The parasites and in-vitro cultivation
Theileria equi and Babesia caballi parasites (Indian isolates) were cultured in horse red blood cells (RBCs) through continuous microaerophilic stationary-phase (MASP) culture system. The culture medium M 199 and RPMI 1640, (Sigma-Aldrich, India) were used for MASP culture of T. equi and B. caballi, respectively. These mediums were supplemented with 40% defibrinated equine serum, antibiotic solution (containing 60 IU/ml penicillin and 60 mg/ml streptomycin) and 200 µM hypoxanthine solution. Theileria equi and B. caballi MASP cultures were maintained at a temperature of 37 °C with micro-aerophilic atmosphere of 5% CO2, 3% O2, and 95% N.
In-vitro growth inhibition assay
Theileria equi and B. caballi parasitized RBCs were obtained from primary MASP cultures at 10% and 6% parasitaemia respectively. These parasitized RBC’s (T. equi and B. caballi) were adjusted to 1% parasitemia by diluting with uninfected RBCs obtained from a healthy horse and used for in-vitro evaluation of drug. The parasite growth inhibition assay was performed in 48 well culture plates. Fifty microliter of T. equi or B. caballi parasite infected RBCs (at 1% parasitaemia) were dispensed per well (in triplicate) together with 500 µl of the culture complete medium containing the indicated drug concentrations. Novobiocin drug (cat no.: N1628-1G; Sigma–Aldrich, India) was tested at 1 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM concentrations against T. equi and 10 µM, 20 µM, 50 µM, 100 µM, 200 µM against B. caballi parasites. Cultures without novobiocin drug molecule and cultures containing only DMSO (0.005% and 0.5%) were also prepared and initiated in MASP system for control experiments. ID drug was diluted to 0.5 µg/ml, 1.0 µg/ml and 10.0 µg/ml concentration and kept as positive drug control experiment against T. equi and B. caballi parasites in MASP in-vitro culture system. These in-vitro cultures with or without drug molecules concentrations were incubated at 37°C in an atmosphere of 5% CO2, 3% O2, and 95% N, for a period of 96 hours (h). The overlaid culture medium was replaced daily with fresh medium containing indicated drug molecule concentration. IC50 value was calculated by standard curve fitting technique. [2].
In-vitro viability test
After 96 h of in-vitro treatment with novobiocin, 20 µL of drug-treated/un-treated parasitized RBCs were collected (at different concentration) and transferred to a fresh culture plate (48 wells) containing 30 µL of parasite-free fresh equine RBCs in 500 µl of growth medium (without any drug molecule). The overlaid growth medium was replaced after every 24 h for the next 72 h, and parasite recrudescence was determined by examining its stained blood smears [2].
In-vitro cytotoxicity assay on equine PBMCs
Cytotoxicity of each concentration of drug molecules was analysed on peripheral blood mononuclear cells (PBMCs) by resazurin-based cell viability assay [8]. Briefly, freshly collected equine PBMCs were suspended in 1 ml complete growth medium consisting of RPMI-1640 supplemented with 2 mM L-glutamine, 60 µg/ml penicillin, 100 µg/ml streptomycin and 10% foetal bovine serum (Sigma Aldrich, India). Enriched PBMCs suspension with complete growth media was adjusted to a final concentration of 3 × 107 cells/100 µL and distributed to each well (100 µL volume) in 96 well culture plate. Simultaneously, phytohaemagglutinin–A (PHA, at the concentration of 10 µg/ml) in 50 µL volume was also added to each of these well. The culture plate was incubated at 37 °C having 5% CO2 in air for 48 h. After 48 h, PBMCs in cultured wells were treated with 100 µl volume of the different respective concentration of novobiocin drug molecule – 1 µM, 5 µM, 10 µM, 25 µM, 50 µM, 100 µM, 1000 µM, 2000 µM. The 96 well culture plate was again incubated (as above) for another 24 h, followed by addition of 25 µL of resazurin dye (150 µg/ml) and culture plate was kept in an incubator for another 4 h. The change of dye colour was monitored by measuring optical density (OD) at 570 nm and 650 nm. The effective OD value for each well was calculated by deducting OD570 value from its respective OD650 value. The IC50 of each drug molecule on PBMCs was calculated from a regression equation based on the effective OD value, as mentioned above. Effect of drug molecules on PBMCs in terms of per cent viable cell population was determined as below:
