Molecular Detection of Leishmania (Leishmania) mexicana in Sandflies from the State of Yucatan, Mexico.

Background: Leishmaniases are a group of vector-borne zoonotic diseases of public health relevance within the tropical and subtropical regions of the world. The state of Yucatan is a vulnerable and receptive area to localized cutaneous leishmaniasis (LCL) due to its proximity to the high-transmission endemic states of Campeche and Quintana Roo. Autochthonous cases of LCL caused by Leishmania (Leishmania) mexicana have been documented in the state, showing a geographical expansion of the disease. Materials and Methods: Using CO2-supplemented Centers for Disease Control and Prevention light traps and Shannon traps, we captured anthropophilic sandflies in the surroundings of a locality with recent records of autochthonous cases of LCL. Sandflies carrying Leishmania DNA were evidenced using PCR. Results: A total of 140 Phlebotominae (Diptera: Psychodidae) females of four species were captured: Lutzomyia (Tricholateralis) cruciata (Coquillett), Psathyromyia (Psathyromyia) shannoni (Dyar), Lutzomyia (Lutzomyia) longipalpis (Lutz and Neiva), and Dampfomyia (Coromyia) deleoni (Fairchild and Hertig). Molecular results showed that 6.1% (95% confidence interval [CI] = 2.3-12.9%) of Lu. cruciata and 43.8% (95% CI = 19.8-70.1%) of Pa. shannoni showed evidence of carrying L. (L.) mexicana DNA. Conclusion: We provide evidence of anthropophilic sandflies carrying L. mexicana DNA in a municipality with recorded autochthonous cases of LCL caused by this parasite species in the state of Yucatan, suggesting the emergence of new focus of LCL in Mexico.

Leishmania amazonensis: characterization of an ecto-3'-nucleotidase activity and its possible role in virulence.

Ecto-3'-nucleotidase/nuclease (3'NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 3'mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-3'-nucleotidase activity (La3'-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La3'-nucleotidase inhibitor and approach the possible involvement of ecto-3'-nucleotidase in cellular adhesion. La3'-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La3'-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu(2+) ions. Interestingly, ecto-3'-nucleotidase activity is 60-fold higher than that of ecto-5'-nucleotidase in L. amazonensis. Additionally, ecto-3'-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage-parasite attachment/invasion was increased by 400% in the presence of adenosine 3'-monophosphate (3'AMP); however, this effect was reverted by TTM treatment. We believe that La3'-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection.

Two phylogenetically divergent isocitrate dehydrogenases are encoded in Leishmania parasites. Molecular and functional characterization of Leishmania mexicana isoenzymes with specificity towards NAD+ and NADP.

Leishmania parasites are of great relevance to public health because they are the causative agents of various long-term and health-threatening diseases in humans. Dependent on the manifestation, drugs either require difficult and lengthy administration, are toxic, expensive, not very effective or have lost efficacy due to the resistance developed by these pathogens against clinical treatments. The intermediary metabolism of Leishmania parasites is characterized by several unusual features, among which whether the Krebs cycle operates in a cyclic and/or in a non-cyclic mode is included. Our survey of the genomes of Leishmania species and monoxenous parasites such as those of the genera Crithidia and Leptomonas (http://www.tritrypdb.org) revealed that two genes encoding putative isocitrate dehydrogenases (IDHs) -with distantly related sequences- are strictly conserved among these parasites. Thus, in this study, we aimed to functionally characterize the two leishmanial IDH isoenzymes, for which we selected the genes LmxM10.0290 (Lmex_IDH-90) and LmxM32.2550 (Lmex_IDH-50) from L. mexicana. Phylogenetic analysis showed that Lmex_IDH-50 clustered with members of Subfamily I, which contains mainly archaeal and bacterial IDHs, and that Lmex_IDH-90 was a close relative of eukaryotic enzymes comprised within Subfamily II IDHs. 3-D homology modeling predicted that both IDHs exhibited the typical folding motifs recognized as canonical for prokaryotic and eukaryotic counterparts, respectively. Both IDH isoforms displayed dual subcellular localization, in the cytosol and the mitochondrion. Kinetic studies showed that Lmex_IDH-50 exclusively catalyzed the reduction of NAD+, while Lmex_IDH-90 solely used NADP+ as coenzyme. Besides, Lmex_IDH-50 differed from Lmex_IDH-90 by exhibiting a nearly 20-fold lower apparent Km value towards isocitrate (2.0 µM vs 43 µM). Our findings showed, for the first time, that the genus Leishmania differentiates not only from other trypanosomatids such as Trypanosoma cruzi and Trypanosoma brucei, but also from most living organisms, by exhibiting two functional homo-dimeric IDHs, highly specific towards NAD+ and NADP+, respectively. It is tempting to argue that any or both types of IDHs might be directly or indirectly linked to the Krebs cycle and/or to the de novo synthesis of glutamate. Our results about the biochemical and structural features of leishmanial IDHs show the relevance of deepening our knowledge of the metabolic processes in these pathogenic parasites to potentially identify new therapeutic targets.

