Tafenoquine succinate inhibits the growth of the equine piroplasmosis hemoparasites Theileria equi and Babesia caballi

Tafenoquine succinate inhibits the growth of the equine piroplasmosis hemoparasites Theileria equi and Babesia caballi | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tafenoquine succinate inhibits the growth of the equine piroplasmosis hemoparasites Theileria equi and Babesia caballi Natalia N. Cardillo, Nicolas F. Villarino, Lowell S. Kappmeyer, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7769436/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jan, 2026 Read the published version in Parasites & Vectors → Version 1 posted 8 You are reading this latest preprint version Abstract Background Equine piroplasmosis (EP) is a tick-borne disease of equids caused by intraerythrocytic apicomplexan parasites Theileria equi , Babesia caballi , and the recently identified Theileria haneyi . Acute cases can be severe, with anemia, jaundice, abortion, or sudden death. Survivors remain lifelong carriers, serving as reservoirs for tick-borne and iatrogenic transmission. No vaccines are currently available, and control strategies rely heavily on accurate diagnostics and chemotherapeutic intervention. Imidocarb dipropionate (ID) is the current standard of care for both acute treatment and radical cure. However, growing concerns regarding ID-resistant parasite strains and its associated toxicity have highlighted the urgent need for novel, safer, and more effective antiparasitic agents. Here, we assessed the in vitro efficacy of tafenoquine succinate (TFQ), a synthetic 8-aminoquinoline with broad antiparasitic activity, against T. equi and B. caballi as a potential treatment for equine piroplasmosis. Methods The effect of TFQ on T. equi and B. caballi was evaluated in vitro in parasite cultures. The percentage of parasitized erythrocytes was measured by flow cytometry, and the effect of TFQ on parasite growth was compared to that of ID. TFQ toxicity on horse peripheral blood mononuclear cells (PBMC) was assessed via a colorimetric metabolic assay. Results TFQ reduced T. equi parasitemia dose-dependently, matching ID efficacy at 72 hours. For B. caballi , TFQ had no effect at 5–10 µM but inhibited growth at 15 µM, similar results to ID. TFQ exhibited approximately threefold greater potency against T. equi [IC₅₀: 5.90 µM (95% CI: 4.99–5.96); IC₉₉: 60.74 µM (95% CI: 37.41–113.3)] compared to B. caballi [IC₅₀: 14.5 µM (95% CI: 13.81–15.23); IC₉₉: 20.44 µM (95% CI: 17.77–28.84)]. The narrower confidence intervals for T. equi suggest a more consistent antiparasitic response across replicates. Cytotoxicity assays showed no toxic effects on equine PBMCs at 2.5–5 µM (p > 0.05), while concentrations ≥ 10 µM indicated potential toxicity. These findings suggest TFQ selectively targets parasites over host cells, supporting its therapeutic potential. Conclusions TFQ significantly inhibited T. equi and B. caballi growth at doses tolerated by equine PBMCs, supporting its potential as an alternative treatment for EP and warranting further in vivo study. therapeutics horses tafenoquine succinate imidocarb dipropionate Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Equine piroplasmosis (EP) is a significant tick-borne disease that affects horses worldwide, caused by the protozoan apicomplexan hemoparasites Theileria equi , Babesia caballi , and the recently identified Theileria haneyi [ 1 , 2 ]. The disease poses a serious threat to equine health and significant economic losses in the equine industry due to its impact on animal performance and restriction on international movement [ 3 , 4 ]. In addition to tick transmission, iatrogenic and transplacental transmissions of EP parasites have also been reported [ 5 , 6 ]. Currently, no vaccines are available against EP, and control relies on diagnostics testing, antiparasitic drugs, and supportive treatments [ 1 ]. Current antiparasitic treatment options for EP are limited, with imidocarb dipropionate (ID) being the drug of choice [ 7 ]. However, ID-linked toxicity in horses, lack of effectiveness against T. haneyi , and drug-resistant field isolates of T. equi and B. caballi have been documented [ 8 ]. Additionally, a previous study demonstrated that co-infection of T. haneyi and T. equi reduces the efficacy of ID against T. equi [ 9 ]. This is particularly concerning, as ID remains the primary drug used globally to manage the clinical signs of acute disease and to achieve the complete cure of EP [ 10 , 11 ]. Taken together, these factors underscore the urgent need for new, safer, and more effective therapeutic strategies to control the causative agents of EP [ 8 , 11 ]. Tafenoquine succinate (TFQ) is an 8-aminoquinoline compound discovered by the Walter Reed Army Institute of Research in 1978 as a potential replacement for primaquine in the treatment and prevention of malaria caused by Plasmodium parasites [ 12 , 13 ]. TFQ is effective not only against P. falciparum but also in achieving the radical cure of P. vivax , owing to its pharmacological properties and long half-life [ 14 , 15 ]. In addition, recent studies have demonstrated that TFQ is effective against Babesia microti , one of the major causative agents of human babesiosis, an emerging tickborne disease in the United States, Europe and Asia [ 16 , 17 ]. While the mechanism of action of TFQ is not fully understood, it is known that the drug can disrupt mitochondrial function [ 18 , 19 ], inhibit hematin polymerization [ 20 ], and induce oxidative stress [ 15 , 21 ]. These factors can contribute to the drug’s efficacy against various Plasmodium species and parasite stages, including pre-erythrocytic (liver stages), erythrocytic (asexual stages), and gametocytes, thereby helping to provide prophylactic protection, prevent relapses and achieve radical cure [ 19 , 22 , 23 ]. Safety studies have demonstrated that TFQ is well tolerated when administered orally to humans and laboratory animals [ 24 ]. Considering both the safety and efficacy data of TFQ, the drug was approved in 2018 by the United State Food and Drug Administration (FDA) for prophylactic use and the radical cure of human malaria [ 25 ]. While generally well-tolerated, TFQ can cause asymptomatic hemoglobin decline and is contraindicated in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals [ 26 , 27 ]. Although G6PD deficiency affects approximately 400 million people globally, the alteration is considered rare or non-existent in agricultural animal species [ 28 ]. To the best of our knowledge, no studies have been so far reported on the effect of TFQ on Theileria and Babesia species that affect agricultural animals. Considering the broad efficacy of TFQ against apicomplexan parasites, including Plasmodium and Babesia species, we evaluated the in-vitro inhibitory effect of the drug on the growth of T. equi and B. caballi . This comparative efficacy study between tafenoquine (TFQ) and imidocarb dipropionate (ID) aimed to evaluate TFQ as a potential new treatment for EP, with the goal of overcoming limitations of currently approved drugs. Methods Drug compounds Lyophilized TFQ, with ≥ 95% purity determined by high-performance liquid chromatography (HPLC), was purchased from Millipore-Sigma (catalog number TSML0396, St. Louis, MO, USA). The compound was suspended in dimethyl sulfoxide (DMSO), following the manufacturer’s recommendations. ID (VETRANAL™, Supelco® Buchs, Switzerland) was used as a reference compound in the in vitro inhibition assays for T. equi and B. caballi , following the same protocol described below for TFQ. Purity of ID was determined to be > 98% by proton-nuclear magnetic resonance spectroscopy and HPLC, according to the certificate of analysis. TFQ and ID were diluted in 100% DMSO to prepare stock solutions, which were stored at room temperature until use. Working solutions were freshly prepared in parasite culture medium on each test day prior to being added to the parasite cultures. T. equi and B. caballi in vitro culture The T. equi Florida isolates and B. caballi Puerto Rico isolates were maintained in long-term microaerophilic stationary-phase cultures at 37°C under atmospheric condition of 5% CO 2 , 5% O 2 , and 90% N 2 , as previously described [ 29 – 31 ]. Both parasites were cultured in 24-well culture plates, containing 20% (v:v) red blood cells (RBC) in 1 ml per well of HL-1 culture medium (pH 7.2). Parasite growth inhibition assay Theileria equi and B. caballi cultures were initiated at parasitized erythrocyte percentages (PPE) of 1% and 0.5%, respectively. Parasites were cultured in media containing TFQ and ID at concentrations of 5, 10, and 15 µM, diluted