Volume 28, 2021
Special Issue – NexGard® Combo (esafoxolaner, eprinomectin, praziquantel): A new endectocide spot-on formulation for cats. Invited Editor: Frédéric Beugnet
Article Number 21
Number of page(s) 6
Published online 02 April 2021

© E. Tielemans 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.


Some of the most frequent parasitic disorders of felines are caused by fleas, major ectoparasites of carnivores and other mammalian species worldwide [8, 20, 21, 23]. Ctenocephalides felis is the main flea species found in both cats and dogs globally. The species is not fully host-specific and it can infest or take blood meals from other mammals such as wild canids and felids, ruminants, rodents or humans [8, 9, 20, 21]. Flea infestations represent a significant veterinary and public health concern. Flea bites induce discomfort and skin inflammatory reactions that can result in dermatological signs such as alopecia, erythema, or moist dermatitis, and systemic disorders such as anemia [8, 22, 23]. Flea bite hypersensitivity, also called flea allergy dermatitis (FAD), is one of the most common dermatological conditions in companion animals and includes signs such as pruritus, crusts, alopecia, and miliary dermatitis [11, 22]. The cat flea can also transmit zoonotic diseases such as Rickettsia felis, the agent of flea-borne spotted fever, and Bartonella henselae, the agent of cat scratch disease [14, 9, 13, 16, 18]. Additionally, fleas are the intermediate host for Dipylidium caninum [7, 10], a common cestode of cats and dogs.

Pet owners often treat their animal only when they see fleas or flea infestation-related symptoms, but this approach cannot break the epidemiological cycle as adult fleas infesting animals represent a minor part of the total flea population [14, 23]. In an infested environment, the vast majority of the flea population is composed of eggs, larvae and pupae. Thus, sustained efficacy against new infestations is important for an optimal flea control program, as well as the inhibition of flea egg production and/or development.

NexGard® Combo, a novel topical combination of esafoxolaner, eprinomectin and praziquantel has been designed to offer a wide spectrum of antiparasitic activity.

The objectives of the four studies presented here were to assess the efficacy of this novel formulation for the treatment and control of adult C. felis and for the inhibition of flea egg production and larval development, when administered topically to cats.

Materials and methods


The study protocols were reviewed and approved by the Sponsor’s and local Institutional Animal Care and Use Committees. Cats were managed and handled with due regard for their wellbeing.

Study designs

The four 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” [19], and 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”.

The four studies had different objectives, contexts and designs, as summarized in Tables 1 and 2. The studies were conducted in three licensed laboratories, in South Africa (two studies), in France, and in California, United States. Overall, the objectives were to measure adulticide efficacy and egg production inhibition, for at least one month, of one application of the novel formulation at the minimum recommended dose against flea isolates of European or American origin.

Table 1

Study objectives and contexts.

Table 2

Study designs.

Each study was conducted using a randomized design, with cats allocated to either a placebo control or a novel formulation treated group, with blocking based on pre-treatment flea infestation or larval hatching levels.

The efficacy assessments against adult fleas were based on comparison of adult flea counts in the control and treated group 24 h after treatment (curative efficacy), or 24 h after subsequent weekly infestations (preventive efficacy). The efficacy assessments against immature stages were based on two variables: egg production inhibition, i.e. comparison of number of eggs found in the control and treated group at identical weekly timepoints; and larval hatching inhibition, i.e. comparison of larvae hatched from collected eggs in the control and treated groups after incubation.

All personnel collecting animal health and efficacy data were blinded to treatment.

Animals and housing

The cats were healthy, purpose-bred laboratory Domestic Short/Long-hair cats, belonging to the local colony of each laboratory, and are described in Table 3. Cats were single-housed (and placed on wire mesh flooring when individual egg collection was performed) at least during the periods of flea infestation and flea removal (up to 48 h).

Table 3

Animal details.

Flea strains

All fleas were laboratory-maintained C. felis colonies originally sourced from the field. The flea colonies were not known to be resistant to ectoparasiticide compounds and had not previously been subjected to ectoparasiticide challenge. Genetic enrichments of the colonies were performed at least every 5 years. The different flea isolates had an unknown specific pathogen-free status.

The isolate used in Study #1 originated from Germany and was maintained on an artificial membrane device. The isolate used in Study #2 originated from Stanislaus County, California, USA, and was maintained on cats. The isolate used in the two South African studies (Studies #3 and #4) originated from the field in Germany, and was maintained on cats.

