Comparison of the Methods Fixation and Staining of Infected Macrophages with Recovery of Promastigotes in Culture to Evaluate Phagocytosis and Amastigote Proliferation of Leishmania sp. in vitro

Abstract

Purpose

Infection of macrophages is a mandatory step for Lesihmania to promote mammalian infection and the evaluation of parasites proliferating inside macrophages reveals important information about parasites virulence and leishmanicidal drugs. Here we compare macrophage phagocytosis ability and amastigote proliferation of L. major or L. braziliensis by two different methods: fixation and staining of infected macrophages or recovery of promastigotes in culture.

Methods

Promastigote parasites were used to infect thioglycolate-elicited BALB/c mice peritoneal macrophages. Phagocytosis was evaluated at 3 h after infection and amastigote proliferation was evaluated directly or indirectly at 3, 6, and 9 days after infection.

Results

Phagocytosis of L. major and L. braziliensis by murine macrophages was respectively 54.38 ± 10.41% and 62.54 ± 19.01%, according to the fixation and staining method. The infection index (product of the percentage of infected cells X the number of parasites per cell) obtained by fixation and staining method showed proliferation of L. major inside macrophage from 108.42 ± 25.57 (3 h) to 510.09 ± 99.13 (9 days) and decrease of the number of L. braziliensis from 223.01 ± 58.46 (3 h) to 101.37 ± 20.06 (9 days). The parasites recovered/ mL in culture increased for L. major infected macrophage from 2,38 × 10⁶ ± 2,31 × 10⁶ (3 h) to 16,8 × 10⁶ ± 8,3 × 10⁶ (9 days) and decreased from 8.43 × 10⁶ ± 6.8 × 10⁶ (3 h) to 0.19 × 10⁶ ± 0.21 × 10⁶ (9 days) for L. braziliensis infected macrophage. The fixation and staining method allowed to observe that both parasite species proliferated inside of some macrophages, while other macrophages maintain the parasite number unaltered or even kill the parasite.

Conclusion

Both methods showed that L. major proliferates in vitro inside of murine macrophages while L. braziliensis are mostly eliminated by these cells. Only fixation and staining allowed to identify L. braziliensis susceptible macrophages in vitro.