SYBR Green I (SGI) nucleic acid stain (Lonza, Rockland, United States; 10,000 x) was stored at −20 °C and thawed before use for Babesia inhibition assay in vitro and in vivo. A lysis buffer was prepared in advance and stored at 4 °C. Resveratrol (Sigma-Aldrich, Japan) stock solution was dissolved in double distilled water (DDW) at 0.0022 g/100 μL and kept at −30°C until use. The commonly used antibabesial drugs DA (Ganaseg, Ciba-Geigy Japan Ltd., Tokyo, Japan) and ID (Sigma-Aldrich, Japan) were utilized as positive control drugs. Furthermore, the compound MMV396693 (MolPort, Latvia) and AZM (Tocris Bioscience, UK) were used for combination inhibition assays.

Babesia bovis (Texas strain), B. bigemina (Argentina strain), B. divergens (German strain), and B. caballi and Theileria equi (USDA strain) were grown and maintained in purified bovine or equine red blood cells (RBCs) using a microaerophilic stationary-phase culture technique at 37°C under the environmental condition of 5% CO₂, 5% O₂ and 90% N2 as detailed in our previous studies (Rizk et al., 2015; Rizk et al., 2017b; Rizk et al., 2021, 2023).

The inhibitory effect of RVT on the screened piroplasm growth was investigated using an SGI-based fluorescence assay (Rizk et al., 2015; Rizk et al., 2016; Rizk et al., 2020). Double 96-well plates (Nunc, Roskilde, Denmark) were used to cultivate bovine Babesia and equine Babesia/Theileria pRBCs with either media alone or medium with the appropriate doses of RVT ranging from 0.005 to 200 μM. DA was used as a positive control with concentrations ranging from 0.25 to 10 μM. Meanwhile, cultures without the drug and/or cultures containing only the used solvent DDW (0.02%) were used as negative controls. A viability assay was performed as previously detailed in our studies to assess the antipiroplasm efficacy of RVT following the stop of the treatment (Rizk et al., 2016; Rizk et al., 2017b; Rizk et al., 2020; Rizk et al., 2021).

A pharmaceutical combination experiment was carried out using the Chou-Talalay approach at a constant ratio (Rizk et al., 2023). A two-drug combination (RVT + DA, RVT + ID, RVT + MMV396693) was added in triplicate to the wells containing B. bovis-, B. bigemina- and T. equi-infected RBC in a 96-well plate at concentrations of 0.25 x IC50, 0.5 x IC50, IC50, 2 x IC50 and 4 x IC50.

All of the in vitro experiments were performed at 1% parasitemia, and 2.5% hematocrit (HCT) for B. bovis and B. bigemina and 5% HCT for other screened parasites (Rizk et al., 2017b; Rizk et al., 2020; Rizk et al., 2021). After 4 days of incubation, 100 μL a lysis buffer mixed with a 2× SGI was added directly to each well in the 96- wells plates. Then, the emitted fluorescence signals were determined using a fluorescence spectrophotometer. The experiments were repeated thrice.

Reverse transcription-PCR (RT-PCR) was performed to investigate the impact of RVT administration on BbKATs and BbKDAC3 mRNA transcription based on the previously described method (Aboulaila et al., 2012) with some modifications. Resveratrol was added to B. bovis in 24-well culture plates for 8 h at the 99% inhibitory concentration (IC99) (58.42 μM). The solvent served as a negative control. A commercial RNeasy minikit (QIAGEN) was used to extract total RNA from RBCs based on the manufacturer’s instructions. The extracted RNA (150 ng) from the treated- and controlled-cultures was used to amplify BbKATs and BbKDAC3 genes using a One Step RNA Kit (AMV) (Takara, Japan) following the manufacturer’s protocol. The B. bovis profilin (BbPROF) gene is used as the control gene. The used forward and reverse primers are listed in Table 1. The used PCR conditions were as follows: 30 min at 50°C for reverse transcription, denaturation at 94°C for 2 min then 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s for BbKDAC3, BbPROF and 57°C for BbKATs then, extension at 72°C for 1 min, and final extension at 72°C for 5 min. Finally, the amplified products were analysed by electrophoresis on 2.0% (w/v) agarose gels and visualised by a UV transilluminator after staining with ethidium bromide (Nippon Gene, Tokyo, Japan). The experiment was repeated thrice.

