In vitro and in vivo acaricidal properties of orally delivered ivermectin against the blacklegged tick, Ixodes scapularis

In vitro and in vivo acaricidal properties of orally delivered ivermectin against the blacklegged tick, Ixodes scapularis | 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 In vitro and in vivo acaricidal properties of orally delivered ivermectin against the blacklegged tick, Ixodes scapularis Jolieke van Oosterwijk, Luciana Richer, Laura Beimfohr-Griffing, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7744931/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Apr, 2026 Read the published version in Parasites & Vectors → Version 1 posted 9 You are reading this latest preprint version Abstract Background The lack of effective and affordable new host-targeted tick control products is among major challenges for the existing control strategies against the blacklegged tick ( Ixodes scapularis ), the vector of Lyme disease affecting public health in the United States. Ivermectin is systemic acaricide that has been used successfully to control biting flies and ticks infesting livestock. Ivermectin-treated corn has also been shown to be effective against adult ticks feeding on deer. The goal of this study was to assess acaricidal properties of orally delivered ivermectin against the blacklegged tick, Ixodes scapularis , for development of new mouse bait formulation to control immature stages of the blacklegged tick. Methods The oral toxicity of ivermectin against I. scapularis was evaluated through in vitro capillary feeding tick feeding experiments and in vivo animal trials using laboratory bred white-footed mouse, Peromyscus leucopus . Capillary feeding of adult females and nymphs with different concentrations (18.8–600 ppb) of ivermectin resolved in rabbit blood were performed to ascertain necessary ivermectin plasma levels to kill feeding adult and nymphal ticks. Mouse baits dosed with two different ivermectin concentrations (24 and 48 ppm) were fed to mice to ascertain resulting pharmacokinetic properties of ivermectin in mouse serum via HPLC analysis. Subsequent tick-challenge trials were conducted to determine impacts of ivermectin from ingested the mouse diet against larval or nymphal ticks in the mouse model. Results I. scapularis females capillary-fed with rabbit blood containing 300 and 600 ppb demonstrated a significantly higher tick mortality starting at 72 h after the start of capillary feeding. Such Ivermectin concentrations also significantly reduced blood feeding of the females, as determined by female excretion and engorgement scores. Nymphal capillary feeding experiments were unsuccessful as nymphal in all treatment groups died like in the control group, likely due to desiccation. In the mouse trials, ivermectin reached peak serum concentrations, 650 ppb and 6715 ppb, respectively at 2 hours after consumption of a single treated pellet containing 80 µg and 160 µg ivermectin by mice but was rapidly depleted from mouse blood with a half-life less than six hours. When mice were infested with nymphal and larval ticks at different times relative to mice’s access to diet pellets containing ivermectin (48 ppm) ad libitum, a 45.5% to 100% reduction in the number of blood-fed nymphs and larvae was observed in the treatment groups in comparison to ticks fed on untreated mouse pellets. Conclusions Result of in vitro and in vivo experiments from this study demonstrated the oral toxicity of ivermectin against different developmental stages of the blacklegged tick. Given the acaricidal effects of ivermectin against I. scapularis nymphs and larvae feeding on white-footed mice observed from the mouse trials and how inexpensive ivermectin is, it is feasible to develop new commercial ivermectin-based mouse bait products to add to the tick control toolbox. Further laboratory and field studies are necessary to validate the utility of ivermectin-based mouse-targeted tick control products. Ixodes scapularis ivermectin white-footed mouse Peromyscus leucopus capillary feeding mouse bait HPLC tick challenge Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The United States is facing a rapid range expansion of major tick species of medical and veterinary importance, indicating elevated risk of tick-borne diseases [ 1 , 2 , 3 ]. The blacklegged tick, Ixodes scapularis , is responsible for the transmission of at least seven human pathogens, including Borrelia burgdorferi , the causative agent of Lyme disease (LD) [ 4 ]. Lyme disease, transmitted only by I. scapularis , accounts for > 80% of reported tickborne diseases, making it the most important vector-borne disease affecting humans in the United States [ 5 ]. With I. scapularis posing the biggest threat in the northeast and upper-Midwest of the United States, the lone star tick, Amblyomma americanum , is posing the biggest threat in the Southeast United States, with increasing reports of red meat allergies (or Alpha-Gal Syndrome) in humans induced by bites of lone star ticks [ 6 , 7 ]. In an earlier study by the DHHS Tick-Borne Disease Working Group, Lyme disease, by itself, is estimated to be a $ 50B - $ 100B challenge to the United States healthcare System [ 8 ]. Various personal protection measures, such as repellents and protective clothing, and tick control products have been studied to reduce the risk of tick bites in the past decades [ 9 , 10 ]. Chemical acaricides can be effective when directed at localized tick habitats (wooded edges of suburban home sites) as sprays and granules [ 11 , 12 , 13 , 14 ], but safety and environmental concerns limit regular use of acaricides for tick control. Host-targeted tick control products, including the “4-Poster” deer bait and treatment station and rodent bait boxes have been successfully used to control ticks on white-tailed deer and white-footed mice [ 15 , 16 , 17 , 18 , 19 , 20 ]. The white-footed mouse, Peromyscus leucopus , is the important host for immature stages of I. scapularis and the reservoir host for B. burgdorferi and other tick-borne pathogens. Current mouse-targeted tick control products on market include mouse bait boxes containing passively applied topical acaricide fipronil and tick tubes with permethrin-treated cotton as mouse nest material [ 17 , 21 , 22 ]. In recent years, oral bait formulations containing systemic acaricides, fipronil or Fluralaner, have been developed and evaluated to control immature I. scapularis ticks feeding on white-footed mice and break the life cycle of I. scapularis [ 23 , 24 , 25 ]. This is part of the effort to re-purpose the newer active acaricidal compounds that are successfully used in veterinary ectoparasite control products for public health use. The high cost associated with new acaricidal compounds remains one of major limiting factors that hinder their adoption for public health use. Therefore, researchers have examined older active acaricidal compounds, such as ivermectin, that have been shown to be effective against veterinary pests, have favorable safety profiles, and are inexpensive to formulate. Ivermectin is a antiparasitic drug that has been used as veterinary medicine to control biting flies and ticks infesting livestock [ 26 , 27 , 28 , 29 , 30 ]. Systemic treatment of white-tailed deer with ivermectin-medicated bait has been shown to be effective in suppressing populations of A. americanum and I. scapularis in field studies [ 31 , 32 ]. Use of ivermectin in mouse bait formulation to control immature I. scapularis ticks feeding on white-footed mice has not been attempted. We conducted a study to explore the feasibility of developing an ivermectin-based mouse bait to break the life cycle of I. scapularis . We conducted a study between 2019 and 2000 to explore the feasibility of developing an ivermectin-based mouse bait to break the life cycle of I. scapularis. The goal of this study was to assess the acaricidal properties of orally delivered ivermectin against the blacklegged tick I. scapularis through both in vitro and in vivo laboratory experiments. Methods Animals Adult females and nymphs of I. scapularis used for capillary feeding experiments were purchased from the Oklahoma State University National Tick Research and Education Resource (Stillwater, Oklahoma). Nympha and larvae of I. scapularis used for mouse infestation were obtained the CDC National Center for Emerging and Zoonotic Infectious Diseases Division of Vector-Borne Diseases, Rickettsial Zoonoses Branch (Atlanta, Georgia). Ticks were maintained at 22 ± 2 o C with a relative humidity (R.H.) of 95 ± 2% on a photoperiod of 12:12 h (L:D) before use in experiments. A total of 41 white-footed mice, P. leucopus , were obtained from the Peromyscus Genetic Stock Center at University of South Carolina (Columbia, South Carolina). Mice were maintained at the animal facility of TriMetis Life Sciences (City, State). Laboratory maintenance of mice and mouse experiments followed the animal use protocol (IACUC 19–0081) approved by the Institutional Animal Care and Use Committee (IACUC) of University of Tennessee Health Center (UTHSC). During challenge, mice were maintained individually in microisolator caging, with half the cage positioned on a heating pad, allowing the mice to keep warm and move away from the heating source when desired. Mice had access to water ad libitum at all times and to feed ad libitum unless stated otherwise, and health checks were performed daily. Tick capillary feeding experiments Capillary feeding experiments were performed at the USDA, ARS, Invasive Insect Biocontrol and Behavior Laboratory, Beltsville, Maryland, USA. Technical ivermectin (96.3% a.i., Chem Service Inc., West Chester, PA, USA) was dissolved in Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) which was further diluted in purified water to generate stock dilutions containing 0.625 ppm to 10 ppm ivermectin and 1% DMSO. The stock solutions were further diluted in deiminated rabbit blood (Hemostat Laboratories, Dixon, California) to desired test concentration of ivermectin in rabbit blood: 18.75, 37.5, 75, 150, 300, and 600 ppb with 0.03% DMSO. The control blood contained only 0.03% DMSO. In vitro blood feeding of adult females and nymphs of I. scapularis were achieved using 5 µl and 2 µl MicroCaps® capillary tube (Drummond