Immunology Letters, 31 (1991)91 - 96
Elsevier IMLET 01719
Trypanosoma cruzi infection in immunosuppressed mice K a t i a S. C a l a b r e s e 1, P. G. B a u e r 1, P. H . L a g r a n g e 2 a n d S. C. Gon~alves d a C o s t a 2 ~Laboratorio de Imunomodulacao, Departamentode Protozoologia, lnstituto Oswaldo Cruz, Rio de Janeiro, and 2Laboratoire Central de Microbiologie, Hotel-Dieu de Paris, Paris, France
(Received 16 April 1991; revisionreceived20 August 1991; accepted21 August 1991)
1. Summary Groups of 10 mice were challenged with 104 trypomastigotes of the Y strain of Trypanosoma cruzi. Cyclophosphamide (200 mg per kg) was used for immunosuppression and administered two days before infection in one group and on day 5 after infection in a second group. Enhancement of infection was more drastic in the second group, with a uniform acceleration of mortality and no clearance of the blood parasites. Electron microscopy showed that the hepatocytes were colonized by T. cruzi and that the invasion of the liver, spleen and lungs was associated with secondary infection by bacteria, which may be one of the cases of acceleration of mortality in infected mice. It was suggested that immunosuppressive therapy may favor unexpected symptoms and tissue localization of parasites in patients with Chagas' disease.
2. Introduction In cyclophosphamide (CY)-treated mice infected with Trypanosoma cruzi, an enhancement of the infection [1, 2] and of chronic myocarditis [3] has been shown to occur. It was suspected, however, that the mortality of animals infected with T. cruzi and treated with CY was associated with secondary infections [2]. Furthermore, experimental Chagas' disease in inbred strains of mice was associated with
severe diarrhea, and secondary infections were also suspected of accelerating the death of infected animals [4]. In the present paper we report the association of bacteria in the liver and lungs colonized by T. cruzi in immunosuppressed mice.
3. Materials and Methods 3.1. Parasite
The Y strain of T. cruzi isolated from a patient in the acute phase [5] was maintained by serial passages in mice and used for experimental protocols. 3.2. Animals Specific-pathogen-free outbred female OFI mice 4 - 6 weeks old were originally purchased from IFFA-CREDO (Domaine des Oncins, St. Germain Sur L'Arbresle, France), and then introduced at the Instituto Oswaldo Cruz in 1980 by one of us (S.C.G.C.). 3.3. Cyclophosphamide (Endoxan) Cyclophosphamide (CY) was purchased from Pravaz Division, Abbott Laboratories (Brazil), dissolved in saline, Millipore-filtered (0.22#m) and injected intraperitoneally (i.p.) at a dose of 200 m g / kg.
Key words: Trypanosoma cruzi; Immunosuppression; Cyclo-
phosphamide; Tissuecolonization;Secondaryinfection
3.4. Histopathology
Correspondenceto: K. S. Calabrese, Laboratorio de Imuno-
modulagao, Departamento de Protozoologia, Instituto Oswaldo Cruz, CP 926, CEP 21045, Rio de Janeiro, Brazil.
Fragments of organs were removed and immediately fixed in Millonig's fluid [6]. Part of this
0165-2478 / 91 / $ 3.50 © 1991 ElsevierSciencePublishers B.V. All rights reserved
91
material was then routinely processed for paraffin embedding. Sections (5/zm thick) were stained with hematoxylin-eosin. 3.5. Electron microscopy studies Aiming to define the presence of secondary infections as well as the nature of the T. cruzi-infected cells in the liver, some fragments were processed for transmission electron microscopy. Specimens were then pre-fixed in Millonig's fluid, minced into thin pieces, washed in 0.1 M cacodylate buffer and subsequently fixed in 2.50/0 glutaraldehyde and 1% osmium tetroxide. Material was processed for epon embedding and ultrathin sections were stained with uranil acetate and lead citrate.
