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Antigenic differences of Leishmania amazonensis isolates causing diffuse cutaneous leishmaniasis
678
TRANSACT~NS OF THE ROYALSOCIETY OF TROPICAL. MEDNX.IEAND HYGIENE(1990) 84, 678-680
Antigenic differences of Leishmania cutaneous leishmaniasis
amazonensis
isolates causing diffuse
Leonor L. Leon, Gerzia M. C. Machado, Luis E. de Carvalho Paes and Gabriel Grimaldi, Jr Departamento de Imunologia, Instituto Oswald0 Cruz, C.P. 926, 20.000 Rio de Janeiro, R. J., Brazil 1 Abstract Six geographically distinct isolates of Leishmania amazonensis causing diffuse cutaneous leishmaniasis (DCL) (from Bahia and Maranhfio, in Brazil and Guarico, in Venezuela) were characterized by immunoblot analysis to see whether any geographical or strain-related differences existed in antigenic composition. Western blots of promastigote homogenates were reacted with polyclonal sera from patients infected with L. amazonensis with the various forms of clinical disease. The pattern of antigenic reactivity of these strains revealed the presence of shared antigenic components between geographically distinct L. amazonensis isolates causing DCL, when tested with the sera of the infected patients. In certain cases, however, some polyclonal sera also detected antigenic fractions unique to the strains examined. Variation was observed between the antigenic components of some isolates of L. amazonensis that were recognized by a single serum, and between the antigenic components of a single isolate of L. amazonensis that were recognized by the different patients’ sera. However, no constant association was found between the antigenic components identified in these isolates and the geographical area of isolation. These results indicate that, although these parasites appear to be closely related antigenically, they also possess some strain-related antigenic differences.
Introduction American cutaneous leishmaniasis (ACL) is caused mainly by species classified into two broad complexes: Leishmania braziliensis and L. mexicana (see LAINSON & SHAW, 1987). Self-healing ulcerative cutaneous leishmaniasis (CL), accompanied by positive cellular immune responses, has been associated with all the New World dermatotropic Leishmania species. Diffuse cutaneous leishmaniasis (DCL), the anergic form, however, is produced by parasite species of the L. mexicana complex only (GRIMALDI et al., 1989). This condition originates as a single lesion which, with time, spreads metastatically giving rise to multiple non-ulcerated nodules. The parasitism is typically intense with a heavy accumulation of parasitized and vacuolated macrophages in the lesions and there is poor response to treatment. Immunologically, there is a positive antibody response, but a negative cellular or delayed type hypersensitivity response to leishmanial antigen (CONVIT et al., 1972). It has been suggested that the development of the disease is not due to an inherent (immunogenetic) specific anergy to Address offprint requests to: Gabriel Grimaldi, Jr, Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College Street, P.O. Box 3333, New Haven, CT 06510, USA.
the parasite by the host; however, the defect is more likely to be due to induction of specific immunological effector mechanisms that fail to control infection (HOWARD,~~S;MAUEL& BEHIN, 1987).Inprevious studies we have shown that L. amazonensis infection can be associated with distinct clinical forms of ACL (BARRAL et al., 1986, 1990). Antigenic variation of L. amuzonensis, associated with these distinct clinical forms of the disease, was also demonstrated (Leon et al., paper in preparation). Here, we have extended these investigations using 6 geographically distinct isolates of this parasite species causing DCL to see whether any strain-related differences exist in their antigenic composition.