Scientific Company, Broomall, PA). Each ivermectin concentration included 10 females or nymphs evenly positioned in two standard 9-cm diameter Petri dishes (5 ticks / dish). After each tick was attached with ventral side up to the petri dish using double-sided tape, a capillary tube containing blood containing a known concentration of ivermection was placed over the mouthparts (hypostome and palps) of the tick. The distal end of each capillary tube was placed on a strip of utility wax to secure the capillary tube. After closing the Petri dish with lid, the Petri dish was placed in an incubator and maintained at 85–99% R.H. and 36 o C. Capillary tubes are changed twice each with fresh blood with the same the Ivermectin-blood mixture for ticks in each treatment group. Blood consumption and tick mortality were documented at each change of capillary tube. Mortality status was categorized as “alive”, “moribund” or “dead. Ticks were rated based on engorgement level (0–5) with 0 being flat/unfed and five being fully engorged, as documented using Leica S6D compound microscope equipped with a digital Leica camera (Flexaam C1) (Deerfield, IL, USA; Fig. 1 ). Fecal pellets were counted and used as an indicator of how well ticks were or were not feeding. Ticks were fed for four-five consecutive days, then removed from the petri dish and placed into labeled plastic snap cap vials with holes poked into the lids. The snap cap vials were then placed into a room temperature humidor. Ticks were then monitored for mortality, up to 11-days post treatment. The capillary feeding experiments were replicated three times. Mouse trials Pharmacokinetic study To determine the pharmacokinetic properties in P. leucopus after oral administration, feed pellets were treated with ivermectin (I8898, Millipore Sigma, Burlington, Massachusetts). Pellets containing either 24ppm (80ug/pellet) or 48ppm (160ug/pellet) of ivermectin were created in the US BIOLOGIC laboratory. Mice were housed individually and fasted for eight hours prior to ivermectin administration. Each mouse was presented with one pellet of either 24ppm or 48ppm ivermectin, and time started after full consumption of the pellet. Two mice were sacrificed at two, three, four, five, six, 12-, and 24-hours post administration, at which point blood was collected in non-heparinized tubes using a terminal cardiac bleed. One mouse was used to collect baseline blood. As ivermectin is metabolized by the liver and can be detected in the blood stream, HPLC analysis was performed at the University of Memphis to detect plasma concentrations in the mice. Detection of plasma ivermectin concentration was performed according to a previously described protocol [ 33 ]. and adapted for use with mouse sera. In short, four-parts of acetonitrile and one-part ddH 2 O were added to four-parts sera and mixed for 30 minutes. After five minutes of centrifugation at 2000g, supernatant was transferred into a clean tube, and solid phase was derived by drying using nitrogen flow. For derivatization, residue was dissolved in N-methylimidazole/acetonitrile (1:2 v/v). Samples were incubated with trifluoracetic acid / acetonitrile (1:2 v/v) and incubated for 10 minutes before injection into the chromatograph. Tick challenge experiments : To establish the acaricidal activity of ivermectin to I. scapularis ticks, a total of 12 P. leucopus mice were used to determine impact of ivermectin to nymphal and larval ticks in relation to timing of mouse ingestion of ivermectin pellets. The mice were divided into four treatment groups with three mice in each group: (1) three mice were subjected to a tick challenge without ivermectin exposure, (2) three mice received ivermectin one-day prior to tick challenge, (3) three mice at one-day post ivermectin, and (4) three mice at two-days post ivermectin. All mice in a treatment group had 24 hours to consume ivermectin treated pellets, after which their pellets were replaced with normal food. Mice had access to food and water ad libitum. Mice were anaesthetized during the challenges, and challenged with eight nymphs and 30 larvae, according to the methods described in Bouchard et al. [ 34 ] During tick challenges, mice were housed separately, in FIC microisolator caging, with wire bottoms for eight days. Detached ticks were collected from the bottoms of the cages and inspected for engorgement status. Over the course of eight days, cages were inspected twice a day for detached ticks and to perform health checks. Dat analysis Figures were created using GraphPad Prism Software (San Diego, California, USA). The same software was used to compare means of capillary feeding parameters (unpaired two-tailed t -test) presented in Figs. 2 and 3 . Tick control efficacy of ivermectin-treated bait pellets from the mouse trial was determined using the Henderson–Tilton formula [ 35 ] Results ivermectin ingested through capillary tube killed female ticks and reduced tick blood-feeding Nymphal and female adult I. scapularis ticks were exposed to increasing ivermectin concentrations in capillary tubes over the course of 4–5 days. Thirty ticks of either life stage were exposed to each concentration over three trials. Adult I. scapularis females showed significantly higher mortality at 150 ppb and 600 ppb of ivermectin than those at lower ivermectin concentrations or the untreated control (Fig. 2 A, p < 0.05, independent two-tailed t -test). There was a clear concentration-dependent mortality response in female ticks in response to different concentrations of ivermectin in blood. The negative impact of ivermectin on tick blood feeding was similarly demonstrated in female ticks feeding on rabbit sera with 300 ppb or 600 ppb of ivermectin showing significantly lower levels of engorgement ( p > 0.05 and p < 0.0005, respectively, independent two-tailed t -test; Fig. 2 B). In contrast, no concentration-dependent effect of ivermectin on mortality or engorgement score was observed in nymphal I. scapularis ticks, which could in part be due to the reduced viability of these nymphal ticks throughout the experiment (Fig. 2 C, D). Examination of tick excretions during capillary blood feeding showed that adult female I. scapularis ticks had significantly less fecal pellets, ( p < 0.05, and p < 0.0005, in ticks fed on blood with 300 ppb and 600 ppb ivermectin, respectively), indicating that ivermectin is inhibiting tick feeding behavior (Fig. 3 A). No significant differences were found among different treatment groups in nymphal fecal excretions (Fig. 3 B). Orally ingested ivermectin was rapidly depleted from mouse serum HPLC analysis of serum samples prepared using blood plasma collected at different times after mice were given a single diet pellet containing either 24 ppm or 48 ppm of ivermectin and standard noncompartmental pharmacokinetic analysis was performed to estimate ivermectin bioavailability in the mouse after oral consumption of ivermectin. The peak concentration (Cmax) of Ivermectin observed in the mouse blood plasma was during the first collection time point, at two hours post consumption, and ivermectin was rapidly eliminated from the bloodstream. For the high dose, 48 ppm delivered orally, the initial ivermectin concentration measured was 6715ng/ml at two hours post ingestion (AUC 3881, 95% CI 3780–3982), and for the low dose, 24 ppm delivered orally, the initial ivermectin concentration measured was 650 ng/ml at two hours post ingestion (AUC 622.8, 95% CI 566.3–679.3) (Fig. 4 ). The relative bioavailability (Frel) of doubling the dose of ivermectin orally from 24 ppm to 48 ppm, in this study led to a 1.7 increase in the total bioavailability over time as calculated by the AUC. The most rapid decline was observed during the first 6 hours, at which point mice ingesting 24 ppm ivermectin had an average blood plasma level of 6 ng/ml, and mice ingesting 48 ppm ivermectin had an average blood plasma level of 14 ng/ml. At 24 hours, the serum concentration of ivermectin was ranging from 2–4 ng/ml and continued to decline around 1–3 ng/ml (Fig. 4 ). Ivermectin orally ingested by mice inhibited feeding of both larval and nymphal ticks Figure 5 summarizes the results of vivo tick challenge experiments. Mice that had been started on ivermectin pellets one day prior and one-day post to tick infestation (Fig. 5 ) showed a 100% inhibition of larval and 45.5% to 81.8% nymphal feeding as illustrated by the reduction of fed nymphs and larvae observed at the bottom of the wire cages after placement (Fig. 5 A and 5 B). A similar level (81.8%) of inhibition on nymphal feeding was also observed when nymphs were placed on mice two days after mice were given ivermectin-treated pellets. The only exception to the above observations was that the number of engorged larvae at the 2 days post treatment group was s the same as in the control group (Fig. 5 B). Discussion The goal of capillary feeding of nymphs with five concentrations of ivermectin in blood and a blood only control was to determine minimal lethal concentration to nymphs when fed continuously for 4 to 5 days. Unfortunately, nymphs did not do well when fed through capillary tubes and nymphs in all treatment groups died similarly over the course of four and half days. The similar mortality observed in nymphs of all treatment groups suggested nymphs may have died from repetitive handling and other unknown factors other than the action of ivermectin in blood. Result from capillary feeding of adult females showed the minimal lethal ivermectin concentration was 300 ppb. Blood feeding of female ticks was repetitively interrupted by change of capillary tubes, leading to reduced amount of blood and ivermectin ingested by ticks. It is conceivable that the lethal ivermectin concentration to adults feeding on live host is expected to be much lower than 300 ppb observed in this study. Similarly, the lethal ivermectin concentration to immature ticks is expected to be much further lower although we were able to determine due to the nymphal mortality issue encountered. Relatively low serum concentration (5–8 ng/ml ) of ivermectin has been shown to be effective against lone star ticks infesting goat and deer [ 36 ]. A following field study by Pound et al. [ 37 ] found white-tailed deer fed corn treated with ivermectin at the