3.6. Experimental design Two series of experiments were developed employing groups of 6 mice, one series for histopathology studies and the other for parasitaemia and mortality, each being carried out in triplicate. Groups of normal and CY-pretreated mice were infected with 104 trypomastigotes injected i.p. One additional group of normal mice was infected with the same inoculum, and was treated on day 5 after the challenge, with 200 mg/kg of CY injected i.p. Parasitemia was estimated by counting 50 highpower fields of 5 t~l tail blood under a 22 x 22 mm cover slip; this figure was converted to the number of parasites per ml of blood [7]. Mice were checked daily and mortality was recorded by cumulative percentage of 6 animals. Tissue sections were prepared from the second series of experiments on day 12 after infection. Cardiac and skeletal muscles, brain, lungs, kidneys, liver and spleen were removed and fixed in Millonig's fluid.
3.7. Stat&tical analysis Results are expressed as arithmetic means and respective standard errors of the mean. Statistical significance was determined by the Student's test for non-paired data. 4. Results
The present study shows an enhancement of T. 92
cruzi infection by treatment of mice with CY injected two days before or five days after injection, being more drastic in the latter case. CY pre-treated mice presented a peak of parasitaemia equal to control on day 8. Nonetheless, the ability of the first group to clear parasites from blood was delayed by two days (Fig. 1). The peak of parasitaemia of infected mice treated with CY on day + 5 was delayed by one day, being somewhat higher and without clearance of trypanosomes. Fig. 1 also shows the cumulative mortality in different groups of OF1 mice inoculated intraperitoneally with T. cruzi in which mice treated with CY on day + 5 died earlier than mice of other groups; on 16th day post-infection 80°7o of deaths occurred in this group against 40°70 in mice treated on day - 2 and 30% in non-CY-treated T. cruz# infected animals. In experimental groups selected for histopathological studies, we observed a higher colonization of the liver in CY-treated mice (day + 5) than in normal mice (Fig. 2a). Another point observed was that under immunosuppression by CY, colonization of the liver by T. cruzi was associated with secondary infections. We observed large nests of filamentous bacteria in the liver (Fig. 2b) and spleen. Ultrastructural studies of infected liver showed that besides Kupffer cells, hepatocytes were colonized by T. cruzi (Fig. 2c). Evaluation of lungs also showed secondary bacterial infection but in the forms of cocci or short rods and more scattered forms (Fig. 3). In contrast, untreated infected mice (control group) showed the characteristic pattern of tissue colonization of the strain assayed, as previously described [1]. 5. Discussion
Recently, with the introduction of cardiac transplantation in chagasic patients, strong immunosuppressive therapy was applied, and some rare clinical aspects, such as nodular paniculite containing amastigotes were described [8]. In addition, concomitant infection occurs [9, 10] and studies have been suggested for a better understanding of particular problems in chagasic patients under immunosuppression. CY has also been employed two days before infection as immunomodulator in experimental Chagas' disease to enhance delayed type
,0'
.100%
"I0 0
o
JD
o
E
> ,m O. 111 @
E
E
-
4
5
6
7
8
9 Days
10 after
11
12
infection
13
14
15
16
17
50~*
4 |
Fig. 1. Parasitaemia and cumulative mortality of different groups of mice infected with l04 T. cruzi (Y strain): mice treated with cyclophosphamide on day + 5 (black circles) or on day - 2 (white circles) or not treated (gray circles). Cumulative mortality showing 80°7o of death in mice treated with CY on day - 5 (black bars). No significant difference was observed between control groups (gray bars) and group treated on day - 2 (white bar).
