Topical permethrin may increase blacklegged tick (Ixodes scapularis) repellency but is associated with cutaneous irritation in horses

Karen C. Poh Department of Entomology, Pennsylvania State University, University Park, PA

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Zoey T. Cole Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, PA

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Danielle N. Smarsh Department of Animal Science, Pennsylvania State University, University Park, PA

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Hayley R. Springer Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, PA
Penn State Extension, Pennsylvania State University, University Park, PA

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Kathleen Kelly Animal Diagnostic Laboratory, Pennsylvania State University, University Park, PA

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Laura B. Kenny Penn State Extension, Pennsylvania State University, University Park, PA

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Erika T. Machtinger Department of Entomology, Pennsylvania State University, University Park, PA

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Abstract

OBJECTIVE

To evaluate the safety of repeated applications of permethrin concentrations (0% control, 1.5%, 5%, and 10%) to the necks and faces of horses and assess the efficacy and longevity of permethrin as an equine tick repellent.

ANIMALS

5 healthy adult Quarter Horses.

PROCEDURES

Each treatment was applied to the neck of each horse (0.01 m2) 4 times a day, for up to 10 days. An 8-mm biopsy was taken to evaluate postexposure dermal responses. Any treatments that were not withdrawn were applied to a quadrant of the horse’s face 4 times a day, for up to 5 days. For tick bioassays, a treatment was applied to 1 leg of a horse and 5 female blacklegged ticks (Ixodes scapularis) were evaluated as “repelled” or “not repelled” by the treatment. The bioassays were repeated up to 5 days, but treatment application took place only on the first day of the experiment.

RESULTS

Histological results of neck biopsies indicated that more repeated exposures or higher concentrations resulted in more dermal damage. Tick bioassays showed that 5% and 10% permethrin had the greatest efficacy and longevity as a tick repellent, but the differences in tick repellency were not significant overall.

CLINICAL RELEVANCE

While there was a nonsignificant trend of higher permethrin concentrations repelling more ticks with longer-lasting residual repellent effects, higher concentrations also produced greater skin damage after repeated exposures. These opposing findings emphasize the need for better tick prevention and control methods that balance safety and efficacy for the equine community.

Abstract

OBJECTIVE

To evaluate the safety of repeated applications of permethrin concentrations (0% control, 1.5%, 5%, and 10%) to the necks and faces of horses and assess the efficacy and longevity of permethrin as an equine tick repellent.

ANIMALS

5 healthy adult Quarter Horses.

PROCEDURES

Each treatment was applied to the neck of each horse (0.01 m2) 4 times a day, for up to 10 days. An 8-mm biopsy was taken to evaluate postexposure dermal responses. Any treatments that were not withdrawn were applied to a quadrant of the horse’s face 4 times a day, for up to 5 days. For tick bioassays, a treatment was applied to 1 leg of a horse and 5 female blacklegged ticks (Ixodes scapularis) were evaluated as “repelled” or “not repelled” by the treatment. The bioassays were repeated up to 5 days, but treatment application took place only on the first day of the experiment.

RESULTS

Histological results of neck biopsies indicated that more repeated exposures or higher concentrations resulted in more dermal damage. Tick bioassays showed that 5% and 10% permethrin had the greatest efficacy and longevity as a tick repellent, but the differences in tick repellency were not significant overall.

CLINICAL RELEVANCE

While there was a nonsignificant trend of higher permethrin concentrations repelling more ticks with longer-lasting residual repellent effects, higher concentrations also produced greater skin damage after repeated exposures. These opposing findings emphasize the need for better tick prevention and control methods that balance safety and efficacy for the equine community.

Ticks are common ectoparasites on horses in the United States.1 In the United States, over 15% of equids and 29% of equine operations have reported tick infestations.2 In particular, the blacklegged tick (Ixodes scapularis) poses significant risks to equine health as it is responsible for transmitting Borrelia burgdorferi, the causative agent for Lyme disease, and Anaplasma phagocytophilum, the causative agent for equine anaplasmosis.3,4

Tick-borne pathogens cause disease that could permanently disable horses and affect their performance and value. For example, clinical signs of Lyme disease in horses include sporadic lameness, chronically poor performance, muscle tenderness, swollen joints, and uveitis (inflammation of the eye), which can lead to neuroborreliosis, the neurologic form of Lyme disease.510 Neurologic disease caused by B burgdorferi affects the nervous system and impacts facial and spinal nerves, which can lead to difficulty in eating, facial drooping, unstable gaits, or complete paralysis.3,11,12 Equine anaplasmosis signs include depression, limb swelling, reluctance to move, and ataxia.4 While antimicrobials can treat both diseases, diagnoses of equine tick-borne diseases (TBDs) are challenging, and symptoms may progress into irreversible chronic conditions.