rate of 10 mg per 0.45 kg corn (22.2 ppm) led to 83.4% and 92.4% reduction in adults and nymphs, respectively, of a free-living population of the lone star tick, Amblyomma americanum in a field location in Texas. In another field study conducted on an isolated island of Maine, 90% control of female blacklegged tick infestation, subsequent oviposition, and larval eclosion was obtained in sampled deer with serum ivermectin levels of $ 15 ng/ml (ppb) after the deer population wad baited with similar ivermectin-treated corn (10 mg ivermectin / 0.45 kg corn [ 32 ]. Similarly, oral administration of sustained release bolus of ivermectin to cattle led to 5–10 ppb serum ivermectin concentration in cattle for more than two weeks, leading to 84.4% control of engorging female cattle ticks [ 38 ]. Ivermectin and other systemic acaracides are known to be metabolized slower in larger animals, such as dogs, deer and cattle. In our study, ingestion of a single oral dose (80 µg, 160 µg) of ivermectin in a pellet by mice can lead to 650 ppb and 6715 ppb serum concentration of ivermectin at 2 hours after pellet ingestion (Fig. 4 ). Ivermectin serum concentration dropped rapidly to 10 ± 5 ppb in 6 hours after pellet ingestion. This ivermection concentration range in this relatively short period of time could be sufficient to kill and severely impeding blood feeding of immature I. scapularis ticks attached on mice. Results of the tick challenge experiment of the current study demonstrated that a 45.5% to 81.8% reduction in blood-fed nymphs when achieved in the three-treatment group in comparison to the untreated control group. It is worth noting that all nymphs collected from the treatment groups were partially fed while nymphs from the control group were fully fed. The observation of 100% inhibition of larval feeding observed for the 1-day prior and 1-day post groups was expected. However, the failure to inhibit blood-feeding of larvae in the 2-day post group was unexpected. This could be caused by unknown experimental errors. We demonstrated in our study that ivermectin was metabolized and cleared out of the system at a much faster pace than other systemic acaricides, such as fipronil, reported by other researchers. Poche et al. [ 24 ] reported feeding fipronil-mediated diet to white-footed mice for two days can achieve a fipronil plasma concentration of 949 ppb, which declined slowly to 101ppb, and 79 ppb at 9 days and 15 days post oral ingestion of fipronil-treated diet. This treatment completely interrupted blood feeding of I. scapularis larvae, leading to a100% repletion efficacy for 15 days. In a similar study, Pelletier et al. [ 23 ] demonstrated that ingestion of fluralaner-treated diet by white-footed mice led to significant reduction in attachment of I. scapularis larvae and significant increase in larval mortality. The larval control efficacy was reported at 93% to 97% at two days posttreatment for the different doses tested respectively. However, the larval control efficacy dropped significantly to a neglectable level of 3 to 4% at 28 days post-treatment. This is westly different from the monthly control efficacy of oral fluralaner formulation against ticks infesting dogs. It is generally known that rodents, like the white-footed mouse, metabolize and remove drugs and pesticides from their blood, tissues and organs at much faster pace than larger animals. Although the tick-killing efficacy of ivermectin was short-lasting after a single oral dose. Ingestion of multiple doses within several days could elevate serum ivermectin concentration which could also persist for a longer time. This could explain the 45.5% to 100% inhibition of blood feeding of I. scapularies nymphs observed from our study. Based on the positive results generated from our study, we can expect a positive outcome if the same ivermectin bait formulation (48 ppm) is tested under a semi-field or full field evaluation when sufficient ivermectin-treated mouse pellets are distributed using bait boxes. In comparison to newer active ingredients, like fluralaner. use of Ivermectin as the active systemic acaricide in mouse bait products have several advantages. First, it is cost-effective. Based on relevant references, we calculated the cost per pellet of ivermectin ( $ 0.0018), in comparison to Spinosad ( $ 0.08), fipronil ( $ 0.1), imidacloprid ( $ 0.04), and Fluralaner ( $ 0.74). Based on a application rate of 200 pellets per acre, the cost per acre per application would be $ 0.36 for ivermection, $ 8 for imidacloprid, $ 16 for Spinosad, $ 20 for fipronil and $ 148 for fluralaner. This would make an ivermectin product much more affordable for the end users, particularly homeowners. Secondly, it is relatively safe. Like pest / vector control products, safety and regulatory approval must be considered for product development. Ivermectin products have been used extensively on livestock to control internal and ectoparasites. Ivermectin products have been used to treat headlice and even tested to kill I. scapularis ticks feeding on humans [ 39 , 40 ]. In addition, ivermectin kills ticks and insect pests by blocking the glutamate-gate chloride channel [ 41 , 42 ]. This mode of action is different from those of other commonly used acaricides, such as pyrethroids, fipronil, or imidacloprid. Given the low cost and known safety and environmental profiles of ivermectin, further investigations to fully characterize its acaricidal properties when multiple oral doses are administered in a period of multiple days would allow a better judgement on its potential utility for product development. Declarations Acknowledgments We would like to thank Caterina Torres and Samantha Berman of USDA-ARS for technical assistance with capillary feeding experiments; Michael Harris and Amber Jennings at the University of Memphis for assisting with HPLC analysis of mouse serum ivermectin concentrations. This article reports the results of research only. Mentioning a proprietary product does not constitute an endorsement or a recommendation by the USDA for its use. The USDA is an equal opportunity provider employer. Authors ’ contributions AYL designed the study. AYL and LKB wrote the capillary feeding protocol. JGvO and AYL prepared the mouse trial protocols. LKB and AYL conducted capillary feeding experiments and analyzed the experiment results. JGvO and LR conducted mouse trials and analyzed serum concentration data, collected and analyzed tick control efficacy data jointly with AYL. JGvO conducted statistical analysis of both capillary feeding and mouse trial data. AYL and JGvO wrote the manuscript and LKB reviewed the manuscript. Funding The research described was supported by Non-Assistance Cooperative Agreement (NADA) (# 58-8042-058) between USDA Agricultural Research Service (ARS) and U.S. Biologic, Inc. Original source of funds came from United States Department of Defense, Armed Forces Pest Management Board, Deployed Warfare Fighter Protection Program Interagency Reimbursable Agreement (# 60-0208-720) with USDA, ARS. Availability of data and materials Data supporting the conclusions of this article are included in the article. The datasets generated during and/or analyzed during the present study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate The test protocol and all procedures performed during this study involving white-footed mice were approved by the Animal Care and Use Committee (IACUC) of University of Tennessee Health Center (UTHSC) (IACUC 19-0081; 2019). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details a U.S. Biologic, Inc., 20 Dudley, Suite 900, Memphis, TN 38103, USA b USDA-ARS Invasive Insect Biocontrol & Behavior Laboratory, 10300 Baltimore Avenue, Beltsville, MD 20705, USA References Eisen RJ, Eisen L. The Blacklegged Tick, Ixodes scapularis : An increasing public health concern. Trends in Parasitology 2018;34(4): 295–309. Eisen L, Eisen R. Changes in the geographic distribution of the blacklegged tick, Ixodes scapularis, in the United States. 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Journal of Medical Entomology 2021;58(6): 2358–2367. Tiffin HS, Green KD, Burgess ER, Machtinger ET Maximizing and sustaining the efficacy of tick tubes for management of Ixodes scapularis through optimized deployment strategies. Journal of Medical Entomology 2024;61(6): 1459–1469. Pelletier J, Rocheleau J-P, Aenishaenslin C., Beaudry F, Masson GD, Lindsay R, Ogden NH, Bouchard C, Leighton PA Evaluation of fluralaner as an oral acaricide to reduce tick infestation in a wild rodent reservoir of Lyme disease. Parasites & Vectors 2020;13(1), 73. Poché DM, Franckowiak G, Clarke T, Tseveenjav B, Polyakova L., Poché RM Efficacy of a low dose fipronil bait against blacklegged tick ( Ixodes scapularis ) larvae feeding on white‑footed mice ( Peromyscus leucopus ) under laboratory conditions. Parasites & Vectors 2020;13, 391. Williams SC, Linske MA, Stafford KC 2023. Orally delivered fipronil-laced bait reduces juvenile blacklegged tick ( Ixodes scapularis ) burdens on wild white-footed mice ( Peromyscus leucopus ). Ticks and Tick-borne Diseases 2023;14, 102189. Jackson HC Ivermectin as a systemic insecticide. Parasitology Today 1989;5(5): 146-156. Byford RL, Craig ME, DeRouen SM, Kimball MD, Morrison DG, Wyatt WE, Foil LD Influence of permethrin, diazinon and ivermectin treatments on insecticide resistance in the horn fly (Diptera: Muscidae). International Journal for Parasitology 1999;29(1): 125-135. Miller JA, Davey RB, Oehler DD, Pound JM, George JE The Ivomec SR bolus for control of Boophilus annulatus (Acari: Ixodidae) on cattle in south Texas. J. Econ. Entomol. 2000;94(6): 1622-1627 Lifschitz A, Virkel G, Ballent M, Pis, A., Lanusse, C. Ivermectin (3.15%) long-acting formulations in cattle: absorption pattern and pharmacokinetic considerations. Vet. Parasitol. 2007;147(3-4): 303-310. Hanafi HA, Szumlas DE, Fryauff DJ, Furman BD, Hoel DF Effects of ivermectin on blood-feeding Phlebotomus papatasi , and the promastigote stage of Leishmania major. Vector Borne Zoonotic Dis. 2011;11(1): 43-52. Pound JM, Miller JA, George JE, Oehler DD, Harmel DE Systemic treatment of white-tailed deer with ivermectin-medicated bait to control free-living populations of lone star ticks (Acari: Ixodidae). Journal of Medical Entomology 1996;33(3): 385–394. Rand PW, Lacombe EH, Holman MS, Lubelczyk C., Smith Jr. RP Attempt to control ticks (Acari: Ixodidae) on deer on an isolated island using ivermectin-treated corn. J. Med. Entomol. 