The leishmanicidal effect of Lucilia sericata larval saliva and hemolymph on in vitro Leishmania tropica

Abstract

Background: Leishmaniasis is a major parasitic disease worldwide, except in Australia and Antarctica, and it poses a significant public health problem. Due to the absence of safe and effective vaccines and drugs, researchers have begun an extensive search for new drugs. The aim of the current study was to investigate the in vitro leishmanicidal activity of larval saliva and hemolymph of Lucilia sericata on Leishmania tropica.

Methods: The effects of different concentrations of larval products on promastigotes and intracellular amastigotes of L. tropica were investigated using the mouse cell line J774A.1 and peritoneal macrophages as host cells. The 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and direct observation and counting method were used to assess the inhibitory effects and cell cytotoxicity of the larval products. The effects of larval products on the amastigote form of L. tropica were quantitatively estimated by calculating the rate of macrophage infection, number of amastigotes per infected macrophage cell, parasite load and survival index.

Results: The 50% cytotoxicity concentration (CC₅₀) value of both larval saliva and hemolymph was 750 µg/ml, and the 50% inhibitory concentration (IC₅₀) values were 134 µg/ml and 60 µg/ml for larval saliva and larval hemolymph, respectively. The IC₅₀ for Glucantime, used a positive control, was (11.65 µg/ml). Statistically significant differences in viability percentages of promastigotes were observed for different doses of both larval saliva and hemolymph when compared with the negative control (p ≤ 0.0001). Microscopic evaluation of the amastigote forms revealed that treatment with 150 µg/ml larval hemolymph and 450 µg/ml larval saliva significantly decreased the rate of macrophage infection and the number of amastigotes per infected macrophage cell.

Conclusion: Larval saliva and hemolymph of L. sericata have acceptable leishmanicidal properties against L. tropica.

Keywords: Anti-leishmanial activity; Hemolymph; Intracellular amastigote; Leishmania tropica; Promastigote; Saliva.

Fig. 1

SDS-PAGE analyses of salivary gland lysates of field- and laboratory-bred L. sericata third-instar larvae. L: pre-stained protein ladder (page-ruler); lanes 1 and 2: laboratory-bred larvae; lanes 3 and 4: field larvae collected from Tehran Province, Iran

Fig. 2

Dose-response curves regarding the effect of Lucilia sericata larval-derived products on Leishmania tropica promastigotes (IC₅₀). a Larval saliva IC₅₀ vs larval hemolymph IC₅₀ at 24 h. b Larval saliva IC₅₀ vs larval hemolymph IC₅₀ at 48 h. c Larval saliva IC₅₀ vs larval hemolymph IC₅₀ at 72 h. d Larval saliva IC₅₀ vs larval hemolymph IC₅₀ at 96 h and compared with standard drug

Fig. 3

The number of alive promastigotes exposed to larval-derived products in different dosages at different time points

Fig. 4

The number of viable cells treated with larval-derived products by trypan blue in different dosages at different time points. a The number of peritoneal viable cells treated with larva-derived products in different concentrations at different time points. b The number of J774 viable cells treated with larva-derived products in different concentrations at different time points

Fig. 5

Leishmania tropica amastigote susceptibility to Lucilia sericata larval products at 72 and 120 h compared with Glucantime. a Decrease in infection percentage (DI %) in L. tropica amastigote peritoneal cells. b Decrease in amastigote viability (DV %) in L. tropica amastigote peritoneal cells. c Decrease in infection percentage (DI %) in L. tropica amastigote J774A.1 cells. d Decrease in amastigote viability (DV %) in L .tropica amastigote J774A.1 cells