Axenic Cultivation And Determination Of Leishmania Amazonensis Amastigotes

Objective: To establish the axenic culture system for the amastigotes of Leishmania amazonensis and so as to generate large quantity of amastigotes for immunological and molecular biology studies; To further assess the antigenic characterization and to detect the expression levels of stage-specific genes and to monitor the infectivity to the host in different amastigote preparations (promastigote-derived amastigotes). Methods: We generated amastigotes via in vitro transformation from promastigotes and maintained amastigotes in axenic culture using a modified protocol by increasing the FBS from 20% to 50%. Amastigotes generated from three different sources ( from animal lesions,infected-macrophages and promastigote trasmormation) were analyzed by indirect immunofluorescent analysis (IFA) employing mAbs reactive for either amastigotes (P-4 and P-8) or promastigotes (GP46/M-2); and designed the specific primers from the genes encoding P-4/single-strand nucleases, GP46, and beta-tubulin for RT-PCR analysis;At the same time we did the experiments to compare the infectivity of these amastigotes to macrophages. Results: Amastigotes generated from three different sources were similar morphological features under the optical microscopy of Giemsa-staining; .And these three different sources amastigotes were analyzed by indirect immunofluorescent analysis employing mAbs reactive for either amastigotes (P-4 and P-8) or promastigotes (GP46/M-2). P-4 and P-8 mAbs displayed positive staining of all three types of amastigotes with similar intensity and localization patterns. P-8, but not P-4, showed weak staining for promastigotes. In contrast, GP46/M-2 mAb was reactive strongly to promastigote antigens but weakly to amastigotes. A P-4-specific band (273bp) was observed in all three types of amastiotes, with similar intensity, but it was almost undetectable in promastigotes. In contract, a GP46-specific band (325bp) was expressed in higher levels in promastigotes than in three types of amastigotes. The infection rates of these three types of amastigotes in macrophages were close to 100%. Conclusion: The axenic culture system has been established to transform the promastigotes into the amastiotes by increasing the temperature and the transformed amastiotes were maintained in the modified media by increasing the FBS from 20% to 50%. Furthermore, our results indicate that amastigotes generated via three different methods display similar characteristics in morphology, antigenicity, P-4 gene expression and infectivity to the macrophage. And promastigote-derived amastigotes can serve as an excellent source of amastigotes for further in vivo and in vitro immunological studies of L. amazonensis.