J. Cardiol., 10: 277-290, 1986.

[17] 17. GILI, M.; GRANA, D.; MARESO, E.; GARCILAZO, E. & FALASCA, C.A. Electrocardiograma normal y patológico, en el mono Cebus apella, un modelo experimental de cardiopatia chagasica cronica. Rev. argent. Cardiol., 55(4), 1987.

[18] 18. GILI, M.; GRANA, D.; GARCILAZO, E. & FALASCA, C.A. Alteraciones electro y ecocardiograficas (Modo M) en un modelo experimental crônico de la enfermedad de Chagas, el primate Cebus apella. Medicina (B. Aires), 46: 553, 1986.

[19] 19. HELD, J.R. Appropiate animal models. Ann. N. Y. Acad. Sci., 406: 13-19, 1983.

[20] 20. JORG, M.E. Tripanosomiasis cruzi; anarquia angiotopografica por descapilarization mesenquimorreactiva, co-factor patogenico de la miocardiopatia crônica. Pren. méd. argent., 61: 94-106, 1974.

[21] 21. KÖBERLE, F. Pathologic anatomy of enteromegaly in Chagas' disease. In: MEETING BOCKUS ALUMNI INTERNATIONAL SOCIETY OF GASTROENTEROLOGY, 2., Rio de Janeiro, 1960. Proceedings. Rio de Janeiro, 1961. p. 92-103.

[22] 22. LAGUENS, R.P.; MECKERT, P.C. & GELPI, R.J. Chronic Chagas' disease in the mouse. I. Electrocardiographic and morphological patterns of the cardiopathy. Medicina (B. Aires), 41: 35-30, 1981.

[23] 23. MARSDEN, P. & HAGSTROM, J.W.C. Experimental Trypanosoma cruzi infection in beagle puppies. The effect of variations in the dose and source of infecting trypanosomes and the route of inoculations of the course of the infection. Trans. roy. Soc. trop. Med. Hyg., 62: 816-824, 1968.

[24] 24. MARSDEN, P.; SEAH, S.K.K.; DRAPER, C.C.; PETTITT, L.E.; MILES, M.A. & VOLLER, A. Experimental T. cruzi infections in Rhesus monkeys II. The early chronic phase. Trans. roy Soc. trop. Med. Hyg., 70: 247-251, 1976.

[25] 25. MOLINA, H.A.; MILEI, J.; RIMOLDI, M.T.; GONZALEZ CAPPA, S.M. & STORINO, R.A. - Histopathology of the heart conducting system in experimental Chagas disease in mice. Trans. roy. Soc. trop. Med. Hyg., 82: 241-246, 1988.

[26] 26. OLSEN, E.G.J. Examination of the heart. In: OLSEN, E.G.J. The pathology of the heart. 2 nd. ed. London, Macmillan Press, 1980. p. 5-10.

[27] 27. PAN AMERICAN HEALTH ORGANIZATION. Report of a study group on Chagas' disease. Washington, 1970 (Scientific Publication No. 195).

[28] 28. REVELLI, S.S.; AMERIO, N.; MORENO, H.S.; VALENTI, J.L.; BALBARREY, H. & MORINI, J.C. Enfermedad de Chagas cronica en la rata. Características serologicas, electrocardiograficas e histopatologicas. Medicina (B. Aires), 40 (Suppl. 1): 69-76, 1980.

[29] 29. ROSEMBAUM, M.B. & ALVAREZ, A. The electrocardiogram in chronic chagasic myocarditis. Amer. Heart J., 50: 492-527, 1955.

[30] 30. STEFANINI, M.; DE MARTINO, C. & ZAMBONI, L. Fixation of ejeculated spermatozoa for electron microscopy. Nature, 216: 173, 1976.

[31] 31. STROUT, R.G. A method for concentrating hemoflagellates. J. Parasit., 48: 100, 1962.

[32] 32. TEIXEIRA, A.R.L.; TEIXEIRA, L. & SANTOS-BUSH, C.A. The immunology of experimental Chagas' disease. IV. The production of lesions in rabbits similar to those of chronic Chagas' disease in man. Amer. J. Path, 80: 163-177, 1975.

[33] 33. TEIXEIRA, A.R.L.; FIGUEIREDO, F.; REZENDE, F.J. & MACEDO, V. Chagas' disease: a clinical, parasitological, immunological and pathological study in rabbits. Amer. J. trop. Med. Hyg., 32: 258-272, 1983.

[34] 34. TORRES, C.M. Miocitolise e fibrose do miocárdio na doença de Chagas. Mem. Inst. Oswaldo Cruz, 58: 161-182, 1960.

[35] 35. TORRES, C.M. & TAVARES, B.M. Miocardite no macaco Cebus após inoculaçőes repetidas com Schizotrypanum cruzi. Mem. Inst. Oswaldo Cruz, 5: 85-152, 1958.

[36] 36. VIANNA, G. Contribuiçăo para o estudo da anatomia patológica da moléstia de Carlos Chagas. Mem. Inst. Oswaldo Cruz, 3: 276-294, 1911.

[37] 37. VOLLER, A.; DRAPER, C.C.; BIDWELL, D.F. & BARTLETT, A. Microplate enzyme-Linked immunosorbent assay for Chagas' disease. Lancet, 1: 426-427, 1975.

[38] 38. WESSLER, S. Introduction: What is a model? Animal models of thrombosis and hemorrhagic disease. Bethesda. National Institutes of Health, 1976.

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