Volume 28, 2021
Special Issue – NexGard® Combo (esafoxolaner, eprinomectin, praziquantel): A new endectocide spot-on formulation for cats. Invited Editor: Frédéric Beugnet
Article Number 23
Number of page(s) 6
Published online 02 April 2021

© J. Prullage et al., published by EDP Sciences, 2021

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Ixodes ricinus is the most common tick species infesting companion animals and is present throughout Europe, especially in central Europe [5, 6, 11, 34, 44]. Ixodes scapularis is among the main tick species infesting cats in North America [7, 13, 23, 45]. Ixodes spp. infestations are an important concern for pet owners and public health [1, 4, 12, 35, 36]. In addition to causing local discomfort and local inflammatory reactions, they may also transmit pathogens that can cause severe disease in animals and humans such as anaplasmosis, babesiosis and borreliosis [8, 9, 15, 17, 19, 21, 26, 27, 3742]. Tick prevalence, as well as the risk for transmission of tick-borne diseases, has been increasing over the last three decades in both Europe and the United States [16, 18, 22, 28, 29, 31].

A novel topical combination of (S)-afoxolaner, eprinomectin and praziquantel has been designed to offer a wide spectrum of antiparasitic activities. Esafoxolaner is the active purified (S)-enantiomer of afoxolaner, the racemic mixture. Afoxolaner has already been proven effective in dogs against several Ixodidae, including I. ricinus and I. scapularis when administered orally as a single-compound (Nexgard®) or combination product (Nexgard® Spectra) [14, 20, 24, 25, 32, 33].

The objectives of the four studies presented here were to assess the efficacy of this novel formulation for the treatment and control of I. ricinus and I. scapularis infestations, when administered topically to cats at the minimum recommended label dose in a laboratory setting.

Materials and methods


The study protocols were reviewed and approved by the Sponsor’s and Investigator’s Institutional Animal Care and Use Committees or by the responsible governmental authority as per legal requirements. Cats were managed and handled similarly within each study and with due regard for their wellbeing.

Study designs

The studies were designed in accordance with the “World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of parasiticides for the treatment, prevention and control of flea and tick infestation on dogs and cats” [30]. The three I. ricinus studies were also designed in accordance with the Committee for Medicinal Products for Veterinary Use (CVMP) “Guideline for the Testing and Evaluation of the Efficacy of Antiparasitic Substances for the Treatment and Prevention of Tick and Flea Infestation in Dogs and Cats”, EMEA/CVMP/EWP/005/2000-Rev.3. Two of the I. ricinus studies and the two I. scapularis studies were conducted in accordance with Good Clinical Practices as described in “International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guideline GL9”.

Each study was conducted using a randomized design based on a pre-treatment parasite infestation and count, for allocation to either a placebo/untreated negative control group or a novel formulation treated group. Personnel collecting animal health and efficacy data were blinded to treatment group. Cats were single housed during the periods of tick infestation/tick removal. The efficacy assessment was based on comparison of live ticks found in the control versus the treated group at identical time-points after treatment on Day 0. All efficacy assessments were performed 48 h after treatment (immediate efficacy) or subsequent infestations (persistent efficacy).

The five studies were conducted at different locations, I. ricinus studies in Europe and I. scapularis studies in the United States. Individual specifics of the studies are detailed in Tables 1 and 2.

Table 1

Regulatory context, location, timing and animals by study.

Table 2

Tick colonies (origin, number and sex), infestations and tick counts for efficacy evaluations by study.


The cats were healthy, purpose-bred laboratory Domestic Short-hair cats. Sex, age and bodyweight are described in Table 1.


Ticks used in all I. ricinus studies and in one of the I. scapularis studies originated from laboratory-maintained colonies genetically enriched at least every 5 years, not previously exposed and not known to be resistant to any ectoparasiticide compound, and were pathogen-free. Ticks used in the second I. scapularis study were collected from the wild. The origin of the ticks is described in Table 2.


Cats were treated once on Day 0. The treatment was applied as one spot directly on the skin, after parting the hair, in the midline of the neck between the base of the skull and the shoulder blades. Cats assigned to the placebo control group were treated with mineral oil at 0.12 mL/kg (in one of the I. ricinus studies, cats were sham dosed). Cats assigned to the novel formulation group were treated with the minimum recommended dose of 0.12 mL/kg, delivering 1.44 mg/kg esafoxolaner, 0.48 mg/kg eprinomectin and 10.0 mg/kg praziquantel. The formulation used in the first of the I. ricinus studies was an early-stage experimental formulation with identical concentration and composition of active ingredients, but with some differences in the solvent system. The formulation used in the other four studies was the final novel formulation.

Health observations were conducted daily and at hourly intervals for 4 h after treatment in all studies to detect any adverse reaction or health abnormality.

Tick infestations and counts

The timepoints of infestation, count and the number of ticks used at each individual infestation are described in Table 2. In the five studies, ticks were placed on the side or the head of each cat, avoiding the application site. To minimize grooming and scratching of ticks, cats were fitted with Elizabethan collars (except in the second I. ricinus study) at the time of infestation. The collars were removed before treatment on Day 0, or when ticks were counted on Day 9 and afterwards. In the I. ricinus studies, all cats were sedated for infestations and counts; in the I. scapularis studies, cats were only sedated when necessary.

