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Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms

Published online by Cambridge University Press:  06 April 2009

P. A. Bates ,
C. D. Robertson ,
L. Tetley  and
G. H. Coombs
P. A. Bates
Affiliation:
Laboratory for Biochemical Parasitology, Department of Zoology, University of Glasgow, Glasgow G12 8QQ
C. D. Robertson
Affiliation:
Laboratory for Biochemical Parasitology, Department of Zoology, University of Glasgow, Glasgow G12 8QQ
L. Tetley
Affiliation:
Electron Microscopy Centre, Department of Zoology, University of Glasgow, Glasgow G12 8QQ
G. H. Coombs
Affiliation:
Laboratory for Biochemical Parasitology, Department of Zoology, University of Glasgow, Glasgow G12 8QQ
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Summary

A new method is described which has made possible the long-term axenic cultivation of Leishmania mexicana amastigotelike forms in Schneider's Drosophila medium supplemented with 2% (v/v) foetal calf serum. Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotes in vitro. Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion amastigotes. The axenically cultured forms grew optimally at a temperature of 32–33 °C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5·4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial amastigotes are acidophiles.

Keywords

Leishmania mexicanaaxenic cultureamastigotesmegasomescysteine proteinaseinfectivity.
Type
Research Article
Information
Parasitology , Volume 105 , Issue 2 , October 1992 , pp. 193 - 202
Copyright
Copyright © Cambridge University Press 1992