Materials and Methods Stocks of Leishmania Six Leishmania isolates from oatients with DCL in Brazil and Venezuela were ex&ined (see Figure). These stocks have been typed as L. amazonensis by both indirect radioimmune binding assay, using specific monoclonal antibodies, and enzyme electrophoresis (GRIMALDI et al., 1987, 1989). Preparation of samples Procedures for growing Leishmania promastigotes in vitro have been reported previously (GRIMALDI et al., 1987). When the promastigotes were in the log phase of growth in Schneider’s medium they were harvested by centrifugation (1500 g for 10 min at CC) and washed twice in phosphate-buffered saline (PBS), pH 7.3. The final pellet was resuspended in an antiproteolytic buffer containing 0.04 M NaCl, 0.01 M sodium ethylenediaminetetraacetate, 0.001 M phenylmethylsulphonylfluoride, 0.01 M lodoacetamide, 0.005 M l,lO-phenanthroline, and 0.01 M Tris, pH 8.0. Before analysis, the samples were briefl sonicated using a bath sonicator (RAI Researc B , model 250 ultrasonic cleaner) to homogenize the antigens, and then centrifuged (2000 g for 10 min at 4°C). The supernatants were divided into aliquots and stored at -20°C until used for analysis. Protein concentration was measured by the protein assay method of LOWRY et al. (1951), and all samples were resuspended to the same concentration before analysis. Western blot analysis The soluble antigen extracts were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using 10% slab gels in non-reducing conditions, and electrophoretically transferred to nitrocellulose paper (Schleicher and Schuell, Keene, New Hampshire, USA) according to the procedure described by TOWBIN et al. (1979). The nitrocellulose strips were then incubated overnight at 4°C with
679
Mr -
-’
180 .O
84 .O
48.
26. 6,
12
3456
123456
123456
123456
Figure.Western blotanalyses of promastigote homogenates of geographically distinctLeishmonia omazaensis isolates causingdiffusecmanemts leishmaniasis (seelist below),usingL. amozonensis spech%polyclonalantibodies:immuneseraobtainedfrom patientswith A: visceral leishmaniasis, B: diffusecutaneous leishmaniasis, C:mucocutaneous leishmaniasis, andD: cutaneous leishmaniasis. Track1:MHOMIBR/86IBA106(isolated fromBrazil,Bahia,Ilheus);track2: MHOMIBRI83IJGL (Brazil,Maranhlo,Cavalcante); track3: MHOMNEIOOIL54 (Venezuela, (Brazil,MaranhPo,?); track 6: Guarico);track4: MHOiWBRI83IDAY (Brazil,MaranhPo. Buriticupu);track 5: MHOMIBRI86ILCD MHOMIBRi79113035 (Brazil,Maranhao,Rosario). anti-L. amazonensis polyclonal antibodies. The immune sera were obtained from patients infected with L. amazonensis, but with different clinical forms of leishmaniasis (Figure). Following incubation with the primarv antibody, the strips were washed 3 times in wash buffer (PBS containing 0.05% Tween-20; Fisher Scientific). and incubated with rabbit anti-human immunogiobulin G conjugated to peroxidase (Sigma) for 1 h. After rinsing with wash buffer, the strips were incubated for 15 min in a saturated solution of 3,3’-diaminobenzidine in Tris buffer, pH 7.4, containing 0.01% H202 (GRAHAM& KARNOVSKY,1966). Results Characterization of L. amazonensis antigens by immune Sl??U
The Figure shows Western blots prepared from L. umuzonensis isolates causing DCL, from different endemic areas, reacted with polyclonal sera from patients infected with L. amuzoncnsis. Certain antigenie components in individual strains were recognized by sera from all the patients grouped by clinical presentation; the spectra of antigens recognized by these groups of sera, however, were not identical. Differences were observed between the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum, and between the antigenic components of a single isolate of L. amazonensis that were recognized by the different
patients’ sera. The blots showed no correlation, however, between the antigenic components present in the various strains of L. amazunensis and the geographical area of isolation. Discussion Genetic factors expressed in some races or individuals, like those hefined in the murine model system (HOWARD, 1985; BLACKWELL& ALEXANDER, 1986), are likely to-play a role in determining some of the variations in the course of infection seen in human leishmaniasis (CONVITet aE., 1972; MAUEL 8t BEHIN, 1987). Clinical and experimental evidence suggests that the intrinsic characteristics of the parasite also influence the course of infection. For example, in the New World, DCL (the anergic, non-ulcerative form which is accompanied by defective cellular immune responses) is produced by parasite snecies of the L. mexicana comnlex only ~GRIMALD~et al., 1989; VELASCOet ai., 1989). Therefore, it is believed that the outcome of Leishmania infection is dependent on the genetically determined immunoregulatory mechanisms of the host as well as the virulence of the infecting parasite
(HOWARD,1985; BLACKWELL& ALEXANDER,1986; MAUEL St BEHIN, 1987).