The whole body was examined for tick counts. The hair was pushed manually against its natural nap to expose the ticks. The whole body was thoroughly searched. All ticks found were removed using a tick extractor. After the visual and digital examination was complete, the hair coat was thoroughly combed using a fine-toothed flea comb for secondary confirmation that all ticks had been found and counted. The live or dead status was determined. In one of the I. ricinus studies, the attached or free status of each collected tick was also determined and any tick found in a comb was classified by default as attached. In the I. scapularis studies, dead ticks found in the enclosures were also counted.

Statistical analysis

The efficacy variable was the live tick count (or live attached tick count in one of the I. ricinus studies). Arithmetic means were calculated for each treatment group at each efficacy timepoint. The percent efficacy was calculated as [(C − T)/C] × 100, where C was the arithmetic mean of the counts among the control cats and T was the arithmetic mean among the treated cats.

To compare the groups statistically, the logarithm of (counts + 1) was analyzed. The log-counts of each treated group were compared to the log-counts of the control group using an F-test adjusted for the allocation blocks used to randomize the animals to the treatment groups at each timepoint separately. The MIXED procedure in SAS Version 9.4 was used for the analysis, with group listed as a fixed effect and the allocation blocks listed as a random effect. All statistical comparisons used a 5% significance level.


Efficacy against I. ricinus

The efficacy results obtained in I. ricinus studies are summarized in Table 3.

Table 3

Ixodes ricinus studies: mean live tick counts per group and efficacy results.

In the I. ricinus studies, the curative efficacies of the novel formulation, 48 h after treatment, were 91.1%, 91.9% and 100%, respectively; the preventive efficacies 48 h after subsequent infestations were at least 91.4% for five weeks, 93.9% for six weeks, and 100.0% for five weeks, respectively. At each timepoint and in the three studies, the number of live ticks was significantly lower on treated animals than on control animals (p < 0.0001).

In all three studies and considering all timepoints, mean tick retention in the control animals ranged from 36% to 78%, demonstrating robustness of the challenge.

Efficacy against I. scapularis

The percent efficacy results obtained in studies I. scapularis #1 and #2 are summarized in Table 4.

Table 4

Ixodes scapularis studies: mean live tick counts per group and efficacy results

The curative efficacy of the novel formulation, 48 h after treatment, was 95.1% and 98.8% in the two studies. The preventive efficacy 48 h after subsequent infestations was at least 98.8% for one month in the first and was at least 98.2% for one month in the second study. At each timepoint and in both studies, the number of live ticks was significantly lower on treated animals than on control animals (p < 0.0001). Significantly fewer dead ticks were found in the control animals in both studies, (p ≤ 0.0005 and p ≤ 0.02, respectively). In both studies and considering all timepoints, mean tick retention in the control animals ranged from 44% to 65%, demonstrating robustness of the challenge.

No adverse reactions related to treatment were observed in any of the five studies.


Companion animals may contribute to the circulation and spread in the environment of ticks, fleas, and tick and flea-borne pathogens, and require control and protection by year-round use of ectoparasiticides with both insecticidal and acaricidal activity [2, 12, 43, 45].

The results of these five studies illustrate the high level of efficacy of the novel topical formulation of esafoxolaner, eprinomectin and praziquantel against I. ricinus and I. scapularis infestations. This high level of efficacy is achieved within 48 h of treatment and is maintained within 48 h following infestation for at least one month after application.

The control of multiple and various concurrent parasitic infestations by a range of cat parasites is important for cats but also public health [6, 10, 46]. In a European survey on domestic cats, co-infestation with endo- and ectoparasites was observed in 14% of the subjects [3]. This novel formulation offers a broad spectrum of efficacy against the main parasites of cats including ecto- and endoparasites.

In addition to a high level of efficacy and safety, owner compliance with a regular treatment schedule is an important feature for successful control of ticks. The simple conditions of use and of treatment application of this product should ensure a high level of compliance.

This novel formulation provides pet owners and veterinarians with an effective solution for an integrated approach for cats presenting multiple parasitic infestations, or presenting risks associated with these parasitic infestations.

Competing interest

The work reported herein was funded by Boehringer-Ingelheim. The authors are current employees of Boehringer-Ingelheim. Other than this, the authors declare no conflict of interest. This document is provided for scientific purposes only. Any reference to a brand or trademark herein is for information purposes only and is not intended for any commercial purposes or to dilute the rights of the respective owners of the brand(s) or trademark(s).

NexGard® is a registered trademark of the Boehringer-Ingelheim Group.


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Cite this article as: Prullage J, Pfefferkorn A, Knaus M, Frost J, Mitchell E & Tielemans E. 2021. Efficacy of a novel topical combination of esafoxolaner, eprinomectin and praziquantel against Ixodes ricinus and Ixodes scapularis in cats. Parasite 28, 23.

All Tables

Table 1

Regulatory context, location, timing and animals by study.

Table 2

Tick colonies (origin, number and sex), infestations and tick counts for efficacy evaluations by study.

Table 3

Ixodes ricinus studies: mean live tick counts per group and efficacy results.

Table 4

Ixodes scapularis studies: mean live tick counts per group and efficacy results

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