Calmodulin Polymerase Chain Reaction-Restriction Fragment Length Polymorphism for Leishmania Identification and Typing.

A precise identification of Leishmania species involved in human infections has epidemiological and clinical importance. Herein, we describe a preliminary validation of a restriction fragment length polymorphism assay, based on the calmodulin intergenic spacer region, as a tool for detecting and typing Leishmania species. After calmodulin amplification, the enzyme HaeIII yielded a clear distinction between reference strains of Leishmania mexicana, Leishmania amazonensis, Leishmania infantum, Leishmania lainsoni, and the rest of the Viannia reference species analyzed. The closely related Viannia species: Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, are separated in a subsequent digestion step with different restriction enzymes. We have developed a more accessible molecular protocol for Leishmania identification/typing based on the exploitation of part of the calmodulin gene. This methodology has the potential to become an additional tool for Leishmania species characterization and taxonomy.

Study of Leishmania mexicana electrokaryotype by clamped homogeneous electric field electrophoresis.

In spite of the wide use of electrokaryotypes for Leishmania identification, the number, ploidy and associated functions of the chromosomal bands still remain controversial topics. In the present work, we studied these problems in the pathogenic organism Leishmania mexicana using the clamped homogeneous electric field electrophoresis (CHEF) technique, which allows the separation of uniform chromosomal bands in one run. We arrived at the following general conclusions: (i) a comparative densitometric study using haploid Saccharomyces cerevisiae cells as standard reveals that although L. mexicana is an aneuploid organism, its larger bands are diploid; (ii) a total of 18 chromosomal bands ranging from 3.2 to 0.245 Mbp were resolved. These molecules summed to 1.34 X 10(8) bp, a value within the range of the Leishmania genome; (iii) in hybridisation experiments using different housekeeping gene probes, the majority hybridised with chromosomal band 17 or 18 of L. mexicana, with additional locations for some genes; (iv) the presence of the ubiquitous leishmanial (CA/GT)n sequence in the DNA probes could lead to erroneous gene localisation.

The ribosomal gene spacer as a tool for the taxonomy of Leishmania.

A recombinant DNA ribosomal gene spacer of Leishmania braziliensis Y was used as probe to test different Leishmania species. Based on the similarity of their restriction patterns, three groups were distinguished with respect to international Leishmania references: first a group with a similar restriction pattern to L. braziliensis Y and the reference organism L. mexicana garnhami JAP78; a second group with restriction patterns similar to the reference organism L. mexicana mexicana M379; and finally a group where all the restriction patterns were related to the reference organism L. braziliensis braziliensis M2903. These results support the existence of L. garnhami as an independent Leishmania species; they confirm previous studies on L. mexicana and L. braziliensis and open the way for the more exact diagnosis of New World Leishmaniasis.

Molecular karyotype of species and subspecies of Leishmania.

