Leishmaniasis in Brazil: XII. Observations on cross-immunity in monkeys and man infected with Leishmania mexicana mexicana, L. m. amazonensis, L. braziliensis braziliensis, L. b. guyanensis and L. b. panamensis.

Cross-immunity trials in monkeys (Cebus apella apella) and observations on experimental and natural infections in man confirm the separate identity of L. mexicana mexicana, L. m. amazonensis, L. b. braziliensis, L. b. guyanensis and L. b. panamensis. Neither L. m. mexicana nor L. m. amazonensis infections gave protection against subsequent challenge with parasites of the L. braziliensis complex; but recovery from infection with subspecies of L. braziliensis in most cases gave firm resistance to infection with the mexicana parasites. The failure of certain New World leishmanias to immunize against each other has an important bearing on taxonomy, future attempts to prepare vaccines against Leishmania, epidemiology and diagnosis.     

IgE antibody against surface antigens of Leishmania promastigotes in American cutaneous leishmaniasis

As IgE-mediated immune mechanisms participate in the host defence against some types of parasites, we evaluated sera from American cutaneous leishmaniasis (ACL) patients for the presence of this antibody against Leishmania. Using monoclonal antibodies against human IgE and an immunoperoxidase staining technique, 48% of the patients sera tested were found to contain IgE antibody that bound strongly to Leishmania promastigotes. A much lower proportion of sera from non-symptomatic subjects from either endemic or non-endemic areas of the disease contained significant levels of anti-Leishmania IgE antibody (6.5% and 0% respectively). The results indicated that the IgE antibody bound predominantly to surface components of the promastigotes, and reactivity against the intracellular amastigote form of the parasite was rarely detected. Somewhat unexpectedly, in a small proportion of the sera, the IgE antibody showed apparent specificity for L. mexicana or L. braziliensis. This study demonstrates that ACL patients can develop anti-Leishmania IgE antibody responses, that seem to be directed preferentially against surface antigens of promastigotes, and that can be strain specific. This raises the question as to the possible contribution of this antibody to the immune defence mechanisms against the parasite.     

Subcellular and taxonomic specificity of monoclonal antibodies to New World Leishmania

Murine monoclonal antibodies to flagellar, surface membrane and cytoplasmic antigens of New World Leishmania were assessed for their taxonomic specificity in enzyme-linked immunosorbent assays with three genera of the family Trypanosomatidae and three species and seven subspecies of the genus Leishmania. Antibodies exhibiting exclusive reactivity with either the flagellum, flagellar pocket, kinetoplast, or nucleus lacked specificity at all phylogenetic levels and, in fact, recognized epitopes common to cultured mammalian cells. Monoclonals to intracellular antigens were capable of distinguishing Leishmania from Trypanosoma and Endotrypanum. Antibodies reactive at the surface membrane could separate six isolates of L. braziliensis from three isolates of L. mexicana but the differences in antigen expression were frequently quantitative rather than qualitative. Antigenic variability within species and/or subspecies often exceeded that which was observed between species and/or subspecies. At least one monoclonal antibody was specific for a surface antigen peculiar to a subpopulation of promastigotes of an L. braziliensis panamensis isolate.     

Development of metacyclic Leishmania promastigotes is associated with the increasing expression of GP65, the major surface antigen

Using immunofluorescence techniques and flow microfluorometry analysis, we have demonstrated that the binding of a monoclonal antibody (VD5/25) produced against GP65, the major surface antigen of Leishmania braziliensis, increased on the surface of stationary-phase promastigotes from all the New World Leishmania species causing mucocutaneous or cutaneous disease as compared with the log-phase parasites. In addition, a sequential development of Leishmania amazonensis promastigotes from a non-infective to an infective stage was demonstrated. Indeed, promastigotes in the stationary phase (days 6-7) were found to be far more infective than those in the logarithmic phase of growth (day 3) both in vitro for mouse peritoneal macrophages and in vivo for BALB/c mice. The intracellular survival and multiplication of L. amazonensis were significantly inhibited when infective promastigotes were treated with the VD5/25 monoclonal antibody. The increasing expression of GP65 on the promastigote surface may thus contribute to Leishmania infectivity. This seems to represent a characteristic mechanism applicable to all New World Leishmania species studied.     

Differentiation and limited proliferation of isolated Leishmania mexicana amastigotes at 27 degrees C.

