Axenic cultivation and partial characterization of Leishmania braziliensis amastigote-like stages

Leishmania braziliensis strain M2903 was adapted for growth and serially maintained as amastigotes at 34 degrees C in modified UM-54 medium, with growth curves exhibiting typical log and stationary phases. In late passages, amastigote growth took place in the absence of supplementary haemin and was unaffected when the initial medium pH was adjusted between 5.4 and 6.3. In contrast to promastigotes, which were elongated and exhibited very long free flagella endowed with the paraflagellar rod (PFR), axenic amastigotes were rounded to ovoid and displayed a short flagellum restricted to the pocket area. The absence of PFR in axenic amastigotes was confirmed in Western blots and confocal immunofluorescence microscopy, by lack of reactivity with mAb 1B10. The antibody, which specifically labelled the paraflagellar structure, recognized a 70/72 kDa doublet in Trypanosoma cruzi epimastigotes and two 70/74 kDa related proteins in L. braziliensis promastigotes. Surface 125I-labelling experiments identified promastigote-specific components (> 100, 74, 45/47 and 28 kDa) and at least 1, a 76 kDa polypeptide was specific for the amastigote stage. While axenic amastigotes were agglutinated by both peanut (PNA) and Lens culinaris (LCA) agglutinins, respectively at 50 and 12.5 micrograms/ml, promastigotes were not agglutinated by PNA and agglutinated in the presence of LCA at concentrations of 100 micrograms/ml and higher. Axenic amastigotes infected rat bone marrow-derived macrophages and were avidly taken up by J774 cells, from which numerous organisms, able to proliferate at 34 degrees C in UM-54 medium, could be recovered 48 h later.