2000;37(1): 126-133. Morbidelli E, Rambaldi J, Ricci Bitti L, Zaghini A, Barbarossa A. A quick and simple method for the determination of ivermectin in dog plasma by LC–MS/MS. Methods X. 2018; 5:1503-1507. Bouchard KR, Wikel SK. Care, maintenance, and experimental infestation of ticks in the laboratory setting. In, Biology of Disease Vectors, Second ed. W. C. Marquart, ed. Elsevier Academic Press, San Diego, 2005. Henderson CF, Tilton EW Tests with acaricides against the brow wheat mite, J. Econ. Entomol. 1955;48: 157-161. Miller JA, Garris GI, George JE, Oehler, DD Control of lone star ticks (Acari: Ixodidae) on Spanish goats and white-tailed deer with orally administered ivermectin. Journal of Economic Entomology 1989;82(6): 1650–1656. Pound JM, Miller JA, George JE, Oehler DD, Harmel DE Systemic treatment of white-tailed deer with ivermectin-medicated bait to control free-living populations of lone star ticks (Acari: Ixodidae). Journal of Medical Entomology 1996;33(3): 385–394. Miller JA, Davey RB, Oehler DD, Pound JM, George JE The Ivomec SR bolus for control of Boophilus annulatus (Acari: Ixodidae) on cattle in south Texas. J. Econ. Entomol. 2001;94(6): 1622-1627. Strycharz, J.P., Berge, N.M., Alves, A.-M., Clark, J.M. 2011. Ivermectin acts as a posteclosion nymphicide by reducing blood feeding of human head lice (Anoplura: Pediculidae) that hatched from treated eggs. Journal of Medical Entomology 2011;48(6): 1174–1182. Sheele, J.M., Ford, L.R., Tse, A., Chidester, B., Byers, P.A., Sonenshine, D.E. 2014. The use of ivermectin to kill Ixodes scapularis ticks feeding on humans. Wilderness Environ. Med. 25(1): 29-34. Bloomquist JR Chloride channels as tools for developing selective insecticides. Arch. Insect Biochem. Physiol. 2003;54: 145-156. Waldman J, Klafke GM, Tirloni L, Logullo C, da Silva Vaz Jr I Putative target sites in synganglion for novel ixodid tick control strategies. Ticks and Tick-borne Diseases 2023;14: 102123. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 08 Apr, 2026 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 12 Dec, 2025 Reviews received at journal 12 Dec, 2025 Reviewers agreed at journal 22 Nov, 2025 Reviews received at journal 30 Oct, 2025 Reviewers agreed at journal 09 Oct, 2025 Reviewers invited by journal 07 Oct, 2025 Editor assigned by journal 03 Oct, 2025 Submission checks completed at journal 03 Oct, 2025 First submitted to journal 29 Sep, 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7744931","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":531371167,"identity":"da09c1ca-5385-4647-8280-fd075c761ddb","order_by":0,"name":"Jolieke van Oosterwijk","email":"","orcid":"","institution":"U.S. Biologic, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Jolieke","middleName":"van","lastName":"Oosterwijk","suffix":""},{"id":531371168,"identity":"f236fca5-1867-4b30-90a7-911747e9f99d","order_by":1,"name":"Luciana Richer","email":"","orcid":"","institution":"U.S. Biologic, 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09:27:42","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100517,"visible":true,"origin":"","legend":"","description":"","filename":"464e09330d36447eaeda8beda3ec0c1e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7744931/v1/a2a09144cac4414d46435195.xml"},{"id":93920188,"identity":"15c4cda7-d080-444a-97ef-c4fdc293f0cd","added_by":"auto","created_at":"2025-10-20 09:35:41","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110509,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7744931/v1/faa2a465a9effc1e334666b0.html"},{"id":93919629,"identity":"2feb9f69-790b-4ae5-a7bf-a168f97a2a94","added_by":"auto","created_at":"2025-10-20 09:27:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1248447,"visible":true,"origin":"","legend":"\u003cp\u003eObserved changes associated with capillary feeding of blood in females (top panel) and nymphs\u003cem\u003e \u003c/em\u003e(lower panel) of \u003cem\u003eI. scapularis. \u003c/em\u003eThe score, ranging from 0 to 5, assigned to each image represents an estimated stage of engorgement.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7744931/v1/c00de46ff376d05cfdaf3b3d.png"},{"id":93919630,"identity":"e840eaba-36f6-4150-843c-94c9484b676b","added_by":"auto","created_at":"2025-10-20 09:27:41","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":224041,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ivermectin ingested with blood through capillary feeding on the survival (A, C) and engorgement (B, D) of adult females and nymphs of \u003cem\u003eI. scapularis. \u003c/em\u003eData points in graph represent the mean and standard deviations. Lines of different colors represent different concentrations (ppm) of ivermection in blood. * and *** represent significant difference between means at \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0005 level, respectively.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7744931/v1/640db01a68ef3f4b9f98f8a8.jpeg"},{"id":93920186,"identity":"dcd586b0-0b6a-42c0-908d-25ab2efbec38","added_by":"auto","created_at":"2025-10-20 09:35:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112812,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ivermectin ingested with blood through capillary feeding on tick feeding, as measures by fecal excretion, in adult females (A) and nymphs (B) of \u003cem\u003eI. scapularis. \u003c/em\u003eData points in graph represent the mean and standard deviations. Lines of different colors represent different concentrations (ppm) of ivermection in blood. *, ** and *** represent significant difference between means at \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.005 and \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0005 level, respectively.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7744931/v1/e61feafb617b9c630d25ae18.jpeg"},{"id":93919639,"identity":"e55362b1-a2a5-478e-9f68-114d9b5f2b02","added_by":"auto","created_at":"2025-10-20 09:27:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":162147,"visible":true,"origin":"","legend":"\u003cp\u003eChange in\u003cstrong\u003e \u003c/strong\u003eserum ivermectin concentration\u003cstrong\u003e \u003c/strong\u003e(ng/mL or ppb)\u003cstrong\u003e \u003c/strong\u003eat different times (hours) after mice ingested a single diet pellet containing 24 ppm (80 µg) or 48 ppm (160 µg) of ivermection. Each data point represents one mouse.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7744931/v1/d4fdf7d03e6d1c8c6c6ce887.png"},{"id":93919637,"identity":"844628bc-cb24-42c2-bf7d-84d1cb0b59d1","added_by":"auto","created_at":"2025-10-20 09:27:41","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":247075,"visible":true,"origin":"","legend":"\u003cp\u003eResults of \u003cem\u003ein vivo\u003c/em\u003e mouse trial on effect of ingestion of ivermectin-treated pellets (48 ppm) on the blood-feeding success of nymphs and Larvae of \u003cem\u003eI. scapularis\u003c/em\u003e in relation to timing of tick infestation and mouse access to ivermectin-treated pellets. \u003cem\u003e2 days post\u003c/em\u003e: tick infestation occurred at 2 days after mice feeding on ivermectin-treated pellets; \u003cem\u003e1 day post\u003c/em\u003e: tick infestation occurred at 1day after mice feeding on ivermectin-treated pellets; \u003cem\u003e1 day prior\u003c/em\u003e: tick infestation occurred at 1day prior to mice feeding on ivermectin-treated pellets; \u003cem\u003eNo ivermectin\u003c/em\u003e: tick infestation on mice feeding on regular untreated pellets (untreated control).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7744931/v1/8370867f6f20467fbb67c58b.jpeg"},{"id":106808821,"identity":"bf51835d-8d05-4431-88b1-6511b353f38b","added_by":"auto","created_at":"2026-04-13 16:02:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3026250,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7744931/v1/c4afb2d2-1d80-41e8-be00-f0e5ba372e96.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"In vitro and in vivo acaricidal properties of orally delivered ivermectin against the blacklegged tick, Ixodes scapularis","fulltext":[{"header":"Background","content":"\u003cp\u003eThe United States is facing a rapid range expansion of major tick species of medical and veterinary importance, indicating elevated risk of tick-borne diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The blacklegged tick, \u003cem\u003eIxodes scapularis\u003c/em\u003e, is responsible for the transmission of at least seven human pathogens, including \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e, the causative agent of Lyme disease (LD) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Lyme disease, transmitted only by \u003cem\u003eI. scapularis\u003c/em\u003e, accounts for \u0026gt;\u0026thinsp;80% of reported tickborne diseases, making it the most important vector-borne disease affecting humans in the United States [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. With \u003cem\u003eI. scapularis\u003c/em\u003e posing the biggest threat in the northeast and upper-Midwest of the United States, the lone star tick, \u003cem\u003eAmblyomma americanum\u003c/em\u003e, is posing the biggest threat in the Southeast United States, with increasing reports of red meat allergies (or Alpha-Gal Syndrome) in humans induced by bites of lone star ticks [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In an earlier study by the DHHS Tick-Borne Disease Working Group, Lyme disease, by itself, is estimated to be a \u003cspan\u003e$\u003c/span\u003e50B - \u003cspan\u003e$\u003c/span\u003e100B challenge to the United States healthcare System [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eVarious personal protection measures, such as repellents and protective clothing, and tick control products have been studied to reduce the risk of tick bites in the past decades [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Chemical acaricides can be effective when directed at localized tick habitats (wooded edges of suburban home sites) as sprays and granules [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], but safety and