The in vivo inhibitory effect of RVT on the growth of B. Microti (Munich strain) was evaluated twice in BALB/c mice aged 8 weeks (CLEA, Tokyo, Japan) using a fluorescence-based SGI assay (Rizk et al., 2017a; Rizk et al., 2020). All mice were housed in pathogen-free environments. A total of 50 female BALB/c mice were equally divided into 10 groups. All animals were intraperitoneally inoculated with 1 × 107 B. Microti-infected RBCs, except for the mice in the first group which remained uninfected and served as negative control. When the parasitemia in the infected mice reached 1%, mice in the experimental groups were treated with the specific drug. Each RVT, DA and ID was dissolved in DDW (12.5%) before inoculation. Meanwhile, AZM was dissolved in DMSO (0.02%).

As a positive placebo control, mice in the second group were given intraperitoneal doses of the used solvent. In addition, mice in the third group were given DA subcutaneously at 25 mg/kg. Meanwhile, mice in the fourth group were given 25 mg/kg AZM intraperitoneally. Moreover, mice in the fifth group were given 8.5 mg/kg ID. Furthermore, mice in the sixth group were intraperitoneally administered a non-toxic dose (5 mg/kg) from RVT. The remaining groups (7th and 8th) were injected with drug combinations of RVT 5 mg/kg + AZM 25 mg/kg or RVT 5 mg/kg + ID 8.5 mg/kg. All drugs were administrated for 5 successive days except those treated with AZM monotherapy or AZM combined with RVT, which were treated for 14 successive days.

A venous tail blood sample (2.5 μL) was drawn from each mouse every 48 h until 28 days post-inoculation or the end of parasitemia and placed in a 96-well plate with RPMI 1640 Medium previously mixed with 50 μL lysis buffer. Afterwards, each well is properly mixed with 50 μL of lysis buffer containing 2X SG I nucleic acid stain. Then, the plate was incubated in the dark for 1 h. Subsequently, the emitted fluorescence signals as indicators of the parasitemia were determined as previously detailed.

A nested PCR assay targeting the B. Microti small subunit rRNA (ss-rRNA) gene was performed to determine the presence of parasite nucleic acid remnant in the blood and tissue of the treated mice (Rizk et al., 2023). On day 28 post-infection, blood and tissue samples (heart, lung, liver, kidney and spleen) were collected from B. Microti–infected mice treated with 25 mg/kg DA, RVT 5 mg/kg + ID 8.5 mg/kg, the solvent (positive control) and non-treated mice (negative control). NucleoSpin tissue kit (Macherey-Nagel, Düren, Germany) and a QIAamp DNA Blood Mini Kit (Qiagen, Tokyo, Japan) were used for DNA extraction from tissues and blood, respectively. PCR cycling was carried out as previously detailed in our study (Rizk et al., 2017a). The experiment was repeated twice.

For the cardioprotective effect of RVT to be determined, cardiac troponin T (cTnT) level in the plasma, and heart tissue of B. microti-infected mice treated with DA 25 mg/kg, AZM 25 mg/kg, AZM 25 mg/kg + RVT 5 mg/kg and RVT 5 mg/kg as well as in those treated with the solvent (positive control) and non-treated mice (negative control). The plasma was collected on day 0 (pre-infection), day 10 and day 28 post-infection. Meanwhile, heart tissue samples were collected on day 28 post-infection. Cardiac troponin T level was determined using commercially available mouse ELISA reagent kits (MyBioSource, Inc., United States) based on the manufacturer’s recommendations. The experiment was repeated twice.

The significant differences between the groups in the present study were determined using GraphPad Prism (version 5.0 for Windows; GraphPad Software, Inc., San Diego, CA, United States). In addition, a one-way ANOVA test was used and a p-value lower than 0.05 was considered statistically significant.

All experimental protocols used in this study were authorized by the Obihiro University of Agriculture and Veterinary Medicine’s Animal Care and Use Committee (Approval No. 27-65). All research was done following the Fundamental Guidelines for the Proper Conduct of Animal Experiments and Related Activities at Academic Research Institutions published by the Ministry of Education, Culture, Sports, Science and Technology of Japan. The following were the IDs for the pathogen experiment: Babesia microti: 20170905; equine piroplasm parasites: 201910-2; and bovine Babesia: 201708-4.

The in vitro growth of Babesia bovis, B. bigemina and B. divergens was significantly inhibited (p < 0.05) by RVT treatments of 25 μM, 50 μM and 100 μM, respectively (Figure 1). The estimated IC50 values revealed that RVT has the greatest inhibitory effects on B. bovis growth (Table 2). The in vitro growth of Theileria equi and B. caballi was significantly inhibited (p < 0.05) by RVT treatments of 10 μM and 200 μM, respectively (Figure 2).