Treatment and health observations

Cats were treated once on Day 0. The treatment was applied on 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; cats assigned to the novel formulation group were treated at 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.

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

Flea infestations and counts

Each cat was infested weekly with approximately 100 unfed adult C. felis. Fleas were removed and counted via thorough combing of all body areas with a fine-tooth flea comb. The schedule of flea infestation and count per study is detailed in Table 2.

Flea eggs collection and larval hatch assessment

Flea eggs falling from each cat were collected from a pan under a wire mesh floor over a period of two days following infestation. Collected eggs were placed in individual containers for incubation at approximately 25 °C and 80% relative humidity. After 4–6 days of incubation, hatched flea larvae were counted.

Statistical analysis

For the evaluation of efficacy against adult fleas, arithmetic means were calculated for each treatment group at each weekly timepoint. The percent efficacy was calculated as [(C − T)/C] × 100, where C was the arithmetic mean of the flea counts among the placebo-treated cats, and T was the arithmetic mean among the novel formulation-treated cats.

For the evaluation of efficacy against immature flea stages, geometric means of collected eggs were calculated for each treatment group at each weekly timepoint. The percent efficacy was calculated as [(C − T)/C] × 100, where C was the geometric mean of the flea counts among the placebo-treated cats, and T was the geometric mean among the novel formulation-treated cats.

For the evaluation of viability of eggs, the proportion of larval hatch from the total number of eggs collected was calculated at each timepoint. If both treatment groups at a given timepoint had more than two animals for which eggs were collected, the proportion of larval hatch of the novel formulation-treated group was compared to the proportion of larval hatch from the placebo-treated group.

For counts of adult fleas and eggs, the log-counts of the two groups were compared using the MIXED procedure in SAS Version 9.4 with treatment as a fixed effect and allocation block as a random effect. For the evaluation of viability of eggs, the proportions of larval hatches of the two groups were compared using the GLIMMIX procedure in SAS Version 9.4. For this analysis, block was included as a random effect, treatment group was the fixed effect, and the link function was the logit.

All treatment comparisons utilized a (two-sided) 5% significance level.


Efficacy against adult fleas (Studies #1, #2 and #4)

The weekly mean flea counts and percent efficacy results obtained in the three studies are summarized in Table 4.

Table 4

Ctenocephalides felis adulticide efficacy.

At all timepoints, the arithmetic means of live fleas in the control group were at least 49.2, confirming consistent robustness of the challenges in the three studies.

Curative efficacy on Day 1, 24 h after treatment, was 92.1%, 98.3% and 99.7% for each study, respectively. Preventive weekly efficacy, 24 h after weekly infestations, was at least 95.5% for one month in the three studies. One study was extended to eight weeks and at least 96.9% reduction was demonstrated for seven weeks; one study was extended to five weeks when the reduction was 90.2%. At all timepoints and in all studies, the number of live fleas was significantly lower on treated animals than on control animals (p < 0.0001).

Flea egg production and larval hatch assessment (Studies #2 and #3)

The weekly mean egg counts, percent reduction of egg production, and larval hatching from collected eggs after incubation during the two studies are summarized in Table 5.

Table 5

Ctenocephalides felis egg production reduction and larval hatching inhibition.

In the first week after treatment, the reduction of egg production in Study #3 during Days 1 to 3 was 93.2% (p = 0.0019). In Study #2, a higher number of eggs was found in the treated group on Day 1 because in this study, the first flea infestation had been performed 24 h before treatment, on Day −1. Therefore, fleas were already producing eggs when cats were treated on Day 0 and continued to lay eggs until their death, resulting in lower egg reduction of 75.9%. From the second week after treatment to the end of the month, efficacy of the novel formulation to reduce production of C. felis eggs in both studies was at least ≥ 99.8%.

The proportion of larval hatching from collected eggs in the control groups in both studies ranged from 34% to 84%. Because of the low number of eggs in the treated groups, statistical analysis was only meaningful for the eggs collected and incubated on the week of treatment. Larval hatching from these eggs in the untreated control group and in the treated group was significantly different (p = 0.0058 in Study #2; p = 0.0001 in Study #3).

Results of the flea egg analyses indicate that the novel formulation was highly effective in reducing the number and the viability of flea eggs shed in the environment for at least one month in both studies.