environmental concerns limit regular use of acaricides for tick control. Host-targeted tick control products, including the \u0026ldquo;4-Poster\u0026rdquo; deer bait and treatment station and rodent bait boxes have been successfully used to control ticks on white-tailed deer and white-footed mice [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe white-footed mouse, \u003cem\u003ePeromyscus leucopus\u003c/em\u003e, is the important host for immature stages of \u003cem\u003eI. scapularis\u003c/em\u003e and the reservoir host for \u003cem\u003eB. burgdorferi\u003c/em\u003e and other tick-borne pathogens. Current mouse-targeted tick control products on market include mouse bait boxes containing passively applied topical acaricide fipronil and tick tubes with permethrin-treated cotton as mouse nest material [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In recent years, oral bait formulations containing systemic acaricides, fipronil or Fluralaner, have been developed and evaluated to control immature \u003cem\u003eI. scapularis\u003c/em\u003e ticks feeding on white-footed mice and break the life cycle of \u003cem\u003eI. scapularis\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This is part of the effort to re-purpose the newer active acaricidal compounds that are successfully used in veterinary ectoparasite control products for public health use. The high cost associated with new acaricidal compounds remains one of major limiting factors that hinder their adoption for public health use. Therefore, researchers have examined older active acaricidal compounds, such as ivermectin, that have been shown to be effective against veterinary pests, have favorable safety profiles, and are inexpensive to formulate.\u003c/p\u003e\u003cp\u003eIvermectin is a antiparasitic drug that has been used as veterinary medicine to control biting flies and ticks infesting livestock [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Systemic treatment of white-tailed deer with ivermectin-medicated bait has been shown to be effective in suppressing populations of \u003cem\u003eA. americanum\u003c/em\u003e and \u003cem\u003eI. scapularis\u003c/em\u003e in field studies [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Use of ivermectin in mouse bait formulation to control immature \u003cem\u003eI. scapularis\u003c/em\u003e ticks feeding on white-footed mice has not been attempted. We conducted a study to explore the feasibility of developing an ivermectin-based mouse bait to break the life cycle of \u003cem\u003eI. scapularis\u003c/em\u003e. We conducted a study between 2019 and 2000 to explore the feasibility of developing an ivermectin-based mouse bait to break the life cycle of I. scapularis. The goal of this study was to assess the acaricidal properties of orally delivered ivermectin against the blacklegged tick \u003cem\u003eI. scapularis\u003c/em\u003e through both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e laboratory experiments.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eAdult females and nymphs of \u003cem\u003eI. scapularis\u003c/em\u003e used for capillary feeding experiments were purchased from the Oklahoma State University National Tick Research and Education Resource (Stillwater, Oklahoma). Nympha and larvae of \u003cem\u003eI. scapularis\u003c/em\u003e used for mouse infestation were obtained the CDC National Center for Emerging and Zoonotic Infectious Diseases Division of Vector-Borne Diseases, Rickettsial Zoonoses Branch (Atlanta, Georgia). Ticks were maintained at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u003csup\u003eo\u003c/sup\u003eC with a relative humidity (R.H.) of 95\u0026thinsp;\u0026plusmn;\u0026thinsp;2% on a photoperiod of 12:12 h (L:D) before use in experiments.\u003c/p\u003e\u003cp\u003eA total of 41 white-footed mice, \u003cem\u003eP. leucopus\u003c/em\u003e, were obtained from the \u003cem\u003ePeromyscus\u003c/em\u003e Genetic Stock Center at University of South Carolina (Columbia, South Carolina). Mice were maintained at the animal facility of TriMetis Life Sciences (City, State). Laboratory maintenance of mice and mouse experiments followed the animal use protocol (IACUC 19\u0026ndash;0081) approved by the Institutional Animal Care and Use Committee (IACUC) of University of Tennessee Health Center (UTHSC). During challenge, mice were maintained individually in microisolator caging, with half the cage positioned on a heating pad, allowing the mice to keep warm and move away from the heating source when desired. Mice had access to water ad libitum at all times and to feed ad libitum unless stated otherwise, and health checks were performed daily.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTick capillary feeding experiments\u003c/h3\u003e\n\u003cp\u003eCapillary feeding experiments were performed at the USDA, ARS, Invasive Insect Biocontrol and Behavior Laboratory, Beltsville, Maryland, USA. Technical ivermectin (96.3% a.i., Chem Service Inc., West Chester, PA, USA) was dissolved in Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) which was further diluted in purified water to generate stock dilutions containing 0.625 ppm to 10 ppm ivermectin and 1% DMSO. The stock solutions were further diluted in deiminated rabbit blood (Hemostat Laboratories, Dixon, California) to desired test concentration of ivermectin in rabbit blood: 18.75, 37.5, 75, 150, 300, and 600 ppb with 0.03% DMSO. The control blood contained only 0.03% DMSO.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e blood feeding of adult females and nymphs of \u003cem\u003eI. scapularis\u003c/em\u003e were achieved using 5 \u0026micro;l and 2 \u0026micro;l MicroCaps\u0026reg; capillary tube (Drummond Scientific Company, Broomall, PA). Each ivermectin concentration included 10 females or nymphs evenly positioned in two standard 9-cm diameter Petri dishes (5 ticks / dish). After each tick was attached with ventral side up to the petri dish using double-sided tape, a capillary tube containing blood containing a known concentration of ivermection was placed over the mouthparts (hypostome and palps) of the tick. The distal end of each capillary tube was placed on a strip of utility wax to secure the capillary tube. After closing the Petri dish with lid, the Petri dish was placed in an incubator and maintained at 85\u0026ndash;99% R.H. and 36 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\u003cp\u003eCapillary tubes are changed twice each with fresh blood with the same the Ivermectin-blood mixture for ticks in each treatment group. Blood consumption and tick mortality were documented at each change of capillary tube. Mortality status was categorized as \u0026ldquo;alive\u0026rdquo;, \u0026ldquo;moribund\u0026rdquo; or \u0026ldquo;dead. Ticks were rated based on engorgement level (0\u0026ndash;5) with 0 being flat/unfed and five being fully engorged, as documented using Leica S6D compound microscope equipped with a digital Leica camera (Flexaam C1) (Deerfield, IL, USA; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Fecal pellets were counted and used as an indicator of how well ticks were or were not feeding. Ticks were fed for four-five consecutive days, then removed from the petri dish and placed into labeled plastic snap cap vials with holes poked into the lids. The snap cap vials were then placed into a room temperature humidor. Ticks were then monitored for mortality, up to 11-days post treatment. The capillary feeding experiments were replicated three times.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMouse trials\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacokinetic study\u003c/strong\u003e\u003cp\u003eTo determine the pharmacokinetic properties in \u003cem\u003eP. leucopus\u003c/em\u003e after oral administration, feed pellets were treated with ivermectin (I8898, Millipore Sigma, Burlington, Massachusetts). Pellets containing either 24ppm (80ug/pellet) or 48ppm (160ug/pellet) of ivermectin were created in the \u003cem\u003eUS BIOLOGIC\u003c/em\u003e laboratory. Mice were housed individually and fasted for eight hours prior to ivermectin administration. Each mouse was presented with one pellet of either 24ppm or 48ppm ivermectin, and time started after full consumption of the pellet. Two mice were sacrificed at two, three, four, five, six, 12-, and 24-hours post administration, at which point blood was collected in non-heparinized tubes using a terminal cardiac bleed. One mouse was used to collect baseline blood. As ivermectin is metabolized by the liver and can be detected in the blood stream, HPLC analysis was performed at the University of Memphis to detect plasma concentrations in the mice.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eDetection of plasma ivermectin concentration was performed according to a previously described protocol [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. and adapted for use with mouse sera. In short, four-parts of acetonitrile and one-part ddH\u003csub\u003e2\u003c/sub\u003eO were added to four-parts sera and mixed for 30 minutes. After five minutes of centrifugation at 2000g, supernatant was transferred into a clean tube, and solid phase was derived by drying using nitrogen flow. For derivatization, residue was dissolved in N-methylimidazole/acetonitrile (1:2 v/v). Samples were incubated with trifluoracetic acid / acetonitrile (1:2 v/v) and incubated for 10 minutes before injection into the chromatograph.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTick challenge experiments\u003c/span\u003e: To establish the acaricidal activity of ivermectin to \u003cem\u003eI. scapularis\u003c/em\u003e ticks, a total of 12 \u003cem\u003eP. leucopus\u003c/em\u003e mice were used to determine impact of ivermectin to nymphal and larval ticks in relation to timing of mouse ingestion of ivermectin pellets. The mice were divided into four treatment groups with three mice in each group: (1) three mice were subjected to a tick challenge without ivermectin exposure, (2) three mice received ivermectin one-day prior to tick challenge, (3) three mice at one-day post ivermectin, and (4) three mice at two-days post ivermectin. All mice in a treatment group had 24 hours to consume ivermectin treated pellets, after which their pellets were replaced with normal food. Mice had access to food and water ad libitum. Mice were anaesthetized during the challenges, and challenged with eight nymphs and 30 larvae, according to the methods described in Bouchard et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] During tick challenges, mice were housed separately, in FIC microisolator caging, with wire bottoms for eight days. Detached ticks were collected from the bottoms of the cages and inspected for engorgement status. Over the course of eight days, cages were inspected twice a day for detached ticks and to perform health checks.