AXENIC CULTIVATION AND PARTIAL CHARACTERIZATION OF LEISHMANIA-BRAZILIENSIS AMASTIGOTE-LIKE STAGES

Leishmania braziliensis strain M2903 was adapted for growth and serial ly maintained as amastigotes at 34 degrees C in modified UM-54 medium, with growth curves exhibiting typical log and stationary phases. In l ate passages, amastigote growth took place in the absence of supplemen tary haemin and was unaffected when the initial medium pH was adjusted between 5.4 and 6.3. In contrast to promastigotes, which were elongat ed and exhibited very long free flagella endowed with the paraflagella r rod (PFR), axenic amastigotes were rounded to ovoid and displayed a short flagellum restricted to the pocket area. The absence of PFR in a xenic amastigotes was confirmed in Western blots and confocal immunofl uorescence microscopy, by lack of reactivity with mAb 1B10. The antibo dy, which specifically labelled the paraflagellar structure, recognize d a 70/72 kDa doublet in Trypanosoma cruzi epimastigotes and two 70/74 kDa related proteins in L. braziliensis promastigotes. Surface I-125- labelling experiments identified promastigote-specific components (>10 0, 74, 45/47 and 28 kDa) and at least 1, a 76 kDa polypeptide was spec ific for the amastigote stage. While axenic amastigotes were agglutina ted by both peanut (PNA) and Lens culinaris (LCA) agglutinins, respect ively at 50 and 12.5 mu gl/l, promastigotes were not agglutinated by P NA and agglutinated in the presence of LCA at concentrations of 100 mu g/ml and higher. Axenic amastigotes infected rat bone marrow-derived macrophages and were avidly taken up by J774 cells, from which numerou s organisms, able to proliferate at 34 degrees C in UM-54 medium, coul d be recovered 48 h later.