Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060
Vol. 93, Num. 3, 1998, pp. 283-287
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 93 (3), May/June 1998, pp. 283-287

Zymodeme and Serodeme Characterization of Leishmania Isolates Obtained from Costa Rican Patients

Johnny Peraza^+, Andrea Urbina, Rodrigo Zeledon

Laboratorio de Zoonosis, Escuela de Medicina Veterinaria, Universidad Nacional, Apartado Postal 304-3000, Heredia, Costa Rica
^+Corresponding author. Fax: +506-237.9735

Received 9 September 1997; Accepted 18 December 1997

Code Number:OC98054
Sizes of Files:
      Text: 15.8K
      Graphics: Line drawings and photographs (jpg) - 45.4K
                       Tables (jpg) - 171.4K

Thirty-four Leishmania isolates obtained from Costa Rican patients, from different geographical areas, were characterized by isoenzyme electrophoresis and indirect immunofluorescense with monoclonal antibodies. Thirty-two were characterized as L. panamensis strains and two were L. braziliensis variants. We confirm the evident predominance of L. panamensis as the main etiological agent of leishmaniasis in Costa Rica and the existence of L. braziliensis in the country.

Key words: leishmaniasis - Costa Rica - Leishmania panamensis - characterization - etiological agent

Human leishmaniasis is widespread throughout Central America and constitutes an important public health problem. Annual incidence is estimated to be about 20,000 cases (Carreira et al. 1995), of which 4,000 to 5,000 correspond to Costa Rica (Zeledon 1992). The disease exists under different clinical manifestations and in a variety of ecological and epidemiological patterns (Zeledon 1985) making it a complex problem that is difficult to manage.

The identity of the Leishmania species involved is a factor that largely determines the clinical manifestations of the disease (Lainson & Shaw 1987, Alexander & Russell 1992) and its response to treatment (Navin et al. 1992, Grogl et al. 1992). The World Health Organization (WHO 1990) recommends isoenzyme electrophoresis and the use of specific monoclonal antibodies as appropriate methods for characterizing Leishmania.

In Costa Rica, although it is known that L. panamensis species is the principal etiological agent of leishmaniasis (Zeledon 1992), there are no detailed and extensive taxonomic studies of the implicated parasites in different areas of the country. Here, we report the identification by isoenzyme electrophoresis and indirect immunofluorescence (IFA) with monoclonal antibodies of 34 Leishmania strains, isolated from different geographical and ecological areas of Costa Rica, from patients with cutaneous leishmaniasis.

MATERIALS AND METHODS

Reference strains - The WHO Leishmania reference strains used in this study are shown in Table I.

Parasite isolation and cultivation - The primary isolation was made from patients with cutaneous lesions by inoculation of material obtained by needle aspiration in biphasic Senekjie's medium with Locke's solution (Tobie & Rees 1948). Promastigotes were expanded in enriched Schneider's Drosophila liquid medium (Gibco, Grand Island, NY) suplemented with 20% fetal bovine serum (FBS), and incubated at 27 C. All strains were stored in liquid nitrogen in Schneider' medium contanining 30% FBS and 7.5% dimethylsulfoxide.

Preparation of extracts for electrophoresis - Cultured Leishmania were harvested during exponential growth by centrifugation at 1,000 x g at 4 C for 10 min. The pellet was washed three times in phosphate buffered saline (PBS), pH 7.2 and resuspended in an equal volume of membrane buffer (14 parts distilled H2O: 1 part 0.1 M Tris/Maleic Acid/EDTA/MgCl2, pH 7.4). The material was stored at -70 C until use. For electrophoresis, the pellets were lysed by three cycles of freezing and thawing in liquid nitrogen.

Isoenzyme electrophoresis - Electrophoresis was carried out in cellulose acetate as described by Kreutzer and Christensen (1980) and Kreutzer et al. (1983).

The following eight enzymatic systems, allowing a good separation of all reference strains, were used: 6-phosphogluconate dehydrogenase (E.C.1.1.1.44 6-PGDH), phosphoglucomutase (E.C.2.7.5.1 PGM), glucose phosphate isomerase (E.C.5.3.1.9 GPI), proline iminopeptidase (E.C.3.4.11.5 PEP-D), alanine aminotransferase (E.C.2.6.1.2 ALAT), aspartate aminotransferase (E.C.2.6.1.1 ASAT), mannose phosphate isomerase (E.C.5.3.1.8 MPI), and nucleoside hydrolase (E.C.3.2.2.1 NH).