in 100% DMSO. Experiments were conducted in triplicate wells for each drug concentration and corresponding controls over an 8-day period. Cultures maintained in drug-free medium and uninfected RBCs served as positive and negative controls, respectively. Drug-containing media were replaced daily for the first 3 days of culture. Seventy-two hours after the final treatment, the medium in all culture wells was replaced with fresh drug-free medium, and 100 µL of fresh RBCs were added to each well. Only fresh media (drug-free) was replaced daily for 4 more days to determine if the parasite cultures were viable and could continue growing in the absence of the drug. Parasitemia level was assessed daily using flow cytometry, between days 1 through 8, as described below. IC₅₀ and IC₉₉ determination for TFQ Cultures of both T. equi and B. caballi were initiated at 1% PPE. T. equi was treated with TFQ at concentrations of 1.25, 2.5, 3.75, 6.25, 7.5, 12.5, 15, and 25 µM, while B. caballi was treated with concentrations of 2.5, 3.75, 6.25, 7.5, 12.5, 15, 25, and 35 µM. All experiments were conducted in triplicate wells for each concentration, including the corresponding controls. The drug-containing medium was replaced daily for 72 hours (3 days) of culture, after which PPE was assessed by flow cytometry. In both experiments, DMSO-treated parasites (without drug) served as positive controls, and uninfected equine RBCs served as negative controls. Flow cytometric analysis PPE was determined by flow cytometry analysis after staining the parasite cultures with hydroethidine (HE), as previously described (4,32). Briefly, 5 µL of the cultures were collected from the bottom of the wells and suspended in 150 µL of phosphate-buffered saline (PBS) at pH 7.2. The sample was then centrifuged at 450 xg for 1 minute at 4 ℃. This procedure was repeated twice, using the same buffer. The pellet was then resuspended in 200 µl of HE at a concentration of 25 µg/µl (Invitrogen, Carlsbad, CA, USA) and incubated at 37 ℃ in 5% CO 2 atmosphere for 20 min in the dark. Following incubation, the cells were washed twice with 200 µl of PBS to remove excess HE. The supernatant was then discarded, and the cell pellet was resuspended in 200 µl of fresh PBS. The resuspended cells were analyzed by flow cytometry using a Guava® easyCyte flow cytometer (Luminex Corp., Austin, TX, USA) at a ratio of 800–1000 cells/µl with 20,000 events collected. The results were analyzed by FCS Express v6 (De Novo Software, Glendale, CA, USA). Uninfected horse RBC were used as a negative control for the flow cytometric analysis. PBMC cytotoxicity assay Cytotoxicity assay was conducted to evaluate the potential effects of TFQ on the viability of equine PBMC, which were used as surrogates for nucleated vertebrate host cells. The assays were performed in in vitro cultures using 2.5, 5, 10, 15, 20, and 50 µM of TFQ. PBMC viability was assessed by monitoring cellular metabolic activity using a colorimetric WST-1 assay, as previously described (4). Briefly, peripheral blood was collected from healthy horses via jugular venipuncture into Vacutainer® tubes containing acid citrate dextrose (ACD) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). PBMCs were isolated according to standard procedure using Histopaque® (Sigma-Aldrich). The cells were plated at a density of 2 × 10 5 cells/well in 96-well plates in complete Dulbecco’s Modified Eagle Medium (cDMEM) (Sigma-Aldrich), containing 10% fetal bovine serum, 24 mM HEPES, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The cells were then incubated with the respective TFQ concentrations. Cell proliferation reagent WST-1 (catalog number 05 015 944 001 Roche Applied Science, Penzberg, Germany) was added at 24-, 48-, and 72-hours post-exposure, according to the manufacturer’s instructions. Absorbance was measured at 440 nm using an ELISA plate reader 4 hours after adding WST-1. Negative controls included cells cultured in cDMEM without TFQ, as well as cells treated with DMSO alone (1:400 dilution, equivalent to the maximum volume used for TFQ compound dilutions). As a positive control for cytotoxicity, PBMCs were treated with 5 µg/ml concanavalin A (Con A) diluted in cDMEM. Specific selectivity index (SSI) The selectivity of TFQ against T. equi or B. caballi in comparison to mammalian cells (equine PBMCs) was assessed at the respective IC 50 concentrations using the standard formula described by Ndjakou Lenta et al. (2007) [ 32 ]: SSI = IC 50 ​ (parasite) / CC 50 ​ (host cells)​, where CC₅₀ represents the concentration that reduces host cell viability by 50% and IC₅₀ denotes the concentration that inhibits parasite growth by 50% [ 32 ]. Statistical analysis PPE levels were assessed daily and comparisons among TFQ-treated, ID-treated, and untreated controls were performed using the Kruskal-Wallis nonparametric test. The drug concentrations required to IC₅₀ relative to the untreated control, as well as the concentration needed to IC₉₉, were estimated for TFQ using nonlinear regression analysis. The normality of PBMC viability data was assessed using the Shapiro–Wilk test. Since the data did not meet the assumption of normality, the Kruskal–Wallis test was used for statistical analysis. GraphPad Prism v7 software for Windows (GraphPad Software, Inc., San Diego, CA, USA) was used for the statistical analysis. The significance level was set at p < 0.05. Results Tafenoquine exposure significantly inhibits the in vitro growth of T. equi TFQ exposure led to a progressive and consistent reduction in the T. equi PPE in a dose-dependent manner, resulting in significantly lower parasitemia compared to untreated control (Fig. 1 ). Moreover, TFQ demonstrated efficacy comparable to that of ID at 72 h post-treatment across all tested concentrations, with no significant differences ( p > 0.05) in inhibitory effect observed over time (Fig. 2 ). The results of in vitro growth of T. equi during a 3-day drug treatment period with either TFQ or ID, followed by a 5-day drug-free incubation period, are shown in Fig. 1 and Fig. 2 . Tafenoquine significantly suppress B. caballi growth During the first 72 h, the growth of B. caballi was significantly inhibited by ID at all tested concentrations. In contrast, TFQ did not show any inhibitory effect at 5 µM and 10 µM. However, at 15 µM, TFQ demonstrated a significant inhibitory effect on the parasite growth (Fig. 3 and Fig. 4 ). At 72 h post-treatment, TFQ showed limited efficacy against B. caballi at both 5 µM and 10 µM, with parasite growth rates similar to those observed in untreated control cultures. In contrast, ID exhibited significantly higher inhibitory activity at these concentrations, which was sustained throughout the observation period ( p < 0.01). At a concentration of 15 µM, both TFQ and ID demonstrated significant efficacy against B. caballi beginning on the second day of treatment, with no significant differences observed between the two through day 5. After this point, parasitemia in TFQ-treated cultures continued to decline, whereas recrudescence was noted in the ID-treated cultures. Figures 3 and 4 show the results of B. caballi in vitro growth during a 3-day exposure to either TFQ or ID, followed by a 5-day drug-free incubation period. Distinct TFQ potency and dose-response in T. equi than against B. caballi The mean and range of IC₅₀ values were calculated to compare the in vitro potency of TFQ against B. caballi and T. equi ( Table 1 and Fig. 5 ) . TFQ exhibited approximately threefold greater potency against T. equi than B. caballi , indicating species-specific differences in drug susceptibility. Notably, the IC₅₀ values of TFQ against T. equi exhibited a narrower 95% confidence interval, indicating a more consistent response across replicates. In contrast, the broader IC₅₀ range observed for B. caballi may reflect greater biological variability or differences in assay sensitivity. The remarkable difference between IC₅₀ and IC₉₉ values against T. equi suggests a steep dose-response curve, whereas the relatively smaller difference against B. caballi indicates a more gradual inhibition profile. Table 1 IC₅₀ and IC₉₉ values (95% CI) of Tafenoquine succinate (TFQ) for inhibition of T. equi and B. caballi after 72-hour in vitro cultures. IC 50 (µM) IC 99 (µM) T. equi 5.904 µM (IC 95 = 4.99–5.96) 60.74 µM (IC 95 = 37.41–113.3) B. caballi 14.5 µM (IC 95 = 13.81 to 15.23) 20.44 µM (IC 95 = 17.77 to 28.84) IC95% Confident Interval Cytotoxicity of Tafenoquine in Equine PBMCs To evaluate the potential cytotoxic effect of TFQ on host cells, equine PBMC were exposed to varying concentrations of the drug. During the post-treatment period (24, 48, and 