The Texas strain of B. bovis (Hines et al. 1992), the Argentine strain of B. bigemina (Hotzel et al. 1997), the USDA strain of B. caballi (Avarzed et al. 1997), Theileria equi (Bork et al. 2004), and the Munich strain of B. microti (AbouLaila et al. 2012) were used in this study.
The Babesia parasites used in this study were maintained in bovine or equine red blood cells (RBCs) using a microaerophilic stationary-phase culture system (Igarashi et al. 1994; AbouLaila et al. 2010a). Briefly, Medium 199 was used for B. bovis, B. bigemina, and T. equi, while RPMI-1640 was used for B. caballi (both from Sigma-Aldrich, Tokyo, Japan). Media were supplemented with 40 % normal bovine serum for bovine Babesia or 40 % normal equine serum for equine Babesia, and 60 U/ml of penicillin G, 60 μg/ml of streptomycin, and 0.15 μg/ml of amphotericin B (all three drugs from Sigma-Aldrich) were prepared and used in the culture media. Additionally, 13.6 μg of hypoxanthine (ICN Biomedicals Inc., Aurora, OH) per milliliter was added to the T. equi culture as a vital supplement.
Allicin was purchased from iHerb.com and used for examining babesicidal effects. A stock solution of 100 mM was dissolved in dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industrial, Ltd., Osaka, Japan) and then stored at 4 °C until use. Ten millimolars of stock solution of diminazene aceturate (Ciba-Geigy Japan Limited, Tokyo, Japan) was prepared in distilled water and stored at −30 °C until use; it was used as a comparator drug in vitro and used in combination with allicin either in vivo or in vitro.
In vitro growth inhibition assay
The inhibitory effect of allicin on the growth of Babesia parasites was tested using an assay previously described (Bork et al. 2003a; AbouLaila et al. 2010a) with slight modification. Briefly, parasite-infected RBCs were diluted with uninfected RBCs to obtain 1 % parasitemia. From this stock, 20 μl was dispensed into a 96-well microtiter plate (Nunc, Roskilde, Denmark) with 200 μl of the culture medium containing the indicated concentration of allicin (5, 50, 200, 500, 1,000, and 4,000 μM) for B. bovis, (1, 100, 200, 500, 2,000, and 4,000 μM) for B. bigemina, (1, 10, 100, 500, 1,000, 4,000, and 5,000 μM) for B. caballi, and (5, 50, 100, 200, 500, 2,000, and 5,000 μM) for T. equi. Diminazene aceturate was used at concentrations of 5, 25, 50, 100, 500, 1,000, and 2,000 nM for all four parasites from the same cultures used for the allicin experiment and then incubated at 37 °C in a humidified multi-gas water-jacketed incubator. For experimental control, cultures without the drug and cultures containing only DMSO (0.06 %, for allicin) or distilled water (0.02 %, for diminazene aceturate) were prepared. Parasitemia was monitored daily by counting the parasitized RBCs to approximately 1,000 RBCs in Giemsa-stained thin blood smears. The IC50 values (50 % inhibitory concentration) for the two drugs upon growth of all parasites tested were calculated based on the parasitemia recorded on day 3 in the in vitro cell culture system using interpolation after the curve fitting technique (AbouLaila et al. 2010a).
In vitro drug combination test
Combination therapies of allicin and diminazene aceturate were tested in the in vitro cultures of B. bovis and B. caballi. Allicin/diminazene aceturate combinations (M1, M2, M3, M4, M5, M6, M7, and M8) were prepared as previously described (AbouLaila et al. 2010a) with some modifications. Combinations were based on the calculated IC50 values obtained from the in vitro inhibition assay (Table 1). For experimental control, drug-free cultures were used. Cultures containing only diminazene aceturate IC50 of each parasite were used as positive drug controls. Three separate trials were performed, consisting of triplicate experiments for individual drug concentrations over a period of 4 days. During the incubation period, the overlaying culture medium was replaced daily with 200 μl of fresh medium containing the indicated concentrations of allicin. Parasitemias were monitored daily by counting the parasitized RBCs to approximately 1,000 RBCs in Giemsa-stained thin blood smears. The growth of all parasites was calculated based on the parasitemia observations recorded on 4 days of the in vitro cell culture system (AbouLaila et al. 2010a).
