Superiority of Hemoglobin to Hemin for Cultivation of Leishmania tropica Promastigotes in Serum-Free Media1

Leishmania tropica promastigotes grew slowly but could be maintained for long periods in serum-free hemin-containing media formulated previously for other Leishmania species or in slightly simplified versions of these media. Replacement of hemin in the medium by hemoglobin resulted in a much longer log phase and a significant reduction in the doubling time. Cell counts in cultures started at 1 times 10⁵ cells/ml increased 400-fold in less than 140 h in the hemoglobin-containing media. These media also proved suitable for growing L. donovani and L. enriettii promastigotes.     

Leishmania (Viannia) braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania) amazonensis

We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania) amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia) braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V.) braziliensis replication rates and decreased the time of maximum replication to 48 h.     

Infectivity and virulence of Leishmania donovani promastigotes: a role for media,source, and strain of parasite

Transformation of promastigotes of Leishmania donovani strain AG83 from amastigotes derived from an infected animal was studied in three media, Schneider's Drosophila medium (SDM), Medium 199 (M199), and biphasic M199 (B-M199) with 10% fetal bovine serum. The media, SDM and B-M199, both supported a more efficient transformation of promastigotes in comparison with M199. Infectivity studies in hamsters and BALB/c mice showed that promastigotes isolated in B-M199 were several folds more infective than those obtained from M199. Comparison of the infectivity and virulence of promastigotes of AG83, with a recent isolate of kala-azar, SL94, harvested under similar conditions, revealed greater infectivity of SL94 for both macrophages and animal models. The present study demonstrates that the medium used for the conversion of amastigotes to promastigotes plays a major role in determining the infectivity of the freshly transformed L. donovani promastigotes in hamsters and BALB/c mice. The source and the strain of the parasite also influence the outcome of L. donovani infection.     

Two separate growth phases during the development of Leishmania in sand flies: implications for understanding the life cycle

The life cycle of Leishmania alternates between two main morphological forms: intracellular amastigotes in the mammalian host and motile promastigotes in the sand fly vector. Several different forms of promastigote have been described in sandfly infections, the best known of these being metacyclic promastigotes, the mammal-infective stages. Here we provide evidence that for Leishmania (Leishmania) mexicana and Leishmania (Leishmania) infantum (syn. chagasi) there are two separate, consecutive growth cycles during development in Lutzomyia longipalpis sand flies involving four distinct life cycle stages. The first growth cycle is initiated by procyclic promastigotes, which divide in the bloodmeal in the abdominal midgut and subsequently give rise to non-dividing nectomonad promastigotes. Nectomonad forms are responsible for anterior migration of the infection and in turn transform into leptomonad promastigotes that initiate a second growth cycle in the anterior midgut. Subsequently, leptomonad promastigotes differentiate into non-dividing metacyclic promastigotes in preparation for transmission to a mammalian host. Differences in timing, prevalence and persistence of the four promastigote stages were observed between L. mexicana and L. infantum in vivo, which were reproduced in cultures initiated with lesion amastigotes, indicating that development is to some extent governed by a programmed series of events. A new scheme for the life cycle in the subgenus Leishmania (Leishmania) is proposed that incorporates these findings.     

Haemoflagellates: commercially available liquid media for rapid cultivation.

The successful cultivation of a variety of haemoflagellates in three different liquid media is reported. These media include medium 199, Grace's insect tissue-culture medium and Schneider's drosophila medium, each in combination with 30% (v/v) foetal calf serum. These media were used to cultivate Old and New World species of visceral and cutaneous human Leishmania, as well as Leishmania species isolated from sandflies, rodents, and reptiles. Four strains of Trypanosoma cruzi, an isolate of T. R-angeli and and an isolate of T. lewisi have also been cultivated in these media. One or more of these media have been used to cultivate 121 strains of haemoflagellates, including at least 14 different species (11 Leishmania and 3 Trypanosoma) and many geographic isolates or strains. The Leishmania include L. braziliensis, L. peruviana, L. mexicana, L. tropica, L. donovani, L. chagasi, L. enriettii, L. hertigi, L. hoogstraali, L. adleri, and L. agamae. Using the Schneider's based medium, we have obtained primary isolates of both cutaneous and visceral Leishmania of man and of experimentally infected laboratory rodents and canines. Freeze-dried preparations of the Schneider's based medium that were reconsituted with distilled water after 24 months of storage at ambient temperature have proven to be suitable cultivation media. This feature makes the media valuable field tools. The various species of human Leishmania cultivated in these media have in our experience demonstrated no differences in growth rate, viability after liquid nitrogen preservation, or infectivity for laboratory animals and tissue-culture cells compared with promastigotes derived from blood-agar cultivation.     

