2.1. Animals and parasites:
Syrian golden hamsters (Mesocricetus auratus) of both sexes, aged 4–6 weeks were purchased form the Schistosome Biological Supply Program (SBSP), Theodor Bilharz Research Institute (TBRI), Giza, Egypt, and kept under favorable conditions of 25˚C temperature, 70% humidity, 12-hr light and 12-hr dark cycle and adapted for 1 week before infection. Animals were infected percutaneously with freshly shed Egyptian strain of S. hematobium cercariae from experimentally infected Bulinus trucatus snails according to [10].
Early stages Schistosomulae were obtained by mechanical transformation of S. hematobium cercariae [11, 12] while that of hepatic stages were recovered by portal-mesenteric perfusion at the 42nd day post infection. Moreover, adult S. hematobium worms were recovered by perfusion of the vesical venous plexus at the 90th day post infection [13].
2.2. Drugs and reagents:
SynriamTM (Arterolane maleate, 150mg and Piperaquine phosphate, 750mg) obtained from Ranbaxy Laboratory Limited, India. Praziquantel (PZQ) tablets (Distocide®) purchased from EIPICO (Cairo, Egypt). Chemicals and solvents were delivered by Sigma, St. Louis, USA. RPMI 1640 medium supplemented with 2 mM of L-glutamine, 25 mM Hydroxyethyl-piperazine-ethanesulfonic acid, 20%foetal calf serum, 300 μg/ml streptomycin, 160 μg/ml gentamycin and 300 IU/ml penicillin was used for the in vitro culture [14]. Synriam was dissolved in dimethyl sulfoxide (DMSO) (10 mg/ml), and then diluted in the culture medium.
2.3. Preparation of the parasites and experimental design for in vitro culturing:
Recovered juvenile and adult (male and female) worms were washed 10 times in RPMI medium, taking great care for parasite tegument integrity. Worms were placed in wells of sterile, flat-bottom, 24-well plates (Corning, NY) containing 2 ml RPMI 1640 medium /well and incubated at 37°C and 5% CO2 atmosphere [9]. To mimic an in-vivo physiological environment, serum albumin (SA) and human alpha acidic glycoprotein (AGP) were added to the culture medium [15].
Before the beginning of the experiment, Worms were being assessed hourly, using light microscope (Olympus Inverted Microscope Model IX70; Olympus, Tokyo, Japan). Only viable, transparent, contractile worms showing total tegument integrity were included in the experiment while parasites that were contracted or had acquired an opaque appearance were considered dead and discarded.
The crowds of worms (adult stage, early and late juvenile stages) were grouped as follows; Experiment I: adult S. hematobium (90 days), experiment II: early schistosomula S. hematobium (3-hours), experiment III: late schistosomula S. hematobium (42 days).
Each experiment has been tested as 3 groups (12/well); group a: Medium + 0.5% DMSO (negative control), group b: Medium + 1 μg/ml PZQ (positive control) and group c: medium + SYN (test group). Group c is further subdivided into subgroups by using different concentrations of the test drug (SYN was used to obtain final concentrations of 5 to 80 μg/ml [5, 10, 20, 40, 60, and 80 μg/ml]).
In each experiment, the survival was monitored in (1st, 3rd, 24th and 48th hours) post-incubation with SYN, for body motility and occurrence of death using the dissecting microscope. Worms showed no body movement for at least 30 seconds observation was considered as dead [8]. All tests were repeated three times, viability data from one experiment was presented as range of scores (0-3) based on the motility of the worms; 0 (all worms are dead), 1 (minimal motor activity), 2 (slow motor activity) and 3 (normal motor activity) [16, 17]. The mean ±SD were determined for each independent experiment over the groups.
2.4. Ultrastructural studies using scanning electron microscope:
Ultrastructural features of samples of juvenile and adult Shistosomes were examined and compared to those of the control groups for the effect of the tested drugs. Worms were washed out in PBS from the culture solution, fixed in Karnovsky’s solution, for 10 hours (hrs), and then cleared by keeping them overnight at 4˚C in PBS. The samples were immediately processed according to [18]. The samples were fixed in equal volumes of glutaraldehyde 4% + cacodylate 0.2 % for 2 hrs. Then, washed in equal volumes of sucrose 0.4 % and cacodylate 0.2 % for 2 hrs followed by fixation in equal volumes of osmic acid 2% and cacodylate 0.3 % for 1 hr and then washed with distilled water.
Finally, the samples were dehydrated in ascending grades of ethyl alcohol for 5 minutes (min) each (30%, 50%, 70% and 90%) then 100% absolute alcohol for 10 min. thrice. Examination was done using Environmental Scanning Electron Microscope (Inspect S; FEI, Holland) at Electron Microscopy Unit, TBRI.
2.5. Statistical analysis of data:
Data was entered and analyzed using SPSS Version 21 (IBM Corporation, NY, USA). Survival analysis was done using the Kaplan-Meier method. The log-rank test was used for pairwise comparison between different groups in each experiment. P value less than or equal to 0.05 was considered significant. Viability scoring was calculated using Kruskal-Wallis Test (non parametric test) and displayed as Viability score box-plot. Viability data from one experiment having average replicate was presented as range of scores. Mean values were determined for each independent experiment over the group. The mean ± SD of three viability mean values (of three independent experiments) for each group was calculated at different time points and used to examine if differences between the groups were statistically significant.