Cultivation and characterization of stable Leishmania guyanensis complex axenic amastigotes derived from infected U937 cells.

The study of the differential regulation of several genes, in both Leishmania parasite life cycle forms, has been simplified by the development of in vitro axenic amastigote culture. Different reports have described extracellular amastigote production and maintenance from several Leishmania spp. A general approach to induce amastigote-like transformation includes progressive pH and temperature changes. Production of axenic amastigotes in continuous cultures using amastigotes recovered from macrophages is described in this report. Leishmania (Viannia) panamensis (M/HOM/PA/71/LS94) and Leishmania (V). guyanensis (M/HOM/BR/75/M4147) intracellular amastigotes were recovered from the human macrophage-like U937 cell line previously infected with promastigotes. The parasites were immediately adapted for growth and kept as axenic amastigotes at 34 degrees C and acidic pH. These organisms were able to infect macrophage cell lines, maintain amastigote morphologic features, and express stage-specific transcripts. The relevance of axenic amastigotes in characterizing virulence factors in American leishmaniasis is discussed.