Efficacy of afoxolaner or a combination of afoxolaner and milbemycin oxime against fleas, ticks, lice and/or intestinal nematodes in dogs naturally infested in Brazil

Efficacy of afoxolaner or a combination of afoxolaner and milbemycin oxime against fleas, ticks, lice and/or intestinal nematodes in dogs naturally infested in Brazil | 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 Efficacy of afoxolaner or a combination of afoxolaner and milbemycin oxime against fleas, ticks, lice and/or intestinal nematodes in dogs naturally infested in Brazil Andre A. Cutolo, Luis G. R. Pelissoni, Tassia Lopes Vale, Livio Martins Costa-Junior, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9294152/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background Infestations with fleas, ticks and intestinal nematodes are commonly identified in domestic dogs and have significant veterinary and public health importances. The study described in this manuscript was conducted to verify the natural parasitism of domestic dogs in two Brazilian regions, and to confirm the efficacy of the isoxazoline afoxolaner, formulated alone (NexGard®) or in combination with the macrocyclic lactone milbemycin oxime (NexGard Spectra®), for the control of fleas and ticks, and to confirm the efficacy of the milbemycin oxime combination for the control of intestinal nematodes. Methods The trial was conducted in Mato Grosso (MT) and Maranhão (MA) states, Center-West and Northeast region. Three hundred and forty-five (345) domestic dogs, 170 in MT and 175 in MA, infested with at least one of these parasites were studied. Dogs were treated once and evaluated approximately two and four weeks later for ectoparasites, and two weeks later for intestinal nematodes. Results Approximately half (47.5%) of the dogs were infested with more than one parasite, more than one third (38.6%) with intestinal nematodes. All fleas were identified as Ctenocephalides felis , ticks as Rhipicephalus linnaei and intestinal nematodes eggs as Ancylostoma, Toxocara or Trichuris . Eleven dogs were also diagnosed infested with the chewing louse Heterodoxus spiniger. Both products had an identical efficacy against fleas (98.4% to 100%), ticks (96.9% to 100%) and lice (100%). The MO formulation had an efficacy of ≥ 97.8% against Ancylostoma , ≥ 94.7% against Toxocara and ≥ 97.7% against Trichuris. Conclusions This study demonstrated a significant parasitic abundance in domestic dogs in Brazil, and confirmed a high efficacy of afoxolaner against fleas, ticks and lice and a high efficacy of MO against intestinal nematodes. Dog efficacy afoxolaner milbemycin oxime Ctenocephalides felis Rhipicephalus linnaei Heterodoxus spiniger Ancylostoma Toxocara Trichuris Background Fleas, ticks, lice and intestinal nematodes are commonly identified in domestic dogs and have a significant veterinary and public health importance [ 1 – 3 ]. These parasites can have a direct pathogenic effect on the host, associated to the immune reactions caused by their presence [ 4 ], their life cycle, and their feeding on host tissue [ 5 ]. Many ectoparasites have an indirect pathogenic effect, being vector of filarial nematode, protozoan, bacterial and viral diseases. Importantly, several canine parasites and several vector-borne agents are zoonotic [ 3 , 6 – 10 ]. Afoxolaner is an insecticide and acaricide isoxazoline compound formulated as an oral chewable ectoparasiticide tablet for dogs (NexGard®, Boehringer Ingelheim). Milbemycin oxime (MO) is a macrocyclic lactone nematicide compound formulated in combination with afoxolaner as an oral chewable endectoparasiticide tablet for dogs (NexGard Spectra®, Boehringer Ingelheim). Isoxazolines antagonize the GABA-gated chloride ion channels [ 11 – 13 ], while macrocyclic lactones, mainly agonize the Glutamate-gated chloride ion channel [ 14 , 15 ] of the parasites. Afoxolaner, alone or in combination with MO has been reported efficacious several times against fleas [ 16 – 18 ], ticks [ 18 – 21 ], mites [ 22 – 25 ], flies [ 26 , 27 ] and lice [ 28 , 29 ], in laboratory or in field conditions. Milbemycin oxime, in combination with afoxolaner has also been reported efficacious several times against intestinal nematodes [ 30 – 32 ]. This manuscript describes a field study conducted in two cities in different Brazilian states, Mato Grosso and Maranhão, located respectively in the Center-West and Northeast administrative regions of Brazil, for the investigation of the efficacy and tolerance of NexGard and NexGard Spectra in dogs naturally infested with fleas, ticks and/or intestinal helminths. The objectives of this study were to verify the absence of local effects on sensitivity to the parasiticide molecules