\u003c/p\u003e\n\u003ch3\u003eDat analysis\u003c/h3\u003e\n\u003cp\u003eFigures were created using GraphPad Prism Software (San Diego, California, USA). The same software was used to compare means of capillary feeding parameters (unpaired two-tailed \u003cem\u003et\u003c/em\u003e-test) presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Tick control efficacy of ivermectin-treated bait pellets from the mouse trial was determined using the Henderson\u0026ndash;Tilton formula [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eivermectin ingested through capillary tube killed female ticks and reduced tick blood-feeding\u003c/h2\u003e\u003cp\u003eNymphal and female adult \u003cem\u003eI. scapularis\u003c/em\u003e ticks were exposed to increasing ivermectin concentrations in capillary tubes over the course of 4\u0026ndash;5 days. Thirty ticks of either life stage were exposed to each concentration over three trials. Adult \u003cem\u003eI. scapularis\u003c/em\u003e females showed significantly higher mortality at 150 ppb and 600 ppb of ivermectin than those at lower ivermectin concentrations or the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, independent two-tailed \u003cem\u003et\u003c/em\u003e-test). There was a clear concentration-dependent mortality response in female ticks in response to different concentrations of ivermectin in blood. The negative impact of ivermectin on tick blood feeding was similarly demonstrated in female ticks feeding on rabbit sera with 300 ppb or 600 ppb of ivermectin showing significantly lower levels of engorgement (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0005, respectively, independent two-tailed \u003cem\u003et\u003c/em\u003e-test; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eIn contrast, no concentration-dependent effect of ivermectin on mortality or engorgement score was observed in nymphal \u003cem\u003eI. scapularis\u003c/em\u003e ticks, which could in part be due to the reduced viability of these nymphal ticks throughout the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Examination of tick excretions during capillary blood feeding showed that adult female \u003cem\u003eI. scapularis\u003c/em\u003e ticks had significantly less fecal pellets, (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0005, in ticks fed on blood with 300 ppb and 600 ppb ivermectin, respectively), indicating that ivermectin is inhibiting tick feeding behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). No significant differences were found among different treatment groups in nymphal fecal excretions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eOrally ingested ivermectin was rapidly depleted from mouse serum\u003c/h3\u003e\n\u003cp\u003eHPLC analysis of serum samples prepared using blood plasma collected at different times after mice were given a single diet pellet containing either 24 ppm or 48 ppm of ivermectin and standard noncompartmental pharmacokinetic analysis was performed to estimate ivermectin bioavailability in the mouse after oral consumption of ivermectin.\u003c/p\u003e\u003cp\u003eThe peak concentration (Cmax) of Ivermectin observed in the mouse blood plasma was during the first collection time point, at two hours post consumption, and ivermectin was rapidly eliminated from the bloodstream. For the high dose, 48 ppm delivered orally, the initial ivermectin concentration measured was 6715ng/ml at two hours post ingestion (AUC 3881, 95% CI 3780\u0026ndash;3982), and for the low dose, 24 ppm delivered orally, the initial ivermectin concentration measured was 650 ng/ml at two hours post ingestion (AUC 622.8, 95% CI 566.3\u0026ndash;679.3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The relative bioavailability (Frel) of doubling the dose of ivermectin orally from 24 ppm to 48 ppm, in this study led to a 1.7 increase in the total bioavailability over time as calculated by the AUC. The most rapid decline was observed during the first 6 hours, at which point mice ingesting 24 ppm ivermectin had an average blood plasma level of 6 ng/ml, and mice ingesting 48 ppm ivermectin had an average blood plasma level of 14 ng/ml. At 24 hours, the serum concentration of ivermectin was ranging from 2\u0026ndash;4 ng/ml and continued to decline around 1\u0026ndash;3 ng/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eIvermectin orally ingested by mice inhibited feeding of both larval and nymphal ticks\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e summarizes the results of \u003cem\u003evivo\u003c/em\u003e tick challenge experiments. Mice that had been started on ivermectin pellets one day prior and one-day post to tick infestation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) showed a 100% inhibition of larval and 45.5% to 81.8% nymphal feeding as illustrated by the reduction of fed nymphs and larvae observed at the bottom of the wire cages after placement (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). A similar level (81.8%) of inhibition on nymphal feeding was also observed when nymphs were placed on mice two days after mice were given ivermectin-treated pellets. The only exception to the above observations was that the number of engorged larvae at the 2 days post treatment group was s the same as in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe goal of capillary feeding of nymphs with five concentrations of ivermectin in blood and a blood only control was to determine minimal lethal concentration to nymphs when fed continuously for 4 to 5 days. Unfortunately, nymphs did not do well when fed through capillary tubes and nymphs in all treatment groups died similarly over the course of four and half days. The similar mortality observed in nymphs of all treatment groups suggested nymphs may have died from repetitive handling and other unknown factors other than the action of ivermectin in blood. Result from capillary feeding of adult females showed the minimal lethal ivermectin concentration was 300 ppb. Blood feeding of female ticks was repetitively interrupted by change of capillary tubes, leading to reduced amount of blood and ivermectin ingested by ticks. It is conceivable that the lethal ivermectin concentration to adults feeding on live host is expected to be much lower than 300 ppb observed in this study. Similarly, the lethal ivermectin concentration to immature ticks is expected to be much further lower although we were able to determine due to the nymphal mortality issue encountered.\u003c/p\u003e\u003cp\u003eRelatively low serum concentration (5\u0026ndash;8 ng/ml\u0026thinsp;\u0026lt;\u0026thinsp;ppb\u0026gt;) of ivermectin has been shown to be effective against lone star ticks infesting goat and deer [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. A following field study by Pound et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] found white-tailed deer fed corn treated with ivermectin at the rate of 10 mg per 0.45 kg corn (22.2 ppm) led to 83.4% and 92.4% reduction in adults and nymphs, respectively, of a free-living population of the lone star tick, \u003cem\u003eAmblyomma americanum\u003c/em\u003e in a field location in Texas. In another field study conducted on an isolated island of Maine, 90% control of female blacklegged tick infestation, subsequent oviposition, and larval eclosion was obtained in sampled deer with serum ivermectin levels of \u003cspan\u003e$\u003c/span\u003e15 ng/ml (ppb) after the deer population wad baited with similar ivermectin-treated corn (10 mg ivermectin / 0.45 kg corn [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, oral administration of sustained release bolus of ivermectin to cattle led to 5\u0026ndash;10 ppb serum ivermectin concentration in cattle for more than two weeks, leading to 84.4% control of engorging female cattle ticks [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Ivermectin and other systemic acaracides are known to be metabolized slower in larger animals, such as dogs, deer and cattle.\u003c/p\u003e\u003cp\u003eIn our study, ingestion of a single oral dose (80 \u0026micro;g, 160 \u0026micro;g) of ivermectin in a pellet by mice can lead to 650 ppb and 6715 ppb serum concentration of ivermectin at 2 hours after pellet ingestion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Ivermectin serum concentration dropped rapidly to 10\u0026thinsp;\u0026plusmn;\u0026thinsp;5 ppb in 6 hours after pellet ingestion. This ivermection concentration range in this relatively short period of time could be sufficient to kill and severely impeding blood feeding of immature \u003cem\u003eI. scapularis\u003c/em\u003e ticks attached on mice. Results of the tick challenge experiment of the current study demonstrated that a 45.5% to 81.8% reduction in blood-fed nymphs when achieved in the three-treatment group in comparison to the untreated control group. It is worth noting that all nymphs collected from the treatment groups were partially fed while nymphs from the control group were fully fed. The observation of 100% inhibition of larval feeding observed for the 1-day prior and 1-day post groups was expected. However, the failure to inhibit blood-feeding of larvae in the 2-day post group was unexpected. This could be caused by unknown experimental errors.