Effects of Long-Term in Vitro Cultivation on Leishmania donovani Promastigotes

Promastigotes of Leishmania donovani that had been subcultued in modified Tobie's medium for 2 to 3 years showed decreased infectivity and lack of virulence for hamsters and mice compared to newly transformed promastigotes. Amastigotes derived from these long-term promastigote cultures decreased in number rapidly in hamsters, but only slightly in mice, over a 48-day period. In cultured mouse and hamster macrophags infected in vitro, amastigotes derived from long-term cultures rapidly decreased to low numbers, which persisted. The same pattern was seen in macriphages treated with catalase, an inhibitor of the oxygen-dependent killing mechanism of the macrophage. Promastigotes from long-term cultures also differed from virulent first-passage promastigotes in size, growth patterns in Tabie's medium, and in the quantities of some of their antigens.     

Effects of long-term in vitro cultivation on Leishmania donovani promastigotes

Promastigotes of Leishmania donovani that had been subcultured in modified Tobie's medium for 2 to 3 years showed decreased infectivity and lack of virulence for hamsters and mice compared to newly transformed promastigotes. Amastigotes derived from these long-term promastigote cultures decreased in number rapidly in hamsters, but only slightly in mice, over a 48-day period. In cultured mouse and hamster macrophages infected in vitro, amastigotes derived from long-term cultures rapidly decreased to low numbers, which persisted. The same pattern was seen in macrophages treated with catalase, an inhibitor of the oxygen-dependent killing mechanism of the macrophage. Promastigotes from long-term cultures also differed from virulent first-passage promastigotes in size, growth patterns in Tobie's medium, and in the quantities of some of their antigens.     

Infectivity of Leishmania donovani Amastigotes and Promastigotes for Golden Hamsters*

SYNOPSIS. Intracardial injection of hamsters with from 5 to 114 million amastigotes or promastigotes of Leishmania donovani and screening of the 8th-day liver impression smears, provides a rapid and reproducible method for assaying infectivity. Amastigotes are at least 10X more infective than promastigotes, and log-phase promastigotes act as a single infective population for hamsters.     

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.     

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.     

Characterization of Populations of Promastigotes of Leishmania donovani1

Promastigotes of Leismania donovani cultured for either 3 or 10 days in vitro and inoculated intracardially into golden hamsters with an equal number of organisms from either population showed a 7-fold difference in infectivity when compared at both 10 to 16 days post-infection. Reproducible histochemical staining for the promastigote enzymes glucose-6-phosphate dehydrogenase (G6PDH) and peptidase after polyacrylamide gel electrophoresis showed two isoelectric variants of G6PDH (Bands 1 and 2) that displayed a 45% decrease (Band 1) and a 60% increase (Band 2) in total activity when 3- and 10-day-old promastigores were compared. Peptidase activity, present in a single band, increased 7-fold in 10-day-old promastigotes. A decrease in the lectin-induced agglutination of promastigotes by castor bean agglutinin (RCA₆₀), specific for D-galactose and N-acetyl-D-galactosamine, was seen when 3- and 10-day-old promastigotes are compared. Antisera raised against sonicated 10-day-old promastigotes showed a unique precipitin band between the antiserum and sonicated 10-day-old promastigotes not found between the antiserum and sonicated 3-day-old promastigotes.     

Leishmania mexicana: Serial cultivation of intracellular stages in a cell-free medium

A cell-free liquid medium has been devised for serial cultivation of Leishmania mexicana pifanoi amastigotes at 33 and 35 C. It consists of tissue culture Medium 199 fortified primarily with a high concentration of water-soluble vitamins, nucleotides, and inactivated fetal bovine serum. The initial pH of the medium is 7.2. Starting with a population of promastigotes as inoculum and serially cultured at 33 or 35 C at 4- to 10-day intervals, the proportion of amastigotes steadily increased and that of promastigotes gradually decreased during the first subculture. By the end of the incubation period in the second subculture, practically all (99%) of the organisms are amastigotes. The amastigotes thus obtained can be cultured indefinitely by serial transfers. In this medium, amastigotes may reach a density of 8 × 10⁷/ml after 10 days of incubation at 33 C, and 5 × 10⁷/ml at 35 C. The medium was modified to have an initial pH of 8.0 by Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer and higher concentrations of sodium bicarbonate. When amastigotes cultured in the original medium at 33 or 35 C are transferred into the modified medium and incubated at 26 C, the amastigotes entirely transformed into promastigotes after three serial passages. These promastigotes could be serially subcultured indefinitely in the modified medium at 4- to 12-day intervals. The promastigotes cultured at 26 C may reach a population density of 7 × 10⁷/ml after 12 days of incubation.     

