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Ecological studies on the sand fly Lutzomyia longipalpis (Lutz & Neiva) were conducted during 1990-1992 in a small rural community in Colombia where American visceral leishmaniasis (AVL) is endemic. Subsamples of sand flies collected weekly from pigpens, the interior of houses, and natural outdoor resting sites were dissected to determine physiological age and Leishmania chagasi Cunha & Chagas infection rates. Eleven female L. longipalpis had flagellates in their gut, 2 of which were successfully cultured and identified as Leishmania chagasi. The reproductive status, stage of ovarian development, and trophic history of female sand flies varied among sites, habitats, and time of collection. The percentage of parous females ranged from about one-third to two-thirds overall and varied seasonally. Of most relevance to AVL transmission was the finding that 8% of L. longipalpis females were multiparous. In addition, our data suggest that L. longipalpis rest inside houses after blood-feeding outdoors, and that this species can blood-feed more than once during a single gonotrophic cycle.

Parasitic protozoa of the genus Leishmania (Kinetoplastida: Trypanosomatidae) are generally thought to multiply by binary fission; however, data from quantitative microspectrophotometry indicate that nuclear fusion or sexual reproduction takes place in the intracellular amastigote form. Among several different Leishmania species, the mean +/- SD nuclear DNA content of all promastigotes (extracellular form) and of some amastigotes (intracellular form) in macrophages was 0.098 +/- 0.032 relative units; in contrast, other amastigotes within the same macrophage had a mean +/- SD nuclear DNA content of 0.219 +/- 0.050. The latter population of amastigotes are apparently produced when the nuclei of a pair of 0.098 amastigotes fuse. These 0.219 amastigotes later go through what is probably the typical meiotic cycle to reform the 0.098 condition we observed among promastigotes. The demonstration of sexual reproduction in Leishmania has important implications for the future direction of research on this medically important parasite.

Characterization is given of a new parasite, Leishmania equatorensis sp. n., which was isolated from the viscera of a sloth (Choloepus hoffmanni) and a squirrel (Sciurus granatensis), captured in humid tropical forest on the Pacific Coast of Ecuador. Data based on biological and molecular criteria, as well as numerical zymotaxonomical analysis, indicate that this parasite is a new species of the L. braziliensis complex. L. equatorensis is clearly distinguishable from all other known species within this complex, using the following molecular criteria: reactivity patterns with specific monoclonal antibodies, isoenzyme electrophoresis, and restriction-endonuclease fragment patterns of kinetoplast DNA (k-DNA).

Characterization of Leishmania colombiensis sp.n. is presented, which on the basis of biological and molecular criteria, appears to be a new member of the L. braziliensis complex. A total of nine isolates of the new parasite were made in Colombia and Panama between 1980 and 1986: two from human cases of cutaneous leishmaniasis, six from phlebotomine sand flies, and one from a sloth. Although most closely related to L. lainsoni, L. colombiensis sp.n. is clearly distinguishable from other members of the genus by its reactivity with monoclonal antibodies, isoenzyme electrophoresis, and restriction endonuclease fragment patterns of kinetoplast DNA (k-DNA).

A total of 340 Leishmania strains, isolated from humans, animals, and sand flies from various regions of Colombia, were examined by isozyme electrophoresis. Seven different Leishmania species were identified. Leishmania panamensis and L. braziliensis were the most common, representing 53.8% and 30.3% of the total, respectively. Isolation rates of the other species were as follows: L. chagasi, 9.4%; L. guyanensis, 2.6%; L. amazonensis, 1.8%; L. mexicana, 0.8%; and a new species requiring additional study, 1.2%. Statistical analyses of representative L. panamensis and L. braziliensis isolates indicated that the populations of these 2 species are genetically very similar. L. panamensis may have a continuous distribution in Colombia west of the eastern Andes Mountains and L. braziliensis may have a continuous distribution east of the western Andes Mountains. Information is given on disease manifestations of the parasites in human hosts and on isolation records from sand flies and animals.

Previously described monoclonal antibodies IX-IF9-D8, IX-2H7-E10 and IX-5H9-C1 recognize promastigote stage-specific determinants present on externally exposed membrane proteins of axenically cultured Leishmania mexicana amazonensis. In the present study, these antigens were demonstrated by indirect immunofluorescence to be present on promastigotes found in the gut lumen of infected Lutzoymia longipalpis. The presence of these antigens on promastigotes found in infected sandflies suggests that the same antigens should be relevant for protective immunity studies and for species identification of Leishmania encountered in epidemiological studies.

A review of the epidemiologic aspects of the New World leishmaniases, including their known geographic distribution, etiologic agents, zoonotic reservoirs, and insect vectors, based on biological and molecular characterization of Leishmania isolates is presented. Data summarized in this paper on parasite taxonomy and geographic distribution come from our studies of greater than 1,000 New World Leishmania isolates identified by species-specific monoclonal antibodies using an indirect radioimmune binding assay and from scientific literature.

