Experimental animals and P. berghei ANKA reference lines
Male BALB/cByJ mice (6–8 weeks-old) purchased from Charles River Laboratories Inc. (Lyon, France) were used. Two parasite lines were employed in the experimental work, a transgenic parasite line termed PbCSGFP-Luc (RMgm-152),which expresses the fusion gene gfp-luc under the control of the csp promotor (PBANKA_0403200) integrated into the silent 230p gene locus (PBANKA_0306000) , and the transgenic parasite line PbFluo-frmg (RMgm-164), which expresses GFP under control of the ‘male gametocyte-specific’ promoter of PB000791.03.0 (dynein heavy chain, putative) and RFP under the control of the ‘female gametocyte-specific’ promoter PB000504.02.0 (LCCL domain-containing protein CCP2). The rfp and gfp genes are integrated into the genome in the 230p locus (PBANKA_0306000) .
Plasmodium berghei ANKA expressing green fluorescent protein (GFP) and luciferase under the control of the circumsporozoite protein (CSP) promoter (line 784cl1, RMgm-152, PbCSPGFP-Luc) was maintained in Anopheles stephensi mosquitoes and BALB/cByJ mice. To maintain gametocyte infectivity, only up to six passages of parasites from infected to naïve mice were performed. Ookinete in vitro production was performed as previously described . Briefly, BALB/cByJ mice were treated with 0.1 ml phenylhydrazine (25 mg/ml) 3 days prior to infection with 107 P. berghei-infected red blood cells (iRBC) obtained from a donor mouse. On the third day after infection, gametocytemia was monitored by light microscopy for the presence of exflagellating gametocytes in ookinete medium (1:4 dilution). Blood collected by heart puncture was pooled from 2 mice and washed with RPMI at 37 °C, followed by centrifugation at 1100×g for 10 min at 37 °C. After washing, 5 μl of blood containing exflagellating gametocytes was mixed with medium supplemented for ookinete formation [RPMI1640 (Sigma-Aldrich, Saint Louis, USA), 25 mM HEPES, 0.4 mM hypoxanthine, 100 mM xanthurenic acid (85570, Fluka, Saint Gallen, Switzerland), 10% FBS (pH 7.6)] in a final volume of 200 μl, and cultured in 96-well plates for 24 h at 19 °C. Additionally, blood containing exflagellating gametocytes was mixed with the ookinete medium in 1:20 ratio and cultured in T75 flasks for 22–24 h at 19 °C. Following incubation, ookinete enrichment was performed as previously described , with some modifications. Briefly, cultured blood was collected, and erythrocytes were lysed for 15 min on ice with 30 volumes of ice-cold 0.17 M ammonium chloride. Lysed erythrocytes were removed by washing with RPMI, and ookinetes were purified by centrifugation on a 63% Nycodenz cushion at 650×g at 4 °C for 30 min. Following centrifugation, the ookinete-containing interface was collected, washed in ice-cold RPMI and resuspended in 0.5–1.0 ml of oocyst medium.
Purified ookinetes were seeded with Drosophila melanogaster S2 cells (Drosophila Genomics Resource Center, Bloomington, USA) in a 1:10 ratio (104 ookinetes and 105 S2 cells) in Schneider’s medium (S0146, Sigma-Aldrich) supplemented with 15% FBS, penicillin/streptomycin (50 U/ml, 50 µg/ml) and gentamicin (50 µg/ml) to promote oocyst development. Oocysts were co-cultured with D. melanogaster S2 cells in flat bottom 96-well plates (Corning, New York, USA) for up to 15 days at 19 °C. One-quarter of the medium was changed 3 times per week (every 48 to 72 h), and 105 S2 cells were added once per week. In parallel, S2 cells were maintained at 27 °C in Schneider’s medium (S0146, Sigma-Aldrich) supplemented with 10% FBS and penicillin/streptomycin (50 U/ml, 50 µg/ml).
A bioluminescence assay was used to assess the development of the mosquito stages of PbCSGFP-Luc. In order to evaluate the effect of compounds on the development of the parasite’s mosquito stages, samples were collected at 3 different time points to determine the effect on ookinete and oocyst formation, and oocyst maturation, as previously described . The bioluminescence assay was performed using the Firefly Luciferase Assay Kit (Biotium, Hayward, USA) according to the manufacturer’s instructions, with some modifications. Briefly, the whole well contents were collected and spun down, washed with PBS, frozen in 50 µl of lysis buffer (1:5 ratio) and stored at − 20 °C until further use. Collected samples were lysed and 30 µl of the resulting supernatant were transferred into white 96-well plates. Fifty µl of D-luciferin in Firefly luciferase assay buffer (1:50 ratio) were added to the samples and parasite load was determined by measuring luminescence intensity using a microplate reader (Tecan Infinite M200, Zurich, Switzerland).