The current concept is that CL is rarely due to L. amazonensis, but approximately 30% of human infections with this parasite evolve to DCL (LAINSON,
686
1983). In contrast, data from other regions in Brazil show that this anergic form of the disease is unusual, only a few cases l&.&g been reported (GRIMALDI et al.. 19891. Co&mine: this view, we recentlv demonstrated that? despite th; relativeli high frequency of L. amazotwws infection in Bahia (BARRAL et al., 1990), only a few cases of DCL were associated with this parasite (BITTENCOURTet al., 1989). We have also shown that L. amazonensis is capable of producing a wide spectrum of disease in humans (BARRALet al., 1990). Moreover, the L. amazonensis isolates associated with these distinct clinical forms were not antigenically identical (homogenous) and the immune responses (antibodies) observed among infected patients were heterogenous (Leon et al., paper in preparation). In tl& study; we demonstrated that serum from a single individual infected with L. amazonensis recognizes many different epitopes from different strains of this narasite associated with DCL. showing that they also ire not antigenically identical (homogeneous). The heterogeneity of these stocks is confirmed by our results obtained in a parallel study: the kinetoplast deoxyribonucleic acid fragment patterns of these L. amuzunensis strains were different from one another (Grimaldi, unpublished observations) . The immune sera used were obtained from patients infected with L. amazonensis, but with different clinical presentations (BARML et aE., 1990; Leon et al., paper in preparation). We also demonstrated that sera from different individuals recognized different antigens from the same isolate. This, as well as the variation in the clinical manifestations observed among infected patients, may in part be explained by the various genetic factors expressed in different races or individuals, as discussed above. Acknowledgements
This work was supported by PADCT/FINEP, CNPq (Brazil), and the UNDPKVorld BanwHO Special Programme for Research and Training in Trooical Diseases. The study was made possible bygenerous-donations from the culture collections of Dr A. Barral (Universidade Federal da Bahia, Salvador, Brazil), Dr I. Per& (Institute Venezolano de Investigation Cientifica, Caracas, Venezuela), and A. R. Silva (Universidade Federal do Maranhho, SBo Luis, Brazil). Special thanks are given to Dr Aldina Barral for providing the patients’ sera. References Barral, A.,Badar6, R., Barral-Neto, M., Grimaldi, G., Jr, Momen. H. & Carvalho. E. M. (1986). Isolation of Leishmaka mexicana ama&nensis from th; bone marrow in a case of American visceral leishmaniasis. American Journal of Tropical Medicine and Hygiene, 35, 732-734. Barral, A., Pedral-Sampaio, D., Grimaldi, G., Jr Momen, H., McMahon-Pratt, D., Rlbeiro de Jesus, A., Almeida, R., Badar6 R., Barral-Neto, M., Carvalho, E. M. & Johnson, &. D., Jr (1990). Leishmaniasis in Bahia,
Brazil: evidence that Leishmania amazanensis produces a wide spectrum of clinical disease. American Journal of Tropical Medicine and Hygiene, in press. Bittencourt, A.,, Barral, A., Ribeiro de Jesus, Almeida, R. P. & Grimakh, G. Jr (1989). In situ identification of L. arnuzonensis associated with diffuse cutaneous leishmaniasis in Bahia, Brazil. Memtis do Znstituto Oswaldo Crw, 84, 585-586. Blackwell. I. M. & Alexander. I. (1986). Different host genes iecognize and control tie&on wjth taxonomically distinct Leishmania species. In: Leishmania. Taxonomie et phylogenke. Applications &-kpidemiologiques. Colloquium International du CNRSIINSERM. Paris: Editions du CNRS, pp. 211-219. Convit, J., Pinardi, M. E. & Randon, A. J. (1972). Diffuse cut&tebus leishmaniasis: a disease due- to an immunological defect of the host. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 603-610. Graham, R. C. & Karnovsky, M. J. (1966). The early stages of absorntion of iniected horseradish oeroxidase in the proximal tubules ‘of mouse kidney: ultrastructural cytochemistry by a new technique. Journal of Histochemishy and Cytochemisty, 14, 192-302. Grimaldi, G., Jr, David, R. & McMahon-Pratt, D. (1987). Identification of New World Leishmunia species characterized by serodeme analysis using monoclonal antibodies. American Journal of Tropical Medicine and Hygiene, 36, 270-287. Grimaldi, G., Jr, Tesh, R. B. & McMahon-Pratt, D. (1989). A review of the geographic distribution and epidemiology of leishmaniasis in the New World. American Journal of Tropical Medicine and Hygiene, 41, 687-725. Howard, J. G. (1985). Host immunity to leishmaniasis. In: Leishmaniasis, Volume 1, Chang, K.-P. & Bray, R. S. (editors). Amsterdam: Elsevier, pp. 139-162. Lainson, R. (1983). The American leishmaniases: some obseivations on their ecology and epidemiology. Transactions of the Royal Socieh, of TroDical Medicine and Hygiene, _77, 5&P-596.- Lainson, R. & Shaw, J. J. (1987). Evolution, classification and geographical distribution. In: The Leishmaniases in Biology and Medicine, volume 1, Peters, W. & KillickF;$ick, R. (editors). London: Academic Press, pp. Lowry, d. H., Roseburgh, N. J., Farr? A. L. & Randal, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemisq, 193, 265-275. Mauel, J. & Behin, R. (1987). Immumty: clinical and experimental. In: The Leishmaniases in Biology and Medicine, volume 2, Peters, W. & Killick-Kendrick, R. (editors). London: Academic Press, pp. 731-791. Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the USA, 76, 43504354. Velasco, O., Savarino, S. J., Walton, B. C., Gam, A. A. & Neva, F. A. (1989). Diffuse cufaneous leishmaniasis in Mexico. American 3ournal of Tropical Medicine and Hygiene, 41, 280-288. Received accepted
2 January 1990; revised 26 March publication 20 April 1990
for
1990;
TRANSACT~NS OF THE ROYALSOCIETY OF TROPICAL. MEDNX.IEAND HYGIENE(1990) 84, 678-680
Antigenic differences of Leishmania cutaneous leishmaniasis
amazonensis
isolates causing diffuse
Leonor L. Leon, Gerzia M. C. Machado, Luis E. de Carvalho Paes and Gabriel Grimaldi, Jr Departamento de Imunologia, Instituto Oswald0 Cruz, C.P. 926, 20.000 Rio de Janeiro, R. J., Brazil 1 Abstract Six geographically distinct isolates of Leishmania amazonensis causing diffuse cutaneous leishmaniasis (DCL) (from Bahia and Maranhfio, in Brazil and Guarico, in Venezuela) were characterized by immunoblot analysis to see whether any geographical or strain-related differences existed in antigenic composition. Western blots of promastigote homogenates were reacted with polyclonal sera from patients infected with L. amazonensis with the various forms of clinical disease. The pattern of antigenic reactivity of these strains revealed the presence of shared antigenic components between geographically distinct L. amazonensis isolates causing DCL, when tested with the sera of the infected patients. In certain cases, however, some polyclonal sera also detected antigenic fractions unique to the strains examined. Variation was observed between the antigenic components of some isolates of L. amazonensis that were recognized by a single serum, and between the antigenic components of a single isolate of L. amazonensis that were recognized by the different patients’ sera. However, no constant association was found between the antigenic components identified in these isolates and the geographical area of isolation. These results indicate that, although these parasites appear to be closely related antigenically, they also possess some strain-related antigenic differences.
