Leishmania amazonensis: cultivation and characterization of axenic amastigote-like organisms

Extracellular amastigote-like forms of Leishmania amazonensis can be maintained in axenic culture at 32 degrees C, pH 4.6, with a generation time of approximately 17 hr. This species of Leishmania is of particular interest since it has been associated with cutaneous, diffuse cutaneous, and mucocutaneous forms of the disease. Immunofluorescence, Western and Northern blot analyses, and immunoprecipitation have been used to estimate the expression levels of amastigote or promastigote antigens in axenically cultured amastigotes. In these analyses, monoclonal antibodies (mAbs) specific for either the amastigote (A-1, A-2, P-2, P-4, P-5, P-8) or promastigote (M-2, P-9, and F-4) and a DNA probe that was specific for the amastigote gene encoding the protein reactive with mAb P-4 have been employed. The amastigote-like organisms were infective for peritoneal and J774.G8 macrophages and BALB/c mice. While amastigote-like forms maintained at pH 4.6, 32 degrees C transformed to promastigotes when transferred to pH 7.3, 24 degrees C, transformation of promastigotes to amastigote-like organisms required a period of growth at pH 4.6 24 degrees C prior to transfer to 32 degrees C. This is the first report of the axenic cultivation of L. amazonensis amastigote-like organisms. This species grows in continual culture at a lower pH than any other species characterized to date. These organisms will prove useful in further studies of the biochemistry, immunology, developmental biology, and molecular biology of this parasite.