72 hours), significant differences were observed among the various TFQ concentrations. No significant differences were observed between the control group and the treatment groups with 2.5 or 5 µM concentrations (p > 0.05); however, significant difference was observed between the control group and these groups treated with 10 to 50 µM concentrations of TFQ (p < 0.05) ( Fig. 6 ) . PBMC viability data suggests that TFQ at the IC 50 concentration for T. equi does not compromise cell viability. However, cytotoxicity may be more relevant in the case of B. caballi , as its IC 50 was three times higher. Additionally, a significant increase in PBMC proliferation was observed in cells exposed to ConA at 24 and 48 h ( p < 0.05) (Fig. 6 ), confirming the sensitivity of the WST-1 assay used in this study. Taken together, the results indicate that TFQ does not exert significant toxic effects on equine PBMCs cultured in vitro at concentrations of 2.5 and 5 µM but has toxic effects on PBMCs at concentration higher than 10 uM. Additionally, TFQ was 1.69 more selective for T. equi and 0.69 times more selective for B. caballi than for the host cells (Table 2) . Discussion The present study assessed the in vitro efficacy of tafenoquine succinate (TFQ) against Theileria equi and Babesia caballi . Our findings show that TFQ exerted a marked inhibitory effect on parasite growth, particularly on T. equi , with suppression persisting even after treatment withdrawal, suggesting a potential sustained parasiticidal activity. While B. caballi also responded to TFQ, its sensitivity was comparatively lower than that of T. equi . Nonetheless, TFQ at a concentration of 15 µM significantly reduced B. caballi parasitemia by day 3, with sustained suppression observed throughout the study. Notably, B. caballi cultures treated with ID showed recrudescence after initial inhibition, whereas those treated with TFQ did not, suggesting that TFQ may be more effective in preventing parasite recovery. Although the mechanism of action of tafenoquine (TFQ) remains unclear, a recent study using human primary hepatocytes demonstrated that primaquine, an 8-aminoquinoline closely related to TFQ, exerts its anti-Plasmodial effect through a mechanism dependent on cytochrome P450 NADPH:oxidoreductase, accompanied by the production of hydrogen peroxide (H₂O₂) [ 33 ]. Given the structural relatedness between primaquine and TFQ, it is plausible to assume that the latter compound also acts through host-generated oxidative intermediates. While the in vitro system used in this study may not fully reflect the profile of reductases present in vivo, NADPH reductases are known to be present in erythrocytes, the cells used to support the growth of T. equi and B. caballi in culture [ 34 ]. Therefore, it is plausible to speculate that these enzymes could be responsible for generating host-derived oxidative intermediates in the presence of TFQ, which may explain the compound’s parasiticidal effect. The IC₅₀ for T. equi is 5.9 µM, and for B. caballi 14.5 µM, which technically fall within the broad Plasmodium range, but the implication of "comparable sensitivity" is misleading, especially considering P. falciparum can be sensitive at submicromolar levels (e.g., 0.5 µM) [ 35 , 37 ]. A critical aspect in comparing drug efficacy between species lies in analyzing the relationship between IC₅₀ (concentration for 50% inhibition) and IC₉₉ (for near-complete inhibition). The gap between IC₅₀ and IC₉₉ offers insight into the compound’s dose-response dynamics. A wide gap suggests a steep dose-response curve, potentially indicating cooperative or heterogeneous mechanisms, whereas a narrow gap implies a more uniform and predictable response [ 36 ]. In this study, T. equi had a wider IC₅₀–IC₉₉ gap (IC₅₀: 5.9 µM; IC₉₉: 60.7 µM), indicating a steep dose-response curve. This pattern suggests that while low concentrations partially suppress the parasite, much higher concentrations are required for near-complete clearance. Such steep curves may reflect heterogeneous susceptibility within the parasite population, cooperative drug interactions, or differences in drug uptake or metabolism [ 15 , 37 ]. A similar trend was observed in a previous study with novobiocin, where T. equi displayed a steeper curve than B. caballi [ 7 ]. Conversely, B. caballi showed a narrower IC₅₀–IC₉₉ gap (IC₅₀: 14.5 µM; IC₉₉: 20.44 µM), indicating a more gradual and linear response to increasing TFQ concentrations. This may indicate a more homogeneous parasite population or a more consistent interaction between the drug and its target [ 36 , 38 ]. Similarly, narrow-gap profiles have been observed in other protozoan species with uniform drug sensitivity - such as clinical isolates of Plasmodium falciparum [ 39 ]- where limited subpopulation variability affected treatment outcomes. Interestingly, B. caballi has often exhibited greater sensitivity (lower IC₅₀ values) to other drugs than T. equi , with varying data on the IC₅₀–IC₉₉ gap [ 40 ]. The findings in the current study suggest that, although B. caballi exhibits a higher IC₅₀ to TFQ, its narrower IC₅₀–IC₉₉ gap may allow more straightforward dosing strategies. In contrast, the broader range observed in T. equ i requires careful dose optimization to ensure complete parasite clearance. TFQ demonstrated no significant cytotoxicity in equine PBMCs at concentrations up to 5 µM. Cell viability began to decline only at concentrations ≥ 10 µM (p < 0.05), which falls within or slightly above the effective antiparasitic range for T. equi , but remained below the concentration required for complete inhibition of B. caballi . The SSI of TFQ was nearly twice as high for T. equi as for host cells. In contrast, the SSI for B. caballi was only 0.69, indicating that the therapeutic concentrations required to inhibit this species approach or exceed the cytotoxic threshold in PBMCs. These findings highlight TFQ’s more favorable safety margin and selectivity against T. equi , while also underscoring its narrower therapeutic window for B. caballi . This profile is consistent with a previous study in human THP-1 cells, where TFQ demonstrated a relatively high LD₅₀ of 53.57 µM, indicating its preferential toxicity toward parasites over host cells [41]. Although tafenoquine has been associated with hemolytic toxicity in G6PD-deficient human erythrocytes [ 25 ], only a single case of G6PD deficiency has been reported in equines, unrelated to tafenoquine-induced hemolysis (40). This suggests that, although G6PD deficiency may have limited clinical relevance in horses, its significance remains uncertain and warrants further investigation. Taken together, our results support the potential of TFQ as a promising alternative to ID for the treatment of EP, particularly for T. equi . Its sustained activity, low micromolar efficacy, and low cytotoxicity make it a compelling candidate for further evaluation. Notably, TFQ’s broader antiparasitic spectrum and unique dose-response dynamics provide an opportunity for controlling mixed or resistant infections. However, to fully validate its clinical potential, in vivo studies will be required to determine pharmacokinetics, optimal dosing regimens, and therapeutic efficacy in horses. Conclusions This study provides the first evidence of TFQ’s potent in vitro activity against T. equi and moderate activity against B. caballi , along with a mild cytotoxic profile in equine PBMCs. These findings lay the foundation for further preclinical and clinical research and support further validation as a novel chemotherapeutic option for equine piroplasmosis. Abbreviations EP – equine piroplasmosis TFQ – tafenoquine succinate ID – imidocarb dipropionate PBMC – peripheral blood mononuclear cells IC – inhibitory concentration G6PD - glucose-6-phosphate dehydrogenase FDA - the United State Food and Drug Administration DMSO - dimethyl sulfoxide HPLC - high-performance liquid chromatography H₂O₂ - hydrogen peroxide Declarations Acknowledgements We are grateful to Megan Jacks, Shelby Beckner, Sarah Therrian, Elizabeth Hart, Emma Karel, and Kristin Erickson for their excellent technical assistance and animal care. Thanks to Isabelle Brock for maintenance of T. equi and B. caballi tissue cultures. The authors also wish to thank Dr. Geoffrey Dow, CEO of 60 Degrees Pharmaceuticals, Inc) for critically reviewing the manuscript and for fruitful discussions regarding tafenoquine as an anti-hemoparasitic therapeutic. Funding The research was funded by the USDA-ARS CRIS# 2090–32000-044-000-D. Availability of data and materials All data generated or analyzed during this study are included in this article and its supplementary materials. Authors’ contributions NC: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Writing–original draft, Writing–review and editing, Software. NV: Supervision, Writing–review and editing, Conceptualization, Data curation, Formal Analysis, Software. LK: Methodology, Writing–review and editing, Supervision. CS: Writing–review and editing. CC: Funding acquisition, Project administration, Writing–review and editing. RB: Supervision, Conceptualization, Writing–review and editing, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Validation, Visualization. Ethics approval and consent to participate Blood from healthy, hemoparasite-free horses was used in this study to maintain in vitro cultures of T. equi and B. caballi . Blood collections were performed according to protocols approved by the Institutional Animal Care and Use Committees of the University of Idaho (protocol # 2024-26) and Washington State University (protocol # 6982). Consent for publication Not applicable. No personal data or images of individuals were included in this study. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were not an editorial board member of the journal, at the time of submission. 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Cite Share Download PDF Status: Published Journal Publication published 27 Jan, 2026 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 17 Nov, 2025 Reviews received at journal 10 Nov, 2025 Reviewers agreed at journal 17 Oct, 2025 Reviewers agreed at journal 16 Oct, 2025 Reviewers invited by journal 14 Oct, 2025 Editor assigned by journal 06 Oct, 2025 Submission checks completed at journal 06 Oct, 2025 First submitted to journal 02 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":104900,"visible":true,"origin":"","legend":"\u003cp\u003eMean percentage (±StDev) of \u003cem\u003eT. equi\u003c/em\u003e-parasitized erythrocytes (PPE) in triplicate \u003cem\u003ein vitro\u003c/em\u003ecultures following treatment with varying concentrations of tafenoquine succinate (TFQ) and imidocarb dipropionate (ID). Data are presented for the initial 3-day treatment period and the subsequent 5-day drug-free incubation period (days 4–8).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7769436/v1/b9d69e93615aa529ccac497d.png"},{"id":94623224,"identity":"6b550326-b07a-4242-89a5-4dece298cbb9","added_by":"auto","created_at":"2025-10-29 04:19:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":77582,"visible":true,"origin":"","legend":"\u003cp\u003eMean percentage (±StDev) of \u003cem\u003eT. equi\u003c/em\u003e survival in triplicate in vitro cultures after exposure to different concentrations of tafenoquine succinate (TFQ) and imidocarb dipropionate (ID): (a) 5 µM, (b) 10 µM, and (c) 15 µM of TFO or ID. Parasites were treated for an initial 3-day period with drugs, followed by a 5-day drug-free incubation phase (days 4–8).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7769436/v1/61fbb4eea44eb5ce429d53ed.png"},{"id":94640867,"identity":"d660ea10-91d7-43d3-8af7-b602ff39ee00","added_by":"auto","created_at":"2025-10-29 07:50:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":125438,"visible":true,"origin":"","legend":"\u003cp\u003eMean percentage (±StDev) of \u003cem\u003eB. caballi\u003c/em\u003e-parasitized erythrocytes (PPE) in triplicate in vitro cultures following exposure to varying concentrations of tafenoquine succinate (TFQ) and imidocarb dipropionate (ID). Parasites were treated for an initial 3-day period, followed by a 5-day drug-free incubation phase (days 4–8).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7769436/v1/d836079200c6e5530263d22f.png"},{"id":94623501,"identity":"e7eb7817-7b9a-44ad-af9c-e0ebe446b333","added_by":"auto","created_at":"2025-10-29 04:19:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":79978,"visible":true,"origin":"","legend":"\u003cp\u003eMean percentage (±StDev) of \u003cem\u003eB. caballi\u003c/em\u003e survival in triplicate in vitro cultures after exposure to different concentrations of tafenoquine succinate (TFQ) and imidocarb dipropionate (ID): (a) 5 µM, (b) 10 µM, and (c) 15 µM. Parasites were treated for an initial 3-day period, followed by a 5-day drug-free incubation phase (days 4–8).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7769436/v1/aa005d1b39a17ce89bc3d741.png"},{"id":94623265,"identity":"d905a300-44f6-40e7-9d10-d7edc419f3bc","added_by":"auto","created_at":"2025-10-29 04:19:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":100702,"visible":true,"origin":"","legend":"\u003cp\u003eTafenoquine succinate (TFQ) potency against \u003cem\u003eB. caballi\u003c/em\u003e (a) and \u003cem\u003eT. equi\u003c/em\u003e (b) at 72h post-treatment.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7769436/v1/d052692977fc0856c210b202.png"},{"id":94623435,"identity":"28df711f-f5e5-4a51-a65f-199808950d12","added_by":"auto","created_at":"2025-10-29 04:19:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":95367,"visible":true,"origin":"","legend":"\u003cp\u003ePBMC viability (%) at various tafenoquine succinate (TFQ) concentrations measured at 24-, 48-, and 72-h post-treatment. Group columns marked with an asterisk (*) indicate statistically significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) compared to unmarked columns.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7769436/v1/d44eb9b62edc2c6515f0b447.png"},{"id":101692324,"identity":"4be3769d-3644-4bd2-84ee-8deb3ee76269","added_by":"auto","created_at":"2026-02-02 16:17:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1285561,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7769436/v1/b72dd923-7cff-473d-9fef-58f8c983eaf9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tafenoquine succinate inhibits the growth of the equine piroplasmosis hemoparasites Theileria equi and Babesia caballi","fulltext":[{"header":"Background","content":"\u003cp\u003eEquine piroplasmosis (EP) is a significant tick-borne disease that affects horses worldwide, caused by the protozoan apicomplexan hemoparasites \u003cem\u003eTheileria equi\u003c/em\u003e, \u003cem\u003eBabesia caballi\u003c/em\u003e, and the recently identified \u003cem\u003eTheileria haneyi\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The disease poses a serious threat to equine health and significant economic losses in the equine industry due to its impact on animal performance and restriction on international movement [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition to tick transmission, iatrogenic and transplacental transmissions of EP parasites have also been reported [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Currently, no vaccines are available against EP, and control relies on diagnostics testing, antiparasitic drugs, and supportive treatments [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Current antiparasitic treatment options for EP are limited, with imidocarb dipropionate (ID) being the drug of choice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, ID-linked toxicity in horses, lack of effectiveness against \u003cem\u003eT. haneyi\u003c/em\u003e, and drug-resistant field isolates of \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e have been documented [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, a previous study demonstrated that co-infection of \u003cem\u003eT. haneyi\u003c/em\u003e and \u003cem\u003eT. equi\u003c/em\u003e reduces the efficacy of ID against \u003cem\u003eT. equi\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This is particularly concerning, as ID remains the primary drug used globally to manage the clinical signs of acute disease and to achieve the complete cure of EP [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Taken together, these factors underscore the urgent need for new, safer, and more effective therapeutic strategies to control the causative agents of EP [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTafenoquine succinate (TFQ) is an 8-aminoquinoline compound discovered by the Walter Reed Army Institute of Research in 1978 as a potential replacement for primaquine in the treatment and prevention of malaria caused by \u003cem\u003ePlasmodium\u003c/em\u003e parasites [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. TFQ is effective not only against \u003cem\u003eP. falciparum\u003c/em\u003e but also in achieving the radical cure of \u003cem\u003eP. vivax\u003c/em\u003e, owing to its pharmacological properties and long half-life [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, recent studies have demonstrated that TFQ is effective against \u003cem\u003eBabesia microti\u003c/em\u003e, one of the major causative agents of human babesiosis, an