Introduction American cutaneous leishmaniasis (ACL) is caused mainly by species classified into two broad complexes: Leishmania braziliensis and L. mexicana (see LAINSON & SHAW, 1987). Self-healing ulcerative cutaneous leishmaniasis (CL), accompanied by positive cellular immune responses, has been associated with all the New World dermatotropic Leishmania species. Diffuse cutaneous leishmaniasis (DCL), the anergic form, however, is produced by parasite species of the L. mexicana complex only (GRIMALDI et al., 1989). This condition originates as a single lesion which, with time, spreads metastatically giving rise to multiple non-ulcerated nodules. The parasitism is typically intense with a heavy accumulation of parasitized and vacuolated macrophages in the lesions and there is poor response to treatment. Immunologically, there is a positive antibody response, but a negative cellular or delayed type hypersensitivity response to leishmanial antigen (CONVIT et al., 1972). It has been suggested that the development of the disease is not due to an inherent (immunogenetic) specific anergy to Address offprint requests to: Gabriel Grimaldi, Jr, Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College Street, P.O. Box 3333, New Haven, CT 06510, USA.
the parasite by the host; however, the defect is more likely to be due to induction of specific immunological effector mechanisms that fail to control infection (HOWARD,~~S;MAUEL& BEHIN, 1987).Inprevious studies we have shown that L. amazonensis infection can be associated with distinct clinical forms of ACL (BARRAL et al., 1986, 1990). Antigenic variation of L. amuzonensis, associated with these distinct clinical forms of the disease, was also demonstrated (Leon et al., paper in preparation). Here, we have extended these investigations using 6 geographically distinct isolates of this parasite species causing DCL to see whether any strain-related differences exist in their antigenic composition.
Materials and Methods Stocks of Leishmania Six Leishmania isolates from oatients with DCL in Brazil and Venezuela were ex&ined (see Figure). These stocks have been typed as L. amazonensis by both indirect radioimmune binding assay, using specific monoclonal antibodies, and enzyme electrophoresis (GRIMALDI et al., 1987, 1989). Preparation of samples Procedures for growing Leishmania promastigotes in vitro have been reported previously (GRIMALDI et al., 1987). When the promastigotes were in the log phase of growth in Schneider’s medium they were harvested by centrifugation (1500 g for 10 min at CC) and washed twice in phosphate-buffered saline (PBS), pH 7.3. The final pellet was resuspended in an antiproteolytic buffer containing 0.04 M NaCl, 0.01 M sodium ethylenediaminetetraacetate, 0.001 M phenylmethylsulphonylfluoride, 0.01 M lodoacetamide, 0.005 M l,lO-phenanthroline, and 0.01 M Tris, pH 8.0. Before analysis, the samples were briefl sonicated using a bath sonicator (RAI Researc B , model 250 ultrasonic cleaner) to homogenize the antigens, and then centrifuged (2000 g for 10 min at 4°C). The supernatants were divided into aliquots and stored at -20°C until used for analysis. Protein concentration was measured by the protein assay method of LOWRY et al. (1951), and all samples were resuspended to the same concentration before analysis. Western blot analysis The soluble antigen extracts were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using 10% slab gels in non-reducing conditions, and electrophoretically transferred to nitrocellulose paper (Schleicher and Schuell, Keene, New Hampshire, USA) according to the procedure described by TOWBIN et al. (1979). The nitrocellulose strips were then incubated overnight at 4°C with
679
Mr -
-’
180 .O
84 .O
48.