Leishmania major: culture media, mouse strains, and promastigote virulence and infectivity

Promastigotes of Leishmania major were isolated from an infected mouse in two media, blood agar and Schneider's medium + 30% fetal calf serum, and maintained continuously for over 1 year. Infectivity studies in two strains of mice, outbred CD1 strain and inbred BALB/c strain, showed that promastigotes grown in Schneider's medium maintained infectivity to BALB/c mice throughout the period of cultivation. Infectivity to CD1 strain mice was progressively lost. Promastigotes grown in blood agar medium, however, lost infectivity to both strains of mice at a faster rate than promastigotes grown in Schneider's medium.     

Differential survival of Leishmania donovani amastigotes in human monocytes

Leishmania donovani is an important intracellular protozoal pathogen of man; it is found solely within macrophages in its amastigote stage in humans, and exists in its extracellular, flagellated promastigote stage in the sandfly, its arthropod vector. To determine if either stage of L. donovani was capable of surviving within monocytes--the oxidatively active precursors of tissue macrophages--interactions of the parasite with human monocytes were studied in vitro. Amastigotes and promastigotes were ingested to a comparable degree by monocytes; whereas 79% of promastigotes were killed within 48 hr, however, amastigotes survived and multiplied threefold over 5 days. Promastigotes, which have been shown to be sensitive to hydrogen peroxide-peroxidase-halide microbicidal mechanisms, elicited a phagocytic oxidative burst that was 49% of the response to serum-opsonized zymosan, as assessed by luminol-enhanced chemiluminescence. NBT was reduced to formazan in 71% of monocytes exposed to promastigotes. The death of promastigotes within monocytes could be attributed at least in part to oxidative microbicidal mechanisms because there was no significant decrease in the number of cell-associated parasites in monocytes from donors with chronic granulomatous disease of childhood. In contrast to promastigotes, amastigotes survived within monocytes, despite eliciting an oxidative response that was 27% of the response produced by serum-opsonized zymosan; this response was not significantly different from that produced by promastigotes. In a phagocyte-free system, amastigotes were found to be sevenfold more resistant than were promastigotes to the lethal effects of hydrogen peroxide. The survival of L. donovani in human monocytes is thus dependent on the parasite stage; promastigotes are ingested, they elicit an oxidative burst, and the majority are killed by oxidative microbicidal mechanisms, whereas amastigotes are ingested and survive to parasitize human monocytes successfully, despite eliciting a phagocytic oxidative burst.     

Leishmania donovani: A Chemically Defined Medium Suitable for Cultivation and Cloning of Promastigotes and Transformation of Amastigotes to Promastigotes

A chemically defined medium using commercially available α-MEM supplemented with HEPES, L-glutamine, D-glucose, folic acid, D-biotin and adenine supports the luxuriant growth and propagation of Leishmania donovani promastigotes. A peak parasite population of about 7.0 × 10⁷/ml at stationary phase and a population doubling time of 11.4 h for high-subpassage promastigotes were obtained. The medium was suitable for transformation of isolated amastigotes from infected hamster spleen. Promastigotes could be detected by culturing kala-azar patients’bone-marrow aspirate or spleen puncture material in this medium. Four out of six freshly transformed isolates gradually adapted and grew well in this medium. Macroscopic colonies appeared on agar plates prepared with the medium within 16–20 days after inoculation. The cloning efficiency was increased about five-fold by glycerol supplementation.     

Leishmania donovani metacyclic promastigotes: transformation in vitro, lectin agglutination, complement resistance, and infectivity

Freshly transformed Leishmania donovani amastigotes from hamster spleen were used to establish axenic cultures at high density in a modified Grace's medium, which was only partly replenished when cultures were fed. Small, free-swimming, highly active stationary phase promastigotes with a short cell body and long flagellum were induced in this medium. The freshly transformed stationary phase promastigotes so induced were less able to bind peanut agglutinin, had more than 40-fold increased resistance to killing by normal human serum, and 15-fold increased infectivity both in vivo and in vitro when compared to freshly transformed logarithmic phase or long term culture promastigotes. These short form promastigotes may correspond to the metacyclic promastigote forms in the sand fly vector.     