in the field and to evaluate the parasitism of domestic dogs in these regions. Methods This field trial was conducted during the period October 2018 - February 2019, in urban areas, in two states in Brazil, Mato Grosso (Sinop and Ipiranga do Norte cities) and Maranhão (metropolitan region of São Luís). Although both São Luís and Sinop have tropical climates with high temperatures throughout the year, their environmental conditions differ markedly due to their geographic separation (~ 1,600 km) and distinct climatic influences. São Luís experiences a humid tropical coastal climate characterized by high relative humidity and a short dry winter (Aw, Köppen classification), whereas Sinop, located inland in the Amazon–Cerrado transition zone, presents a tropical monsoon climate influenced by the South American monsoon system, with a pronounced dry season (Am) [ 33 ]. Study design This study was conducted in accordance with the relevant WAAVP (World Association for the Advancement of Veterinary Parasitology) guidelines for the evaluation of efficacy of parasiticides on dogs and cats against fleas and ticks [ 34 ] and anthelmintics [ 35 ]; the relevant VICH (Veterinary Internation Conference in Harmonization) International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products guidelines: VICH GL7, “Efficacy of Anthelmintics: General Requirements”, VICH GL19 “Efficacy of Anthelmintics: Specific Recommendations for Canines”, and VICH GL9 “Good Clinical Practices”. This study was designed to test the efficacy of NexGard® and NexGard Spectra® against natural infestations with fleas, ticks and intestinal nematodes (NexGard Spectra® only) in the field in two regions of Brazil, and to confirm their safety. The study was fully conducted in households. The households were recruited on voluntary basis, without other veterinary-related purposes. To qualify for the study, a dog was naturally infested with fleas, ticks or intestinal nematode. A maximum of three dogs per household were included for efficacy assessment, and a maximum of five dogs per household were acceptable (households with dogs and cats were excluded). The same treatment was administered to all dogs in a household. Dogs infected with intestinal nematodes were assigned to the NexGard Spectra® group. Dogs only infested with fleas and/or ticks were assigned to the NexGard® group (except for dogs within a household of a dog assigned to NexGard Spectra®, in which case treated with the same treatment). In a household, dogs that were not included for efficacy evaluation received the same treatment and were evaluated for safety. No masking procedure was implemented during this study. Following treatment, dogs were assessed for ectoparasites and/or intestinal nematodes based on their diagnosed parasitism at inclusion day. No parasiticide compound with remanent activity within the three previous months of inclusion had been used on dogs or in their environment. Animals Three-hundred and forty-five (345) domestic dogs (177 males, 168 females) of various age (2 months to 15 years), bodyweight (2 to 46 kg) and breed (85% mongrels) were included for efficacy assessment. These dogs belonged to 84 households in Mato Grosso, and 130 households in Maranhão. All dogs were infested with at least six live fleas and/or four live ticks and/or were infected with intestinal nematode (hookworm, roundworm, whipworm) through identification of eggs in feces. Dogs were maintained at home during the study in their usual routine, environmental and husbandry conditions. Treatment On Day 0, dogs were treated with NexGard® (when they had been included on the basis of ectoparasite infestation, only) or with NexGard Spectra® (when they or any other dog from their household had been included on the basis of endoparasite infestation, namely). The products were administered per label recommendations (orally and with a tablet strength corresponding to their bodyweight, delivering 2.5 to 7.1 mg/kg afoxolaner and 0.5 to 1.1 mg/kg milbemycin oxime in the combined product). In Mato Grosso, 86 and 84 dogs and in Maranhão, 63 and 112 dogs were treated with NexGard® and NexGard Spectra®, respectively (exclusive of the 21 dogs that were not evaluated for efficacy). Parasite evaluations Live flea counts were performed by combing all parts of the animal’s haircoat using a fine-tooth comb (11 to 13 teeth per centimeter) for at least 5 minutes after the encounter of the last ectoparasite on the animal’s skin. Live ticks found during the flea combing process or haircoat search were counted and removed manually with fingertips or forceps. The interdigital and pad areas, internal face of ears, pubic, perineal and axillary areas were individually checked. Samples