Leishmania (Leishmania) amazonensis LV79 (MPRO/BR/72/M1841) has been adapted to grow at 33 C as amastigote-like (AL) organisms in modified UM-54 medium initially adjusted to a pH of 4.8–5.0. Axenic cultures could be routinely restarted from parasites recovered from footpad lesions obtained by inoculation of BALB/c mice with preadapted culture stages. Morphological features, proteinase activities, and infectivity of AL organisms were examined during the in vitro growth cycle, and differences were found between log- and stationary-phase parasites. Stationary-phase AL organisms were morphologically similar to lesion amastigotes, did not react with a paraflagellar rod-specific monoclonal antibody in western blots, and contained proteinase activities resolving identically to the enzymes of lesion amastigotes in gelatin gels. Whereas typical megasomes could be identified in about a third of the stationary-phase AL population, the organelles were rarely seen in log-phase organisms. Azocaseinolytic activity progressively increased during the exponential growth phase and reached its highest values (∼65–70% of those determined in lesion amastigotes) at the stationary phase; the association of total proteinase activity with increased expression of cysteine proteinases was indicated by the strong inhibition of azocasein hydrolysis by E-64, the intensified banding of the 28-, 31-, and 35-kDa proteinases in gelatin gels, and the higher susceptibility of stationary-phase AL organisms to l-leucine methyl ester. Although overall axenic amastigotes were less infective to BALB/c mice than were lesion-derived parasites, stationary-phase AL organisms were more infective than were log-phase parasites. Medium pH increased during the exponential growth phase, but dropped in the stationary phase, when the observed morphological, biochemical, and biological changes became apparent.

American Society of Parasitologists
2003
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