94
Fig. 3. Ultrastructure of mouse lung cells colonized by T. cruzi Y strain showing amastigote (large arrow) and bacteria in a capillary vessel (thin arrow) 8 days after infection. Magnification 3150 x .
hypersensitivity to T. cruzi antigens associated with BCG [11, 12], and a correlation between DTH and resistance was observed [12]. When low doses of CY were injected alone in mice before [13] or after infection [3], an enhancement of myocarditis was reported and, in chronic phase of the infection, several low doses of CY promoted a new acute phase [14]. A depressed resistance to T. cruzi occurs after treatment with cortisone or hydrocortisone [15, 16] and X-irradiation [4]. In the case of CY a marked difference was detected, depending on whether in-
jection was made before or after infection. When CY was used 8 days before T. cruzi challenge a relative protection occurred against the acute phase since 70% of mice survived, in spite of the high parasitemia observed [1]. This can be contrasted with the present results in which 100% of deaths occurred if CY (200mg/kg) was injected 5 days after. The present report confirmed the hypothesis that the increased mortality of CY-treated mice might be related to secondary infection [2]. We have observed bacterial infection in the liver, spleen and
Fig. 2. Histopathology of liver infected with Y strain of T. cruzi after 12 days of infection. Mice were treated by CY (200mg/kg) on day + 5. (a) Amastigotes nests (arrows) in the liver parenchyma without inflammatory infiltrate. Haematoxylin and eosin staining, 160 x . (b) Colonization of liver by T. cruzi Y strain showing amastigotes (arrow). Nests of filamentous bacteria were also observed near a sinusoid. Magnification 380 ×. (c) Ultrastructure of an hepatocyte colonized by T. cruzi amastigote (arrow). Host cell ultrastructure shows the characteristic components (M, mitochondrion; BC, bile canaiicular domain of plasma membrane; T J, tight junction; RER, rough endoplasmic reticulum). Magnification 3150 x .
95
lungs. In a previous report [17] we showed that immunodeficient mice, like humans who have received cardiac transplantation [9], present nodular tegumentary lesions with T. cruzi amastigotes. However, little investigation has been done upon T. cruzi tissue invasion under immunosuppression. Liver and lungs, generally lightly affected by T. cruzi colonization, are heavily infected in irradiated [4] or athymic mice [18]. In the present report we show that cyclophosphamide treatment favored liver colonization and that hepatocytes and Kupffer cells were also colonized by the parasite. It has been known, since the classical report of Carlos Chagas in 1916 [19], that the colonization of the liver is not observed in patients who die during the acute phase of the disease. Degeneration of the liver was described as a consequence of the transitory liver colonization by the parasite leading at least to a more or less focal inflammatory reaction [18]. Although reports showing that hepatocytes are parasitized by T. cruzi are scarce, the present data does suggest that this occurs in both human and murine infection under immunosuppressor therapy. Although differences exist between experimental murine models and the human disease, it is suggested that unexpected symptoms in chagasic patients under immunosuppressive therapy should receive attention.
Acknowledgements This work was partially supported by a grant from CNPq (Brazil) and INSERM (France) EC/ MB number 5297. We are grateful to Dr. Leonidas M. Deane and Dr. Wilson Savino for their critical reading of the manuscript; to Ruy Quintella and Sonia Quintella for technical assistance; to Mauro Mauricio Carneiro Campello, Roberto Jesus Oscar and George Carvalho Cruz for the graphs and photographic assistance; and to Carlos Costa for his help with the computer.