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


The results of these four experimental studies illustrate the high level of efficacy of NexGard® Combo for the reduction of adult flea infestations, and for the prevention of flea egg production. The three adulticide studies demonstrated that the new formulation is highly effective against existing flea infestations within 24 h of treatment and against subsequent infestations for at least one month, as previously reported for afoxolaner-based products used on dogs [6, 15].

The two studies targeting flea eggs demonstrated that the novel formulation causes a significant reduction in egg production, as previously found in dogs treated with afoxolaner [17]. This novel formulation quickly kills the fleas before they are able to lay eggs, which is necessary, in combination with the adulticide effect, to break the flea lifecycle at multiple stages [14, 23].

In a European survey on domestic cats examined in veterinary clinics for reasons unrelated to parasitic disorders, co-infestation with endo- and ectoparasites was observed in 14% of the subjects [5]. The control of multiple and various concurrent parasitic infestations by a range of cat parasites is important for cats but also public health [12, 24, 25].

This novel association of esafoxolaner, eprinomectin and praziquantel offers a broad spectrum of efficacy against the main parasites of cats including ecto- and endoparasites. Esafoxolaner is the purified and active (S)-enantiomer of afoxolaner, the racemic mixture. Afoxolaner has been proven effective against adult fleas and flea egg production in dogs [6, 15, 17]. In this novel formulation, the use of a purified enantiomer enables lowering of the dose and thus the potential for side effects, and interactions with the other active substances of the combination.

In addition to a high level of efficacy and safety, owner and cat compliance is an important feature for successful control of fleas. The simple conditions of use and of treatment application of this product should guarantee 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 of such parasitic infestations.

Competing interest

The work reported herein was funded by Boehringer-Ingelheim. The authors are current employees of Boehringer-Ingelheim Animal Health or external organizations. Other than that, 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.