With no available vaccine for Lyme disease or anaplasmosis for horses, tick bite prevention is the recommended method to prevent TBD transmission. On-animal prevention of tick bites on horses is generally reliant on chemical sprays. While permethrin is promoted as an active ingredient in many fly and sometimes tick-repellent sprays, current formulations of permethrin and other pyrethrins degrade rapidly when exposed to sunlight, lasting only 1 to 2 days before being degraded.1316 Even when not exposed to the environment, equine insect repellents lose efficacy over time.17 To mitigate this loss of efficacy, horse caretakers may resort to off-label use of permethrin concentrate, which could affect the health of the horse if multiple applications of incorrect dilutions are applied. Currently, 4-poster feeders are used to reduce ticks on white-tailed deer by self-applying 10% permethrin to the deer’s necks and faces.18,19 A similar tool could be used for horses, but there are currently no published studies that have evaluated the effect of repeated permethrin exposures at similar concentrations on equine dermal health.

To maintain healthy horse populations, horses should be protected against ticks and tick-borne pathogens. Even though permethrin is a common active ingredient in many registered insecticides used on horses, the safety and efficacy of permethrin use on horses against ticks have not been fully evaluated. Therefore, this study had 2 major objectives. First, evaluate the dermal responses to permethrin at different concentrations on the body and face of horses. Second, conduct tick bioassays to compare the effectiveness and longevity of permethrin at various concentrations against ticks. We hypothesized that horses exposed to higher concentrations of permethrin will show signs of greater dermal damage compared to lower concentrations. In addition, tick repellency will remain effective and more ticks will be repelled at higher concentrations of permethrin.

Materials and Methods

Dermal responses to permethrin on the neck and face and biopsy collection

All animal experiments were approved by the Pennsylvania State University Institutional Animal Care and Use Committee (PROTO202101835).

Three concentrations of permethrin (1.5%, 5%, and 10%) and water (control) were evaluated to determine if they caused dermal responses in 5 adult horses. A 10% concentration of permethrin was diluted with water to achieve the 1.5% and 5% concentrations (Durvet Permethrin). For reference, the recommended permethrin concentration is 0.05% for normal infestations of ticks or 0.1% for severe infestations.20 American Quarter Horses (3 geldings and 2 mares) from the Pennsylvania State University Equine Facilities were used in these experiments, with a mean ± SD age of 11.6 ± 2.3 years and a mean weight of 516.8 ± 48.9 kg.

Because ticks were previously found on the chin, neck, and chest regions of horses,21 the permethrin application experiments were conducted on the neck and the face of the horse. Dermal responses were tested first on the neck and then the face of each horse. Four treatment areas on the horse’s neck (approx 0.01 m2 each), with 2 areas on each lateral side of the neck, were washed and shaved with a No. 40 blade at least 24 hours prior to the first day of the experiment. Permethrin treatments were randomly assigned to 1 treatment area and reapplied for up to 10 days of the experiment unless the treatment area was withdrawn during dermal evaluation. Treatments were reapplied every 2 hours for a total of 6 hours per day to evaluate dermal response to repeated exposures to permethrin. Using a flat-edge paintbrush (size 11) assigned to each treatment to avoid cross-contamination, treatments were applied to the respective treatment area to form a thin layer on the skin (approx 5 ml).

Dermal evaluations occurred prior to the first application of the day, before every subsequent reapplication of treatments, and at the end of the day (2 hours after the final treatment reapplication of the day). The horses’ treatment areas were evaluated by trained personnel using dermal response criteria presented in the Draize scoring system22 (Supplementary Table S1). If a treatment area on the horse had well-defined and visible redness or swelling (edema or erythema score of “2” or higher), treatments were not reapplied to those areas for the remainder of the study. To prevent bias during the dermal evaluation, evaluators were blinded to the treatments assigned to each treatment area. Treatments were applied outdoors where the equine subjects were naturally grazing to replicate realistic field conditions in which horses may be exposed to permethrin applications.