emerging tickborne disease in the United States, Europe and Asia [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While the mechanism of action of TFQ is not fully understood, it is known that the drug can disrupt mitochondrial function [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], inhibit hematin polymerization [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and induce oxidative stress [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These factors can contribute to the drug\u0026rsquo;s efficacy against various \u003cem\u003ePlasmodium\u003c/em\u003e species and parasite stages, including pre-erythrocytic (liver stages), erythrocytic (asexual stages), and gametocytes, thereby helping to provide prophylactic protection, prevent relapses and achieve radical cure [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Safety studies have demonstrated that TFQ is well tolerated when administered orally to humans and laboratory animals [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Considering both the safety and efficacy data of TFQ, the drug was approved in 2018 by the United State Food and Drug Administration (FDA) for prophylactic use and the radical cure of human malaria [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. While generally well-tolerated, TFQ can cause asymptomatic hemoglobin decline and is contraindicated in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Although G6PD deficiency affects approximately 400\u0026nbsp;million people globally, the alteration is considered rare or non-existent in agricultural animal species [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To the best of our knowledge, no studies have been so far reported on the effect of TFQ on \u003cem\u003eTheileria\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e species that affect agricultural animals.\u003c/p\u003e\u003cp\u003eConsidering the broad efficacy of TFQ against apicomplexan parasites, including \u003cem\u003ePlasmodium\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e species, we evaluated the in-vitro inhibitory effect of the drug on the growth of \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e. This comparative efficacy study between tafenoquine (TFQ) and imidocarb dipropionate (ID) aimed to evaluate TFQ as a potential new treatment for EP, with the goal of overcoming limitations of currently approved drugs.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDrug compounds\u003c/h2\u003e\u003cp\u003eLyophilized TFQ, with \u0026ge;\u0026thinsp;95% purity determined by high-performance liquid chromatography (HPLC), was purchased from Millipore-Sigma (catalog number TSML0396, St. Louis, MO, USA). The compound was suspended in dimethyl sulfoxide (DMSO), following the manufacturer\u0026rsquo;s recommendations. ID (VETRANAL\u0026trade;, Supelco\u0026reg; Buchs, Switzerland) was used as a reference compound in the \u003cem\u003ein vitro\u003c/em\u003e inhibition assays for \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e, following the same protocol described below for TFQ. Purity of ID was determined to be \u0026gt;\u0026thinsp;98% by proton-nuclear magnetic resonance spectroscopy and HPLC, according to the certificate of analysis. TFQ and ID were diluted in 100% DMSO to prepare stock solutions, which were stored at room temperature until use. Working solutions were freshly prepared in parasite culture medium on each test day prior to being added to the parasite cultures.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eT. equi\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eB. caballi\u003c/b\u003e \u003cb\u003ein vitro culture\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eT. equi\u003c/em\u003e Florida isolates and \u003cem\u003eB. caballi\u003c/em\u003e Puerto Rico isolates were maintained in long-term microaerophilic stationary-phase cultures at 37\u0026deg;C under atmospheric condition of 5% CO\u003csub\u003e2\u003c/sub\u003e, 5% O\u003csub\u003e2\u003c/sub\u003e, and 90% N\u003csub\u003e2\u003c/sub\u003e, as previously described [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR30\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Both parasites were cultured in 24-well culture plates, containing 20% (v:v) red blood cells (RBC) in 1 ml per well of HL-1 culture medium (pH 7.2).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eParasite growth inhibition assay\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eTheileria equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e cultures were initiated at parasitized erythrocyte percentages (PPE) of 1% and 0.5%, respectively. Parasites were cultured in media containing TFQ and ID at concentrations of 5, 10, and 15 \u0026micro;M, diluted in 100% DMSO. Experiments were conducted in triplicate wells for each drug concentration and corresponding controls over an 8-day period. Cultures maintained in drug-free medium and uninfected RBCs served as positive and negative controls, respectively. Drug-containing media were replaced daily for the first 3 days of culture. Seventy-two hours after the final treatment, the medium in all culture wells was replaced with fresh drug-free medium, and 100 \u0026micro;L of fresh RBCs were added to each well. Only fresh media (drug-free) was replaced daily for 4 more days to determine if the parasite cultures were viable and could continue growing in the absence of the drug. Parasitemia level was assessed daily using flow cytometry, between days 1 through 8, as described below.\u003c/p\u003e\n\u003ch3\u003eIC₅₀ and IC₉₉ determination for TFQ\u003c/h3\u003e\n\u003cp\u003eCultures of both \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e were initiated at 1% PPE. \u003cem\u003eT. equi\u003c/em\u003e was treated with TFQ at concentrations of 1.25, 2.5, 3.75, 6.25, 7.5, 12.5, 15, and 25 \u0026micro;M, while \u003cem\u003eB. caballi\u003c/em\u003e was treated with concentrations of 2.5, 3.75, 6.25, 7.5, 12.5, 15, 25, and 35 \u0026micro;M. All experiments were conducted in triplicate wells for each concentration, including the corresponding controls. The drug-containing medium was replaced daily for 72 hours (3 days) of culture, after which PPE was assessed by flow cytometry. In both experiments, DMSO-treated parasites (without drug) served as positive controls, and uninfected equine RBCs served as negative controls.\u003c/p\u003e\n\u003ch3\u003eFlow cytometric analysis\u003c/h3\u003e\n\u003cp\u003ePPE was determined by flow cytometry analysis after staining the parasite cultures with hydroethidine (HE), as previously described (4,32). Briefly, 5 \u0026micro;L of the cultures were collected from the bottom of the wells and suspended in 150 \u0026micro;L of phosphate-buffered saline (PBS) at pH 7.2. The sample was then centrifuged at 450 xg for 1 minute at 4 ℃. This procedure was repeated twice, using the same buffer. The pellet was then resuspended in 200 \u0026micro;l of HE at a concentration of 25 \u0026micro;g/\u0026micro;l (Invitrogen, Carlsbad, CA, USA) and incubated at 37 ℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere for 20 min in the dark. Following incubation, the cells were washed twice with 200 \u0026micro;l of PBS to remove excess HE. The supernatant was then discarded, and the cell pellet was resuspended in 200 \u0026micro;l of fresh PBS. The resuspended cells were analyzed by flow cytometry using a Guava\u0026reg; easyCyte flow cytometer (Luminex Corp., Austin, TX, USA) at a ratio of 800\u0026ndash;1000 cells/\u0026micro;l with 20,000 events collected. The results were analyzed by FCS Express v6 (De Novo Software, Glendale, CA, USA). Uninfected horse RBC were used as a negative control for the flow cytometric analysis.\u003c/p\u003e\n\u003ch3\u003ePBMC cytotoxicity assay\u003c/h3\u003e\n\u003cp\u003eCytotoxicity assay was conducted to evaluate the potential effects of TFQ on the viability of equine PBMC, which were used as surrogates for nucleated vertebrate host cells. The assays were performed in in vitro cultures using 2.5, 5, 10, 15, 20, and 50 \u0026micro;M of TFQ. PBMC viability was assessed by monitoring cellular metabolic activity using a colorimetric WST-1 assay, as previously described (4). Briefly, peripheral blood was collected from healthy horses via jugular venipuncture into Vacutainer\u0026reg; tubes containing acid citrate dextrose (ACD) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). PBMCs were isolated according to standard procedure using Histopaque\u0026reg; (Sigma-Aldrich). The cells were plated at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in 96-well plates in complete Dulbecco\u0026rsquo;s Modified Eagle Medium (cDMEM) (Sigma-Aldrich), containing 10% fetal bovine serum, 24 mM HEPES, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 \u0026micro;g/ml streptomycin. The cells were then incubated with the respective TFQ concentrations. Cell proliferation reagent WST-1 (catalog number 05 015 944 001 Roche Applied Science, Penzberg, Germany) was added at 24-, 48-, and 72-hours post-exposure, according to the manufacturer\u0026rsquo;s instructions. Absorbance was measured at 440 nm using an ELISA plate reader 4 hours after adding WST-1. Negative controls included cells cultured in cDMEM without TFQ, as well as cells treated with DMSO alone (1:400 dilution, equivalent to the maximum volume used for TFQ compound dilutions). As a positive control for cytotoxicity, PBMCs were treated with 5 \u0026micro;g/ml concanavalin A (Con A) diluted in cDMEM.