Development of Leishmania (Viannia) panamensis lesions and relationship of numbers of amastigotes to lesion area on antimony-treated and untreated hamsters

Young adult (60-70-g) male golden hamsters (Mesocricetus auratus) each were injected intradermally at the dorsal base of the tail with 15 x 10(6) promastigotes of Leishmania (Viannia) panamensis (MHOM/PA/83/WR539), and progression and regression of subsequent lesions were evaluated for up to 17 wk postinfection (PI) as to area, weight, and number of amastigotes within lesions in untreated hamsters and in hamsters treated with meglumine antimoniate (Glucantime). In untreated hamsters total area of lesion, weight, and numbers of amastigotes generally increased rapidly and concomitantly up to 3-4 wk PI. Amastigote numbers tended to decrease from 4 to 11 wk PI and subsequently the numbers of amastigotes within the lesions decreased rapidly, whereas relatively little change occurred in the area and weight of the lesions. Meglumine antimoniate treatment of cutaneous hamster lesions resulted in marked concomitant decrease in size of the lesions and numbers of amastigotes within the lesions examined 1 wk after treatment. Measurement of the area of cutaneous leishmanial lesions thus would appear to be a valid method of evaluating the efficacy of promising compounds against L. panamensis in hamsters when measurements are taken 3-5 wk after experimental infection and reflects the number of amastigotes present in the lesion.     

Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics

In this report, we describe an in vitro culture system for the generation and propagation of axenic amastigotes from the well characterised 1S-CL2D line of Leishmania donovani. Fine structure analyses of these in vitro-grown amastigotes demonstrated that they possessed morphological features characteristic of L. donovani tissue-derived amastigotes. Further, these axenic amastigotes (LdAxAm) were shown to synthesise and release a secretory acid phosphatase isoform similar to that produced by intracellular amastigotes. Such LdAxAm also expressed surface membrane 3'-nucleotidase enzyme activity similar to that of tissue-derived amastigotes. Moreover, LdAxAm, in contrast to promastigotes, expressed significant levels of the amastigote-specific A2 proteins. In addition, LdAxAm, derived from long term cultures of Ld 1S-CL2D promastigotes, had significant infectivity for both human macrophages in vitro and for hamsters in vivo. Thus, the in vitro culture system described herein provides a useful tool for the generation of large quantities of uniform populations of axenic amastigotes of the L. donovani 1S-CL2D line. The availability of such material should greatly facilitate studies concerning the cell and molecular biology of this parasite developmental stage.     

Leishmania mexicana: comparative fine structure of amastigotes and promastigotes in vitro and in vivo

Amastigotes of Leishmania mexicana pifanoi were cultivated by serial transfers in cell-free medium UM-54 at 33 and 35 C. Electron microscopy was used to analyze the structural relationships among promastigotes, axenically cultured amastigotes, and amastigotes in footpads of infected hamsters. These studies revealed very close structural similarities between culture and hamster derived amastigotes. However, both of these amastigotes differed from the promastigotes in the following aspects. The flagellum of promastigotes contained a paraxial rod originating at the axosome level within the flagellar pocket, whereas the flagellum of amastigotes lacks this structure. The flagellar pocket of promastigotes was usually small whereas amastigotes had a distended reservoir. Subpellicular microtubules of promastigotes terminated at the posterior end, whereas those of amastigotes ended subterminally. Membrane bounded vesicles were present only in amastigotes. These results along with the biologic and antigenic comparisons indicate that amastigotes obtained from axenic cultures are related very closely to amastigotes from infected hamster footpads and that their relationship to promastigotes is far more distant.     

Leishmania mexicana pifanoi: Analysis of the Antigenic Relationships Between Promastigotes and Amastigotes by Gel Diffusion,Immunoelectrophoresis, and Immunoprecipitation1

ABSTRACT. The antigenic relationships between Leishmania mexicana pifanoi promastigotes, axenically grown amastigotes, and amastigotes isolated from the footpads of infected hamsters or from a J774 macrophage cell line were studied by three serologic methods. Amastigote and promastigote antigens were disrupted by freeze-thawing of intact cells in a lysis buffer. Antisera were prepared in rabbits by repeated subcutaneous inoculations of the parasite antigens in complete Freund's adjuvant and were tested against the homologous and heterologous antigens in a series of gel diffusion experiments. Negative results were obtained in all control experiments. In each instance, the homologous antigen-antiserum reactions yielded the largest numbers of precipitin lines. A pattern of cross-reactivity was also observed in the heterologous systems. Results indicated that the amastigote and promastigote forms had unique and common antigens. The two parasite antigen-antiserum systems were also examined by immunoelectrophoresis. Qualitative and quantitative differences between the promastigote and amastigote antigens were readily demonstrable by this technique. Results indicated that each parasite form had specific and many common antigens. In the homologous system, major proteins, with molecular weights (MW) of 23, 52, and 68 kd, were demonstrated in the promastigotes by immunoprecipitation of lactoperoxidase-catalyzed radioiodinated cells. In a similar (homologous) system, axenically grown amastigotes were found to contain three proteins with MW of 38, 70, and 74 kd and, therefore, different from those demonstrated for the promastigotes. All the results suggested that the three amastigote stages of different origins are antigenically similar to one another, but differ from the promastigote forms.     