The DNA karyotypes of three species and several subspecies of New World Leishmania were found to be distinct. The karyotypes were more similar among closely related isolates than among more distantly related groups. Two classes of chromosomal DNA differences were detected among stocks; +/- 50 kb size differences among DNAs, some of which were shown to contain homologous sequences, and DNAs having no obvious corresponding chromosomal DNA in other isolates. A total of 14-24 chromosomal DNA bands were resolved, depending on the isolate, but densitometric analyses suggest that these isolates contain 26-33 distinct DNA molecules. These molecules total about 2.5 X 10(7) bp, a substantial fraction of the genomic DNA. The chromosomal DNA locations of gene sequences homologous to alpha- and beta-tubulin, ribosomal RNA, thymidylate synthetase-dihydrofolate reductase, and the H-region sequence were determined. The homologous sequences were located on chromosomal DNAs of similar, but not identical sizes among different stocks. We also found species- and some subspecies-specific beta-tubulin chromosomal loci. We conclude that the DNA karyotype is useful for stock identification, taxonomy, and gene localization in Leishmania. Its potential for identifying the species and subspecies in natural infections appears less useful unless applied in conjunction with specific hybridization probes.

Molecular analysis of the cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase in Leishmania mexicana.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in two cell compartments of Leishmania mexicana promastigotes. These activities could be attributed to two different isoenzymes, one residing in glycosomes, the other in the cytosol. We have cloned and sequenced the genes for both isoenzymes. The glycosomal enzyme is encoded by two tandemly linked genes of identical sequence and contains features frequently found in glycosomal enzymes: the presence of peptide insertions, a small carboxy-terminal extension with a potential glycosomal targeting signal (-SKM) and an excess of positively charged residues (net charge +7). Only one open reading frame was detected for the cytosolic enzyme. The amino acid sequences of the two proteins are only 55% identical. We discuss some evolutionary aspects of the observed organization of the GAPDH genes in the Trypanosomatidae and the role of the two isoenzymes in the metabolism of these organisms. The possibility to develop GAPDH-specific inhibitors that will be effective against the enzyme of various parasitic members of this family is explored.

18S rRNA sequences of Leishmania enriettii promastigote and amastigote.

Dideoxy sequencing with reverse transcriptase and universal primers was used to obtain partial sequences of the 18S rRNAs from the promastigote and amastigote life-cycle stages of L. enriettii. Approximately 1400 nucleotides of sequence from the two stages were compared. Unlike Plasmodium berghei, in which 18S rRNAs from the mosquito stage and the mammalian stage of the life cycle are only 96.5% similar, the amastigote and promastigote rRNAs of L. enriettii are identical. In addition, a comparison of 1425 bases of the L. enriettii promastigote sequence with the published sequence of L. donovani revealed only four differences; the two sequences are 99.8% similar. A likely explanation for this high similarity, considering the 97% similarity between L. donovani and the related genus Crithidia fasciculata, is that the two species are closely related and of comparatively recent origin. The low diversity between the 18S rRNA sequences of Leishmania species is similar to that reported for 13 Tetrahymena species, where similarities ranged from 98.1 to 99.9%, but different from the pattern reported in the genus Naegleria, where divergence was greater.

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.

Isoenzyme characterization of Leishmania isolated from human cases with localized cutaneous leishmaniasis from the State of Campeche, Yucatan Peninsula, Mexico.

Seventy-five isolates from the State of Campeche, Mexico, an area endemic for localized cutaneous leishmaniasis (LCL), were characterized by isoenzyme markers (glucose phosphate isomerase, mannose phospate isomerase, nucleoside hydrolase, phosphoglucomutase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase). Seventy (93.3%) were identified as Leishmania (Leishmania) mexicana and 5 (6.7%) as L. (Viannia) braziliensis. This is the first report of authochthonus human LCL caused by L. (V.) braziliensis in the State of Campeche, Yucatan Peninsula, Mexico.

Identification and distribution of New World Leishmania species characterized by serodeme analysis using monoclonal antibodies.