Indirect immunofluorescence with monoclonal antibodies - The monoclonal antibodies, and their respective reactivity, used in this study are shown in Table II. Promastigotes, washed in PBS, pH 7.2 and standardized to a concentration of 1x10^6 parasites/ml, were air dried, in 10 ml volumes, on twelve well microscope slides. The slides were submerged in blocking solution (PBS, pH 7.2 + 5% FBS) during 15 min, washed with PBS, and air dried. Each well was covered with 10 ml of the monoclonal antibodies diluted in PBS (1:1000) for 30 min (McMahon-Pratt et al. 1984). The unreactive protein was removed by three washes in PBS and 10 ul/well of anti-mouse IgG labeled with fluorescein and diluted 1:50 was added. Incubation was continued for 30 min at room temperature, unreactive conjugate was removed by three washes in PBS and the slides were mounted with buffered glycerol and observed with a Olympus fluorescence microscope.

RESULTS

Isolation of strains - Thirty-four strains from a total of 104 patients with suspected cutaneous leishmaniasis from the main endemic areas of Costa Rica were isolated. The details of patients from whom isolates originated as well as the identification obtained by enzyme electrophoresis and monoclonal antibodies are presented in Table III.

Isoenzyme analysis - All of the 34 isolates showed isoenzyme patterns similar to members of the subgenus Viannia. Thirty of them had identical profile to L. panamensis (Table III, Fig. 1). Two strains (CCS3 and CBR3) showed slight variations in the migration of 6-PGDH, PGM, and MPI as compared with L. panamensis reference strain (Fig. 1). The two remaining isolates had a distinct profile from L. panamensis and were closer to L. braziliensis. One of them (CBR2) shared 25% of the alleles with L. braziliensis and the other (CHA9) shared 75%.

    Figure 1: diagramatic representation of the isoenzyme variation observed in eight enzymatic patterns. Lp: Leishmania panamensis reference strain (MHOM/PA/71/LS94); 1: Costa Rican strain (MHOM/CR/95/CCS1); 9: Costa Rican strain (MHOM/CR/95/CBR2); 16: Costa Rican strain (MHOM/CR/95/CBR3); 24: Costa Rican strain (MHOM/CR/95/CHA8); 25: Costa Rican strain (MHOM/CR/95/CHA9); Lb: L. braziliensis reference strain (MHOM/BR/75/M2903); Lm: L. mexicana reference strain (MHOM/BZ/82/BEL21). (Of the strains not shown in the figure, one has pattern similar to track 16 and the rest have patterns similar to tracks 1 and 24).

Indirect immunofluorescence - The reactivities of the eight different monoclonal antibodies were tested with 34 Leishmania strain of Costa Rica. Monoclonal antibody B-4 specific for L. panamensis species reacted with 32 of the 34 isolates. Twenty-seven of them reacted strongly whereas five reacted weakly with this antibody. Monoclonal antibody B-16, which reacts selectively with L. braziliensis, reacted with only two of the 34 Costa Rican strains, although the reactivity was weak in comparison with the reactivity of the L. braziliensis reference strain (M-2903). None of the monoclonal antibodies specific for the L. guyanensis (B-19) or L. mexicana complex (M-3, M-8, M-7) reacted with the strains isolated in this study.

DISCUSSION

Previous studies on leishmaniasis in Costa Rica have revealed usefull information about the epidemiology of the disease. It is known that sloths, Bradypus griseus and Choloepus hoffmanni, are the main reservoirs of L. panamensis both in the Caribbean and Pacific basins (Zeledon et al. 1975). It also has been isolated from a specimen of the rodent Heteromys dermarestianus (Zeledon et al. 1977). Futhermore, L. panamensis has been isolated from the sandflies Lutzomyia ylephiletor and Lu. trapidoi (Zeledon & Alfaro 1973, Zeledon et al. 1977). Taxonomic studies of the parasite implicated have been limited to a few areas, confirming the predominance of L. panamensis (Zeledon 1992). Grimaldi et al. (1987) included five Costa Rican strains in a study of characterization with monoclonal antibodies and all were L. panamensis. In the present study, we confirm L. panamensis as the main etiological agent of cutaneous leishmaniasis in all the endemic areas of the country. We also found L. panamensis enzymatic variants and we confirm the existence of L. braziliensis enzymatic variants in two important foci of leishmaniasis in Costa Rica, one in the Pacific and another in the Caribbean side (Fig. 2). The presence of strains with some genetic (enzyme) polymorphism, suggests the existence of other local populations but, as stated by Kreutzer (1996), probably belonging to "a single reproductive population". The electrophoretic mobilities of our L. panamensis and L. braziliensis variants are different from the L. panamensis/L. braziliensis hybrid as characterized by Belli et al. (1994) in Nicaragua.

    Figure 2: geographical distribution of strains. (l) Leishmania panamensis; (s) L. panamensis variant; (O) L. braziliensis variant.

In Costa Rica, mucocutaneous leishmaniasis (MCL) occurs in 3 to 5% of the cases (Zeledon 1992) and three isolates obtained in cultures directly from the mucosal lesions by biopsy were identifyied by zymodemes as L. panamensis. The only other species of Leishmania recorded from Costa Rica is L. infantum from cutaneous lesions detected in an outbreak in the north-west part of the country (Zeledon et al. 1989). A previous report of L. mexicana from the same area originated from a mislabeled culture (Zeledon 1992).