\u003c/p\u003e\u003cp\u003eWe demonstrated in our study that ivermectin was metabolized and cleared out of the system at a much faster pace than other systemic acaricides, such as fipronil, reported by other researchers. Poche et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] reported feeding fipronil-mediated diet to white-footed mice for two days can achieve a fipronil plasma concentration of 949 ppb, which declined slowly to 101ppb, and 79 ppb at 9 days and 15 days post oral ingestion of fipronil-treated diet. This treatment completely interrupted blood feeding of \u003cem\u003eI. scapularis\u003c/em\u003e larvae, leading to a100% repletion efficacy for 15 days. In a similar study, Pelletier et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] demonstrated that ingestion of fluralaner-treated diet by white-footed mice led to significant reduction in attachment of \u003cem\u003eI. scapularis\u003c/em\u003e larvae and significant increase in larval mortality. The larval control efficacy was reported at 93% to 97% at two days posttreatment for the different doses tested respectively. However, the larval control efficacy dropped significantly to a neglectable level of 3 to 4% at 28 days post-treatment. This is westly different from the monthly control efficacy of oral fluralaner formulation against ticks infesting dogs. It is generally known that rodents, like the white-footed mouse, metabolize and remove drugs and pesticides from their blood, tissues and organs at much faster pace than larger animals.\u003c/p\u003e\u003cp\u003eAlthough the tick-killing efficacy of ivermectin was short-lasting after a single oral dose. Ingestion of multiple doses within several days could elevate serum ivermectin concentration which could also persist for a longer time. This could explain the 45.5% to 100% inhibition of blood feeding of \u003cem\u003eI. scapularies\u003c/em\u003e nymphs observed from our study. Based on the positive results generated from our study, we can expect a positive outcome if the same ivermectin bait formulation (48 ppm) is tested under a semi-field or full field evaluation when sufficient ivermectin-treated mouse pellets are distributed using bait boxes. In comparison to newer active ingredients, like fluralaner. use of Ivermectin as the active systemic acaricide in mouse bait products have several advantages. First, it is cost-effective. Based on relevant references, we calculated the cost per pellet of ivermectin (\u003cspan\u003e$\u003c/span\u003e0.0018), in comparison to Spinosad (\u003cspan\u003e$\u003c/span\u003e0.08), fipronil (\u003cspan\u003e$\u003c/span\u003e0.1), imidacloprid (\u003cspan\u003e$\u003c/span\u003e0.04), and Fluralaner (\u003cspan\u003e$\u003c/span\u003e0.74). Based on a application rate of 200 pellets per acre, the cost per acre per application would be \u003cspan\u003e$\u003c/span\u003e0.36 for ivermection, \u003cspan\u003e$\u003c/span\u003e8 for imidacloprid, \u003cspan\u003e$\u003c/span\u003e16 for Spinosad, \u003cspan\u003e$\u003c/span\u003e20 for fipronil and \u003cspan\u003e$\u003c/span\u003e148 for fluralaner. This would make an ivermectin product much more affordable for the end users, particularly homeowners. Secondly, it is relatively safe. Like pest / vector control products, safety and regulatory approval must be considered for product development. Ivermectin products have been used extensively on livestock to control internal and ectoparasites. Ivermectin products have been used to treat headlice and even tested to kill \u003cem\u003eI. scapularis\u003c/em\u003e ticks feeding on humans [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In addition, ivermectin kills ticks and insect pests by blocking the glutamate-gate chloride channel [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This mode of action is different from those of other commonly used acaricides, such as pyrethroids, fipronil, or imidacloprid. Given the low cost and known safety and environmental profiles of ivermectin, further investigations to fully characterize its acaricidal properties when multiple oral doses are administered in a period of multiple days would allow a better judgement on its potential utility for product development.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Caterina Torres and Samantha Berman of USDA-ARS for technical assistance with capillary feeding experiments; Michael Harris and Amber Jennings at the University of Memphis for assisting with HPLC analysis of mouse serum ivermectin concentrations. This article reports the results of research only. Mentioning a proprietary product does not constitute an endorsement or a recommendation by the USDA for its use. The USDA is an equal opportunity provider employer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAYL designed the study. AYL and LKB wrote the capillary feeding protocol. JGvO and AYL prepared the mouse trial protocols. LKB and AYL conducted capillary feeding experiments and analyzed the experiment results. JGvO and LR conducted mouse trials and analyzed serum concentration data, collected and analyzed tick control efficacy data jointly with AYL. JGvO conducted statistical analysis of both capillary feeding and mouse trial data. \u0026nbsp;AYL and JGvO wrote the manuscript and LKB reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research described was supported by Non-Assistance Cooperative Agreement (NADA) (# 58-8042-058) between USDA Agricultural Research Service (ARS) and U.S. Biologic, Inc. \u0026nbsp;Original source of funds came from United States Department of Defense, Armed Forces Pest Management Board, Deployed Warfare Fighter Protection Program Interagency Reimbursable Agreement (# 60-0208-720) with USDA, ARS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the conclusions of this article are included in the article. The datasets generated during and/or analyzed during the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe test protocol and all procedures performed during this study involving white-footed mice were approved by the Animal Care and Use Committee (IACUC) of University of Tennessee Health Center (UTHSC) (IACUC 19-0081; 2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea \u0026nbsp; U.S. Biologic, Inc., 20 Dudley, Suite 900, Memphis, TN 38103, USA\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eb \u0026nbsp; USDA-ARS Invasive Insect Biocontrol \u0026amp; Behavior Laboratory, 10300 Baltimore Avenue, Beltsville, MD 20705, USA\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEisen RJ, Eisen L. 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Microbiol. 2021;11, 775371.\u003c/li\u003e\n\u003cli\u003eAilsworth SM, Susi A, Workman LJ, Nylund, CM, Wilson JM Alpha-Gal IgE Prevalence patterns in the United States: An investigation of 3,000 military recruits. Journal of Allergy and Clinical Immunology in Practice 2024;12(1): 175\u0026ndash;184.e5.\u003c/li\u003e\n\u003cli\u003eDHHS. Tick Borne Disease Working Group 2018 Report to Congress. https://www.hhs.gov/sites/default/files/tbdwg-report-to-congress-2018.pdf. Accessed 7/15/2020.\u003c/li\u003e\n\u003cli\u003eEisen L Personal protection measures to prevent tick bites in the United States: Knowledge gaps, challenges, and opportunities. Ticks and Tick-borne Diseases 2022;13, 101944. \u003c/li\u003e\n\u003cli\u003eSchwartz AM, Mackeprang JM, Mead PS, Hinckley AF Effectiveness of personal protection measures against Lyme disease: A review of epidemiologic studies from the United States. Zoonoses and Public Health 2022;69: 777\u0026ndash;791. 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Ent. 1991;28(5): 624-629.\u003c/li\u003e\n\u003cli\u003eBron, GM, Lee X, Paskewitz SM Do-It-Yourself tick control: Granular gamma-cyhalothrin reduces \u003cem\u003eIxodes scapularis\u003c/em\u003e (Acari: Ixodidae) nymphs in residential backyards. Journal of Medical Entomology 2021;58(2): 749\u0026ndash;755.\u003c/li\u003e\n\u003cli\u003ePound JM, Miller JA, George JE. Efficacy of amitraz applied to white-tailed deer by the \u0026apos;4- poster\u0026apos; topical treatment device in controlling free-living lone star ticks (Acari: Ixodidae). J. Med. Ent.2000;37(6): 878-884.\u003c/li\u003e\n\u003cli\u003eCarroll JF, Pound, JM, Miller JA, Kramer M Sustained control of Gibson Island, Maryland, populations of \u003cem\u003eIxodes scapularis\u003c/em\u003e and \u003cem\u003eAmblyomma americanum\u003c/em\u003e (Acari: Ixodidae) by community-administered 4-poster deer self-treatment bait stations. Vector Borne and Zoonotic Diseases 2009;9(4): 417\u0026ndash;421.\u003c/li\u003e\n\u003cli\u003eJordan, RA, Schulze TL. Ability of Two commercially available host-targeted technologies to reduce abundance of \u003cem\u003eIxodes scapularis\u003c/em\u003e (Acari: Ixodidae) in a residential landscape. J. Med. Ent. 2019;56(4): 1095-1101.\u003c/li\u003e\n\u003cli\u003eJordan RA, Schulze TL. Availability and nature of commercial tick control services in three Lyme disease endemic states. J. Med. Entomol. 2020;57(3): 807-814.\u003c/li\u003e\n\u003cli\u003eWilliams SC, Stafford KC, Linske MA, Stuber HR, Cozens DW Effective control of the motile stages of \u003cem\u003eAmblyomma americanum\u003c/em\u003e and reduced \u003cem\u003eEhrlichia\u003c/em\u003e spp. prevalence in adults via permethrin treatment of white-tailed deer in coastal Connecticut, USA. Ticks and Tick-Borne Diseases 2011;12(3), 101675.\u003c/li\u003e\n\u003cli\u003eLinske MA, Williams SC, Stafford KC, Li AY Integrated tick management in Guilford, CT: Fipronil-based rodent-targeted bait box deployment configuration and \u003cem\u003ePeromyscus leucopus\u003c/em\u003e (Rodentia: Cricetidae) abundance drive reduction in tick burdens. Journal of Medical Entomology 2011;59(2): 591-597. \u003c/li\u003e\n\u003cli\u003eMandli JT, Lee X, Bron GM, Paskewitz SM Integrated tick management in south central Wisconsin: Impact of invasive vegetation removal and host-targeted acaricides on the density of questing \u003cem\u003eIxodes scapularis\u003c/em\u003e (acari: Ixodidae) nymphs. Journal of Medical Entomology 2021;58(6): 2358\u0026ndash;2367. \u003c/li\u003e\n\u003cli\u003eTiffin HS, Green KD, Burgess ER, Machtinger ET Maximizing and sustaining the efficacy of tick tubes for management of \u003cem\u003eIxodes scapularis\u003c/em\u003e through optimized deployment strategies. Journal of Medical Entomology 2024;61(6): 1459\u0026ndash;1469. \u003c/li\u003e\n\u003cli\u003ePelletier J, Rocheleau J-P, Aenishaenslin C., Beaudry F, Masson GD, Lindsay R, Ogden NH, Bouchard C, Leighton PA Evaluation of fluralaner as an oral acaricide to reduce tick infestation in a wild rodent reservoir of Lyme disease. Parasites \u0026amp; Vectors 2020;13(1), 73. \u003c/li\u003e\n\u003cli\u003ePoch\u0026eacute; DM, Franckowiak G, Clarke T, Tseveenjav B, Polyakova L., Poch\u0026eacute; RM Efficacy of a low dose fipronil bait against blacklegged tick (\u003cem\u003eIxodes scapularis\u003c/em\u003e) larvae feeding on white‑footed mice (\u003cem\u003ePeromyscus leucopus\u003c/em\u003e) under laboratory conditions. Parasites \u0026amp; Vectors 2020;13, 391. \u003c/li\u003e\n\u003cli\u003eWilliams SC, Linske MA, Stafford KC 2023. Orally delivered fipronil-laced bait reduces juvenile blacklegged tick (\u003cem\u003eIxodes scapularis\u003c/em\u003e) burdens on wild white-footed mice (\u003cem\u003ePeromyscus leucopus\u003c/em\u003e). Ticks and Tick-borne Diseases 2023;14, 102189. \u003c/li\u003e\n\u003cli\u003eJackson HC Ivermectin as a systemic insecticide. Parasitology Today 1989;5(5): 146-156. \u003c/li\u003e\n\u003cli\u003eByford RL, Craig ME, DeRouen SM, Kimball MD, Morrison DG, Wyatt WE, Foil LD Influence of permethrin, diazinon and ivermectin treatments on insecticide resistance in the horn fly (Diptera: Muscidae). International Journal for Parasitology 1999;29(1): 125-135.