Five hundred thirty stocks of Leishmania isolated from human and domestic and wild reservoir hosts, representing a wide geographic distribution of endemic foci of American cutaneous (ACL) and visceral leishmaniases (AVL) were characterized and identified at species and/or subspecies levels based on their reactivity to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay. This study confirms and extends our preliminary results on the high specificity of some of these monoclonals for the L. braziliensis, L. mexicana, and L. donovani complexes. This study also demonstrates the relative stability of these molecular markers and the general usefulness of the method for parasite identification. Two hundred ninety-two of 420 isolates of ACL were classified as members of the L. braziliensis complex. Two hundred twenty-seven were L. b. braziliensis; these showed the widest geographical distribution (Brazil: Amazonas, Bahia, Ceara, Espirito Santo, Goias, Minas Gerais, Para, Paraiba, Rio de Janeiro, and Sao Paulo; Honduras: Santa Barbara and Yoko; Peru: Ancash, Piura, and Ucayali; and Venezuela: Cojedes, Distrito Federal, Lara, Portuguesa, Vale Hondo, Yaracuy, and Zulia). Forty-one stocks were identified as L. b. guyanensis (from North Brazil: Amazonas, Amapa, Para, and Rondonia). Twenty-one stocks were identified as L. b. panamensis (from Costa Rica: Alajuela, Guanacasten, Limon, Puntarenas, and San Jose; and Honduras: El Paraiso, and Olancho). Out of 128 isolates classified as members of the L. mexicana complex, 74 were differentiated as L. m. amazonensis (from Bolivia; Brazil: Amazonas, Bahia, Ceara, Goias, Maranhao, Mato Grosso do Norte, and Para; Peru: Pasco Forest and Van Humboldt; and Venezuela: Carabobo, Guarico, and Merida). Forty-four stocks were identified as L. m. venezuelensis (from Venezuela: Lara). Six stocks were L. m. mexicana (from Belize; and Mexico: Campeche [corrected] and Quintana Roo, Yucatan). One hundred ten isolates from AVL were identified as L. donovani chagasi (from Brazil: Bahia, Ceara, Maranhao, Minas Gerais, Mato Grosso do Sul, Piaui, Rio de Janeiro, and Sergipe; and Honduras: Valle). The implications of these results with respect to both the clinical and epidemiological data (including the detection of seven unusual characterized stocks) are discussed.

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.

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.

Characterization and classification of leishmanial parasites from humans, wild mammals, and sand flies in the Amazon region of Brazil.

Ninety-four leishmanial isolates from the Brazilian Amazon Region (Amapá, Amazonas, Pará, and Rondônia) were identified and classified using specific monoclonal antibodies and an indirect radioimmunoassay (serodeme analysis); eighty-two were also characterized by enzyme electrophoresis (zymodeme analysis), the results of which were subjected to a numerical phenetic analysis. Six isolates from humans (3), Didelphis marsupialis (1), Lutzomyia olmeca nociva (1), and Lu, reducta (1) showed reactivity patterns and isoenzyme profiles similar to those obtained with the Leishmania amazonensis reference strains, and were identified as this species. Eighty-six stocks were classified as members of the L. braziliensis complex; of these, 61 were L. guyanensis or variants, which presented three serodeme subtypes, but whose isoenzyme profiles were all similar to the reference strain. A total of 15 isolates were distinguished as L. braziliensis or variants and were classified into five serodeme subtypes. The isolate from Psychodopugus davisi appeared, from the numerical analysis, to be a distinct parasite species. Ten isolates showed reactivity patterns and isoenzyme profiles similar to those obtained with the L. naiffi reference strain. A parasite isolated from Ps. claustrei appeared to be different from all reference strains by both techniques, and was classified as probably being a new species. The importance of these results with respect to the taxonomic status of the New World Leishmania, and their implications for both clinical and epidemiologic data are discussed.

Molecular karyotype characterization of Leishmania panamensis, Leishmania mexicana, and Leishmania major-like parasites: agents of cutaneous leishmaniasis in Ecuador.

Molecular karyotypes of Leishmania isolates from patients with cutaneous leishmaniasis in Ecuador were analyzed by pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization. The DNA karyotypes of L. major-like parasites were similar between two human isolates from a lowland coastal and a highland Andean region, but were apparently different from those of eleven World Health Organization reference strains including L. major. The smallest chromosome of 240 kilobases in L. major-like parasites was found to belong to the 715-class of small linear chromosomal DNAs, which have been shown to appear in some lines of Leishmania. Chromosome banding patterns of L. mexicana isolates exhibited a novel, ordered, chromosomal ladder, and were identical among four human isolates and one canine isolate from a restricted geographic region in the Andes. On the other hand, minor chromosome size polymorphisms were observed among three L. panamensis isolates from different endemic regions near the Pacific Coast. Chromosomal locations of dihydrofolate reductase-thymidylate synthetase and P-glycoprotein genes revealed further differences in chromosomal organizations among these Leishmania species in Ecuador. These results indicate that karyotype analysis by PFGE is useful for epidemiologic studies of leishmaniasis in Ecuador.

