Análise do comportamento in vitro entre uma cepa viscerotrópica e outra dermotrópica de Leishmania amazonensis

Abstract

Leishmaniasis is a complex of serious and neglected diseases caused by protozoa of the genus Leishmania. The two main clinical forms of this disease are: cutaneous leishmaniasis (LC) and visceral (LV). Although not frequent, some LC-causing species can be found in cells of the mononuclear phagocytic system present in visceral organs - such as liver and spleen - leading to a clinical picture suggestive of VL. The objective of this work was to analyze the in vitro behavior of a viscerotrope strain of Leishmania amazonensis isolated from the spleen of a dog with clinical signs of LV and another strain of the same species (MHOM / BR / 1989 / Ba199). The development and multiplication of the promastigote forms was evaluated by means of the growth curve generated on the basis of the number of parasites / ml. Briefly, the parasites were cultured in chemically defined α-MEM medium and incubated at 24 ± 1 ° C, and the cultures were monitored for 7 days. In order to verify the percentages of metacyclic promastigotes in vitro, the strains were incubated (37ºC for 1h) with 10% (viscerotropic strain) and 8% (Ba199) of human serum, the viability of resistant forms being measured by the assay reduction of resazurin. For macrophage infection, 5x105 BMDMs were cultured in 24-well plates (5% CO2) containing coverslips (13mm). Then, stationary phase promastigotes were added to the wells, and the plate was incubated at 26ºC for 18h. After washing, the infected cells were again incubated at 37 ° C for 12h. Subsequently, the coverslips were removed from the wells, stained with Panótico, mounted on slides and analyzed by light microscopy. On the second day of growth, both strains started the exponential phase, with the peak of promastigotes/mL recorded on the fourth day. From that day on, the proliferation decreased, giving rise to the stationary phase and, later, the phase of decline. The profile of the curves was similar, however the number of promastigotes / mL ranged between strains from the second to the seventh day (p <0.05). The percentages of promastigotes resistant to Complement serum proteins were close for both strains. However, an average increase of approximately 1.3 × in the amount of viable (resistant) parasites between the growth phases tested (exponential and stationary) was observed, although there were no statistical differences (p> 0.9 ̅). The percentage of infected macrophages was approximately 2× greater for the viscerotropic strain than for Ba199 (p = 0.1). The infection rate found for the viscerotropic strain was also 3 × greater than that of Ba199 (p = 0.1). The viscerotropic strain apparently exhibits an in vitro behavior similar to that of Ba199, although it has a greater ability to infect the lineage of macrophages used, which may be indicative of the expression of certain virulence factors by this parasite.