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSpecific selectivity index (SSI)\u003c/h2\u003e\u003cp\u003eThe selectivity of TFQ against \u003cem\u003eT. equi\u003c/em\u003e or \u003cem\u003eB. caballi\u003c/em\u003e in comparison to mammalian cells (equine PBMCs) was assessed at the respective IC\u003csub\u003e50\u003c/sub\u003e concentrations using the standard formula described by Ndjakou Lenta et al. (2007) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e]: SSI\u0026thinsp;=\u0026thinsp;IC\u003csub\u003e50\u003c/sub\u003e​ (parasite) / CC\u003csub\u003e50\u003c/sub\u003e​ (host cells)​, where CC₅₀ represents the concentration that reduces host cell viability by 50% and IC₅₀ denotes the concentration that inhibits parasite growth by 50% [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003ePPE levels were assessed daily and comparisons among TFQ-treated, ID-treated, and untreated controls were performed using the Kruskal-Wallis nonparametric test. The drug concentrations required to IC₅₀ relative to the untreated control, as well as the concentration needed to IC₉₉, were estimated for TFQ using nonlinear regression analysis.\u003c/p\u003e\u003cp\u003eThe normality of PBMC viability data was assessed using the Shapiro\u0026ndash;Wilk test. Since the data did not meet the assumption of normality, the Kruskal\u0026ndash;Wallis test was used for statistical analysis. GraphPad Prism v7 software for Windows (GraphPad Software, Inc., San Diego, CA, USA) was used for the statistical analysis. The significance level was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eTafenoquine exposure significantly inhibits the\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003egrowth of\u003c/b\u003e \u003cb\u003eT. equi\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTFQ exposure led to a progressive and consistent reduction in the \u003cem\u003eT. equi\u003c/em\u003e PPE in a dose-dependent manner, resulting in significantly lower parasitemia compared to untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Moreover, TFQ demonstrated efficacy comparable to that of ID at 72 h post-treatment across all tested concentrations, with no significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in inhibitory effect observed over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results of in vitro growth of \u003cem\u003eT. equi\u003c/em\u003e during a 3-day drug treatment period with either TFQ or ID, followed by a 5-day drug-free incubation period, are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTafenoquine significantly suppress\u003c/b\u003e \u003cb\u003eB. caballi\u003c/b\u003e \u003cb\u003egrowth\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDuring the first 72 h, the growth of \u003cem\u003eB. caballi\u003c/em\u003e was significantly inhibited by ID at all tested concentrations. In contrast, TFQ did not show any inhibitory effect at 5 \u0026micro;M and 10 \u0026micro;M. However, at 15 \u0026micro;M, TFQ demonstrated a significant inhibitory effect on the parasite growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At 72 h post-treatment, TFQ showed limited efficacy against \u003cem\u003eB. caballi\u003c/em\u003e at both 5 \u0026micro;M and 10 \u0026micro;M, with parasite growth rates similar to those observed in untreated control cultures. In contrast, ID exhibited significantly higher inhibitory activity at these concentrations, which was sustained throughout the observation period (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). At a concentration of 15 \u0026micro;M, both TFQ and ID demonstrated significant efficacy against \u003cem\u003eB. caballi\u003c/em\u003e beginning on the second day of treatment, with no significant differences observed between the two through day 5. After this point, parasitemia in TFQ-treated cultures continued to decline, whereas recrudescence was noted in the ID-treated cultures. Figures\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e show the results of \u003cem\u003eB. caballi\u003c/em\u003e in vitro growth during a 3-day exposure to either TFQ or ID, followed by a 5-day drug-free incubation period.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eDistinct TFQ potency and dose-response in\u003c/b\u003e \u003cb\u003eT. equi\u003c/b\u003e \u003cb\u003ethan against\u003c/b\u003e \u003cb\u003eB. caballi\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe mean and range of IC₅₀ values were calculated to compare the in vitro potency of TFQ against \u003cem\u003eB. caballi\u003c/em\u003e and \u003cem\u003eT. equi\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. TFQ exhibited approximately threefold greater potency against \u003cem\u003eT. equi\u003c/em\u003e than \u003cem\u003eB. caballi\u003c/em\u003e, indicating species-specific differences in drug susceptibility. Notably, the IC₅₀ values of TFQ against \u003cem\u003eT. equi\u003c/em\u003e exhibited a narrower 95% confidence interval, indicating a more consistent response across replicates. In contrast, the broader IC₅₀ range observed for \u003cem\u003eB. caballi\u003c/em\u003e may reflect greater biological variability or differences in assay sensitivity. The remarkable difference between IC₅₀ and IC₉₉ values against \u003cem\u003eT. equi\u003c/em\u003e suggests a steep dose-response curve, whereas the relatively smaller difference against \u003cem\u003eB. caballi\u003c/em\u003e indicates a more gradual inhibition profile.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIC₅₀ and IC₉₉ values (95% CI) of Tafenoquine succinate (TFQ) for inhibition of \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e after 72-hour in vitro cultures.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIC\u003csub\u003e99\u003c/sub\u003e(\u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eT. equi\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.904 \u0026micro;M (IC\u003csub\u003e95\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.99\u0026ndash;5.96)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60.74 \u0026micro;M (IC\u003csub\u003e95\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;37.41\u0026ndash;113.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eB. caballi\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.5 \u0026micro;M (IC\u003csub\u003e95\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13.81 to 15.23)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20.44 \u0026micro;M (IC\u003csub\u003e95\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;17.77 to 28.84)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csub\u003eIC95% Confident Interval\u003c/sub\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCytotoxicity of Tafenoquine in Equine PBMCs\u003c/h2\u003e\u003cp\u003eTo evaluate the potential cytotoxic effect of TFQ on host cells, equine PBMC were exposed to varying concentrations of the drug. During the post-treatment period (24, 48, and 72 hours), significant differences were observed among the various TFQ concentrations. No significant differences were observed between the control group and the treatment groups with 2.5 or 5 \u0026micro;M concentrations (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05); however, significant difference was observed between the control group and these groups treated with 10 to 50 \u0026micro;M concentrations of TFQ (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003ePBMC viability data suggests that TFQ at the IC\u003csub\u003e50\u003c/sub\u003e concentration for \u003cem\u003eT. equi\u003c/em\u003e does not compromise cell viability. However, cytotoxicity may be more relevant in the case of \u003cem\u003eB. caballi\u003c/em\u003e, as its IC\u003csub\u003e50\u003c/sub\u003e was three times higher. Additionally, a significant increase in PBMC proliferation was observed in cells exposed to ConA at 