Comparative real-time kinetic analysis of human complement killing of Leishmania infantum promastigotes derived from axenic culture or from Phlebotomus perniciosus

Although Leishmania metacyclic promastigotes are generally considered resistant to human complement, studies of in vitro-cultured axenic stationary promastigotes using serum concentrations that approximate physiological plasma conditions indicate complement sensitivity. Natural Leishmania infection is caused by sand fly-inoculated promastigotes, whose complement resistance has not been analyzed systematically. We compared Leishmania susceptibility to human complement in L. infantum promastigotes derived from in vitro cultures and from sand flies. Phlebotomus perniciosus sand flies were fed with axenic promastigotes, L. infantum-infected U-937 cells, or spleen cells from L. infantum-infected hamsters. On selected days post-feeding, flies were dissected and promastigotes isolated; in addition, axenic promastigotes were obtained from culture at equivalent days of growth. In near-physiological serum concentration and temperature conditions, measurement of real-time kinetics of propidium iodide uptake showed that approximately 90% of axenic- and sand fly-derived promastigotes were rapidly killed by complement. We found no substantial differences between promastigotes from axenic culture, those isolated from flies on different post-feeding days, or those generated in flies fed with distinct inocula. The results indicate that Leishmania susceptibility to human complement is independent of promastigote developmental stage in the sand fly mid-gut and in axenic culture.     

Fusion between Leishmania amazonensis and Leishmania major parasitophorous vacuoles: live imaging of coinfected macrophages

Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes--which were destroyed--differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation--a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the mechanisms involved in the recognition and fusion of PVs.     

Metacyclogenesis of Leishmania spp: species-specific in vitro transformation, complement resistance, and cell surface carbohydrate and protein profiles

Metacyclic (stationary) and logarithmic (log) forms of promastigotes of Leishmania donovani and Leishmania major were characterized in several ways. The highly active metacyclic forms were larger with more protein and less carbohydrate. The flagellum increased in length 2.4 times in L. major as compared to 1.8 times in L. donovani. Resistance to complement-mediated lysis by normal human serum of in vitro grown Leishmania promastigotes was related to the species, the growth phase in culture, and also the temperature. Metacyclic forms of both species had a much increased resistance to killing by normal serum at different temperatures. Differences in membrane-exposed carbohydrates were detected by fluorescein-conjugated lectins. Peanut agglutinin and Ulex agglutinin I differentiated log and stationary phase promastigotes of L. major. Higher amounts of acid phosphatase were demonstrated in the metacyclic phase. Differences in polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides of approximately 51 and 114 kDa were found exclusively in metacyclic promastigotes of both species, whereas 38- and 23-kDa polypeptides were lost or reduced during transformation from log to metacyclic phase promastigotes of L. donovani. In addition, a 75-kDa polypeptide was expressed only in metacyclic promastigotes of L. major.     

Identification and partial characterization of an extracellular acid phosphatase activity of Leishmania donovani promastigotes.

An extracellular acid phosphatase was detected in the growth media of Leishmania donovani promastigotes. The enzyme was released at all stages of the growth cycle and in amounts which accounted for 90% of the total amount of this enzyme in the culture. The exoenzyme exhibited a pH optimum of 4.5 to 5.0 and was active with a variety of organic phosphates. The enzymatic activity was excluded from Sephacryl S-300 and was retained by ultrafilters with nominal molecular weight cutoffs of up to 300,000. The results of comparative studies indicated that the extracellular enzyme was distinct from a surface membrane-bound acid phosphatase of L. donovani promastigotes which has been previously described.     

An inexpensive easily available replacement for foetal calf serum in media for the in vitro cultivation of Leishmania spp

A replacement for foetal calf serum (FCS), made from heat-treated adult serum, in culture media designed for the in vitro cultivation of leishmanial promastigotes is described. The growth of 12 World Health Organization recommended strains of Leishmania in media containing heat-treated adult serum was compared with that obtained in media containing FCS. Growth of the promastigotes in the media with the FCS replacement was as good as or slightly better than that with FCS.     