96
References [1] Gonqalves da Costa, S. C. and Lagrange, P. H. (1980) in: Cancer Immunology and Parasite Immunology (L. Israel, P.H. Lagrange and J.C. Salomon, Eds.) INSERM 97, 383. [2] Kumar, R., Kline, I. K. and Abelmann, W. H. (1970) Am. J. Trop. Med. Hyg., 19, 932. [3] Andrade, Z. A., Andrade, S. and Sandigursky, M. (1987) Am. J. Pathol. 127,467. [4] Trischmann, T., Tanowitz, H., Winner, M. and Bloom, B. (1972) Exp. Parasitol. 45, 160. [5] Pereira da Silva, L.H. and Nussenzweig, V. (1953) Folia Clin. Biol. (S~o Paulo) 20, 191. [6] Carson, F. L., Martin, D. H. and Lynn, D. A. (1973) Am. J. Clin. Pathol. 53,365. [7] Pizzi, T. and Rubio, M. (1955) Bol. Chil. Parasitol. 10, 4. [8] Bocchi, E . A . (1987) Rev. Soc. Bras. Med. Trop. 20 (Suppl.) M, 39. [9] Jatene, A. D. (1987) Rev. Soc. Bras. Med. Trop. 20 (Suppl. II), C 5. [10] Uip, D. E., Strabelli, T. M. V., Belotti, G., Bocchi, E., Auler Junior, J. O. C., Ioguche, L., Kalil, J., Lopes, E. A., Chamone, D. A. F., Stolf, N., Amato Neto, V., Pilezzi, F. and Jatene, D. (1987) Rev. Soc. Bras. Med. Trop. 20 (Suppl.) 61. [11] Abrahamsohn, I.A., Blotta, M . H . S . L . and Curotto, M.A. (1981) Infect. Immun. 31, 1145. [12] Gonqalves da Costa, S.C. and Lagrange, P.H. (1981) Mem. Inst. Oswaldo Cruz, 76, 367. [13] Silva, J. S. and Rossi, M. A. (1990) J. Exp. Pathol. 71, 33. [14] Brener, Z. and C hiari, E. (1971) Trans. R. Soc. Trop. Med. Hyg. 65,629. [15] Nery-Guimaraes, F. (1971) Hospital 79, 3. [16] Seneca, H. and Ides, D. (1955) Am. J. Trop. Med. Hyg. 4, 833. [17] Gon~alves da Costa, S.C. and Calabrese, K.S. (1990) Mem. Inst. Oswaldo Cruz, 85 (Suppl. 3) in press. [18] Gon~alves da Costa, S.C., Lagrange, P.H., Hurtrel, B., Kerr, I. and Alencar, A.A. (1984) Ann. Immunol. (Inst. Pasteur) 135C, 317. [19] Chagas, C. (1916) Mem. Inst. Oswaldo Cruz 8, 37.
Elsevier IMLET 01719
Trypanosoma cruzi infection in immunosuppressed mice K a t i a S. C a l a b r e s e 1, P. G. B a u e r 1, P. H . L a g r a n g e 2 a n d S. C. Gon~alves d a C o s t a 2 ~Laboratorio de Imunomodulacao, Departamentode Protozoologia, lnstituto Oswaldo Cruz, Rio de Janeiro, and 2Laboratoire Central de Microbiologie, Hotel-Dieu de Paris, Paris, France
(Received 16 April 1991; revisionreceived20 August 1991; accepted21 August 1991)
1. Summary Groups of 10 mice were challenged with 104 trypomastigotes of the Y strain of Trypanosoma cruzi. Cyclophosphamide (200 mg per kg) was used for immunosuppression and administered two days before infection in one group and on day 5 after infection in a second group. Enhancement of infection was more drastic in the second group, with a uniform acceleration of mortality and no clearance of the blood parasites. Electron microscopy showed that the hepatocytes were colonized by T. cruzi and that the invasion of the liver, spleen and lungs was associated with secondary infection by bacteria, which may be one of the cases of acceleration of mortality in infected mice. It was suggested that immunosuppressive therapy may favor unexpected symptoms and tissue localization of parasites in patients with Chagas' disease.
2. Introduction In cyclophosphamide (CY)-treated mice infected with Trypanosoma cruzi, an enhancement of the infection [1, 2] and of chronic myocarditis [3] has been shown to occur. It was suspected, however, that the mortality of animals infected with T. cruzi and treated with CY was associated with secondary infections [2]. Furthermore, experimental Chagas' disease in inbred strains of mice was associated with
severe diarrhea, and secondary infections were also suspected of accelerating the death of infected animals [4]. In the present paper we report the association of bacteria in the liver and lungs colonized by T. cruzi in immunosuppressed mice.
