Leishmaniasis causes significant morbidity and mortality, constituting an important global health problem for which there are few effective drugs. Given the urgent need to identify a safe and effective Leishmania vaccine to help prevent the two million new cases of human leishmaniasis worldwide each year, all reasonable efforts to achieve this goal should be made. This includes the use of animal models that are as close to leishmanial infection in humans as is practical and feasible. Old world monkey species (macaques, baboons, mandrills etc.) have the closest evolutionary relatedness to humans among the approachable animal models. The Asian rhesus macaques (Macaca mulatta) are quite susceptible to leishmanial infection, develop a human-like disease, exhibit antibodies to Leishmania and parasite-specific T-cell mediated immune responses both in vivo and in vitro, and can be protected effectively by vaccination. Results from macaque vaccine studies could also prove useful in guiding the design of human vaccine trials. This review summarizes our current knowledge on this topic and proposes potential approaches that may result in the more effective use of the macaque model to maximize its potential to help the development of an effective vaccine for human leishmaniasis.

A comparison of kDNA restriction-endonuclease fragment patterns from strains representing selected Endotrypanum zymodemes was done by schizodeme analysis. As the degree of heterogeneity within mini-circles varied among species or strains of Endotrypanum, the fingerprint obtained with each of the restriction enzymes was unique for each of these parasites. The data have revealed that this trypanosomatid genus is much more complex than it was originally thought to be.

An atypical case of acquired immunodeficiency syndrome-associated mucocutaneous lesions due to Leishmania braziliensis is described. Many vacuolated macrophages laden with amastigote forms of the parasite were found in the lesions. Leishmanin skin test and serology for leishmaniasis were both negative. The patient was resistant to therapy with conventional drugs (antimonial and amphotericin B). Interestingly, remission of lesions was achieved after an alternative combined therapy of antimonial associated with immunotherapy (whole promastigote antigens). Peripheral blood mononuclear cells were separated and stimulated in vitro with Leishmania antigens to test the lymphoproliferative responses (LPR). Before the combined immunochemotherapy, the LPR to leishmanial antigens was negligible (stimulation index - SI=1.4). After the first course of combined therapy it became positive (SI=4.17). The antigen responding cells were predominantly T-cells (47.5%) most of them with CD8+ phenotype (33%). Very low CD4+ cells (2.2%) percentages were detected. The increased T-cell responsiveness to leishmanial antigens after combined therapy was accompanied by interferon-g (IFN-g) production as observed in the cell culture supernatants. In this patient, healing of the leishmaniasis lesions was associated with the induction of a specific T-cell immune response, characterized by the production of IFN-g and the predominance of the CD8+ phenotype among the Leishmania-reactive T-cells.

This article reviews current concepts of the biology of Endotrypanum spp. Data summarized here on parasite classification and taxonomic divergence found among these haemoflagellates come from our studies of molecular characterization of Endotrypanum stocks (representing an heterogenous population of reference strains and isolates from the Brazilian Amazon region) and from scientific literature. Using numerical zymotaxonomy we have demonstrated genetic diversity among these parasites. The molecular trees obtained revealed that there are, at least, three groups (distinct species?) of Endotrypanum, which are distributed in Central and South America. In concordance with this classification of the parasites there are further newer molecular data obtained using distinct markers. Moreover, comparative studies (based on the molecular genetics of the organisms) have shown the phylogenetic relationships between some Endotrypanum and related kinetoplastid lineages.

Striking similarities at the morphological, molecular and biological levels exist between many trypanosomatids isolated from sylvatic insects and/or vertebrate reservoir hosts that make the identification of medically important parasites demanding. Some molecular data have pointed to the relationship between some Leishmania species and Endotrypanum, which has an important epidemiological significance and can be helpful to understand the evolution of those parasites. In this study, we have demonstrated a close genetic relationship between Endotrypanum and two new leishmanial species, L. (V.) colombiensis and L. (V.) equatorensis. We have used (a) numerical zymotaxonomy and (b) the variability of the internal transcribed spacers of the rRNA genes to examine relationships in this group. The evolutionary trees obtained revealed high genetic similarity between L. (V.) colombiensis, L. (V.) equatorensis and Endotrypanum, forming a tight cluster of parasites. Based on further results of (c) minicircle kDNA heterogeneity analysis and (d) measurement of the sialidase activity these parasites were also grouped together.

