Investigations on the in vitro metacyclogenesis of a visceral and a cutaneous human strain of Leishmania infantum

Abstract

The in vitro metacyclogenesis of a visceral (VL) and cutaneous (CL) human strain of Leishmania infantum was monitored in order to find out the kinetics of this process and the in vitro infective capacity for macrophages of the metacyclic promastigotes developed. To identify, enumerate, and separate the metacyclic population, the complement-dependent lysis by normal serum and the agglutination by peanut agglutinin (PNA) were used, as they were shown to be useful for the purpose of this study. Maximum percentage of metacyclics was detected by both techniques on the 4th day of growth for VL and the 6th day for CL, and was higher for the VL strain. The in vitro infectivity for macrophages of two strains was assayed, and the high parasitization data obtained were transformed in order to determine the increase of the parasite burden for macrophages throughout the incubation time of the experiments (2–72 h post-infection (p.i.)). This parameter is denominated the infectivity ratio (% I) and calculated as follows: (number of intracellular parasites per infected macrophage at ` x' time p.i./number of intracellular parasites per infected macrophage at 2 h p.i.)×100. When % I was calculated for promastigotes unagglutinated by PNA (PNA−)—metacyclic or infective promastigotes—at any time of culture, the % I at 72 h p.i. was always much higher than for agglutinated promastigotes (2.1–12.5 times)—non-infective promastigotes—and unfractionated promastigotes from culture (1.7–9.5 times), especially with VL parasites. Likewise, the % I for VL PNA− promastigotes from the 4th day of culture was 1.9 times higher than for CL PNA− promastigotes from the 6th day of culture. The higher resistance to lysis by serum, percentage of metacyclics (PNA−), and infectivity ratio of VL than CL could be related to a higher spreading capability into the host body associated with higher pathogenic effects of the visceral strain than the cutaneous one.