3. Materials and Methods 3.1. Parasite
The Y strain of T. cruzi isolated from a patient in the acute phase [5] was maintained by serial passages in mice and used for experimental protocols. 3.2. Animals Specific-pathogen-free outbred female OFI mice 4 - 6 weeks old were originally purchased from IFFA-CREDO (Domaine des Oncins, St. Germain Sur L'Arbresle, France), and then introduced at the Instituto Oswaldo Cruz in 1980 by one of us (S.C.G.C.). 3.3. Cyclophosphamide (Endoxan) Cyclophosphamide (CY) was purchased from Pravaz Division, Abbott Laboratories (Brazil), dissolved in saline, Millipore-filtered (0.22#m) and injected intraperitoneally (i.p.) at a dose of 200 m g / kg.
Key words: Trypanosoma cruzi; Immunosuppression; Cyclo-
phosphamide; Tissuecolonization;Secondaryinfection
3.4. Histopathology
Correspondenceto: K. S. Calabrese, Laboratorio de Imuno-
modulagao, Departamento de Protozoologia, Instituto Oswaldo Cruz, CP 926, CEP 21045, Rio de Janeiro, Brazil.
Fragments of organs were removed and immediately fixed in Millonig's fluid [6]. Part of this
0165-2478 / 91 / $ 3.50 © 1991 ElsevierSciencePublishers B.V. All rights reserved
91
material was then routinely processed for paraffin embedding. Sections (5/zm thick) were stained with hematoxylin-eosin. 3.5. Electron microscopy studies Aiming to define the presence of secondary infections as well as the nature of the T. cruzi-infected cells in the liver, some fragments were processed for transmission electron microscopy. Specimens were then pre-fixed in Millonig's fluid, minced into thin pieces, washed in 0.1 M cacodylate buffer and subsequently fixed in 2.50/0 glutaraldehyde and 1% osmium tetroxide. Material was processed for epon embedding and ultrathin sections were stained with uranil acetate and lead citrate.
3.6. Experimental design Two series of experiments were developed employing groups of 6 mice, one series for histopathology studies and the other for parasitaemia and mortality, each being carried out in triplicate. Groups of normal and CY-pretreated mice were infected with 104 trypomastigotes injected i.p. One additional group of normal mice was infected with the same inoculum, and was treated on day 5 after the challenge, with 200 mg/kg of CY injected i.p. Parasitemia was estimated by counting 50 highpower fields of 5 t~l tail blood under a 22 x 22 mm cover slip; this figure was converted to the number of parasites per ml of blood [7]. Mice were checked daily and mortality was recorded by cumulative percentage of 6 animals. Tissue sections were prepared from the second series of experiments on day 12 after infection. Cardiac and skeletal muscles, brain, lungs, kidneys, liver and spleen were removed and fixed in Millonig's fluid.
3.7. Stat&tical analysis Results are expressed as arithmetic means and respective standard errors of the mean. Statistical significance was determined by the Student's test for non-paired data. 4. Results
The present study shows an enhancement of T. 92
cruzi infection by treatment of mice with CY injected two days before or five days after injection, being more drastic in the latter case. CY pre-treated mice presented a peak of parasitaemia equal to control on day 8. Nonetheless, the ability of the first group to clear parasites from blood was delayed by two days (Fig. 1). The peak of parasitaemia of infected mice treated with CY on day + 5 was delayed by one day, being somewhat higher and without clearance of trypanosomes. Fig. 1 also shows the cumulative mortality in different groups of OF1 mice inoculated intraperitoneally with T. cruzi in which mice treated with CY on day + 5 died earlier than mice of other groups; on 16th day post-infection 80°7o of deaths occurred in this group against 40°70 in mice treated on day - 2 and 30% in non-CY-treated T. cruz# infected animals. In experimental groups selected for histopathological studies, we