Leishmania species: mechanisms of complement activation by five strains of promastigotes

The interaction of fresh serum with promastigotes of Leishmania major, L. donovani, L. mexicana mexicana, L. mexicana amazonensis, and L. braziliensis guyanensis results in lysis of all strains tested with either fresh human or guinea pig serum at 37 C for 30 min. Lysis does not occur in the cold and requires divalent cations and complement that is active hemolytically. Serum deficient in the eighth component of complement is not lytic. Lysis of L. major, L. mexicana, and L. braziliensis proceeds fully in human serum containing EGTA/Mg2+ or in guinea pig serum deficient in the fourth complement component. These species consume only small amounts of C4 from human serum and do not require calcium to optimally bind C3. The data indicate that all are activators of the alternative complement pathway and that the classical pathway is not required for the lysis of these organisms. Promastigotes of L. donovani, in contrast, activate the classical pathway. The presence of calcium is required for both optimal C3 binding and parasite lysis, and L. donovani promastigotes consume C4 when incubated in human serum. In high concentrations, human serum agglutinates all tested Leishmania spp. The agglutinating factor does not require divalent cations, is heat stable, and works at 4 C, suggesting that it is an antibody. This "naturally occurring" antibody cross reacts with all Leishmania spp. and agglutinates them. The adsorption of serum with any Leishmania species or with beads that are Protein A coated, removes the agglutinogen. This factor causes a slight enhancement in alternative pathway activation by L. major and mediates the classical activation by L. donovani. In adsorbed serum, L. donovani promastigotes only weakly activate the alternative complement pathway. Increased concentrations of adsorbed serum are therefore necessary for lysis to proceed. The titer can be partially restored by the addition of heat inactivated serum. Using purified components of the classical cascade, we are unable to visualize surface bound C3 on L. donovani promastigotes unless heat inactivated serum is also present. We conclude that all Leishmania spp. promastigotes are susceptible to lysis by normal serum independent of antibody. The presence of small amounts of naturally occurring antibody in human serum enhances the susceptibility of L. donovani promastigotes to lysis by activating the classical complement pathway.     

Inhibition of macrophage invasion by monoclonal antibodies specific to Leishmania (Viannia) braziliensis promastigotes and characterisation of their antigens

Monoclonal antibodies that specifically recognise Leishmania (Viannia) braziliensis promastigotes were produced and termed SST-2, SST-3 and SST-4. SST-2 recognises a conformational epitope present in a 24-28 kDa doublet and in a 72 kDa component, as verified by Western blotting. Indirect immunofluorescence showed that the antigen recognised by SST-2 is distributed homogeneously on the parasite surface. SST-3 recognises a flagellar glycoprotein of approximately 180 kDa. The reactivity of this mAb was abolished by sodium m-periodate treatment, indicating that SST-3 reacts with a carbohydrate epitope of the 180 kDa antigen. SST-4 recognises a conformational epitope of a 98 kDa antigen. SST-2, SST-3 and SST-4 were specific to L. (V.) braziliensis promastigote forms. Indirect immunofluorescence did not show reactivity of SST-2 or SST-3 with amastigotes of L. (V.) braziliensis, or with promastigotes of Leishmania (Viannia) panamensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Viannia) lainsoni, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) major, or Leishmania (Leishmania) chagasi. We also evaluated the involvement of SST-2, SST-3 and SST-4 antigens in parasite-macrophage interaction. Fab fragments of SST-3 and SST-4 significantly inhibited the infectivity of L. (V.) braziliensis promastigotes to mouse peritoneal macrophages.     

The oxidative response of rabbit peritoneal neutrophils to leishmanias and other trypanosomatids

Luminometry has been used to measure the respiratory burst of rabbit peritoneal neutrophils that is elicited by different forms and species of Leishmania and Herpetomonas. Mid-log phase and metacyclic promastigotes of L. major evoked large responses; that due to metacyclics was lower and slower, but they also bound in smaller numbers than mid-log phase cells. Promastigotes of L. mexicana mexicana also stimulated a large respiratory burst whereas amastigotes elicited little or none. Leishmania donovani promastigotes and culture forms of H. muscarum muscarum and H. m. ingenoplastis all evoked large responses by neutrophils. There was, however, very little response to L. mexicana mexicana promastigotes, L. donovani promastigotes or H. muscarum muscarum when they were added in large numbers. This 'inhibition' was not apparent with L. major.     

Analysis of Leishmania proteinases reveals developmental changes in species-specific forms and a common 68-kDa activity

Abstract The proteinases of three species of Leishmania have been analysed by electrophoresis. Amastigotes of L. mexicana mexicana have several high-activity, low- M _r cysteine proteinases which are absent from log-phase promastigotes of L. m. mexicana and from all developmental stages of the other species analysed ( L. donovani and L. major ). Low-activity, low- M _r proteinases were present in populations of stationary-phase promastigotes of L. m. mexicana . All three species of Leishmania had higher M _r proteinases, a number of which showed developmental regulation, some of them being stage-specific. Significantly, at all stages of the life cycle in all three species a 68-kDa proteinase was apparent. In its size, sensitivity to inhibitors and ability to bind concanavalin A-agarose, this resembles the major surface protein thought to be present in all Leishmania species and which has recently been reported to possess proteinase activity in L. major promastigotes.     