The development of Leishmania (Viannia) panamensis in a natural sand fly host, Lutzomyia gomezi, was studied by light and transmission electron microscopy. New aspects of peripylarian parasite behavior and morphology in the sand fly gut, early bloodmeal stages, and ultrastructural development in the anterior gut were documented. Eight distinct morphological forms were observed in the life cycle of the parasite within the insect. In the bloodmeal, amastigotes (1) transformed into stumpy promastigotes (2) which rapidly multiplied, resulting in spatulate-shaped nectomonad promastigotes (3) and elongate nectomonad promastigotes (4). These latter forms migrated primarily into the hindgut, where both were observed attached (=haptomonad phase) to the cuticular intima by hemidesmosomes within extremely shortened flagella. Spatulate haptomonad promastigotes predominated, colonizing the entire length of the hindgut, with the greatest density at 2 disjunct sites: the pylorus/ileum and the anterior rectum/rectal sac. Paramastigotes and dividing flagellates were rare. Some parasites migrated directly to the cardia/stomodeal valve region without a hindgut phase; however, major movement anteriorly was from the hindgut beginning at 6 days postinfection. In the cardia lumen, dividing short Type A promastigotes (5) predominated, intermixed with short Type B promastigotes with longer flagella (6). Paramastigotes (7) were free-swimming in the lumen as well as attached to the stomodeal valve. The primary colonizers of the valve were pear-shaped haptomonad promastigotes (8), with flagella of variable lengths and multi-segmented hemidesmosomal attachment points to the intima. Promastigotes and paramastigotes colonized the esophagus-pharynx region and attached to the foregut lining by flagellar hemidesmosomes. Both forms may represent infective stages of L. (V.) panamensis; however, no parasites were detected in the cibarium or proboscis. L. (V.) panamensis appeared well-adapted to the gut of Lu. gomezi, multiplying extensively at 2 sites, changing morphological form, and adhering to host surfaces by variously modified flagellar hemidesmosomes.

An in vitro assay was developed to study the recognition mechanism for attachment of Leishmania flagella to sand fly midgut epithelium. Frozen sections of sand fly guts were incubated with flagella preparations, and probed with a flagella-specific monoclonal antibody. Tissue-specific adhesion of flagella to midgut epithelium was demonstrated by indirect immunofluorescence. None of the 13 sugars, screened to test for possible lectin-mediation, appeared to significantly inhibit the adhesion of flagella to gut sections. Similarly no inhibition was achieved by incubating flagella with pep 63 which inhibits the promastigote-macrophage recognition mechanism. Significant inhibition was attained by incubating flagella preparations with a monoclonal antibody which binds to a flagellar membrane-component. The possible relevance of the described mechanism for the biology of Leishmania in their sand fly hosts, is discussed.

The development of Leishmania chagasi, etiologic agent of American visceral leishmaniasis, was studied by light and electron microscopy in the gut of the sand fly, Lutzomyia longipalpis, a natural vector. New aspects of suprapylarian Leishmania behavior were elucidated. In the sand fly midgut, amastigotes transformed into promastigotes (division promastigote I) during a first division sequence within the bloodmeal. Secondary division of these promastigotes resulted in a second form (division promastigote II), and these subsequently elongated into nectomonad promastigotes. Nectomonads existed in long and short populations which divided in the bloodmeal and throughout the midgut lumen after escape from the peritrophic membrane. Nectomonads adhered to the midgut cells in a highly organized manner, with their flagella embedded deep into microvilli and cytoplasm. Migration of parasites from the posterior midgut into the cardia/stomodeal valve region at 36 hr was associated with breakdown of the peritrophic membrane anteriorly. Posterior breakdown at 48 hr resulted in a peritrophic tube open at both ends containing some parasites within the digesting bloodmeal for up to 6 days postinfection. At the stomodeal valve, a myriad of slender and rounded promastigotes attached to the intima by flagellar hemidesmosomes; these may represent a transformation sequence from slender nectomonads to pear-shaped haptomonads. Pear-shaped forms appear to be precursors of paramastigotes, which also attached to the valve intima. Both rounded haptomonads and paramastigotes were found in the esophagus, dividing in a complex sequence initiated by posterior cleavage of the cytoplasm producing unique heart-shaped forms. Dividing paramastigotes also colonized the pharynx up to the cibarial valve. The ultrastructure of paramastigotes suggested that they may be infective forms, capable of some motility in the foregut. Free-swimming "infective" promastigotes were observed throughout the midgut and foregut, were attached in the pharynx (armature region), and were associated with the labrum-epipharynx of the proboscis in 3.6% of flies (16 days). The fine structure of hemidesmosomes in the foregut showed regional specializations, including the presence of plasmalemmar bridges in the gap space.

Six strains of Leishmania isolated from wild mammals and humans on the Pacific Coast of Ecuador were identified by isoenzyme electrophoresis and by their reactivity patterns to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay. Single isolates from Sciurus vulgaris, Potos flavus, and Tamandua tetradactyla were identified as Leishmania amazonensis. Three other strains, isolated from cutaneous lesions of humans, were identified as Leishmania panamensis.