On day 11 (1 day after the planned final treatment application), biopsies were collected from all treatment areas of each horse. The treatment areas were washed and shaved again using a No. 40 blade to remove excess dirt and loose hair. Horses were sedated with detomidine hydrochloride (Zoetis) administered intramuscularly using the manufacturer’s recommended dosages. Treatment areas were surgically prepped by scrubbing with gauze soaked in 2% chlorhexidine surgical scrub (VetOne), followed by wiping with gauze soaked in 70% alcohol. This was repeated for a minimum of 3 cycles or until the final alcohol gauze appeared clean. After surgical preparation, approximately 2 to 3 ml of 2% lidocaine was injected subcutaneously using a 22-g needle at each biopsy site as a local anesthetic. A final surgical scrub and rinse cycle was completed and wiped dry with sterile gauze after the lidocaine injection. Biopsies were collected using a sterile disposable 8-mm biopsy punch (Integra LifeSciences) in approximately the center of the treatment area and stored in buffered 10% formalin (Fisher Scientific) until histology preparation. Each treatment site was sutured and monitored for up to 7 days postprocedure for abnormalities at the biopsy site.

Following the neck experiment and biopsy collection, any permethrin treatments that were not withdrawn from the neck experiment due to adverse skin reactions were tested on the face. The face was washed and cleaned and then divided into 4 quadrants (0.2 to 0.5 m2). Each quadrant was assigned a treatment for the 5 days of the experiment. Treatments were applied to the treatment areas using an assigned hand towel to apply a thin layer on the face, approximately 5 ml. Like the neck experiment, treatments were reapplied every 2 hours during a 6-hour period, for a total of 3 treatment applications in 1 day. Dermal evaluations occurred prior to the first application, before every subsequent reapplication of treatments, and at the end of the trial (2 hours after the final treatment reapplication of the day). The horses’ treatment areas were evaluated by trained personnel using the dermal and ocular response criteria in the Draize scoring system (Supplementary Table S1). If horses scored higher than a “2” for any category of the dermal criteria or if they exhibited visible hyperemic blood vessels or chemosis in the eyes or produced discharge from the eyes (score of “1” or greater in any ocular category), treatments were not reapplied to those areas for the remainder of the study. To prevent bias during the evaluation, evaluators were blinded to the treatments assigned to each treatment area. No biopsy samples were collected from the face.

Histology preparation and analysis

Skin biopsy samples from the neck were processed and stained by the Histology Laboratory at the Penn State Animal Diagnostic Laboratory. Hematoxylin and eosin-stained skin sections were scored by a veterinary pathologist certified by the American College of Veterinary Pathologists. During the analysis, the veterinary pathologist was blinded to treatment and application areas and scored each skin sample for the presence and severity (0, none; 1, mild; or 2, severe) of the following criteria:

  1. Superficial dermal edema.

  2. Perivascular leukocyte infiltrates/reactive capillaries.

  3. Superficial dermal leukocyte infiltrates.

  4. Epidermal spongiosis.

  5. Epidermal hyperplasia/acanthosis.

  6. Intradermal leukocytes.

In addition, samples were scored as 0 (not present) or 1 (present) for the following characteristics:

  1. Apoptotic keratinocytes.

  2. Parakeratosis.

A composite score for each biopsy section from the neck was calculated as the sum of the individual criteria, with a higher score representing a greater indication of skin damage. A measurement of the epidermal thickness was also taken but was not included in the composite score.

Tick bioassays to compare permethrin effectiveness and longevity against ticks

To measure the gradient of repellency and longevity of the permethrin treatments on tick behavior, tick bioassays were conducted on the legs of horses. Detomidine hydrochloride was intramuscularly administered to each horse prior to starting bioassays to ensure the safety of personnel and to prevent horses from disturbing the ticks during the experiment. Each leg represented a treatment during the duration of the experiment, and the experiment was a modified design of the “fingertip” bioassay to test nymphal tick repellents on humans.23 All legs of the horse between the carpus and the coronary band were cleaned with water and had 3 lines drawn using a livestock marker (All-Weather Paintstik; LA-CO Industries) (Figure 1). The first line was approximately 5 cm from the top of the coronet of the hoof, and this was designated as the “must cross line.” The next line was 5 cm proximal relative to the first line and was designated the start of the treatment area (“begin treatment line”). The third line was 10 cm proximal to the begin treatment line and designated the end of the treatment area (“end treatment line”). The region between the begin and end treatment lines was the “treatment zone.” After the lines were drawn, treatments (water control, 1.5%, 5%, and 10%) were randomly assigned to each leg and a thin layer of the treatment (∼2 ml) was applied to the treatment zone of the leg using a paintbrush. The treatments were allowed to dry for 5 to 10 minutes prior to the first experiment. The order in which treatments were tested was randomly assigned to prevent biases in tick activity.