We have demonstrated here that while many amastigotes of both Leishmania mexicana amazonensis and Leishmania mexicana mexicana differentiate to promastigotes when placed in culture at 27 degrees C, many others remain as amastigotes in their proliferative cycle. We have used this system to examine the effects of the anti-leishmanial compounds amphotericin B, 4-pentenoate and sodium stibogluconate (Pentostam) on the proliferation and differentiation of isolated extracellular amastigotes. Amphotericin B and 4-pentenoate showed little preferential effect on amastigote proliferation over promastigote proliferation although Leishmania mexicana mexicana (strain M379) was generally more resistant to these compounds than Leishmania mexicana amazonensis (strain LV78). This relative resistance was also observed in axenic cultures of proliferating promastigotes. L.m. mexicana amastigote differentiation was inhibited by amphotericin B and 4-pentenoate. Pentostam displayed a greater effect on amastigote differentiation than proliferation in both sub-species although again, a higher concentration was required to produce the same effect in L.m. mexicana.     

Epidemiological studies on American leishmaniasis in Ceará State, Brazil. Molecular characterization of the Leishmania isolates

Two methods of molecular characterization, using monoclonal antibodies and enzyme electrophoresis, were employed in the identification of 36 stocks of Leishmania isolated from human and canine cases of American visceral (AVL) and cutaneous (ACL) leishmaniases in the northern part of Ceará State, Brazil. Molecular homogeneous strains of Leishmania donovani (chagasi) isolated from both human and canine hosts were detected in 14 municipalities. Two more parasite species, L. braziliensis braziliensis and L. mexicana amazonensis, were also detected in the state. The implication of these results with respect to both the clinical and epidemiological data are discussed.     

Disseminated cutaneous leishmaniasis in a field clinic in Bahia, Brazil: a report of eight cases

Eight Bahian patients with cutaneous leishmaniasis who had 20 or more ulcerative lesions of short duration are described. Of five identifications of isolated parasites, four were Leishmania braziliensis braziliensis and one was L. mexicana amazonensis. All but one had positive Montenegro tests initially, and all did after treatment. All had circulating anti-leishmanial antibodies and five responded well to glucantime therapy suggesting a functioning immune response. This is quite different to the anergic hansenoid leishmaniasis seen with L. mexicana amazonensis infections in Brazil. Possible reasons for the occurrence of this type of leishmaniasis are briefly discussed.     

Antibody response in patients with cutaneous leishmaniasis infected by Leishmania (Viannia) braziliensis or Leishmania (Viannia) guyanensis in Brazil

The antibody response against Leishmania (Leishmania) amazonensis crude antigen was measured through the indirect immunofluorescent assay (IFA) and the immunoenzymatic assay (ELISA) in 114 patients with cutaneous leishmaniasis (CL) in Brazil. Fifty-four patients were infected by Leishmania (Viannia) braziliensis, and 60 patients had L. (V.) guyanensis infection. Patients were comparable by age, sex, disease duration and the Montenegro skin test diameter. L. (V.) braziliensis-infected patients showed significant lower number of ulcerated lesions, greater ulcerated area and higher proportion of lymph node enlargement. Sensitivity of IFA was 79.6% (95% CI 66.1-88.9) and 71.7% (95% CI 58.4-82.2) for L. (V.) braziliensis and L. (V.) guyanensis-infected patients, respectively (P=0.324). Sensitivity of ELISA was 98.2% (95% CI 88.8-99.9) and 85.0% (95% CI 72.9-92.5) for L. (V.) braziliensis and L. (V.) guyanensis-infected patients, respectively (P=0.018). Significant differences were observed in the magnitude of the antibody response before treatment with higher levels detected in L. (V.) braziliensis-infected patients by both serologic techniques. Eighty-four patients had serologic evaluations before and 12 weeks after treatment with meglumine antimoniate, 20 mg/kg/day for 20 days. Significant lower optic density values were observed after treatment with both species independent of cure or failure. Our data showed that L. (V.) braziliensis induces a higher antibody response against L. (L.) amazonensis antigens than L. (V.) guyanensis and that down-modulation of the antibody response occurs shortly during disease evolution after treatment. Moreover the data support the use of ELISA as a better tool for detection of antibodies in CL.     

Leishmania mexicana complex: human infections in the Republic of Panama

Parasites of the genus Leishmania responsible for human cutaneous leishmaniasis in the New World form 2 major taxonomic divisions: the Leishmania braziliensis and the L. mexicana complexes. We report the isolation and characterization of the L. mexicana complex among humans in the Republic of Panama. Characterization was based on parasite morphology, pathogenesis in infected golden hamsters, cellulose acetate isoenzyme electrophoretic mobilities, and membrane-specific monoclonal antibodies using the radioimmune binding assay technique.     