of removed ectoparasites for each dog were collected and kept stored in ethanol 70% solution for species identification based on morphological characteristics. Fleas and ticks were examined under stereomicroscope and identified using dichotomous keys according to Linardi and Guimarães [ 36 ], as well as taxonomic references described by Aragão and Fonseca [ 37 ] subsequently updated by Martins et al. [ 38 ]. Lice specimens were mounted on glass slides using a drop of lactophenol solution and identified following the criteria established by Werneck [ 39 , 40 ]. Intestinal nematodes, i.e. hookworms ( Ancylostoma sp.), roundworms ( Toxocara sp.) and whipworms ( Trichuris sp.) were evaluated from fecal samples by copromicroscopy using Centrifugal Flotation and McMaster techniques, for identification at genus level per morphological characteristics and calculation of eggs per gram (EPG). The species of roundworm and whipworm were probably T. canis and T. vulpis , but were not identified at molecular level, which is a limitation of this study. Fecal material was not obtained from all the dogs that were included for ectoparasite efficacy evaluation due to lack of feces available from few animals, by the time of inclusion visit. Before treatment, dogs were evaluated for ectoparasites and intestinal nematodes. After treatment, dogs were evaluated for the parasites for which they had been included, i.e. fleas and/or ticks and/or intestinal nematodes. For ectoparasites the post-treatment evaluations were performed on Days 14 (± 4) and 30 (± 4), for intestinal nematodes on Days 14 (± 4). Tolerance evaluations Owners were requested to observe their dog(s) daily and for two hours after each treatment, and report any abnormality to the veterinarian in charge. At the scheduled visits (on Days 14 (± 4) and 30 (± 4) when applicable), a veterinarian performed a physical examination, which together with any adverse reaction reported by owners (resulting or not in an unplanned veterinary consultation, veterinary care, concurrent medication) was considered for an evaluation of the tolerance. Relatedness to treatment for all adverse reactions and abnormalities were evaluated by the Investigator. This involved the 345 dogs included for efficacy assessment and another 21 dogs from multiple dog households that were treated but not assessed for efficacy, as not diagnosed positive for a parasite at inclusion or being a 4th or 5th dog of the household. Statistical analysis No untreated control group was used in this study, each dog served as its own control, the parasite count obtained before treatment at inclusion served as control value. For each dog, the number of live fleas, live attached ticks and the EPG by nematode genus were transformed to the natural logarithm of (count + 1) for analysis and calculation of the geometric means. The percent efficacy was calculated using the formula 100 x [(B - T) / B], where B = geometric mean of the Day 0 (baseline) visit count and T = geometric mean of the appropriate visit day count. The geometric mean was calculated by taking the anti-logarithm of the average of the log-counts and then subtracting 1, or was computed by taking the anti-logarithm of the least square mean minus 1 from the analysis model. The flea and tick efficacies were also calculated with arithmetic means. Normality was first assessed using the D’Agostino–Pearson test, which indicated non-normal distribution of parasite count data; therefore, pre- and post-treatment comparisons were performed using the Wilcoxon signed-rank test for paired samples with statistical significance set at p < 0.05. Results Parasitism at inclusion The parasites detected at inclusion are detailed in Table 1 and the types of (co)-infestations and are described in Table 2 . Table 1 Parasitism per individual dog at inclusion by infestation pattern and state. Infections Brazilian State Total MA (n = 175) MT (n = 170) Single Infections 181 (52.5%) Ancylostoma sp. 14 6 20 Toxocara sp. 1 0 1 Flea 32 47 79 Tick 37 44 81 Dual Infections 100 (29.0%) Ancylostoma sp., Toxocara sp. 10 0 10 Ancylostoma sp., Trichuris sp. 1 0 1 Ancylostoma sp., Toxocara sp., Trichuris sp. 0 2 2 Flea, Ancylostoma sp. 7 8 15 Flea, Lice 2 0 2 Tick, Ancylostoma sp. 8 5 13 Tick, Flea 37 12 49 Tick, Toxocara sp. 0 7 7 Tick, Lice 1 0 1 Multiple infections (≥ 3 parasites) 64 (18.5%) Flea + Intestinal nematodes 5 4 9 Tick + Intestinal nematodes 1 13 14 Flea, Tick + Intestinal nematodes 11 22 33 Flea, Lice, Ancylostoma sp. 1 0 1 Tick, Flea, Lice 7 0 7 MT = Mato Grosso state; MA = Maranhão state.To qualify for inclusion, a dog was infested with at least six live fleas and/or four live ticks and/or was diagnosed infected with intestinal