Figure 1
Figure 1

Setup for on-animal tick bioassays to test permethrin repellency. Three lines are drawn on the horse’s leg, between the coronet and the fetlock. The first line (must cross line) is drawn 5 cm above the coronet. The second line (begin treatment line) is drawn 5 cm proximal from the must cross line. The third line (end treatment line) is drawn 10 cm from the “begin treatment line.” The permethrin treatment (∼2 mL) is applied with a paintbrush to the treatment zone, in between the begin treatment and end treatment lines. Five female blacklegged ticks (Ixodes scapularis) are applied to the loading zone, the area between the coronet and the must cross line. After 5 minutes, tick responses were recorded and assigned as “repelled” (did not cross the begin treatment line or fell off after exposure to the treatment zone) or “not repelled” (crossed the end treatment line or remained in the treatment zone). Only ticks that crossed the must cross line were included in the calculation for the proportion of ticks that were repelled or not repelled by permethrin.

Citation: American Journal of Veterinary Research 84, 4; 10.2460/ajvr.22.10.0176

Colony-raised, pathogen-free adult female I scapularis ticks from Oklahoma State University were used in the tick bioassays. The tick’s scutum was painted with fluorescent paint (Testors Corporation) to track and recover ticks with a black light (Figure 1). Painting ticks does not significantly affect the activity and survival of ticks and has been previously used for research, such as mark-recapture studies.24 For each leg, 5 ticks were placed in the area between the coronet and the must cross line. Following the application of the final tick on the leg, the ticks were allowed 5 minutes to traverse the leg. Tick responses were recorded and assigned as “repelled” (did not cross the begin treatment line or fell off after exposure to the treatment zone) or “not repelled” (crossed the end treatment line or remained in the treatment zone). Only ticks that demonstrated active climbing and passed the must cross line were included in the data analyses and results reporting. Each day, all permethrin treatments were tested 3 times. At the end of each day, the horse was turned back out to pasture. The trials continued for up to 5 consecutive days, but treatments were only applied on day 1 to assess the longevity of permethrin effectiveness in natural field conditions. The experiment ended on day 5 or once repellency dropped to ≤ 30%.

Tick bioassay statistical analysis

Given that some experiment weeks occurred during a period of precipitation and to account for any permethrin that may have washed off during precipitation events, analyses were divided into weeks with precipitation and weeks without precipitation. Three rounds of bioassays were conducted for “precipitation” (replicates during rain events) and “no precipitation” weather types. Day zero treatments (ie, the day treatments were applied) were not exposed to weather events and thus were analyzed separately with a 1-way analysis of variance (ANOVA) with treatment as the explanatory variable and proportion of ticks repelled as the response variable. Means were separated by Tukey’s honestly significant difference (HSD) test among treatments. A binomial generalized linear model (GLM) with logit link was fitted to repellency data following day 0. The final dataset included 3 replicates for each unique combination of 2 factors (2 levels of weather and 3 levels of permethrin concentration and a control) over 3 time points. The response variable was the proportion of ticks repelled by the permethrin application and explanatory variables included time, concentration, and weather as well as relevant interactions. Because of the small sample size, we used penalized likelihood25 also known as a Firth correction.

Two models were fitted and compared via Akaike information criterion (AIC), with a lower value indicating a better fit of the model. The first included all main effects and interactions and the second included all main effects and no interactions. These were the simplest models constructed that still addressed all the research questions. Significance was determined at α = 0.05. The Pearson χ2 statistic was used to estimate overdispersion. All statistical analyses were conducted using statistical software, JMP v. 12.1.0 (SAS Institute Inc.). Means were separated by Tukey’s HSD test and α-levels were adjusted post hoc with Bonferroni correction due to multiple comparisons made among fixed effects (α = 0.0080).