26. 6,
12
3456
123456
123456
123456
Figure.Western blotanalyses of promastigote homogenates of geographically distinctLeishmonia omazaensis isolates causingdiffusecmanemts leishmaniasis (seelist below),usingL. amozonensis spech%polyclonalantibodies:immuneseraobtainedfrom patientswith A: visceral leishmaniasis, B: diffusecutaneous leishmaniasis, C:mucocutaneous leishmaniasis, andD: cutaneous leishmaniasis. Track1:MHOMIBR/86IBA106(isolated fromBrazil,Bahia,Ilheus);track2: MHOMIBRI83IJGL (Brazil,Maranhlo,Cavalcante); track3: MHOMNEIOOIL54 (Venezuela, (Brazil,MaranhPo,?); track 6: Guarico);track4: MHOiWBRI83IDAY (Brazil,MaranhPo. Buriticupu);track 5: MHOMIBRI86ILCD MHOMIBRi79113035 (Brazil,Maranhao,Rosario). anti-L. amazonensis polyclonal antibodies. The immune sera were obtained from patients infected with L. amazonensis, but with different clinical forms of leishmaniasis (Figure). Following incubation with the primarv antibody, the strips were washed 3 times in wash buffer (PBS containing 0.05% Tween-20; Fisher Scientific). and incubated with rabbit anti-human immunogiobulin G conjugated to peroxidase (Sigma) for 1 h. After rinsing with wash buffer, the strips were incubated for 15 min in a saturated solution of 3,3’-diaminobenzidine in Tris buffer, pH 7.4, containing 0.01% H202 (GRAHAM& KARNOVSKY,1966). Results Characterization of L. amazonensis antigens by immune Sl??U
The Figure shows Western blots prepared from L. umuzonensis isolates causing DCL, from different endemic areas, reacted with polyclonal sera from patients infected with L. amuzoncnsis. Certain antigenie components in individual strains were recognized by sera from all the patients grouped by clinical presentation; the spectra of antigens recognized by these groups of sera, however, were not identical. Differences were observed between the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum, and between the antigenic components of a single isolate of L. amazonensis that were recognized by the different
patients’ sera. The blots showed no correlation, however, between the antigenic components present in the various strains of L. amazunensis and the geographical area of isolation. Discussion Genetic factors expressed in some races or individuals, like those hefined in the murine model system (HOWARD, 1985; BLACKWELL& ALEXANDER, 1986), are likely to-play a role in determining some of the variations in the course of infection seen in human leishmaniasis (CONVITet aE., 1972; MAUEL 8t BEHIN, 1987). Clinical and experimental evidence suggests that the intrinsic characteristics of the parasite also influence the course of infection. For example, in the New World, DCL (the anergic, non-ulcerative form which is accompanied by defective cellular immune responses) is produced by parasite snecies of the L. mexicana comnlex only ~GRIMALD~et al., 1989; VELASCOet ai., 1989). Therefore, it is believed that the outcome of Leishmania infection is dependent on the genetically determined immunoregulatory mechanisms of the host as well as the virulence of the infecting parasite
(HOWARD,1985; BLACKWELL& ALEXANDER,1986; MAUEL St BEHIN, 1987).
The current concept is that CL is rarely due to L. amazonensis, but approximately 30% of human infections with this parasite evolve to DCL (LAINSON,
686
1983). In contrast, data from other regions in Brazil show that this anergic form of the disease is unusual, only a few cases l&.&g been reported (GRIMALDI et al.. 19891. Co&mine: this view, we recentlv demonstrated that? despite th; relativeli high frequency of L. amazotwws infection in Bahia (BARRAL et al., 1990), only a few cases of DCL were associated with this parasite (BITTENCOURTet al., 1989). We have also shown that L. amazonensis is capable of producing a wide spectrum of disease in humans (BARRALet al., 1990). Moreover, the L. amazonensis isolates associated with these distinct clinical forms were not antigenically identical (homogenous) and the immune responses (antibodies) observed among infected patients were heterogenous (Leon et al., paper in preparation). In tl& study; we demonstrated that serum from a single individual infected with L. amazonensis recognizes many different epitopes from different strains of this narasite associated with DCL. showing that they also ire not antigenically identical (homogeneous). The heterogeneity of these stocks is confirmed by our results obtained in a parallel study: the kinetoplast deoxyribonucleic acid fragment patterns of these L. amuzunensis strains were different from one another (Grimaldi, unpublished observations) . The immune sera used were obtained from patients infected with L. amazonensis, but with different clinical presentations (BARML et aE., 1990; Leon et al., paper in preparation). We also demonstrated that sera from different individuals recognized different antigens from the same isolate. This, as well as the variation in the clinical manifestations observed among infected patients, may in part be explained by the various genetic factors expressed in different races or individuals, as discussed above. Acknowledgements