Our results have shown the wide diversity of parasites within New World Leishmania. Biochemical and molecular characterization of species within the genus has revealed that much of the population heterogeneity has a genetic basis. The source of genetic diversity among Leishmania appears to arise from predominantly asexual, clonal reproduction, although occasional bouts of sexual reproduction can not be ruled out. Genetic variation is extensive with some clones widely distributed and others seemingly unique and localized to a particular endemic focus. Epidemiological studies of leishmaniasis has been directed to the ecology and dynamics of transmission of Leishmania species/variants, particularly in localized areas. Future research using molecular techniques should aim to identify and follow Leishmania types in nature and correlate genetic typing with important clinical characteristics such as virulence, pathogenicity, drug resistance and antigenic variation. The epidemiological significance of such variation not only has important implications for the control of the leishmaniases, but would also help to elucidate the evolutionary biology of the causative agents.

A pilot study was undertaken to preliminary illustrate the leishmanin skin test (LST) positivity to distinct antigen preparations (derived from promastigote of either Leishmania major or L. amazonensis, or pooled L. mexicana, L. amazonensis and L. guyanensis) in cutaneous leishmaniasis (CL) patients and healthy subjects living in two endemic foci in Nigeria. The study was designed to provide insights into whether cross-species leishmanin, such as that prepared from New World Leishmania could be useful to detect cases of Old World leishmanial infection and to compare the results with LST using L. major-derived leishmanin. The overall LST positivity in individuals from Keana tested with the cross-species leishmanin was 28.7% (27/94), while the positivity rate in the subjects from Kanana tested with the same leishmanin was 54.5% (6/11). Lower positivity values were obtained when L. major (12.5%; 11/88) or L. amazonensis (15.8%; 9/57) was tested as antigen in grossly comparable populations. Moreover, the pooled leishmanin identified most of the subjects (13/14; 92.9%) with active or healed CL, and the maximum reaction sizes were found among positive subjects in this group. No healthy controls (10 total) showed specific DTH response. The LST was useful for assessing the prevalence of subclinical infection and for measuring CL transmission over time. We report for the first time the occurrence of CL in Kanana village of Langtang South local government area of Plateau State

A large number of Endotrypanum stocks (representing an heterogeneous population of strains) have been screened against a panel of monoclonal antibodies (MAbs) derived for selected species of Endotrypanum or Leishmania, to see whether this approach could be used to group/differentiate further among these parasites. Using different immunological assay systems, MAbs considered specific for the genus Endotrypanum (E-24, CXXX-3G5-F12) or strain M6159 of E. schaudinni (E-2, CXIV-3C7-F5) reacted variably according to the test used but in the ELISA or immunofluorescence assay both reacted with all the strains tested. Analyses using these MAbs showed antigenic diversity occurring among the Endotrypanum strains, but no qualitative or quantitative reactivity pattern could be consistently related to parasite origin (i.e., host species involved) or geographic area of isolation. Western blot analyses of the parasites showed that these MAbs recognized multiple components. Differences existed either in the epitope density or molecular forms associated with the antigenic determinants and therefore allowed the assignment of the strains to specific antigenic groups. Using immunofluorescence or ELISA assay, clone E-24 produced reaction with L. equatorensis (which is a parasite of sloth and rodent), but not with other trypanosomatids examined. Interestingly, the latter parasite and the Endotrypanum strains cross-reacted with a number of MAbs that were produced against members of the L. major-L. tropica complex

Monoclonal antibodies specific for selected species complexes of Leishmania have been employed for the characterization of several representative strains of Leishmania isolated from different hosts and localities in the Americas. In the past 15 years, data have been accumulated concerning (i) the specificities of a number of these monoclonal antibodies and (ii) the antigenic variation (level of the expressed antigenic determinants) occurring among New World Leishmania species or strain variants as recognized by the monoclonal antibodies. This report is an attempt to summarize in brief the data accumulated to date on these points and to indicate the directions for future applications of these specific monoclonal antibodies for identification of leishmanial isolates.