Evaluating the inhibitory effect of resveratrol on the multiplication of several Babesia species and Theileria equi on in vitro cultures, and Babesia microti in mice

Abstract

Introduction: Histone post-translational modification is one of the most studied factors influencing epigenetic regulation of protozoan parasite gene expression, which is mediated by histone deacetylases (KDACs) and acetyltransferases (KATs). Objective and methods: The present study investigated the role of resveratrol (RVT) as an activator of histone deacetylases in the control of various pathogenic Babesia sp. and Theileria equi in vitro, as well as B. microti infected mice in vivo using fluorescence assay. Its role in mitigating the side effects associated with the widely used antibabesial drugs diminazene aceturate (DA) and azithromycin (AZM) has also been investigated. Results: The in vitro growth of B. bovis, B. bigemina, B. divergens, B. caballi and Theileria equi (T. equi) was significantly inhibited (P < 0.05) by RVT treatments. The estimated IC50 values revealed that RVT has the greatest inhibitory effects on B. bovis growth in vitro, with an IC50 value of 29.51 ± 2.46 µM. Reverse transcription PCR assay showed that such inhibitory activity might be attributed to resveratrol's stimulatory effect on B. bovis KDAC3 (BbKADC3) as well as its inhibitory effect on BbKATS. RVT causes a significant decrease (P < 0.05) in cardiac troponin T (cTnT) levels in heart tissue of B. microti- infected mice, thereby indicating that RVT may play a part in reducing the cardiotoxic effects of AZM. Resveratrol showed an additive effect with imidocarb dipropionate in vivo. Treatment of B. microti-infected mice with a combined 5 mg/kg RVT and 8.5 mg/kg ID resulted in an 81.55% inhibition at day 10 postinoculation (peak of parasitemia). Conclusion: Our data show that RVT is a promising antibabesial pharmacological candidate with therapeutic activities that could overcome the side effects of the currently used anti-Babesia medications.

Keywords: T. equi; azithromycin; babesia; cardiac troponin T; diminazene aceturate; imidocarb dipropionate; resveratrol.

FIGURE 1

Correlation between relative fluorescence units (RFUs) and the log concentrations of resveratrol (nM) on bovine Babesia parasites in vitro. (A) B. bovis. (B). B. bigemina. (C) B. divergens. Each value represents the mean of triplicate wells after subtraction of the background fluorescence for non-parasitised RBCs. Asterisks indicate a significant difference (ANOVA; *p < 0.05) between the resveratrol-treated and the control cultures.

FIGURE 2

Correlation between relative fluorescence units (RFUs) and the log concentrations of resveratrol (nM) on equine piroplasm in vitro. (A) T. equi. (B) B. caballi. Each value represents the mean of triplicate wells after subtraction of the background fluorescence for non-parasitised RBCs. Asterisks indicate a significant difference (ANOVA; *p < 0.05) between the resveratrol-treated and the control cultures.

FIGURE 3

Reverse transcription-PCR analysis of B. bovis KATs (BbKATs), B. bovis KDAC3 (BbKDAC3) and B. bovis profilin (BbPROF) genes from B. bovis cultures treated with resveratrol at IC99 concentration and DMSO (0.1%) used as a control for 8 h (A). BbKATs. (B). BbKDAC3. Lanes 1 and 3 from the control culture; lanes 2 and 4 resveratrol-treated culture. M, molecular size marker.

FIGURE 4

In vivo inhibitory efficacy of resveratrol. (A). Inhibitory effect of resveratrol, imidocarb dipropionate and the combination of both drugs on the growth of Babesia microti. Each value represents the mean ± standard deviation of five mice per experimental group. Asterisks indicate significant differences (ANOVA; *p < 0.05) between the resveratrol-treated and control groups. (B). PCR of the ss-rRNA gene in blood and different organs of (B). microti-infected mice treated with 25 mg kg −1 diminazene aceturate (DA), and (C). Imidocarb dipropionate (ID) combined with resveratrol (RVT). Bl, blood; Hr, heart; Lg, lung; Lv, liver; Kd, kidney; Sp, spleen. M indicates a 100 bp DNA ladder.

FIGURE 5

Percentages of inhibition in the growth of B. Microti in mice caused by resveratrol-mono-, and combination therapies on the days with peak parasitemia. (A) resveratrol and azithromycin. (B) resveratrol and imidocarb dipropionate. The % of parasite inhibition in each treated group was calculated as a ratio to the positive control group.

FIGURE 6

Cardiac troponin level in the heart tissues of mice treated with resveratrol and azithromycin combination therapy. Asterisks indicate significant differences (ANOVA; *p < 0.05) between the azithromycin-treated and other groups.