Human cutaneous leishmaniasis in Ecuador: identification of parasites by enzyme electrophoresis

Twenty-six strains of Leishmania were isolated from cutaneous lesions in humans in 3 different geographical areas of Ecuador. The species were identified by enzyme electrophoresis as Leishmania braziliensis, L. panamensis, L. guyanensis, L. mexicana, and L. amazonensis.     

Tubulin biosynthesis in the developmental cycle of a parasitic protozoan, Leishmania mexicana: changes during differentiation of motile and nonmotile stages.

Cytodifferentiation in the transition cycle of the parasitic protozoan Leishmania mexicana amazonensis was studied in vitro. The flagellated motile promastigotes transform into the nonmotile amastigotes in 7 days at 35 degrees C intracellularly in the murine macrophage line J774G8. In medium 199 plus fetal bovine serum, the reverse transformation occurs extracellularly at 27 degrees C in 2 days. Slab gel electrophoresis of leishmanias labeled with [35S]methionine during transformation revealed changes in protein banding patterns. The intensity of two protein species with apparent molecular weights of approximately equal to 55,000 increased in the amastigote-to-promastigote differentiation and decreased during the reverse transformation. These two protein species comigrated approximately with alpha- and beta-tubulin of Chlamydomonas flagella in two-dimensional gel electrophoresis. The lower band was further identified as beta-tubulin by immunoprecipitation using rabbit antiserum specific to the beta-tubulin of Chlamydomonas axonemes. The biosynthetic change of tubulin was found to correlate with the morphological change of microtubules is leishmanial flagella and cytoskeleton during transformation.     

Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae)

DNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei).     

Destruction of Leishmania mexicana amazonensis promastigotes by normal human serum

Fresh normal human serum was observed to have a lethal effect on Leishmania mexicana amazonensis promastigotes obtained from laboratory-bred Lutzomyia longipalpis or on promastigotes grown in liquid culture medium, inoculated with the same isolates. Heat inactivation abolished the Leishmania lytic activity from the sera. Resistance of culture promastigotes to lysis by normal human serum was investigated in three isolates of L. m. amazonensis. Development of resistance (up to 7%) was found in only one isolate, obtained from the bone marrow in a human case of visceral leishmaniasis.     

Diffuse cutaneous leishmaniasis in Mexico

In Mexico, 6 cases of diffuse cutaneous leishmaniasis (DCL) were found in widely separated geographic regions. Information was also available on 2 other cases. In addition to the typical clinical features, half of the patients had evidence of nasopharyngeal mucosal involvement. All isolates from the DCL patients were identified as Leishmania mexicana mexicana by isoenzyme analysis and monoclonal antibody typing. In 1 region of Tabasco state where DCL was found, uncomplicated cutaneous leishmaniasis appeared to be highly endemic, and isolates from a few such patients were identified as L. mexicana mexicana. An incidental finding was the recovery of an isolate of L. braziliensis braziliensis from a patient with chiclero ulcer in Oaxaca state. The clinical and epidemiological significance of the reported cases are discussed.     

Isolation of Leishmania mexicana amazonensis from the bone marrow in a case of American visceral leishmaniasis

The first documented human case of visceral leishmaniasis caused by L. mexicana amazonensis is reported. Leishmania were isolated from bone marrow aspirate material from a typical visceral leishmaniasis patient. Further characterization by isoenzyme electrophoresis and by a panel of species- and subspecies-specific monoclonal antibodies established its classification as L. m. amazonensis.     

Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease

One hundred fourteen Leishmania isolates from patients with different clinical forms of leishmaniasis in the State of Bahia, Brazil, were characterized by indirect radioimmune binding assay using specific monoclonal antibodies (serodeme analysis). Seventy-five of these isolates were also analyzed by enzyme electrophoresis, based on 11 enzyme loci; parasite species were compared, according to their characteristic zymodemes, to those of WHO Leishmania reference strains. All isolates could be classified into one of three species: Leishmania amazonensis (n = 40), L. braziliensis (n = 39) or L. chagasi (n = 35). The most interesting information obtained from this study is the realization that L. amazonensis is capable of producing a wide spectrum of disease in humans. Infection with this parasite was associated with many different clinical presentations, including cutaneous leishmaniasis [CL] (20/49 cases), mucocutaneous leishmaniasis [MCL] (5/13 cases) and, of special note, visceral leishmaniasis [VL] (11/46 cases), as well as four cases of post kalaazar dermal leishmaniasis [PKDL]. In situ tissue parasite characterization, by immunoperoxidase assay and employing anti-L. amazonensis amastigote monoclonal antibodies, confirmed the infection with this species in two cases of CL, one case of DCL, one case of MCL and one case of PKDL. Our results also demonstrate the difficulty of parasite differentiation based on clinical grounds, since at least L. amazonensis infection can be associated with all types of leishmanial diseases, and different Leishmania species may be associated with indistinguishable clinical presentations. Since leishmanial parasites may vary in their biological behavior or in their response to treatment, it is important that their identification be made by reliable methods.     