The calculated IC50 values of RVT treatments on all screened parasites were higher than those of the commonly used antibabesial drug, DA (Table 2). With 50 μM of RVT, subsequent viability tests revealed that there was no regrowth of B. bovis (Supplementary Table S1). These results indicate the higher in vitro efficacy of RVT on B. bovis growth inhibition rather than other bovine and equine piroplasms. However, none of the other piroplasmids displayed regrowth at a concentration of 200 μM (Supplementary Table S1). Thus, RVT was used combined with either DA, ID or MMV396693 to treat the parasites whose growth exhibited a high inhibition in the presence of RVT monotherapy; B. bovis, B. bigemina, and T. equi.

All two-drug combinations RVT + DA, RVT + ID or RVT + MMV396693 showed an antagonist effect on the growth of all selected parasites (Table 3).

Resveratrol at the IC99 concentration decreased the mRNA transcription of the target BbKATs but not the mRNA transcription of the control BbPROF gene within 8 h of treatment (Figure 3A). Furthermore, RVT treatment relatively promotes mRNA transcription of the target gene BbKDAC3 but does not inhibit mRNA transcription of the target gene BbKDAC3 but does not inhibit mRNA transcription of the control gene BbPROF (Figure 3B; Supplementary Figure S1).

Unfortunately, there are no suitable laboratory experimental animals for bovine and equine Babesia infections. Alternatively, a mouse model infected with B. Microti, is commonly used for drug evaluation of the antibabesial drugs against animal babesiosis (Rizk et al., 2017a) since the inhibitory effect of the newly developed drug must be evaluated in laboratory animal to determine the possible side adverse effect of these hits before it is administration to animals under field condition. In the present study, RVT treatment alone displayed significant suppression (p < 0.05) in the emitted fluorescence signals from days 8–28 post-infection as compared with the positive control group (Figure 4A). Resveratrol showed an additive effect to the ID in combined treatment however, RVT showed an antagonistic effect to AZM at day 10 p. i. (Table 4). Peak fluorescence values in the treated groups with RVT 5 mg/kg and ID 8.5 mg/kg combinations reached an average of 395.70 at day 10 post-infection (pi). Meanwhile, fluorescence readings were significantly reduced (p < 0.05) in mice treated with RVT and ID combination from days 8–28 p. i. When compared to positive control mice (infected nontreated) (Figure 4A). Unfortunately, the parasite DNA was identified in the blood and other tissues of mice administered either DA monotherapy or RVT and ID combination based on nested PCR analysis (Figures 4B, C). Babesia microti DNA was identified in the blood and all analysed organs of infected mice treated with the solvent (positive control), however, no bands were found in non-treated non-infected mice (negative control) (Supplementary Figure S2). Although the present study determined the remnant of the parasite nucleic acid in the blood and other tissues of treated mice using nested PCR assay, further experiment as quantitative qPCR assay is necessary to determine the amount of nucleic acid in treated mice.

Notably, treatment with 5 mg/kg RVT+ 8.5 mg/kg ID resulted in 81.55% inhibition at day 10 p. i. (peak of parasitemia) as compared to 66.16% inhibition with 25 mg/kg DA and 76.13% inhibition with 8.5 mg/kg ID (Figure 5A). In addition, 5 mg/kg of RVT boosted the inhibition of B. microti by AZM (25 mg/kg) in mice (Figure 5B; Supplementary Figure S3).

Azithromycin has been suggested to have cardiotoxic effects thus to evaluate the ability of RVT to alleviate this side effect, cTnT levels in the plasma and heart tissues were evaluated by ELISA. At day 0, cTnT was nearly equal in the plasma of all mice groups, which decreased on day 10 (peak of parasitemia) to 99.25, 97.53, 101, and 98 pg/mL, and rise again to reach 110.61, 106.85, 105.18, and 106.73 pg/mL at day 28 post-infection in mice treated with RVT, RVT + AZM, positive control and non-treated mice, respectively (Supplementary Figure S4). Conversely, azithromycin treated group gradually increased plasma cTnT levels from day 0 (102.39 pg/mL) to 104.93 pg/mL at day 10 and 107.72 pg/mL at day 28 (Supplementary Figure S4).

On day 28 post-infection, cTnT levels in heart tissue samples collected from B. microti–infected mice were significantly higher (p < 0.05) in the group treated with 25 mg/kg AZM (120.57 pg/mL) than any of the other groups (Figure 6). Of note, 5 mg/kg RVT significantly reduced (p < 0.05) cTnT level (99.35 pg/mL) in the heart tissue of treated mice as compared with those treated with 25 mg/kg AZM (Figure 6). These findings suggest the possible role of RVT to alleviate the cardiotoxic effects of AZM.