Coexistence of two species of Leishmania in the digestive tract of the vector Lutzomyia ovallesi.

Leishmania isolated from the digestive tract of a naturally infected Lutzomyia ovallesi sand fly were cultured in blood agar for rapid growth, cloning, and subsequent identification through schizodeme analysis, dot-blot hybridization, use of monoclonal antibodies with various specificities and absorbed polyclonal antibodies. Twenty-three clones isolated from the primary culture were identified. The results showed that parasites belonging to some clones corresponded to the L. mexicana complex, while others belonged to the L. braziliensis complex. These results clearly establish the coexistence of two Leishmania species in the digestive tract of a single Lu. ovallesi sand fly.

Flagellate infections of Brazilian sand flies (Diptera: Psychodidae): isolation in vitro and biochemical identification of Endotrypanum and Leishmania.

Flagellate infections were found in 1,063 of 18,895 sand flies collected in the states of Amazonas, Pará, Rondonia and Acre, Brazil. Infection rates were 13.4% (species group Shannoni); 7.5% (subgenus Nyssomyia); 6.7% (subgenus Lutzomyia series Cruciata); 0.5% (genus Psychodopygus) and 3.1% for other sand flies (various subgenera). Leishmania braziliensis guyanensis and L. mexicana amazonensis were isolated, respectively, from the known vectors, Lutzomyia umbratilis and L. flaviscutellata. Single stocks of L. braziliensis-like and L. mexicana-like organisms were isolated, respectively, from L. whitmani and L. yuilli. Thirty-eight flagellate stocks, isolated by direct culture from sand flies were characterized in detail by morphology in culture, behavior in hamsters and mice and by enzyme profiles. Sixteen stocks from Lutzomyia sp. (Shannoni group) were identified as Endotrypanum schaudinni; 8 stocks from Lutzomyia sp. (Shannoni group) were identified as Endotrypanum sp.; 7 stocks from Psychodopygus ayrozai and P. paraensis were identified as Leishmania sp. previously isolated from the armadillo, Dasypus novemcinctus; 2 stocks of Trypanosoma rangeli were isolated from recently fed Lutzomyia sp. (Shannoni group) sand flies; the remaining 5 stocks from L. umbratilis and L. yuilli could not be identified. Observations suggested that Shannoni group sand flies were the natural vectors of Endotrypanum. Leishmania sp. infections in the man-biting flies P. ayrozai and P. paraensis were restricted to the midgut and associated with recent bloodmeals. Unidentified flagellates in L. umbratilis and L. yuilli were distributed throughout the digestive tract with no trace of bloodmeals.

Andean leishmaniasis in Ecuador caused by infection with Leishmania mexicana and L. major-like parasites.

Between 1986 and 1988, epidemiologic studies were carried out in a small rural community in an Andean region of Ecuador, where cutaneous leishmaniasis is highly endemic. A total of 25 human cases, positive for Leishmania parasites by culture and/or smear, were examined. Fourteen of the cases were in infants less than one year of age, suggesting intradomiciliary transmission of the disease. Clinically, many of these cases were similar to descriptions of "uta," a form of cutaneous leishmaniasis which occurs in Andean regions of Peru and is reported caused by L. peruviana. Of the 11 positive cultures obtained from human cases in the present study, eight were identified by molecular characterization as L. mexicana and three were identified as L. major-like. Two additional isolates of L. mexicana were also made from an infected dog and from a sand fly, Lutzomyia ayacuchensis, living in the region, thus implicating the latter species as possible reservoir and vector, respectively, of L. mexicana in this highland community. The significance and validity of recent isolates of L. major-like parasites from the New World are also discussed.

Isoenzyme characterization of 112 Leishmania isolates from French Guiana.

112 Leishmania isolates, obtained in French Guiana from human lesions, phlebotomine sandflies and wild mammals, were characterized by isoenzyme electrophoresis. Leishmania braziliensis guyanensis and L. mexicana amazonensis were found parasitizing different natural hosts. L.b. guyanensis was the dominant species (103 isolates) responsible for most of the human lesions (96.7%). Based on variations observed in 2 enzymes, 3 distinct zymodemes were distinguished within the L.b. guyanensis taxon.