This work was supported by PADCT/FINEP, CNPq (Brazil), and the UNDPKVorld BanwHO Special Programme for Research and Training in Trooical Diseases. The study was made possible bygenerous-donations from the culture collections of Dr A. Barral (Universidade Federal da Bahia, Salvador, Brazil), Dr I. Per& (Institute Venezolano de Investigation Cientifica, Caracas, Venezuela), and A. R. Silva (Universidade Federal do Maranhho, SBo Luis, Brazil). Special thanks are given to Dr Aldina Barral for providing the patients’ sera. References Barral, A.,Badar6, R., Barral-Neto, M., Grimaldi, G., Jr, Momen. H. & Carvalho. E. M. (1986). Isolation of Leishmaka mexicana ama&nensis from th; bone marrow in a case of American visceral leishmaniasis. American Journal of Tropical Medicine and Hygiene, 35, 732-734. Barral, A., Pedral-Sampaio, D., Grimaldi, G., Jr Momen, H., McMahon-Pratt, D., Rlbeiro de Jesus, A., Almeida, R., Badar6 R., Barral-Neto, M., Carvalho, E. M. & Johnson, &. D., Jr (1990). Leishmaniasis in Bahia,
Brazil: evidence that Leishmania amazanensis produces a wide spectrum of clinical disease. American Journal of Tropical Medicine and Hygiene, in press. Bittencourt, A.,, Barral, A., Ribeiro de Jesus, Almeida, R. P. & Grimakh, G. Jr (1989). In situ identification of L. arnuzonensis associated with diffuse cutaneous leishmaniasis in Bahia, Brazil. Memtis do Znstituto Oswaldo Crw, 84, 585-586. Blackwell. I. M. & Alexander. I. (1986). Different host genes iecognize and control tie&on wjth taxonomically distinct Leishmania species. In: Leishmania. Taxonomie et phylogenke. Applications &-kpidemiologiques. Colloquium International du CNRSIINSERM. Paris: Editions du CNRS, pp. 211-219. Convit, J., Pinardi, M. E. & Randon, A. J. (1972). Diffuse cut&tebus leishmaniasis: a disease due- to an immunological defect of the host. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 603-610. Graham, R. C. & Karnovsky, M. J. (1966). The early stages of absorntion of iniected horseradish oeroxidase in the proximal tubules ‘of mouse kidney: ultrastructural cytochemistry by a new technique. Journal of Histochemishy and Cytochemisty, 14, 192-302. Grimaldi, G., Jr, David, R. & McMahon-Pratt, D. (1987). Identification of New World Leishmunia species characterized by serodeme analysis using monoclonal antibodies. American Journal of Tropical Medicine and Hygiene, 36, 270-287. Grimaldi, G., Jr, Tesh, R. B. & McMahon-Pratt, D. (1989). A review of the geographic distribution and epidemiology of leishmaniasis in the New World. American Journal of Tropical Medicine and Hygiene, 41, 687-725. Howard, J. G. (1985). Host immunity to leishmaniasis. In: Leishmaniasis, Volume 1, Chang, K.-P. & Bray, R. S. (editors). Amsterdam: Elsevier, pp. 139-162. Lainson, R. (1983). The American leishmaniases: some obseivations on their ecology and epidemiology. Transactions of the Royal Socieh, of TroDical Medicine and Hygiene, _77, 5&P-596.- Lainson, R. & Shaw, J. J. (1987). Evolution, classification and geographical distribution. In: The Leishmaniases in Biology and Medicine, volume 1, Peters, W. & KillickF;$ick, R. (editors). London: Academic Press, pp. Lowry, d. H., Roseburgh, N. J., Farr? A. L. & Randal, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemisq, 193, 265-275. Mauel, J. & Behin, R. (1987). Immumty: clinical and experimental. In: The Leishmaniases in Biology and Medicine, volume 2, Peters, W. & Killick-Kendrick, R. (editors). London: Academic Press, pp. 731-791. Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the USA, 76, 43504354. Velasco, O., Savarino, S. J., Walton, B. C., Gam, A. A. & Neva, F. A. (1989). Diffuse cufaneous leishmaniasis in Mexico. American 3ournal of Tropical Medicine and Hygiene, 41, 280-288. Received accepted
2 January 1990; revised 26 March publication 20 April 1990
for
1990;