24 and 48 h (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), confirming the sensitivity of the WST-1 assay used in this study. Taken together, the results indicate that TFQ does not exert significant toxic effects on equine PBMCs cultured in vitro at concentrations of 2.5 and 5 \u0026micro;M but has toxic effects on PBMCs at concentration higher than 10 uM. Additionally, TFQ was 1.69 more selective for \u003cem\u003eT. equi\u003c/em\u003e and 0.69 times more selective for \u003cem\u003eB. caballi\u003c/em\u003e than for the host cells \u003cb\u003e(Table\u0026nbsp;2)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study assessed the in vitro efficacy of tafenoquine succinate (TFQ) against \u003cem\u003eTheileria equi\u003c/em\u003e and \u003cem\u003eBabesia caballi\u003c/em\u003e. Our findings show that TFQ exerted a marked inhibitory effect on parasite growth, particularly on \u003cem\u003eT. equi\u003c/em\u003e, with suppression persisting even after treatment withdrawal, suggesting a potential sustained parasiticidal activity. While \u003cem\u003eB. caballi\u003c/em\u003e also responded to TFQ, its sensitivity was comparatively lower than that of \u003cem\u003eT. equi\u003c/em\u003e. Nonetheless, TFQ at a concentration of 15 \u0026micro;M significantly reduced \u003cem\u003eB. caballi\u003c/em\u003e parasitemia by day 3, with sustained suppression observed throughout the study. Notably, \u003cem\u003eB. caballi\u003c/em\u003e cultures treated with ID showed recrudescence after initial inhibition, whereas those treated with TFQ did not, suggesting that TFQ may be more effective in preventing parasite recovery.\u003c/p\u003e\u003cp\u003eAlthough the mechanism of action of tafenoquine (TFQ) remains unclear, a recent study using human primary hepatocytes demonstrated that primaquine, an 8-aminoquinoline closely related to TFQ, exerts its anti-Plasmodial effect through a mechanism dependent on cytochrome P450 NADPH:oxidoreductase, accompanied by the production of hydrogen peroxide (H₂O₂) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Given the structural relatedness between primaquine and TFQ, it is plausible to assume that the latter compound also acts through host-generated oxidative intermediates. While the in vitro system used in this study may not fully reflect the profile of reductases present in vivo, NADPH reductases are known to be present in erythrocytes, the cells used to support the growth of \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e in culture [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, it is plausible to speculate that these enzymes could be responsible for generating host-derived oxidative intermediates in the presence of TFQ, which may explain the compound\u0026rsquo;s parasiticidal effect.\u003c/p\u003e\u003cp\u003eThe IC₅₀ for \u003cem\u003eT. equi\u003c/em\u003e is 5.9 \u0026micro;M, and for \u003cem\u003eB. caballi\u003c/em\u003e 14.5 \u0026micro;M, which technically fall within the broad Plasmodium range, but the implication of \"comparable sensitivity\" is misleading, especially considering \u003cem\u003eP. falciparum\u003c/em\u003e can be sensitive at submicromolar levels (e.g., 0.5 \u0026micro;M) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA critical aspect in comparing drug efficacy between species lies in analyzing the relationship between IC₅₀ (concentration for 50% inhibition) and IC₉₉ (for near-complete inhibition). The gap between IC₅₀ and IC₉₉ offers insight into the compound\u0026rsquo;s dose-response dynamics. A wide gap suggests a steep dose-response curve, potentially indicating cooperative or heterogeneous mechanisms, whereas a narrow gap implies a more uniform and predictable response [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, \u003cem\u003eT. equi\u003c/em\u003e had a wider IC₅₀\u0026ndash;IC₉₉ gap (IC₅₀: 5.9 \u0026micro;M; IC₉₉: 60.7 \u0026micro;M), indicating a steep dose-response curve. This pattern suggests that while low concentrations partially suppress the parasite, much higher concentrations are required for near-complete clearance. Such steep curves may reflect heterogeneous susceptibility within the parasite population, cooperative drug interactions, or differences in drug uptake or metabolism [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. A similar trend was observed in a previous study with novobiocin, where \u003cem\u003eT. equi\u003c/em\u003e displayed a steeper curve than \u003cem\u003eB. caballi\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Conversely, \u003cem\u003eB. caballi\u003c/em\u003e showed a narrower IC₅₀\u0026ndash;IC₉₉ gap (IC₅₀: 14.5 \u0026micro;M; IC₉₉: 20.44 \u0026micro;M), indicating a more gradual and linear response to increasing TFQ concentrations. This may indicate a more homogeneous parasite population or a more consistent interaction between the drug and its target [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Similarly, narrow-gap profiles have been observed in other protozoan species with uniform drug sensitivity - such as clinical isolates of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e39\u003c/span\u003e]- where limited subpopulation variability affected treatment outcomes.\u003c/p\u003e\u003cp\u003eInterestingly, \u003cem\u003eB. caballi\u003c/em\u003e has often exhibited greater sensitivity (lower IC₅₀ values) to other drugs than \u003cem\u003eT. equi\u003c/em\u003e, with varying data on the IC₅₀\u0026ndash;IC₉₉ gap [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The findings in the current study suggest that, although \u003cem\u003eB. caballi\u003c/em\u003e exhibits a higher IC₅₀ to TFQ, its narrower IC₅₀\u0026ndash;IC₉₉ gap may allow more straightforward dosing strategies. In contrast, the broader range observed in \u003cem\u003eT. equ\u003c/em\u003ei requires careful dose optimization to ensure complete parasite clearance.\u003c/p\u003e\u003cp\u003eTFQ demonstrated no significant cytotoxicity in equine PBMCs at concentrations up to 5 \u0026micro;M. Cell viability began to decline only at concentrations\u0026thinsp;\u0026ge;\u0026thinsp;10 \u0026micro;M (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which falls within or slightly above the effective antiparasitic range for \u003cem\u003eT. equi\u003c/em\u003e, but remained below the concentration required for complete inhibition of \u003cem\u003eB. caballi\u003c/em\u003e. The SSI of TFQ was nearly twice as high for \u003cem\u003eT. equi\u003c/em\u003e as for host cells. In contrast, the SSI for \u003cem\u003eB. caballi\u003c/em\u003e was only 0.69, indicating that the therapeutic concentrations required to inhibit this species approach or exceed the cytotoxic threshold in PBMCs. These findings highlight TFQ\u0026rsquo;s more favorable safety margin and selectivity against \u003cem\u003eT. equi\u003c/em\u003e, while also underscoring its narrower therapeutic window for \u003cem\u003eB. caballi\u003c/em\u003e. This profile is consistent with a previous study in human THP-1 cells, where TFQ demonstrated a relatively high LD₅₀ of 53.57 \u0026micro;M, indicating its preferential toxicity toward parasites over host cells [41]. Although tafenoquine has been associated with hemolytic toxicity in G6PD-deficient human erythrocytes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e25\u003c/span\u003e], only a single case of G6PD deficiency has been reported in equines, unrelated to tafenoquine-induced hemolysis (40). This suggests that, although G6PD deficiency may have limited clinical relevance in horses, its significance remains uncertain and warrants further investigation.\u003c/p\u003e\u003cp\u003eTaken together, our results support the potential of TFQ as a promising alternative to ID for the treatment of EP, particularly for \u003cem\u003eT. equi\u003c/em\u003e. Its sustained activity, low micromolar efficacy, and low cytotoxicity make it a compelling candidate for further evaluation. Notably, TFQ\u0026rsquo;s broader antiparasitic spectrum and unique dose-response dynamics provide an opportunity for controlling mixed or resistant infections. However, to fully validate its clinical potential, \u003cem\u003ein vivo\u003c/em\u003e studies will be required to determine pharmacokinetics, optimal dosing regimens, and therapeutic efficacy in horses.