A tissue culture system for the growth of several species of Leishmania: growth kinetics and drug sensitivities

We have developed a simple in vitro method of infecting a continuous human macrophage cell line (U937) with promastigotes of several species of Leishmania. These include L. braziliensis braziliensis, L. b. panamensis, L. donovani, L. mexicana mexicana, L. m. pifanoi, L. tropica, and L. major. The growth kinetics of these species are presented as well as drug sensitivity data. The U937 cell system can be used to determine drug efficacy and eliminates the need to use amastigotes from animal tissues to infect the tissue culture.     

Brazilian Leishmania stocks phenotypically similar to Leishmania major

Screening by enzyme electrophoresis of isolates of New World Leishmania from different geographic areas revealed a number of stocks with enzyme profiles different from those produced by reference strains of described subspecies of L. mexicana, L. braziliensis, and L. donovani. Analysis by six enzymes (aspartate aminotransferase; alanine aminotransferase; malate dehydrogenase; glucose-6-phosphate dehydrogenase; phosphoglucomutase; and glucose-phosphate isomerase) showed that these stocks have identical enzyme profiles and form a distinct zymodeme grouping. These observations were confirmed using the technique of schizodeme analysis and by comparing the k-DNA fingerprints produced by the restriction enzymes MspI, BspRI and AluI. The stocks were further analyzed by monoclonal antibodies and did not react with any of a large panel of L. mexicana, L. braziliensis, and L. donovani species- and/or subspecies-specific monoclonal antibodies using either an indirect radioimmune binding assay or immunofluorescence. These stocks did, however, react with a panel of monoclonal antibodies specific for L. major (formerly L. tropica major). Furthermore, the stocks could not be differentiated from L. major reference strains by enzyme electrophoresis nor could they be distinguished qualitatively from L. major based on their reactivity patterns using 10 Old World cutaneous species- and subspecies-specific monoclonal antibodies. Kinetoplast DNA restriction enzyme profiles, however, were different between these stocks and L. major reference strains. The implications of these results are discussed including the existence of other L. major-like stocks currently misidentified or uncharacterized.     

Micro enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of New World leishmaniasis

Of 21 confirmed cases of New World leishmaniasis, 16 exhibited antibody to antigens of the promastigote of Leishmania braziliensis panamensis by the enzyme-linked immunosorbent assay (ELISA). Comparison of antibody titers obtained by ELISA with titers obtained by indirect immunofluorescence using an amastigote substrate confirmed that the sensitivities of the two techniques were within the same range (r = 0.80). Although sera from patients with New World leishmaniasis failed to react with antigens extracted from epimastigotes of Trypanosoma cruzi, sera from 39 cases of Chagas' disease were reactive with promastigotes of L. braziliensis panamensis. This apparent unidirectional cross-reactivity has been attributed to differences in potency of the antigenic stimulus presented in the two diseases.     

Differential expression of mRNAs for alpha- and beta-tubulin during differentiation of the parasitic protozoan Leishmania mexicana.

The parasitic protozoan Leishmania mexicana amazonensis has two developmental stages: a motile flagellated promastigote stage and a sessile intracellular amastigote stage. In our previous work, cells of the promastigote stage were found to synthesize more tubulin protein than those of the amastigote stage. Here, tubulin mRNAs in these leishmanias were analyzed. Based on dot blot hybridization between total leishmanial RNA and tubulin-specific cDNA probes derived from chicken brain, amastigotes and promastigotes were found to have approximately equal amounts of alpha- and beta-tubulin mRNAs. RNA blotting of leishmanial RNA, using chicken tubulin cDNA probes, showed that amastigotes and promastigotes both gave a single mRNA species of 2100 nucleotides for alpha-tubulin in roughly similar quantities. However, such analysis for beta-tubulin revealed mainly a single mRNA species of 3600 nucleotides for amastigotes and three species of 2800, 3600, and 4400 nucleotides for promastigotes, the smallest mRNA being the most predominant. Thus, regulation of gene expression appears to be different only for beta-tubulin between the two developmental stages of this protozoan.