Evaluation of the in vivo activity of ivermectin
In order to assess the in vivo activity of Iv, five BALB/cByJ (Charles River) mice per experimental group were infected by intraperitoneal injection of 107 P. berghei Fluo-frmg-infected red blood cells. Parasitaemia and gametocyteamia were measured the following days by flow cytometry analysis of 4 µl of tail blood. Blood was collected in 200 µl of PBS and 100 µl stored at 4 °C, while the remaining was transferred to 100 µl of PBS containing 1.25 mM of red fluorescent nucleic acid stain Syto®61 (Thermo Fisher Scientific, Waltham, USA) and incubated for 20 min at room temperature in the dark. The samples were analysed on a LSRFortessa X-20 flow cytometer (Becton, Dickinson and Co., New Jersey, USA). Female and male gametocytes were gated based on the analysis of GFP and Red Fluorescence Protein (RFP) fluorescence, and parasitemia was estimated based on the analysis of Syto®61 and forward scatter. The positive cell population was determined by comparison of infected blood samples with an uninfected blood sample. Results were then analysed with the FlowJoTM Software (Version 10, FlowJoTM Software, Ashland, USA). Exflagellation was also monitored every day, until parasitemia reached 3% by microscopy analysis. To this end, 2.5 µl of tail blood were collected and mounted on a glass slide, and 8 min later the number of exflagellation events present in 4 independent fields of vision at 40× were determined. When parasitemia reached up to 3%, either DMSO or ivermectin were administered by oral gavage, at a concentration of 5 mg/kg, to five mice of each experimental group. Parasitemia, gametocytemia and exflagellation were monitored for 3 days after treatment, following which the mice were euthanized, and the experiment was terminated.
Evaluation of the in vitro activity of avermectin compounds
The effect of eprinomectin, abamectin, ivermectin, moxidectin, doramectin and emamectin on the Plasmodium mosquito stages was evaluated as previously described . The compounds were dissolved in dimethyl sulfoxide (DMSO) and the amount of DMSO equivalent to that present in the highest compound concentration was used as a control. The effect of 10 µM of each compound was assessed on ookinetes, and on oocyst development and maturation. Briefly, after 1 h of incubation, the compounds were added to 5 µl of infected blood cultures, and the bioluminescence intensity of the parasites on the ookinete cultures was assessed 24 h later. To assess the effect of the compounds on oocyst development and maturation, compounds were mixed with the mature ookinetes, and parasites collected after 3 days of culture or compounds were added to the oocyst culture after 3 days of culture, and parasites collected 15 days later.
Compound concentration resulting in 50% inhibition (IC50) for oocyst growth and maturation were estimated for eprinomectin, abamectin, ivermectin, moxidectin, doramectin and emamectin (assayed at 0.05, 0.5, 1, 5, 10, 25 and 50 µM) by nonlinear regression analysis.
Evaluation of avermectin compounds’ in vitro cytotoxicity
Compounds were screened for their in vitro cytotoxicity against D. melanogaster S2 cells, using the AlamarBlue® assay (Invitrogen, Carlsbad, USA). This assay allows to measure metabolic activity based on a fluoremetric/colorimetric indicator . To assess the effect of the compounds on cell development, Drosophila melanogaster S2 cells were seeded in a 1:10 ratio (105 S2 cells) in Schneider’s medium (S0146, Sigma-Aldrich) supplemented with 15% FBS, penicillin/streptomycin (50 U/ml, 50 µg/ml) and gentamicin (50 µg/ml). All the above-mentioned compounds were added to the S2 cell cultures to a final concentration of 10 µM and the amount of DMSO equivalent to that present in the highest compound concentration was used as a control. Cultures were maintained for 7 days and one-quarter of the medium was changed 3 times per week every 48 to 72 h. Samples were collected every day by removing 120 µl of medium and adding 80 µl of AlamarBlue previously diluted in Schneider’s medium (1:10 dilution) to each well. The suspension was transferred to a 96 well flat bottom plate and incubated for one and a half hours at 37 °C. Fluorescence intensity was then measured using a microplate reader (Tecan Infinite M200) at 530 nm excitation wavelength/590 nm emission wavelength to determine cell viability.
Data on the assessment of the compounds’ effect in vitro were analysed using the Kruskal-Wallis test. Data on the compounds’ effect on parasitemia, gametocytemia and exflagellation in vivo were analysed employing non-linear regression analysis. Results were considered significant for P-values < 0.05. Nonlinear regression analysis was employed to fit the normalized results of the dose-response curves for IC50 determination. All statistical tests were performed by GraphPad Prism (version 6.00, GraphPad Software, La Jolla, USA).