\u003c/li\u003e\n\u003cli\u003eMiller JA, Davey RB, Oehler DD, Pound JM, George JE The Ivomec SR bolus for control of \u003cem\u003eBoophilus annulatus\u003c/em\u003e (Acari: Ixodidae) on cattle in south Texas. J. Econ. Entomol. 2000;94(6): 1622-1627\u003c/li\u003e\n\u003cli\u003eLifschitz A, Virkel G, Ballent M, Pis, A., Lanusse, C. Ivermectin (3.15%) long-acting formulations in cattle: absorption pattern and pharmacokinetic considerations. Vet. Parasitol. 2007;147(3-4): 303-310.\u003c/li\u003e\n\u003cli\u003eHanafi HA, Szumlas DE, Fryauff DJ, Furman BD, Hoel DF Effects of ivermectin on blood-feeding \u003cem\u003ePhlebotomus papatasi\u003c/em\u003e, and the promastigote stage of Leishmania major. Vector Borne Zoonotic Dis. 2011;11(1): 43-52.\u003c/li\u003e\n\u003cli\u003ePound JM, Miller JA, George JE, Oehler DD, Harmel DE Systemic treatment of white-tailed deer with ivermectin-medicated bait to control free-living populations of lone star ticks (Acari: Ixodidae). Journal of Medical Entomology 1996;33(3): 385\u0026ndash;394.\u003c/li\u003e\n\u003cli\u003eRand PW, Lacombe EH, Holman MS, Lubelczyk C., Smith Jr. RP Attempt to control ticks (Acari: Ixodidae) on deer on an isolated island using ivermectin-treated corn. J. Med. Entomol. 2000;37(1): 126-133.\u003c/li\u003e\n\u003cli\u003eMorbidelli E, Rambaldi J, Ricci Bitti L, Zaghini A, Barbarossa A. A quick and simple method for the determination of ivermectin in dog plasma by LC\u0026ndash;MS/MS. Methods X. 2018; 5:1503-1507.\u003c/li\u003e\n\u003cli\u003eBouchard KR, Wikel SK. Care, maintenance, and experimental infestation of ticks in the laboratory setting. In, Biology of Disease Vectors, Second ed. W. C. Marquart, ed. Elsevier Academic Press, San Diego, 2005.\u003c/li\u003e\n\u003cli\u003eHenderson CF, Tilton EW Tests with acaricides against the brow wheat mite, J. Econ. Entomol. 1955;48: 157-161.\u003c/li\u003e\n\u003cli\u003eMiller JA, Garris GI, George JE, Oehler, DD Control of lone star ticks (Acari: Ixodidae) on Spanish goats and white-tailed deer with orally administered ivermectin. Journal of Economic Entomology 1989;82(6): 1650\u0026ndash;1656.\u003c/li\u003e\n\u003cli\u003ePound JM, Miller JA, George JE, Oehler DD, Harmel DE Systemic treatment of white-tailed deer with ivermectin-medicated bait to control free-living populations of lone star ticks (Acari: Ixodidae). Journal of Medical Entomology 1996;33(3): 385\u0026ndash;394.\u003c/li\u003e\n\u003cli\u003eMiller JA, Davey RB, Oehler DD, Pound JM, George JE The Ivomec SR bolus for control of \u003cem\u003eBoophilus annulatus\u003c/em\u003e (Acari: Ixodidae) on cattle in south Texas. J. Econ. Entomol. 2001;94(6): 1622-1627.\u003c/li\u003e\n\u003cli\u003eStrycharz, J.P., Berge, N.M., Alves, A.-M., Clark, J.M. 2011. Ivermectin acts as a posteclosion nymphicide by reducing blood feeding of human head lice (Anoplura: Pediculidae) that hatched from treated eggs. Journal of Medical Entomology 2011;48(6): 1174\u0026ndash;1182.\u003c/li\u003e\n\u003cli\u003eSheele, J.M., Ford, L.R., Tse, A., Chidester, B., Byers, P.A., Sonenshine, D.E. 2014. The use of ivermectin to kill Ixodes scapularis ticks feeding on humans. Wilderness Environ. Med. 25(1): 29-34. \u003c/li\u003e\n\u003cli\u003eBloomquist JR Chloride channels as tools for developing selective insecticides. Arch. Insect Biochem. Physiol. 2003;54: 145-156.\u003c/li\u003e\n\u003cli\u003eWaldman J, Klafke GM, Tirloni L, Logullo C, da Silva Vaz Jr I Putative target sites in synganglion for novel ixodid tick control strategies. Ticks and Tick-borne Diseases 2023;14: 102123. \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":"Ixodes scapularis, ivermectin, white-footed mouse, Peromyscus leucopus, capillary feeding, mouse bait, HPLC, tick challenge","lastPublishedDoi":"10.21203/rs.3.rs-7744931/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7744931/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe lack of effective and affordable new host-targeted tick control products is among major challenges for the existing control strategies against the blacklegged tick (\u003cem\u003eIxodes scapularis\u003c/em\u003e), the vector of Lyme disease affecting public health in the United States. Ivermectin is systemic acaricide that has been used successfully to control biting flies and ticks infesting livestock. Ivermectin-treated corn has also been shown to be effective against adult ticks feeding on deer. The goal of this study was to assess acaricidal properties of orally delivered ivermectin against the blacklegged tick, \u003cem\u003eIxodes scapularis\u003c/em\u003e, for development of new mouse bait formulation to control immature stages of the blacklegged tick.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe oral toxicity of ivermectin against \u003cem\u003eI. scapularis\u003c/em\u003e was evaluated through \u003cem\u003ein vitro\u003c/em\u003e capillary feeding tick feeding experiments and \u003cem\u003ein vivo\u003c/em\u003e animal trials using laboratory bred white-footed mouse, \u003cem\u003ePeromyscus leucopus\u003c/em\u003e. Capillary feeding of adult females and nymphs with different concentrations (18.8\u0026ndash;600 ppb) of ivermectin resolved in rabbit blood were performed to ascertain necessary ivermectin plasma levels to kill feeding adult and nymphal ticks. Mouse baits dosed with two different ivermectin concentrations (24 and 48 ppm) were fed to mice to ascertain resulting pharmacokinetic properties of ivermectin in mouse serum via HPLC analysis. Subsequent tick-challenge trials were conducted to determine impacts of ivermectin from ingested the mouse diet against larval or nymphal ticks in the mouse model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003e\u003cem\u003eI. scapularis\u003c/em\u003e females capillary-fed with rabbit blood containing 300 and 600 ppb demonstrated a significantly higher tick mortality starting at 72 h after the start of capillary feeding. Such Ivermectin concentrations also significantly reduced blood feeding of the females, as determined by female excretion and engorgement scores. Nymphal capillary feeding experiments were unsuccessful as nymphal in all treatment groups died like in the control group, likely due to desiccation. In the mouse trials, ivermectin reached peak serum concentrations, 650 ppb and 6715 ppb, respectively at 2 hours after consumption of a single treated pellet containing 80 \u0026micro;g and 160 \u0026micro;g ivermectin by mice but was rapidly depleted from mouse blood with a half-life less than six hours. When mice were infested with nymphal and larval ticks at different times relative to mice\u0026rsquo;s access to diet pellets containing ivermectin (48 ppm) ad libitum, a 45.5% to 100% reduction in the number of blood-fed nymphs and larvae was observed in the treatment groups in comparison to ticks fed on untreated mouse pellets.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eResult of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments from this study demonstrated the oral toxicity of ivermectin against different developmental stages of the blacklegged tick. Given the acaricidal effects of ivermectin against \u003cem\u003eI. scapularis\u003c/em\u003e nymphs and larvae feeding on white-footed mice observed from the mouse trials and how inexpensive ivermectin is, it is feasible to develop new commercial ivermectin-based mouse bait products to add to the tick control toolbox. Further laboratory and field studies are necessary to validate the utility of ivermectin-based mouse-targeted tick control products.\u003c/p\u003e","manuscriptTitle":"In vitro and in vivo acaricidal properties of orally delivered ivermectin against the blacklegged tick, Ixodes scapularis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 09:27:36","doi":"10.21203/rs.3.rs-7744931/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-13T00:33:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T09:19:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27211876443748442612664457462315414640","date":"2025-11-22T08:17:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T21:19:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158413666175987029007716466663184902043","date":"2025-10-09T18:03:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-07T18:05:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-03T21:20:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-03T14:47:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-09-29T19:16:27+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":"c2da7268-a732-4686-a4de-b9ec1ebe2ac5","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:00:53+00:00","versionOfRecord":{"articleIdentity":"rs-7744931","link":"https://doi.org/10.1186/s13071-026-07380-7","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2026-04-08 15:57:57","publishedOnDateReadable":"April 8th, 2026"},"versionCreatedAt":"2025-10-20 09:27:36","video":"","vorDoi":"10.1186/s13071-026-07380-7","vorDoiUrl":"https://doi.org/10.1186/s13071-026-07380-7","workflowStages":[]},"version":"v1","identity":"rs-7744931","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7744931","identity":"rs-7744931","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}