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides the first evidence of TFQ\u0026rsquo;s potent in vitro activity against \u003cem\u003eT. equi\u003c/em\u003e and moderate activity against \u003cem\u003eB. caballi\u003c/em\u003e, along with a mild cytotoxic profile in equine PBMCs. These findings lay the foundation for further preclinical and clinical research and support further validation as a novel chemotherapeutic option for equine piroplasmosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eEP \u0026ndash; equine piroplasmosis\u003c/p\u003e\n\u003cp\u003eTFQ \u0026ndash; tafenoquine succinate\u003c/p\u003e\n\u003cp\u003eID \u0026ndash; imidocarb dipropionate\u003c/p\u003e\n\u003cp\u003ePBMC \u0026ndash; peripheral blood mononuclear cells\u003c/p\u003e\n\u003cp\u003eIC \u0026ndash; inhibitory concentration\u003c/p\u003e\n\u003cp\u003eG6PD - glucose-6-phosphate dehydrogenase\u003c/p\u003e\n\u003cp\u003eFDA - the United State Food and Drug Administration\u003c/p\u003e\n\u003cp\u003eDMSO - dimethyl sulfoxide\u003c/p\u003e\n\u003cp\u003eHPLC - high-performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eH₂O₂ - hydrogen peroxide\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Megan Jacks, Shelby Beckner, Sarah Therrian, Elizabeth Hart, Emma Karel, and Kristin Erickson for their excellent technical assistance and animal care. Thanks to Isabelle Brock for maintenance of \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e tissue cultures. The authors also wish to thank Dr. Geoffrey Dow, CEO of 60 Degrees Pharmaceuticals, Inc) for critically reviewing the manuscript and for fruitful discussions regarding tafenoquine as an anti-hemoparasitic therapeutic. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was funded by the USDA-ARS CRIS# 2090\u0026ndash;32000-044-000-D.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article and its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNC: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Writing\u0026ndash;original draft, Writing\u0026ndash;review and editing, Software. NV: Supervision, Writing\u0026ndash;review and editing, Conceptualization, Data curation, Formal Analysis, Software. LK: Methodology, Writing\u0026ndash;review and editing, Supervision. CS: Writing\u0026ndash;review and editing. CC: Funding acquisition, Project administration, Writing\u0026ndash;review and editing. RB: Supervision, Conceptualization, Writing\u0026ndash;review and editing, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Validation, Visualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood from healthy, hemoparasite-free horses was used in this study to maintain \u003cem\u003ein vitro\u003c/em\u003e cultures of \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e. Blood collections were performed according to protocols approved by the Institutional Animal Care and Use Committees of the University of Idaho (protocol # 2024-26) and Washington State University (protocol # 6982).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. No personal data or images of individuals were included in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were not an editorial board member of the journal, at the time of submission. This had no impact on the peer review process and the final decision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMendoza FJ, P\u0026eacute;rez-\u0026Eacute;cija A, Kappmeyer LS, Suarez CE, Bastos RG. New insights in the diagnosis and treatment of equine piroplasmosis: pitfalls, idiosyncrasies, and myths. Frontiers in Veterinary Science. 2024;11. https://doi.org/10.3389/fvets.2024.1459989\u003c/li\u003e\n\u003cli\u003eWise LN, Kappmeyer LS, Mealey RH, Knowles DP. Review of Equine Piroplasmosis. Journal of Veterinary Internal Medicine. 2013;27:1334\u0026ndash;46. https://doi.org/10.1111/jvim.12168\u003c/li\u003e\n\u003cli\u003eRizk MA, El-Sayed SAE-S, Eltaysh R, Igarashi I. MMV020275 and MMV020490, promising compounds from malaria box for the treatment of equine piroplasmosis. 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Pharmaceuticals. 2022;15:1005. https://doi.org/10.3390/ph15081005\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"therapeutics, horses, tafenoquine succinate, imidocarb dipropionate","lastPublishedDoi":"10.21203/rs.3.rs-7769436/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7769436/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eEquine piroplasmosis (EP) is a tick-borne disease of equids caused by intraerythrocytic apicomplexan parasites \u003cem\u003eTheileria equi\u003c/em\u003e, \u003cem\u003eBabesia caballi\u003c/em\u003e, and the recently identified \u003cem\u003eTheileria haneyi\u003c/em\u003e. Acute cases can be severe, with anemia, jaundice, abortion, or sudden death. Survivors remain lifelong carriers, serving as reservoirs for tick-borne and iatrogenic transmission. No vaccines are currently available, and control strategies rely heavily on accurate diagnostics and chemotherapeutic intervention. Imidocarb dipropionate (ID) is the current standard of care for both acute treatment and radical cure. However, growing concerns regarding ID-resistant parasite strains and its associated toxicity have highlighted the urgent need for novel, safer, and more effective antiparasitic agents. Here, we assessed the in vitro efficacy of tafenoquine succinate (TFQ), a synthetic 8-aminoquinoline with broad antiparasitic activity, against \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e as a potential treatment for equine piroplasmosis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe effect of TFQ on \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e was evaluated in vitro in parasite cultures. The percentage of parasitized erythrocytes was measured by flow cytometry, and the effect of TFQ on parasite growth was compared to that of ID. TFQ toxicity on horse peripheral blood mononuclear cells (PBMC) was assessed via a colorimetric metabolic assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eTFQ reduced \u003cem\u003eT. equi\u003c/em\u003e parasitemia dose-dependently, matching ID efficacy at 72 hours. For \u003cem\u003eB. caballi\u003c/em\u003e, TFQ had no effect at 5\u0026ndash;10 \u0026micro;M but inhibited growth at 15 \u0026micro;M, similar results to ID. TFQ exhibited approximately threefold greater potency against \u003cem\u003eT. equi\u003c/em\u003e [IC₅₀: 5.90 \u0026micro;M (95% CI: 4.99\u0026ndash;5.96); IC₉₉: 60.74 \u0026micro;M (95% CI: 37.41\u0026ndash;113.3)] compared to \u003cem\u003eB. caballi\u003c/em\u003e [IC₅₀: 14.5 \u0026micro;M (95% CI: 13.81\u0026ndash;15.23); IC₉₉: 20.44 \u0026micro;M (95% CI: 17.77\u0026ndash;28.84)]. The narrower confidence intervals for \u003cem\u003eT. equi\u003c/em\u003e suggest a more consistent antiparasitic response across replicates. Cytotoxicity assays showed no toxic effects on equine PBMCs at 2.5\u0026ndash;5 \u0026micro;M (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while concentrations\u0026thinsp;\u0026ge;\u0026thinsp;10 \u0026micro;M indicated potential toxicity. These findings suggest TFQ selectively targets parasites over host cells, supporting its therapeutic potential.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eTFQ significantly inhibited \u003cem\u003eT. equi\u003c/em\u003e and \u003cem\u003eB. caballi\u003c/em\u003e growth at doses tolerated by equine PBMCs, supporting its potential as an alternative treatment for EP and warranting further in vivo study.\u003c/p\u003e","manuscriptTitle":"Tafenoquine succinate inhibits the growth of the equine piroplasmosis hemoparasites Theileria equi and Babesia caballi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 04:11:43","doi":"10.21203/rs.3.rs-7769436/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-17T08:34:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-10T12:28:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213931593587863317486884934983501799960","date":"2025-10-17T20:00:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1878375634136929107838392094101548167","date":"2025-10-16T13:09:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-14T12:40:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-06T07:53:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-06T05:17:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-10-03T00:40:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"466785ad-b6f3-4d51-a063-c18acce82016","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:16:36+00:00","versionOfRecord":{"articleIdentity":"rs-7769436","link":"https://doi.org/10.1186/s13071-026-07262-y","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2026-01-27 15:57:52","publishedOnDateReadable":"January 27th, 2026"},"versionCreatedAt":"2025-10-29 04:11:43","video":"","vorDoi":"10.1186/s13071-026-07262-y","vorDoiUrl":"https://doi.org/10.1186/s13071-026-07262-y","workflowStages":[]},"version":"v1","identity":"rs-7769436","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7769436","identity":"rs-7769436","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}