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References

REFERENCES

Alexander, J. (1988). Sex differences and crossimmunity in DBA/2 mice infected with Leishmania mexicana and L. major. Parasitology 96, 297–302.Google Scholar
Alexander, J. & Vickerman, K. (1975). Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexicana-infected macrophages. Journal of Protozoology 22, 502–8.Google Scholar
Antoine, J.-C., Prina, E., Jouanne, C. & Bongrand, P. (1990). Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH. Infection and Immunity 58, 779–87.Google Scholar
Berens, R. L. & Marr, J. J. (1979). Growth of Leishmania donovani amastigotes in a continuous macrophage-like cell culture. Journal of Protozoology 26, 453–6.CrossRefGoogle Scholar
Berens, R. L., Brun, R. & Krassner, S. M. (1976). A simple monophasic medium for axenic culture of hemoflagellates. Journal of Parasitology 62, 360–5.CrossRefGoogle ScholarPubMed
Berman, J. D., Dwyer, D. M. & Wyler, D. J. (1979). Multiplication of Leishmania in human macrophages in vitro. Infection and Immunity 26, 375–9.CrossRefGoogle ScholarPubMed
Berman, J. D. & Neva, F. A. (1981). Effects of temperature on multiplication of Leishmania amastigotes within human monocyte-derived macrophages in vitro. American Journal of Tropical Medicine and Hygiene 30, 318–21.Google Scholar
Biegel, D., Topper, G. & Rabinovitch, M. (1983). Leishmania mexicana: temperature sensitivity of isolated amastigotes and of amastigotes infecting macrophages in culture. Experimental Parasitology 56, 289–97.CrossRefGoogle ScholarPubMed
Brazil, R. P. (1978). Isolation of the intracellular stages of Leishmania mexicana amazonensis using cellulose column. Annals of Tropical Medicine and Parasitology 72, 579–80.Google Scholar
Chang, K.-P. (1980 a). Human cutaneous leishmania in a mouse macrophage cell line: propagation and isolation of intracellular parasites. Science 209, 1240–2.CrossRefGoogle Scholar
CHANG, K-p. (1980 b). Endocytosis of Leishmania infected macrophages. Fluorometry of pinocytic rate, lysosome phagosome fusion and intralysosomal pH. In The Host Invader Interplay (ed. Van Den Bossche, H.), pp. 231–4. Amsterdam: Elsevier.Google Scholar
Chang, K.-P. & Dwyer, D. M. (1976). Multiplication of a human parasite (Leishmania donovani) in phagolysosomes of hamster macrophages in vitro. Science 193, 678–80.CrossRefGoogle ScholarPubMed
Chang, K.-P. & Dwyer, D. M. (1978). Leishmania donovani. Hamster macrophage interactions in vitro: cell entry, intracellular survival, and multiplication of amastigotes. Journal of Experimental Medicine 147, 515–30.CrossRefGoogle ScholarPubMed
Childs, G. E., McRoberts, M. J. & Foster, K. A. (1976). Partial purification of amastigotes from cutaneous lesions of American leishmaniasis. Journal of Parasitology 62, 676–9.CrossRefGoogle ScholarPubMed
Coombs, G. H. (1982). Proteinases of Leishmania mexicana and other flagellate protozoa. Parasitology 84, 149–55.CrossRefGoogle ScholarPubMed
Coombs, G. H., Tetley, L., Moss, V. A. & Vickerman, K. (1986). Three-dimensional structure of the leishmania amastigote as revealed by computer-aided reconstruction from serial sections. Parasitology 92, 13–23.Google Scholar
Coombs, G. H., Robertson, C. D. & Mottram, J. C. (1991). Cysteine proteinases of leishmanias. In Biochemical Protozoology (ed. Coombs, G. H. & North, M. J.), pp. 208–20. London: Taylor & Francis.Google Scholar
Doyle, P. S., Engel, J. C., Gam, A. A. & Dvorak, J. A. (1989). Leishmania mexicana mexicana: quantitative analysis of the intracellular cycle. Parasitology 99, 311–16.CrossRefGoogle ScholarPubMed
Doyle, P. S., Engel, J. C., Pimenta, P. F., Da Silva, P. P. & Dwyer, D. M. (1991). Leishmania donovani: long-term culture of axenic amastigotes at 37 °C. Experimental Parasitology 73, 326–34.CrossRefGoogle Scholar
Eperon, S. & McMahon-Pratt, D. (1989 a). Extracellular cultivation and morphological characterization of amastigote-like forms of Leishmania panamensis and L. braziliensis. Journal of Protozoology 36, 502–10.CrossRefGoogle ScholarPubMed
Eperon, S. & McMahon-Pratt, D. (1989 b). Extracellular amastigote-like forms of Leishmania panamensis and L. braziliensis. II. Stage- and species-specific monoclonal antibodies. Journal of Protozoology 36, 510–18.Google Scholar
Glaser, T. A., Baatz, J. E., Kreishman, G. P. & Mukkada, A. J. (1988). pH homeostasis in Leishmania donovani amastigotes and promastigotes. Proceedings of the National Academy of Sciences, USA 85, 7602–6.CrossRefGoogle Scholar
Glaser, T. A., Wells, S. J., Spithill, T. W., Pettitt, J. M., Humphris, D. C. & Mukkada, A. J. (1990). Leishmania major and L. donovani: a method for rapid purification of amastigotes. Experimental Parasitology 71, 343–5.CrossRefGoogle ScholarPubMed
Hart, D. T., Vickerman, K. & Coombs, G. H. (1981 a). A quick simple method for purifying Leishmania mexicana amastigotes in large numbers. Parasitology 82, 345–55.Google Scholar
Hart, D. T., Vickerman, K. & Coombs, G. H. (1981 b). Transformation in vitro of Leishmania mexicana amastigotes to promastigotes: nutritional requirements and the effect of drugs. Parasitology 83, 529–41.Google Scholar
Hendricks, L. D., Wood, D. E. & Hajduk, M. E. (1978). Haemoflagellates: commercially available liquid media for rapid cultivation. Parasitology 76, 309–16.CrossRefGoogle ScholarPubMed
Infante, R. B., Hernandez, A. G., Riggione, F. & Dawidowicz, K. (1980). A new method for the partial purification of leishmania amastigotes froth cutaneous lesions. Parasitology 80, 105–12.CrossRefGoogle Scholar