observed a higher colonization of the liver in CY-treated mice (day + 5) than in normal mice (Fig. 2a). Another point observed was that under immunosuppression by CY, colonization of the liver by T. cruzi was associated with secondary infections. We observed large nests of filamentous bacteria in the liver (Fig. 2b) and spleen. Ultrastructural studies of infected liver showed that besides Kupffer cells, hepatocytes were colonized by T. cruzi (Fig. 2c). Evaluation of lungs also showed secondary bacterial infection but in the forms of cocci or short rods and more scattered forms (Fig. 3). In contrast, untreated infected mice (control group) showed the characteristic pattern of tissue colonization of the strain assayed, as previously described [1]. 5. Discussion
Recently, with the introduction of cardiac transplantation in chagasic patients, strong immunosuppressive therapy was applied, and some rare clinical aspects, such as nodular paniculite containing amastigotes were described [8]. In addition, concomitant infection occurs [9, 10] and studies have been suggested for a better understanding of particular problems in chagasic patients under immunosuppression. CY has also been employed two days before infection as immunomodulator in experimental Chagas' disease to enhance delayed type
,0'
.100%
"I0 0
o
JD
o
E
> ,m O. 111 @
E
E
-
4
5
6
7
8
9 Days
10 after
11
12
infection
13
14
15
16
17
50~*
4 |
Fig. 1. Parasitaemia and cumulative mortality of different groups of mice infected with l04 T. cruzi (Y strain): mice treated with cyclophosphamide on day + 5 (black circles) or on day - 2 (white circles) or not treated (gray circles). Cumulative mortality showing 80°7o of death in mice treated with CY on day - 5 (black bars). No significant difference was observed between control groups (gray bars) and group treated on day - 2 (white bar).
94
Fig. 3. Ultrastructure of mouse lung cells colonized by T. cruzi Y strain showing amastigote (large arrow) and bacteria in a capillary vessel (thin arrow) 8 days after infection. Magnification 3150 x .
hypersensitivity to T. cruzi antigens associated with BCG [11, 12], and a correlation between DTH and resistance was observed [12]. When low doses of CY were injected alone in mice before [13] or after infection [3], an enhancement of myocarditis was reported and, in chronic phase of the infection, several low doses of CY promoted a new acute phase [14]. A depressed resistance to T. cruzi occurs after treatment with cortisone or hydrocortisone [15, 16] and X-irradiation [4]. In the case of CY a marked difference was detected, depending on whether in-
jection was made before or after infection. When CY was used 8 days before T. cruzi challenge a relative protection occurred against the acute phase since 70% of mice survived, in spite of the high parasitemia observed [1]. This can be contrasted with the present results in which 100% of deaths occurred if CY (200mg/kg) was injected 5 days after. The present report confirmed the hypothesis that the increased mortality of CY-treated mice might be related to secondary infection [2]. We have observed bacterial infection in the liver, spleen and
Fig. 2. Histopathology of liver infected with Y strain of T. cruzi after 12 days of infection. Mice were treated by CY (200mg/kg) on day + 5. (a) Amastigotes nests (arrows) in the liver parenchyma without inflammatory infiltrate. Haematoxylin and eosin staining, 160 x . (b) Colonization of liver by T. cruzi Y strain showing amastigotes (arrow). Nests of filamentous bacteria were also observed near a sinusoid. Magnification 380 ×. (c) Ultrastructure of an hepatocyte colonized by T. cruzi amastigote (arrow). Host cell ultrastructure shows the characteristic components (M, mitochondrion; BC, bile canaiicular domain of plasma membrane; T J, tight junction; RER, rough endoplasmic reticulum). Magnification 3150 x .