  1. Álvarez-Fernández A, Breitschwerdt EB, Solano-Gallego L. 2018. Bartonella infections in cats and dogs including zoonotic aspects. Parasites & Vectors, 11, 624. [PubMed] [Google Scholar]
  2. Barradas PF, de Sousa R, Vilhena H, Oliveira AC, Luz MF, Granada S, Cardoso L, Lopes AP, Gonçalves H, Mesquita JR, Ferreira P, Amorim I, Gärtner F. 2019. Serological and molecular evidence of Bartonella henselae in cats from Luanda city, Angola. Acta Tropica, 195, 142–144. [PubMed] [Google Scholar]
  3. Bergmann M, Hartmann K. 2017. Vector-borne diseases in cats in Germany. Tierärztliche Praxis Ausgabe K Kleintiere Heimtiere, 45, 329–335. [Google Scholar]
  4. Beugnet F. 2013. Guide to vector borne diseases of pets. Printed in France: Ed Ferreol, 425 p. [Google Scholar]
  5. Beugnet F, Bourdeau P, Chalvet-Monfray K, Cozma V, Farkas R, Guillot J, Halos L, Joachim A, Losson B, Miró G, Otranto D, Renaud M, Rinaldi L. 2014. Parasites of domestic owned cats in Europe: Co-infestations and risk factors. Parasites & Vectors, 7, 291. [CrossRef] [PubMed] [Google Scholar]
  6. Beugnet F, de Vos C, Liebenberg J, Halos L, Fourie J. 2014. Afoxolaner against fleas: Immediate efficacy and resultant mortality after short exposure on dogs. Parasite, 2014(21), 42. [Google Scholar]
  7. Beugnet F, Halos L. 2015. Parasitoses & vector borne diseases of cats. In: Beugnet F, Halos L (Scientific Editors). Printed by Ferreol: Lyon, France. 381 p. ISBN 978-2-9550805-0-4. Chapter: Flea Infestation. pp. 210–220. [Google Scholar]
  8. Beugnet F, Halos L, Guillot J. 2018. Textbook of Clinical Parasitology in dogs and cats. Zaragoza, Spain: Ed. Grupo Asis. [Google Scholar]
  9. Beugnet F, Marié JL. 2009. Emerging arthropod-borne diseases of companion animals in Europe. Veterinary Parasitology, 163, 298–305. [CrossRef] [PubMed] [Google Scholar]
  10. Beugnet F, Meyer L, Fourie JJ, Larsen D. 2017. Preventive efficacy of NexGard Spectra® against Dipylidium caninum infection in dogs using a natural flea (Ctenocephalides felis) infestation model. Parasite, 24, 16. [EDP Sciences] [PubMed] [Google Scholar]
  11. Carlotti DN, Jacobs DE. 2001. Therapy, control and prevention of flea allergy dermatitis in dogs and cats. Veterinary Dermatology, 11, 83–98. [Google Scholar]
  12. Deplazes P, van Knapen F, Schweiger A, Overgaauw PA. 2011. Role of pet dogs and cats in the transmission of helminthic zoonoses in Europe, with a focus on echinococcosis and toxocarosis. Veterinary Parasitology, 182(1), 41–53. [CrossRef] [PubMed] [Google Scholar]
  13. Dryden MW, Hodgkins E. 2010. Vector-borne diseases in pets: the stealth health threat. Compendium on Continuing Education for the Practicing Veterinarian, 32, E1–E4. [Google Scholar]
  14. Halos L, Beugnet F, Cardoso L, Farkas R, Franc M, Guillot J, Pfister K, Wall R. 2014. Flea control failure? Myths and realities. Trends in Parasitology, 30, 228–233. [PubMed] [Google Scholar]
  15. Hunter JS, Dumont P, Chester TS, Young DR, Fourie JJ, Larsen DL. 2014. Evaluation of the curative and preventive efficacy of a single oral administration of afoxolaner against cat flea Ctenocephalides felis infestations on dogs. Veterinary Parasitology, 201, 207–211. [CrossRef] [PubMed] [Google Scholar]
  16. Just FT, Gilles J, Pradel I, Pfalzer S, Lengauer H, Hellmann K, Pfister K. 2008. Molecular evidence of Bartonella spp. in cats and dog fleas from Germany and France. Zoonoses and Public Health, 55, 514–522. [PubMed] [Google Scholar]
  17. Kunkle BN, Drag MD, Chester TS, Larsen DL. 2014. Assessment of the onset of action of afoxolaner against existing adult flea (Ctenocephalides felis) infestations on dogs. Veterinary Parasitology, 201, 204–206. [PubMed] [Google Scholar]
  18. Lappin MR. 2018. Update on flea and tick associated diseases of cats. Veterinary Parasitology, 254, 26–29. [PubMed] [Google Scholar]
  19. Marchiondo AA, Holdsworth PA, Fourie LJ, Rugg D, Hellmann K, Snyder DE, Dryden MW. 2013. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) second edition: Guidelines for evaluating the efficacy of parasiticides for the treatment, prevention and control of flea and tick infestations on dogs and cats. Veterinary Parasitology, 194, 84–97. [CrossRef] [PubMed] [Google Scholar]
  20. Otranto D, Dantas-Torres F, Breitschwerdt EB. 2009. Managing canine vector-borne diseases of zoonotic concern: part one. Trends in Parasitology, 25, 157–163. [CrossRef] [PubMed] [Google Scholar]
  21. Otranto D, Dantas-Torres F, Breitschwerdt EB. 2009. Managing canine vector-borne diseases of zoonotic concern: part two. Trends in Parasitology, 25, 228–235. [CrossRef] [PubMed] [Google Scholar]
  22. Plant JD. 1991. Recognizing the manifestation of flea allergy in cats. Veterinary Medicine, 10, 482–486. [Google Scholar]
  23. Rust MK. 2017. The biology and ecology of cat fleas and advancements in their pest management: a review. Insects, 8, 118. [Google Scholar]
  24. Traversa D. 2012. Pet roundworms and hookworms: a continuing need for global worming. Parasites & Vectors, 5, 91. [CrossRef] [PubMed] [Google Scholar]
  25. Traversa D. 2013. Fleas infesting pets in the era of emerging extra-intestinal nematodes. Parasites & Vectors, 6, 59. [PubMed] [Google Scholar]

Cite this article as: Tielemans E, Buellet P, Young D, Viljoen A, Liebenberg J & Prullage J. 2021. Efficacy of a novel topical combination of esafoxolaner, eprinomectin and praziquantel against adult cat flea Ctenocephalides felis and flea egg production in cats. Parasite 28, 21.

All Tables

Table 1

Study objectives and contexts.

Table 2

Study designs.

Table 3

Animal details.

Table 4

Ctenocephalides felis adulticide efficacy.

Table 5

Ctenocephalides felis egg production reduction and larval hatching inhibition.

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