nematode ( Ancylostoma sp., Toxocara sp., Trichuris sp.). It was not possible to evaluate feces from all included animals during inclusion, so the co-infection with helminths by dogs harboring ectoparasites is underestimated Table 2 Overall distribution of parasitism categories at inclusion (n = 345). Ectoparasite n %* 311 90.1 Endoparasite 133 38.6 Ectoparasite only 212 61.4 Endoparasite only 34 9.9 Ecto and endoparasite 99 28.7 Flea 195 56.5 Tick 205 59.4 Flea and tick 40 11.6 * percentage relative to the 345 included dogs. It was not possible to evaluate feces from all included animals during inclusion, so occurrence of intestinal helminths in dogs harboring ectoparasites and co-infection by helminths is underestimated Among the 311 dogs infested with ectoparasites, 66% were infested with ticks and 63% with fleas. All the ticks were determined to be Rhipicephalus linnaei and the fleas Ctenocephalides felis . Among the 133 dogs harboring endoparasites, 110 (83%), 45 (34%) and 21 (19%) were shedding hookworm, roundworm and whipworm eggs, respectively. Interestingly, 11 dogs belonging to nine different households in the Maranhão state were also infested with lice identified as Heterodoxus spiniger. Parasiticide efficacy All the results described below are inclusive of all dogs in both regions and of both treatments for ectoparasites. No regional or treatment effect were observed. For the 204 dogs evaluated for ticks, the efficacy ranged from 96.9% to 100% on Day 14, and from 97.7% to 100% on Day 30. For the 196 dogs evaluated for fleas, the efficacy ranged from 98.4% to 100% on Day 14, and from 98.8% to 100% on Day 30. In Maranhão, Heterodoxus spiniger lice were found on 11 dogs from 9 households. Seven were treated with NexGard and four with NexGard Spectra. The efficacy was 100% on Days 14 and 30. For the 110 dogs were evaluated for hookworm the efficacy was ≥ 97.8%, for the 44 dogs evaluated for roundworm the efficacy was ≥ 94.7%, and for the 23 dogs evaluated for whipworm, the efficacy was ≥ 97.7%. All efficacy evaluations were significant (P < 0.001). Safety There was no adverse event related to treatment reported by the owners, post-treatment or during the study. No significant abnormality deemed related to treatment was observed during this study. Discussion This study allowed the confirmation that, in two regions in Brazil and in field conditions, NexGard® and NexGard Spectra® were efficacious against C. felis and R. linnaei natural infestations, and that NexGard Spectra® is also highly effective against Ancylostoma, Toxocara , and Trichuris infestations. A high efficacy of both products against the chewing louse H. spiniger , with a total elimination of the parasite two weeks after the treatment, was also demonstrated and confirms a previous report of efficacy of afoxolaner in Europe [ 29 ]. All the parasites diagnosed and eliminated in this study have a significant veterinary importance. The ectoparasites, have a direct pathogenic effect on their host with a level of severity correlated to their number, duration of infestation and inherent host conditions (e.g. age, immune status). Ticks, fleas and lice may provoke mild to severe inflammatory cutaneous reactions in relation to their bite wounds that may also cause allergic reactions, such as the Flea Allergic Dermatitis, often leading to pruritus-related damages and secondary infections [ 4 , 41 ]. Systemic effects such as anemia may occur because of spoliation caused by the hematophagous actions of fleas and ticks in high number. Importantly ectoparasites have a significant indirect effect on their vertebrate host, through their ability of vector borne disease (VBD) transmission. This is especially true for ticks but also a reality with fleas and lice [ 6 , 42 ]. Rhipicehalus linnaei previously identified as Rhipicephalus sanguineus s.l. is a recognized vector of the protozoan Babesia vogeli , the bacteria Rickettsia rickettsii (an agent of spotted fever), Anaplasma platys, Ehrlichia canis (the agent of the canine monocytic ehrichiosis), Mycoplasma haemocanis [ 1 , 43 ]. Ctenocephalides felis and H. spiniger namely carry and transmit Rickettsia, Bartonella, Dipylidium [ 44 , 45 ]. Identically, the intestinal nematodes identified in this study provoke pathogenic effects by spoliation of nutrients (roundworms and whipworms) and hematophagous action (hookworms), enteritis through irritation or damage of intestinal mucosa, and very importantly tissue damage associated to their migratory cycle (hookworms and roundworms), namely in the liver or in lungs [ 7 , 8 ]. Several of these vector-borne agents have a high public health significance, namely R. rickettsii Bartonella, D. caninum. Ancylostoma