Results

Dermal responses to permethrin on the neck and face

For the neck application experiment, none of the horses remained enrolled in the 5% and 10% permethrin treatments for the full experimental period of 10 days, but all horses remained in the experiment for the water control and 1.5% permethrin treatments (Supplementary Figure S1). Interestingly, the average number of days after which horses were withdrawn from the 5% permethrin treatment was 3.2 days, but the average number of days after which horses were withdrawn from the 10% permethrin treatment was 6 days. For 3 of the 5 horses, the 5% permethrin treatment areas had a visibly raised area on the first day of application. This was indicative of a dermal score of 2 under the edema category, prompting withdrawal of these horses from the 5% treatment. As horses continued to receive 5% and 10% treatments throughout the experimental period, dry, cracking skin was noted. On day 9, all remaining horses were withdrawn from the 5% and 10% treatments due to having a score ≥ 2 for either category in the dermal scoring system.

Since all horses were withdrawn from the 5% and 10% permethrin treatments before the end of the experiment, they did not receive these treatments on the face. Instead, horses only received the water control and 1.5% permethrin treatments on the face because they showed little to no visible dermal reaction to these treatments when applied to the neck. After 5 days of experimental treatments using water and 1.5% permethrin, all horses remained in the experiment and experienced no visible dermal reaction to either treatment on the face.

Histology findings from neck biopsies

For the neck biopsies, individual histologic criteria and composite scores were compared between permethrin treatment and control water samples. Control water samples had composite scores ranging 0 to 1 for several characteristics and lacked histologic abnormalities (Figure 2; Supplementary Table S2). Apoptotic keratinocytes, epidermal hyperplasia, and parakeratosis were observed with permethrin treatment but not in control sites. Two horses showed evidence of apoptotic keratinocytes, both from the 5% treatment area. Interestingly, these 2 horses had different and early withdrawal days, with 1 horse being withdrawn on day 1 and the other horse withdrawn on day 4.

Figure 2
Figure 2

Example photomicrographs of dermal biopsies from equines exposed to 0% (water control), 1.5%, 5%, or 10% permethrin. A—Normal equine skin at 0% permethrin treatment (control) site. H&E stain; bar = 200 μm. B—Individual keratinocytes are rounded up, hypereosinophilic, and apoptotic at 5% permethrin treatment site. H&E stain; bar = 50 μm. C—Segments of keratinocytes in the stratum corneum are parakeratotic with retained nuclei (arrow) at 1.5% permethrin treatment site. H&E stain; bar = 50 μm. D—Perivascular leukocytes (arrow) and intraepidermal leukocytes (arrowheads) transmigrating through a spongiotic epidermis at 10% permethrin treatment site. H&E stain; bar = 50 μm. E—The epidermis is diffusely acanthotic and thickened more than twice normal (area between arrows) at 10% permethrin treatment site. H&E stain; bar = 200 μm.

Citation: American Journal of Veterinary Research 84, 4; 10.2460/ajvr.22.10.0176

For 3 out of the 5 horses, parakeratosis was evident in the biopsy samples from the 1.5% treatment areas on the neck (Figure 2). No horse was withdrawn from this treatment during the duration of the neck experiment. Parakeratosis was not apparent in horses with other treatments, regardless of when they were withdrawn from the treatment.

All horses with 1.5%, 5%, or 10% permethrin treatments scored mild or severe for epidermal hyperplasia/acanthosis (Figure 2), except for 1 horse that was withdrawn from the 5% and 10% treatments on day 1 due to severe swelling. However, this same horse did have mild epidermal hyperplasia for the 1.5% treatment, which was applied for the entirety of the experiment. Horses with 10% treatments that were applied for more than 1 day generally had increasing severity of epidermal hyperplasia compared to the 5%, 1.5%, and control water. Similarly, measured epidermal thickness generally increased as the treatment concentration and/or length of application days increased (Supplementary Table S2).

Tick bioassays

A total of 6 replicates of equine tick bioassays were conducted between July and October 2021. Regardless of precipitation, repellency generally decreased as the number of days postapplication increased (Figure 3). During the replicates without precipitation, the 10% permethrin treatment repelled over 80% of ticks for up to 48 hours. Repellency decreased to below 60% at 72 hours and to less than 40% after 96 hours. The 5% permethrin treatment demonstrated approximately 70% repellency at 24 hours, but this declined to below 60% and then below 30% at 48 and 72 hours, respectively. There was little effect of the 1.5% permethrin treatment with less than 40% of ticks being repelled at 24 to 48 hours and dropping under 30% repellency at 72 hours. All permethrin treatments without precipitation were similar to the control in repellent effect at 96 hours. In comparison, all permethrin treatments during precipitation replicates were around 80% repellency initially, but this dropped to ≤ 50% at 24 hours and continued to decline throughout the remainder of the trials.