Comparison of the Protein Kinase and Acid Phosphatase Activities of Five Species of Leishmania1

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, historic-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [γ-³²P]ATP and Mg²⁺ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [γ-³²P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA^-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA⁺). The PNA^- and PNA⁺ forms of L. major both phosphorylated a 50-kDa protein when incubated with [γ-³²P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity. With the exception of L. braziliensis panamensis for which late log phase organisms contained 12-fold more tartrate-resistant acid phosphatase activity than did early log phase cells, stationary and log phase parasites contained approximately the same amount of acid phosphatase activity.     

Leishmania donovani: a chemically defined medium suitable for cultivation and cloning of promastigotes and transformation of amastigotes to to promastigotes

A chemically defined medium using commercially available alpha-MEM supplemented with hemin, HEPES, L-glutamine, D-glucose, folic acid, D-biotin and adenine supports the luxuriant growth and propagation of Leishmania donovani promastigotes. A peak parasite population of about 7.0 x 10(7)/ml at stationary phase and a population doubling time of 11.4 h for high-subpassage promastigotes were obtained. The medium was suitable for transformation of isolated amastigotes from infected hamster spleen. Promastigotes could be detected by culturing kala-azar patients' bone-marrow aspirate or spleen puncture material in this medium. Four out of six freshly transformed isolates gradually adapted and grew well in this medium. Macroscopic colonies appeared on agar plates prepared with the medium within 16-20 days after inoculation. The cloning efficiency was increased about five-fold by glycerol supplementation.     

Comparison of the protein kinase and acid phosphatase activities of five species of Leishmania

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, histone-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [gamma-32P]ATP and Mg2+ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [gamma-32P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA+). The PNA- and PNA+ forms of L. major both phosphorylated a 50-kDa protein when incubated with [gamma-32P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential phagocytosis of Leishmania mexicana promastigotes and amastigotes by monocyte‐derived dendritic cells

Leishmania species are dimorphic protozoan parasites that live and replicate in the gut of sand flies as promastigotes or in mammalian hosts as amastigotes. Different immune cells, including DCs, and receptors differ in their involvement in phagocytosis of promastigotes and amastigotes and in recognition of different Leishmania species. In the case of L. mexicana, differences in phagocytosis of promastigotes and amastigotes by DCs and participation of C‐type lectin receptors (CLRs) have not been established. In the present study, flow cytometry and confocal microscopy were used to investigate the phagocytosis by monocyte‐derived dendritic cells (moDCs) of L. mexicana promastigotes and amastigotes in the presence or absence of immune serum during various periods of time. Blocking antibodies against mannose receptors and DC‐SIGN were used to explore the participation of these receptors in the phagocytosis of L. mexicana by moDC. The major differences in interactions of L. mexicana promastigotes and amastigotes with moDC were found to occur within the first 3 hr, during which phagocytosis of promastigotes predominated as compared with opsonization of promastigotes and amastigotes. However, after 6 hr of incubation, opsonized promastigotes were preferentially phagocytosed as compared with unopsonized promastigotes and amastigotes and after 24 hr of incubation there were no differences in the phagocytosis of promastigotes and amastigotes. Finally, after 3 hr incubation, DC‐SIGN was involved in the phagocytosis of promastigotes, but not of amastigotes.     

Leishmania mexicana: enzyme activities of amastigotes and promastigotes and their inhibition by antimonials and arsenicals

A major difference between the metabolism of Leishmania species amastigotes and cultured promastigotes was found in the area of CO2 fixation and phosphoenolpyruvate metabolism. Malate dehydrogenase (EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were at much higher activities in amastigotes than promastigotes of both L. m. mexicana and L. donovani, whereas the reverse was true of pyruvate kinase (EC 2.7.1.40). Pyruvate carboxylase (EC 6.4.1.1) and malic enzyme (carboxylating) (EC 1.1.1.40) could not be detected in L. m. mexicana amastigotes. Promastigotes of L. m. mexicana had a high NAD-linked glutamate dehydrogenase activity in comparison to amastigotes, whereas NADP-linked glutamate dehydrogenase activity was detected only in amastigotes. Leishmania m. mexicana culture promastigotes were killed in vitro by the trivalent antimonial Triostam (LD50, 20 micrograms/ml) and the trivalent arsenical melarsen oxide (LD50, 20 micrograms/ml), but they were unaffected by Pentostam. Neither antimonial drug significantly inhibited leishmanial hexokinase (EC 2.7.1.2), phosphofructokinase (EC 2.7.1.11), pyruvate kinase, malate dehydrogenase or phosphoenolpyruvate carboxykinase, whereas melarsen oxide was a potent inhibitor of all the enzymes tested except phosphoenolpyruvate carboxykinase.