Viability test for in vitro growth inhibition assays
After 4 days of treatment, 6 μl of each of the control and drug-treated (at the various indicated concentrations) infected RBCs was mixed with 14 μl of parasite-free RBCs and suspended in fresh growth medium without allicin supplementation. The plates were incubated at 37 °C for the next 10 days. The culture medium was replaced daily, and parasite recrudescence was determined by light microscopy in order to assess the parasite viability (Bork et al. 2004).
Invasion inhibition assay
The assay was performed as described earlier (Gaffar et al. 2004), but with modification. Briefly, B. bovis-parasitized erythrocytes harvested at the peak parasitemia were pelleted, washed once, and resuspended in an equal volume of GIT (Wako). Merozoites were liberated by five intermittent high voltage pulses (1.25 kV, 200 Ω, 25 μF) with 10 s in ice between pulses in a 4-mm cuvette (Bio-Rad) using a Bio-Rad Gene Pulser with a pulse controller, and then they were resuspended in GIT. A similar method was used for B. bigemina, B. caballi, and T. equi. Merozoites of these infected RBCs were liberated by ten intermittent high voltage pulses (1.20 kV, 300 Ω, 25 μF) with 5 s in ice, and then they were resuspended in M199 for B. bigemina and T. equi and in GIT for B. caballi.
The liberated parasites were incubated at 37 °C in a humidified multi-gas water-jacketed incubator (90 % N2, 5 % CO2, and 5 % O2) with 100 μM and 200 μM of allicin for 30 min. Bovine or equine erythrocytes were then added to final PCV of 10 % and incubated again at 37 °C in a humidified multi-gas water-jacketed incubator (90 % N2, 5 % CO2, and 5 % O2) for 24 h. Giemsa-stained smears were prepared after 1, 3, and 6 h. Invaded and freed parasites were counted on the basis of approximately 3,000 observed erythrocytes. The incubation of merozoites and RBCs without drugs was used as a control.
In vivo growth inhibition and drug combination assays in mice
The in vivo growth inhibition assay for allicin was performed in BALB/c mice as previously described (AbouLaila et al. 2012) in three separate trials. Twenty 8-week-old female BALB/c mice were divided into four groups, each containing five mice, and intraperitoneally inoculated with 1 × 107
B. microti-infected RBCs. In the first group, allicin was administered subcutaneously (s/c) at a dose rate of 30 mg/kg (El-Sabban and Radwan 1997); 100 mg/kg of allicin was administrated s/c to the second group; diminazene aceturate at a concentration of 25 mg/kg was administrated s/c to the third group as a reference drug control; and the fourth group was the DMSO control.
To validate the efficacy of the allicin/diminazene aceturate combination in vivo, thirty-five 8-week-old female BALB/c mice were divided into seven groups, each containing five mice, which were intraperitoneally inoculated with 1 × 107
B. microti-infected RBCs in two separate trials. In the first group, allicin was administered s/c at a dose rate of 15 mg/kg; in the second group, allicin was administered s/c at a dose rate of 30 mg/kg; in the third group, 50 mg/kg of allicin was administered s/c and diminazene aceturate at a dosage of 6.75 mg/kg was administrated s/c to all three previous groups as a combination drug for the same inoculation period; the fourth group was administrated 15 mg/kg of allicin combined with s/c administration of 12.5 mg/kg diminazene aceturate. The fifth and sixth groups were administrated diminazene aceturate at dosages of 6.75 and 25 mg/kg as control groups, and the last group was the DMSO control.
By checking, when the infected mouse parasitemia reached approximately 1 % in all groups, mice were administered daily injections of the specified drugs for 5 days from days 1 to 5 post-inoculation (p.i.). The levels of parasitemia in all mice were monitored daily until 21 days post-inoculation or cessation of parasitemia by examination of the Giemsa-stained thin blood smears prepared from venous tail blood. Chemicals were prepared by dissolving allicin in 0.06 % DMSO and resuspended in 0.1 ml double-distilled water. Diminazene aceturate dissolved in 0.1 ml double-distilled water and DMSO was administered to the control groups in 0.1 ml PBS in all experiments (AbouLaila et al. 2012). All animal experiments were conducted in accordance with the Standard Relating to the Care and Management of Experimental Animals set by the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
The differences in the percentage of parasitemia for the in vitro cultures, drug combination test, and in vivo inhibition assay were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA) using the independent Student’s t test and one-way ANOVA (AbouLaila et al. 2012).