Lockwood, B. C., North, M. J., Mallinson, D. J. & Coombs, G. H. (1987). Analysis of Leishmania proteinases reveals developmental changes in species- specific forms and a common 68-kDa activity. FEMS Microbiology Letters 48, 345–50.Google Scholar
Looker, D. L., Martinez, S., Horton, J. M. & Marr, J. J. (1986). Growth of Leishmania donovani amastigotes in the continuous human macrophage cell line U937: studies of drug efficacy and metabolism. Journal of Infectious Diseases 154, 323–7.CrossRefGoogle ScholarPubMed
Mallinson, D. J. & Coombs, G. H. (1986). Molecular characterisation of the metacyclic forms of Leishmania. IRCS Medical Science 14, 557–8.Google Scholar
Mallinson, D. J. & Coombs, G. H. (1989). Biochemical characteristics of the metacyclic forms of Leishmania major and L. mexicana mexicana. Parasitology 98, 7–15.Google Scholar
Marr, J. J. (1980). Carbohydrate metabolism in Leishmania. In Biochemistry and Physiology of Protozoa, Vol. 3 (ed. Levandowsky, M. and Hutner, S. H.), pp. 313–40. London: Academic Press.Google Scholar
Martinez, S., Looker, D. L. & Marr, J. J. (1988). A tissue culture system for the growth of several species of Leishmania: growth kinetics and drug sensitivities. American Journal of Tropical Medicine and Hygiene 38, 304–7.Google Scholar
Meade, J. C., Claser, T. A., Bonventre, P. F. & Mukkada, A. J. (1984). Enzymes of carbohydrate metabolism in Leishmania donovani amastigotes. Journal of Protozoology 31, 156–61.Google Scholar
Mukkada, A. J., Meade, J. C., Glaser, T. A. & Bonventre, P. F. (1985). Enhanced metabolism of Leishmania donovani amastigotes at acid pH: an adaptation for intracellular growth. Science 229, 1099–101.CrossRefGoogle ScholarPubMed
North, M. J. & Coombs, G. H. (1981). Proteinases of Leishmania mexicana amastigotes and promastigotes: analysis by gel electrophoresis. Molecular and Biochemical Parasitology 3, 293–300.CrossRefGoogle ScholarPubMed
Pan, A. A. (1984). Leishmania mexicana: Serial cultivation of intracellular stages in a cell-free medium. Experimental Parasitology 58, 72–80.CrossRefGoogle Scholar
Pan, A. A. & Honigberg, B. M. (1985). Leishmania mexicana pifanoi: in vivo and in vitro interactions between amastigotes and macrophages. Zeitschrift für Parasitenkunde 71, 3–13.CrossRefGoogle ScholarPubMed
Pan, A. A. & McMahon-Pratt, D. (1988). Monoclonal antibodies specific for the amastigote stage of Leishmania pifanoi. I. Characterization of antigens associated with stage- and species-specific determinants. Journal of Immunology 140, 2406–14.CrossRefGoogle ScholarPubMed
Pan, A. A., McMahon-Pratt, D. & Honigberg, B. M. (1984). Leishmania mexicana pifanoi: antigenic characterization of promastigote and amastigote stages by solid-phase radioimmunoassay. Journal of Parasitology 70, 834–5.CrossRefGoogle ScholarPubMed
Pan, A. A. & Pan, S. C. (1986). Leishmania mexicana: comparative fine structure of amastigotes and promastigotes in vitro and in vivo. Experimental Parasitology 62, 254–65.CrossRefGoogle ScholarPubMed
Pupkis, M. F. & Coombs, G. H. (1984). Purification and characterization of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes. Journal of General Microbiology 130, 2375–83.Google Scholar
Pupkis, M. F., Tetley, L. & Coombs, G. H. (1986). Leishmania mexicana: amastigote hydrolases in unusual lysosomes. Experimental Parasitology 62, 29–39.Google Scholar
Rainey, P. M., Spithill, T. W., McMahon-Pratt, D. & Pan, A. A. (1991). Biochemical and molecular characterization of Leishmania pifanoi amastigotes in continuous axenic culture. Molecular and Biochemical Parasitology 49, 111–18.CrossRefGoogle ScholarPubMed
Rivas, L. & Chang, K.-P. (1983). Intraparasitophorous vacuolar pH of Leishmania mexicana-infected macrophages. Biological Bulletin 165, 536–7.Google Scholar
Robertson, C. D. & Coombs, G. H. (1990). Characterisation of three groups of cysteine proteinases in the amastigotes of Leishmania mexicana mexicana. Molecular and Biochemical Parasitology 42, 269–76.Google Scholar
Sacks, D. L., Barral, A. & Neva, F. A. (1983). Thermosensitivity patterns of Old vs. New World cutaneous strains of Leishmania growing within mouse peritoneal macrophages in vitro. American Journal of Tropical Medicine and Hygiene 32, 300–4.CrossRefGoogle ScholarPubMed
Saraiva, E. M. B., Pimenta, P. F. P., Pereira, M. E. A. & De Souza, W. (1983). Isolation and purification of amastigotes of Leishmania mexicana amazonensis by a gradient of metrizamide. Journal of Parasitology 69, 627–9.CrossRefGoogle ScholarPubMed
Scott, P. A., James, S. & Sher, A. (1985). The respiratory burst is not required for killing of intracellular and extracellular parasites by a lymphokine-activated macrophage cell line. European Journal of Immunology, 15, 553–8.Google Scholar
Shapira, M., McEwen, J. G. & Jaffe, C. L.. (1988). Temperature effects on molecular processes which lead to stage differentiation in Leishmania. EMBO Journal 7, 2895–901.CrossRefGoogle ScholarPubMed
Smejkal, R. M., Wolff, R. & Olenick, J. G. (1988). Leishmania braziliensis panamensis: increased infectivity resulting from heat shock. Experimental Parasitology 65, 1–9.CrossRefGoogle ScholarPubMed
Zilberstein, D., Blumenfeld, N., Liveanu, V., Gepstein, A. & Jaffe, C. L. (1991). Growth at acidic pH induces an amastigote stage-specific protein in Leishmania promastigotes. Molecular and Riochemical Parasitology 45, 175–8.CrossRefGoogle ScholarPubMed