ACKNOWLEDGEMENT

To Dr Diane McMahon-Pratt for providing the monoclonal antibodies. To Mr Fernando Retana for technical assistance. To Dr Paul Hanson for revising the manuscript. To all the medical and laboratory personnel of the Costa Rican Health System who facilitated the obtention of the samples.

REFERENCES

  • Alexander J, Russell DG 1992. The interaction of Leishmania species with macrophages. Advan Parasitol 31: 158-166.
  • Belli A, Miles MA, Kelly JM 1994. A putative Leishmania panamensis/Leishmania braziliensis hybrid is a causative agent of human cutaneous leishmaniasis in Nicaragua. Parasitology 109: 435-442.
  • Carreira PF, Maingon R, Ward RD, Noyes H, Ponce C, Belli A, Arana B, Zeledon R, Sousa OE 1995. Molecular techniques in the characterization of Leishmania isolates from Central America. Ann Trop Med Parasitol 89: 31-36.
  • Grimaldi Jr G, David JR, McMahon-Pratt D 1987. Identification and distribution of New World Leishmania species characterized by serodeme analysis using monoclonal antibodies. Am J Trop Med Hyg 36: 270-287.
  • Grogl M, Thomason TN, Franke ED 1992. Drug resistance in leishmaniasis: its implication in systemic chemotherapy of cutaneous and mucocutaneous disease. Am J Trop Med Hyg 47: 117-126.
  • Kreutzer RD 1996. Genetic similarity among Central and South American populations of Leishmania (Viannia) braziliensis. Am J Trop Med Hyg 55: 106-110.
  • Kreutzer RD, Christensen HA 1980. Characterization of Leishmania spp. by isozyme electrophoresis. Am J Trop Med Hyg 29: 199-208.
  • Kreutzer RD, Semko ME, Hendricks LD, Wright N 1983. Identification of Leishmania spp. by multiple isozyme analysis. Am J Trop Med Hyg 32: 703-715.
  • Lainson R, Shaw JJ 1987. Evolution, classification and geographical distribution, p. 1-120. In W Peters, R Killick-Kendrick (eds), The Leishmaniasis in Biology and Medicine, Academic Press Ltd, London.
  • McMahon-Pratt D, Jaffe CL, Grimaldi G 1984. Application of monoclonal antibodies to the classification of Leishmania species, p. 121-135. In CM Morel, Genes and Antigens of Parasites, Fundacao Oswaldo Cruz, Rio de Janeiro, Brazil.
  • Navin TR, Arana BA, Arana FE, Berman JD, Chajon JF 1992. Placebo-controlled clinical trial of sodium stibogluconate (Pentostam) versus ketoconazole for treating cutaneous leishmaniasis in Guatemala. J Infect Dis 165: 528-534.
  • Tobie EJ, Rees C 1948. The cultivation of Trypanosoma cruzi in dialysate medium. J Parasitol 34: 162-163.
  • WHO-World Health Organization Expert Committee 1990. Control of the leishmaniasis. Tech Rep Ser 793, WHO, Geneva.
  • Zeledon R 1985. Leishmaniasis in North America, Central America and the Caribbean Islands, p. 313-351. In KP Chang, RS Bray (eds), Leishmaniasis, Elsevier Biomedical Press, Amsterdam.
  • Zeledon R 1992. Leishmaniasis en el istmo centro-americano e islas del Caribe, p. 135-148. In H Cosensa, A Kroeger (eds), Enfermedades Parasita-rias de Mayor Prevalencia y Transmitidas por Vectores en Centroamerica, Programa de Coopera-cion Cientifica Internacional.
  • Zeledon R, Alfaro M 1973. Isolation of Leishmania braziliensis from a Costa Rican sandfly and its possible use as a human vaccine. Trans R Soc Trop Med Hyg 67: 416-417.
  • Zeledon R, Hidalgo H, Viquez A, Urbina A 1989. Atypical cutaneous leishmaniasis in a semiarid region of north-west Costa Rica. Trans R Soc Trop Med Hyg 83: 786.
  • Zeledon R, McPherson B, Ponce C 1977. Isolation of Leishmania braziliensis from a wild rodent in Costa Rica. Am J Trop Med Hyg 26: 1044-1045.
  • Zeledon R, Ponce C, De Ponce E 1975. The isolation of Leishmania braziliensis from sloths in Costa Rica. Am J Trop Med Hyg 24: 706-707.

Copyright 1998 Fundacao Oswaldo Cruz - Fiocruz


The following images related to this document are available:

Photo images

[oc98054d.jpg] [oc98054b.jpg] [oc98054a.jpg] [oc98054e.jpg] [oc98054c.jpg]
© Bioline International, 1989 - 2004, Site last up-dated on 17-Ago-2006.