95
lungs. In a previous report [17] we showed that immunodeficient mice, like humans who have received cardiac transplantation [9], present nodular tegumentary lesions with T. cruzi amastigotes. However, little investigation has been done upon T. cruzi tissue invasion under immunosuppression. Liver and lungs, generally lightly affected by T. cruzi colonization, are heavily infected in irradiated [4] or athymic mice [18]. In the present report we show that cyclophosphamide treatment favored liver colonization and that hepatocytes and Kupffer cells were also colonized by the parasite. It has been known, since the classical report of Carlos Chagas in 1916 [19], that the colonization of the liver is not observed in patients who die during the acute phase of the disease. Degeneration of the liver was described as a consequence of the transitory liver colonization by the parasite leading at least to a more or less focal inflammatory reaction [18]. Although reports showing that hepatocytes are parasitized by T. cruzi are scarce, the present data does suggest that this occurs in both human and murine infection under immunosuppressor therapy. Although differences exist between experimental murine models and the human disease, it is suggested that unexpected symptoms in chagasic patients under immunosuppressive therapy should receive attention.
Acknowledgements This work was partially supported by a grant from CNPq (Brazil) and INSERM (France) EC/ MB number 5297. We are grateful to Dr. Leonidas M. Deane and Dr. Wilson Savino for their critical reading of the manuscript; to Ruy Quintella and Sonia Quintella for technical assistance; to Mauro Mauricio Carneiro Campello, Roberto Jesus Oscar and George Carvalho Cruz for the graphs and photographic assistance; and to Carlos Costa for his help with the computer.
96
References [1] Gonqalves da Costa, S. C. and Lagrange, P. H. (1980) in: Cancer Immunology and Parasite Immunology (L. Israel, P.H. Lagrange and J.C. Salomon, Eds.) INSERM 97, 383. [2] Kumar, R., Kline, I. K. and Abelmann, W. H. (1970) Am. J. Trop. Med. Hyg., 19, 932. [3] Andrade, Z. A., Andrade, S. and Sandigursky, M. (1987) Am. J. Pathol. 127,467. [4] Trischmann, T., Tanowitz, H., Winner, M. and Bloom, B. (1972) Exp. Parasitol. 45, 160. [5] Pereira da Silva, L.H. and Nussenzweig, V. (1953) Folia Clin. Biol. (S~o Paulo) 20, 191. [6] Carson, F. L., Martin, D. H. and Lynn, D. A. (1973) Am. J. Clin. Pathol. 53,365. [7] Pizzi, T. and Rubio, M. (1955) Bol. Chil. Parasitol. 10, 4. [8] Bocchi, E . A . (1987) Rev. Soc. Bras. Med. Trop. 20 (Suppl.) M, 39. [9] Jatene, A. D. (1987) Rev. Soc. Bras. Med. Trop. 20 (Suppl. II), C 5. [10] Uip, D. E., Strabelli, T. M. V., Belotti, G., Bocchi, E., Auler Junior, J. O. C., Ioguche, L., Kalil, J., Lopes, E. A., Chamone, D. A. F., Stolf, N., Amato Neto, V., Pilezzi, F. and Jatene, D. (1987) Rev. Soc. Bras. Med. Trop. 20 (Suppl.) 61. [11] Abrahamsohn, I.A., Blotta, M . H . S . L . and Curotto, M.A. (1981) Infect. Immun. 31, 1145. [12] Gonqalves da Costa, S.C. and Lagrange, P.H. (1981) Mem. Inst. Oswaldo Cruz, 76, 367. [13] Silva, J. S. and Rossi, M. A. (1990) J. Exp. Pathol. 71, 33. [14] Brener, Z. and C hiari, E. (1971) Trans. R. Soc. Trop. Med. Hyg. 65,629. [15] Nery-Guimaraes, F. (1971) Hospital 79, 3. [16] Seneca, H. and Ides, D. (1955) Am. J. Trop. Med. Hyg. 4, 833. [17] Gon~alves da Costa, S.C. and Calabrese, K.S. (1990) Mem. Inst. Oswaldo Cruz, 85 (Suppl. 3) in press. [18] Gon~alves da Costa, S.C., Lagrange, P.H., Hurtrel, B., Kerr, I. and Alencar, A.A. (1984) Ann. Immunol. (Inst. Pasteur) 135C, 317. [19] Chagas, C. (1916) Mem. Inst. Oswaldo Cruz 8, 37.