Figure 3
Figure 3

Mean percentage (% ± SE) of repelled female blacklegged ticks (Ixodes scapularis) to permethrin exposure (0%, 1.5%, 5%, and 10% concentrations) and longevity of treatments during on-animal tick bioassays in periods of no precipitation (left) and with precipitation (right). Permethrin treatments were applied only on the first day of tick bioassay experiments (hour 0) and repellency was evaluated for up to 96 hours or until repellency for all treatments dropped below 30%.

Citation: American Journal of Veterinary Research 84, 4; 10.2460/ajvr.22.10.0176

Overall, treatment influenced the response of the ticks (F = 14.11; df = 3, 60; P < .0001) on the initial day of treatment (day 0). The control treatment (water) resulted in 20.3% ± 0.08 ticks being repelled. This was less than the permethrin treatments of 1.5%, 5%, and 10%, which had 58.9% ± 0.09, 78.7% ± 0.08, and 87.8% ± 0.06 of ticks repelled, respectively (Table 1; Figure 3). While none of the treatments were statistically different from each other in terms of percent of ticks repelled, nearly 40% more ticks were repelled by the 10% permethrin than the 1.5% permethrin on average.

Table 1

Mean (% ± SE) adult blacklegged tick (Ixodes scapularis) repelled by 3 concentrations of permethrin and assayed on equine legs on initial day of testing (day 0).

Treatment Mean (%) ± SE Lower 95% CI Upper 95% CI
Control (water) 20.2 ± 8.0a 4.13 36.29
1.5% Permethrin 58.93 ± 8.6a,b 41.74 76.12
5% Permethrin 78.70 ± 7.6b 63.54 93.86
10% Permethrin 87.81 ± 8.0b 71.73 103.89
a,b

Means followed by the same letter are not significantly different (Tukey’s HSD test, α = 0.008).

The AIC for the model including all main effects and interactions was 229.05 and the AIC for the model with main effects and no interactions was 195.80, which suggests that the latter model had a better fit (Table 2). The estimated overdispersion parameter of the selected model was 1.0 (χ2 = 91.97; P = 1.0). The best-fitting model included precipitation, time after treatment application, and treatment type as important factors influencing I scapularis repellency. In particular, precipitation influenced effectiveness of the treatments on I scapularis repellency (χ2 = 4.22; P = .0399). After 24 hours, the repellent effect of the 5% and 10% trials after exposure to precipitation was nearly half of the effect of the same treatments without precipitation exposure (Table 3). Conversely, the repellent effect of the 1.5% permethrin concentration in the no precipitation and precipitation trials remained consistent. By 72 hours postapplication, the repellent effect of all the permethrin concentrations was like the day 0 observed repellency of the water control.

Table 2

Binomial generalized linear model estimates and 95% confidence limits with associated SE and P values for comparing hours after application, weather (precipitation or no precipitation), and permethrin concentration (1.5, 5, and 10%) on repellency to blacklegged ticks (Ixodes scapularis).

Term Estimate SE χ2 P Lower 95% CI Upper 95% CI
Intercept −0.83 0.18 25.64 < .0001 −1.20 −0.49
Weather (no precipitation) 0.34 0.17 4.22 .04 0.01 0.67
Hours (24) 0.59 0.23 6.95 .01 0.14 1.05
Hours (48) 0.17 0.24 0.58 .45 −0.28 0.63
Treatment (control) −1.30 0.35 18.48 < .0001 −2.05 −0.66
Treatment (1.5% permethrin) −0.20 0.29 0.48 .49 −0.79 0.35
Treatment (10% permethrin) 0.43 0.28 2.59 .11 −0.11 0.97
Table 3

Mean (% ± SE) adult blacklegged tick (Ixodes scapularis) repelled by 3 concentrations of permethrin and assayed on equine legs after exposure to precipitation events.