Characterisation of a new Leishmania META gene and genomic analysis of the META cluster

The META1 gene of Leishmania is upregulated in metacyclic promastigotes and encodes a 12 kDa virulence-related protein, conserved in all Leishmania species analysed. In this study, the genomic region adjacent to the Leishmania amazonensis META1 gene was characterised and compared to the Leishmania major META1 locus as well as to syntenic loci identified in Trypanosoma brucei and Trypanosoma cruzi. Three new genes expressed with increased abundance of steady state mRNA in L. amazonensis promastigotes were identified, two of which are upregulated in stationary phase promastigotes, sharing the pattern of expression previously described for the META1 mRNA. One of these new genes, named META2, encodes a polypeptide of 444 amino acid residues with a repetitive structure showing three repeats of the META domain (defined as a small domain family found in the Leishmania META1 protein and in bacterial proteins hypothetically secreted and/or implicated in motility) and a carboxyl-terminal region similar to several putative calpain-like proteins of Trypanosoma and Leishmania.     

Nutrient broth for the cultivation of Leishmania

Nutrient broth containing fetal calf serum was used successfully in isolating Leishmania donovani from animals and cryopreserving Leishmania major, Leishmania donovani, and Leishmania adleri. It also supported heavy growth of promastigotes of laboratory strains of L. donovani, L. major, L. adleri, and uncharacterized reptilian Leishmania-like flagellates.     

Carbocyclic inosine as a potent anti-leishmanial agent: the metabolism and selective cytotoxic effects of carbocyclic inosine in promastigotes of Leishmania tropica and Leishmania donovani

Carbocyclic inosine is a potent inhibitor for the growth of the promastigote form of Leishmania tropica and Leishmania donovani. In culture, the EC50 values of carbocyclic inosine are 8.3 X 10(-8) and 1.3 X 10(-7) M for the promastigotes of L. tropica and L. donovani, respectively. On the other hand, it is less toxic towards mouse mammary tumor FM3A cells: the EC50 value is 2.7 X 10(-4) M. Carbocyclic inosine is metabolized by Leishmania promastigotes to give carbocyclic adenosine-5'-triphosphate (aristeromycin-5'-triphosphate) and carbocyclic guanosine-5'-triphosphate. This metabolic conversion provides a mechanism for the parasite-selective toxicity of carbocyclic inosine.     

Axenic promastigote forms of Leishmania (Viannia) lainsoni as an alternative source for Leishmania antigen production

The present study demonstrates that axenic cultures of Leishmania (Viannia) lainsoni produce larger cell masses in NNN-LIT medium, as well as higher amounts of total proteins in cell extracts, than Leishmania (Leishmania) amazonensis. Antigenicity of L. (V.) lainsoni whole promastigotes is similar to that of L. (L.) amazonensis, as demonstrated by an indirect immunofluorescence diagnostic test using sera from human patients and dogs infected with visceral leishmaniasis. Infectivity of the L. (V.) lainsoni strain used in the present work was demonstrated by the detection by transmission-electron microscopy of tissue amastigotes in skin lesion samples from an experimentally infected hamster. Incubation of lesion fragments in NNN-LIT medium allowed us to obtain promastigote forms, which could be cultivated successfully in vitro. lsoenzyme analysis of such promastigotes confirmed the parasite strain as L. (V.) lainsoni, as compared to other Leishmania reference strains. Our data indicate that L. (V.) lainsoni is a useful alternative source for antigen production as well for use in assays that depend on large cell volumes of Leishmania spp. parasites.     

Electrophoretic analysis of the polypeptides of Leishmania amastigotes and promastigotes

The polypeptides of Leishmania mexicana mexicana (M379), L. m. amazonensis (LV78), L. major (LV39) and L. d. donovani (LV39) amastigotes and cultured promastigotes have been analysed by SDS-polyacrylamide gel electrophoresis. The polypeptide banding patterns of the promastigotes of the four species were quite similar, but distinct differences were detected between those of amastigotes. The results suggest that the various species of Leishmania are adapted differently for survival and growth in the mammalian host. The polypeptides of L. m. mexicana amastigotes were very rapidly hydrolysed unless protected by the cysteine proteinase inhibitor leupeptin.