$$\text{P}\text{B}\text{M}\text{C}\text{s} \text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y} \left(\text{%}\right)=\frac{\text{O}\text{D} \text{o}\text{f} \text{t}\text{e}\text{s}\text{t} \text{s}\text{a}\text{m}\text{p}\text{l}\text{e}-\text{O}\text{D} \text{o}\text{f} \text{p}\text{o}\text{s}\text{i}\text{t}\text{i}\text{v}\text{e} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}{\text{O}\text{D} \text{o}\text{f} \text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{O}\text{D} \text{o}\text{f} \text{p}\text{o}\text{s}\text{i}\text{t}\text{i}\text{v}\text{e} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}} \text{x} 100$$
$$\text{%} \text{C}\text{y}\text{t}\text{o}\text{t}\text{o}\text{x}\text{i}\text{c}\text{i}\text{t}\text{y}=\frac{\text{O}\text{D} \text{o}\text{f} \text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{O}\text{D} \text{o}\text{f} \text{t}\text{e}\text{s}\text{t} \text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{O}\text{D} \text{o}\text{f} \text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}} \text{x} 100$$
$$\text{P}\text{e}\text{r}\text{c}\text{e}\text{n}\text{t}\text{a}\text{g}\text{e} \text{h}\text{e}\text{m}\text{o}\text{l}\text{y}\text{s}\text{i}\text{s}=\frac{\text{O}\text{D} \text{o}\text{f} \text{d}\text{i}\text{f}\text{f}\text{e}\text{r}\text{e}\text{n}\text{t} \text{d}\text{r}\text{u}\text{g} \text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n} – \text{O}\text{D} \text{o}\text{f} \text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}{\text{O}\text{D} \text{o}\text{f} \text{p}\text{o}\text{s}\text{i}\text{t}\text{i}\text{v}\text{e} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} - \text{O}\text{D} \text{o}\text{f} \text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} }$$
$$\text{S}\text{p}\text{e}\text{c}\text{i}\text{f}\text{i}\text{c} \text{S}\text{e}\text{l}\text{e}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \text{I}\text{n}\text{d}\text{e}\text{x} \left(\text{S}\text{S}\text{I}\right)=\frac{\text{I}\text{C}50 \text{o}\text{f} \text{d}\text{r}\text{u}\text{g} \text{m}\text{o}\text{l}\text{e}\text{c}\text{u}\text{l}\text{e} \text{o}\text{n} \text{h}\text{o}\text{r}\text{s}\text{e} \text{P}\text{B}\text{M}\text{C}\text{s}}{\text{I}\text{C}50 \text{o}\text{f} \text{d}\text{r}\text{u}\text{g} \text{m}\text{o}\text{l}\text{e}\text{c}\text{u}\text{l}\text{e} \text{o}\text{n} \text{p}\text{r}\text{o}\text{t}\text{o}\text{z}\text{o}\text{a}\text{n} \text{p}\text{a}\text{r}\text{a}\text{s}\text{i}\text{t}\text{e}}$$
In-vivo
organ toxicity of novobiocin in mice The organ toxicity of novobiocin was analysed in six groups of mice (n = 6, in each group). Each five group was administered (by intraperitoneal route) the drug at respective dose rate of 5, 10, 20, 50, 100 mg/kg body weight, whereas sixth group was kept as no-drug control and administered PBS only. Mice in each group were observed for 14 days. Blood from these group of mice were collected at 0 h, 24 h and 72 h interval from retro-orbital sinus under general anaesthesia. Serum was isolated from these mice and processed for biochemical analysis with-respect-to changes in organ specific biochemical markers. Mice were sacrificed on day 14 post-drug treatment and biopsy sample from vital organs were collected (liver, lung, heart, kidney and spleen) and processed for histopathological examination as per standard protocol.
Statistical analysis
Anti-piroplasmic activity of novobiocin drug molecules against T. equi and B. caballi was computed by Two Way ANOVA test followed by Bonferroni Post-hoc test (p < 0.05). The P values < 0.05 were considered as statistically significant differences between the novobiocin treated wells and control wells. Correlation between novobiocin concentration and its cytotoxicity and haemolytic activity was accessed by using Graphpad prism version 4.0 software (San Diego California, USA).