Hours after application on day 0
Treatment 24 48 72
No precipitation
 Control (water) 15.74 ± 8.61a 5.56 ± 3.67a 5.93 ± 4.07a
 1.5% Permethrin 31.67 ± 15.52a,b 30.00 ± 14.34a,b 18.75 ± 13.15a
 5% Permethrin 71.30 ± 12.81b 53.70 ± 15.66a,b 21.25 ± 6.84a
 10% Permethrin 88.89 ± 11.11b 85.19 ± 11.26b 51.11 ± 11.42a
Precipitation
 Control (water) 22.22 ± 12.11a 16.11 ± 7.67a 0.00 ± 0.022a
 1.5% Permethrin 36.46 ± 14.77a 27.50 ± 13.06a 23.89 ± 8.54a
 5% Permethrin 46.48 ± 11.14a 33.33 ± 16.67a 17.00 ± 4.36a
 10% Permethrin 44.79 ± 12.09a 35.00 ± 8.47a 18.89 ± 8.51a
a,b

Means followed by the same letter in each column for No precipitation and Precipitation separately are not significantly different (Tukey’s HSD test, α = 0.008).

Hours after application was also significant in tick repellent response (χ2 = 10.93; P = .0042), as was treatment (χ2 = 27.94; P < .0001) (Table 2). In trials without precipitation, the 10% permethrin was more effective than the control at the 24- and 48-hour postapplication time points (Table 3). At the 72-hour time point there were no differences among the permethrin treatments and the control. In the trials with precipitation, there were no differences among any of the permethrin treatments or between the permethrin treatments and the control at any time point.

Discussion

The equine industry continues to be threatened by ticks and TBDs that can affect the health and performance of horses. Permethrin is a common insecticide that can be used to control or repel ticks and biting insects that affect people and animals.26 Permethrin continues to be used as an active ingredient in host-targeted tick control tools such as 4-poster feeders2729 and tick control tubes with treated cotton,30,31 but these types of tools do not currently exist for uses against ticks on horses. To develop similar tools specifically that could protect horses from tick bites, this study assessed dermal responses of horses after repeated direct exposures to permethrin and quantified the effect and longevity of permethrin on tick repellency when permethrin is applied to the host.

After applying permethrin to horses, visible skin swelling and redness occurred using the 5% and 10% permethrin on the first day of application, which prompted cessation of treatment application on the horse for the remainder of the experiment. Horses that had these same treatments withdrawn later in the experiment generally had higher composite scores compared to horses that were withdrawn earlier. Biopsy samples indicated that the horses exhibited various lesions typical of irritant contact dermatitis and epidermal cell damage including apoptotic keratinocytes, parakeratosis, and epidermal hyperplasia/acanthosis/thickening. Currently, host-targeted tick treatment devices such as 4-poster feeders for white-tailed deer use a 10% permethrin product,27,28 but these results suggest that this concentration would be inappropriate for horses. It is unknown if lower concentrations of permethrin on a self-treatment device would be as effective for horses.

Interestingly, only 2 horses had signs of mild apoptotic keratinocytes and the 5% treatment was withdrawn from both horses within 4 days. Programmed cell death of keratinocytes is important for many aspects of skin health, including epidermal cell turnover regulation and cancer prevention.32 Elevated or abnormal levels of apoptosis could represent impaired barrier function and dysfunctional cells as a result of an irritant or stress.33

Parakeratosis was also detected in 3 out of the 5 horses enrolled in the experiment. Parakeratosis often indicates increased cell turnover in response to skin/keratinocyte damage and inflammation.34 This is often associated with abnormal hastened keratinocyte maturation, where cells are not maturing at the same rate as cell turnover. Mild parakeratosis was reported for 3 horses exposed to 1.5% permethrin for the length of the experiment, up to 9 days. Parakeratosis was absent from horses that had 5% and 10% permethrin treatments. Parakeratosis often characterizes chronic lesions of irritant reaction and thus may reflect long-term exposure to the 1.5% permethrin treatment in many horses in this cohort. During the neck experiment, all horses were withdrawn from the 5% permethrin treatment within 4 days, except for 1 horse that was withdrawn from the treatment on day 9. Lesions may have resolved during the time from withdrawal of the 5% permethrin treatment to biopsy collection or may have been lost during surgical preparation. Horses exposed to 10% permethrin were enrolled for over half of the experimental time and were also without signs of parakeratosis. This underscores the importance of monitoring the effects of any pesticide application on horses as individuals can demonstrate a range of dermal responses.

Finally, epidermal hyperplasia and acanthosis were present in at least 1 treatment area for all horses. Evidence of epidermal hyperplasia indicates that the skin cells were attempting to regenerate following an inflammatory response and damage to the skin as can be seen with chemical irritants.35,36 While a nonspecific finding, epidermal hyperplasia/acanthosis scores were consistent with the length of time and concentration of treatments in this experiment.

While the horses exhibited these various characteristics indicating skin damage, we noted variability in individual characteristics that may be related to concentration, treatment-biopsy interval, and/or individual horse sensitivity. The sum total of these epidermal and dermal abnormalities can be used to indicate irritant exposure, like permethrin.

Horses demonstrate dermal sensitivity to permethrin, but permethrin is also an effective repellent and pesticide against ticks and biting insects.3739 This study modified the “fingertip assay” typically used to test tick repellent candidates for humans21 and developed a novel way to test tick repellents on animals. The tick bioassay results indicated that initial treatments for 5% and 10% permethrin were more effective than the control at repelling ticks, but there were no significant differences among treatments. More distinct differences may have been observed with larger sample sizes which should be considered in the future.

Precipitation and time postapplication of permethrin influenced tick repellency. Precipitation decreased the repellency effect against ticks within 24 hours of application, presumably due to the permethrin getting washed off or diluted during precipitation events. Similarly, repellency effects generally decreased as postapplication time increased. This phenomenon has been demonstrated previously in other study systems. For example, when testing residual permethrin effects after washing clothing treated with permethrin, researchers found that washing and light exposure significantly decreased the ability of permethrin to knock down mosquitoes.40 Clothes treated with permethrin and then washed showed reduced contact irritancy and repellency to ticks compared to clothes that were not washed.41 In general, permethrin can break down in the presence of UV lighting and has been shown to be less effective on clothes that were washed and dried in the sun compared to those dried under the shade.14,42 Therefore, weather and duration between applications should be considered to determine optimal reapplication intervals.

While an increase in permethrin concentration leads to greater repellency, this finding should be interpreted alongside the permethrin dermal application experiment and histology results, which showed that increased permethrin concentrations generally led to negative consequences in dermal responses on the horses. This highlights the importance of following repellent labels when using them for animals. Anecdotally, equine owners have used permethrin concentrate as tick and insect repellents, which can be useful in repelling ticks as shown with the tick bioassays. On the other hand, there are unintended consequences on the horse’s health when using undiluted permethrin as shown by the permethrin dermal application experiment.

To the authors’ knowledge, this is 1 of the first studies to assess the effects of permethrin after repeated direct contact with equine skin and to demonstrate the practice of conducting on-animal repellency trials. The results of both experiments highlight the importance of equine health and welfare when considering tick repellents. While repellents can protect horses against tick bites and subsequent TBDs such as Lyme disease and equine anaplasmosis, equine owners should consider the safety of the animal as well as the effectiveness of the repellent against ticks. Thus, equine owners should not exceed the recommended permethrin concentrations per the manufacturers’ guidelines to avoid unintended health effects on their animals after exposure to permethrin. Based on these results, permethrin-based products labeled for horses likely need to be applied daily to maintain any protective benefits at the recommended concentrations. These experiments tested concentrations 10 times or more the recommended amount for horses, with the lowest treatment concentration losing efficacy between 24 to 48 hours. Horse owners, especially those who do not or cannot visit their animals daily, may be unable or reluctant to apply products daily as a preventative measure, putting horses at risk for pests. Because of the low response of the blacklegged tick to permethrin concentrations labeled for horses and the short duration of efficacy of these concentrations, this study also emphasizes the need for additional tick control methods for horses including additional low-risk repellents and other on-horse or environmental protective measures.43,44

Supplementary Materials

Supplementary materials are posted online at the journal website: avmajournals.avma.org.

Acknowledgments

The authors extend their gratitude to Baxter Bierbaum, Jessica Brown, Mia Esoldo, Jesse Evans, Kylie Green, Chloe Roberts, Hannah Tiffin, and Anna Marie Wise for their assistance during the experiments. This research would not have been possible without guidance from Drs. Jacob Werner, Edward Jedrzejewski, Sima Lionikaite, and Melissa Welker.

This study was funded by the Pennsylvania State University College of Agricultural Sciences Science to Practice Grant, and the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project PEN04608 and Accession number 1010032.

The authors declare that there were no conflicts of interest.

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