have a high zoonotic potential through cutaneous larva migrans properties and Toxocara have a severe zoonotic potential namely through visceral, cerebellar or ocular tissue larva migrans properties [ 9 , 46 ]. It is thus important in a One-Health perspective to control ecto- and endoparasites, both for veterinary and public health purposes [ 47 ]. The parasiticide effect of afoxolaner, a systemic molecule, requires a blood meal from the arthropod vector. Thus, the direct effect of afoxolaner for blocking of transmission is related to the speed of transmission of the VBD agent. Afoxolaner has been demonstrated efficacious for the prevention of slowly transmitted VBD agents such as Borrelia , and Babesia [ 48 – 51 ]. The efficacy of afoxolaner to block agents transmitted more quickly (e.g. Anaplasma, Rickettsia, Ehrlichia ) is unclear, nevertheless the hypothesis that its use in endemic areas may decrease the local biomass of these VBD agents, through a decrease of their vectors is sound and supports a beneficial effect for a decrease of these VBD presence and transmission. Conclusions This study demonstrated a significant level of parasitism in domestic dogs in two regions of Brazil, and a high efficacy of afoxolaner formulated alone or combined with milbemycin oxime, against ectoparasites (R. linnaei, C. felis and H. spiniger). Comparably, a high efficacy of milbemycin oxime against the intestinal hookworms, roundworm and whipworms was demonstrated. The veterinary and public health importance of these parasites [ 47 , 52 ] advocate consistent treatment of the domestic canine population with these products. Declarations Ethics approval and consent to participate All dog owners were duly informed about study protocol before inclusion, agreeing by writing with all procedures performed after signing the informed consent. The study was approved by Universidade Federal de Mato Grosso (23108.954157/2018-91) and Universidade Federal do Maranhao Institutional (CIAEP: 01.0341.2014) Animal Care and Use Committees (IACUC) with the concurrence of Boehringer Ingelheim. Consent for publication All authors have reviewed this document and consent to publish the data. Competing interest The work reported herein was funded by Boehringer-Ingelheim. AC, LP, ET are current employees or contractors of Boehringer-Ingelheim. 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). Competing Interests A.C, L.P and E.T. are employees of Boehringer Ingelheim, manufacturerof the investigated products (NexGard® and NexGard Spectra®) and sponsor of thisstudy. Funding The work reported herein was funded by Boehringer Ingelheim Animal Health GA, USA. Availability of data and materials All relevant data are provided within the paper. Author Contribution Andre A. Cutolo: study conceptualization, study protocol, study monitoring, funding, writing and language editing, general supervisionLuis. G. R. Pelissoni: study protocol, funding, writing and language editingTássia L. Vale: data gathering Livio M. Costa-Junior: investigation, validation, data analysis, writing and language editingIsabella C. Sousa: data gatheringNaylene C. S. Silva: data gatheringCristiano Grisi do Nascimento: study coordinationMaycon Junior Heidmann: data gatheringBruno Gomes de Castro: investigation, validation, data analysis, writing and language editingEric Tielemans: Manuscript drafting Acknowledgments We gratefully acknowledge the National Institute of Science and Technology in Innovation in Animal Parasitology (INCT-IPA) sponsored by Brazil’s National Council for Scientific and Technological Development (CNPq), grant no. 408812/2024-9. To all R&D Boehringer Ingelheim Animal Health Brazil staff for the support on the logistics and study data review. Data Availability All relevant data are provided within the paper. References Araes-Santos AI, Moraes-Filho J, Peixoto RM, Spolidorio MG, Azevedo SS, Costa MM, Labruna MB, Horta MC. 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Parasit Vectors. 2016;9(1):635. doi: 10.1186/s13071-016-1924-4 . PMID: 27938395; PMCID: PMC5148825. Lebon W, Beccati M, Bourdeau P, Brement T, Bruet V, Cekiera A, Crosaz O, Darmon C, Guillot J, Mosca M, Pin D, Popiel J, Pomorska Handwerker D, Larsen D, Tielemans E, Beugnet F, Halos L. Efficacy of two formulations of afoxolaner (NexGard® and NexGard Spectra®) for the treatment of generalised demodicosis in dogs, in veterinary dermatology referral centers in Europe. Parasit Vectors. 2018;11(1):506. doi: 10.1186/s13071-018-3083-2 . PMID: 30201031; PMCID: PMC6131853. Cutolo AA, Perier N, Menz I, Thyssen P, Silva FO, Beugnet F. Efficacy of afoxolaner (NexGard®) on the treatment of myiasis caused by the New World screwworm fly Cochliomyia hominivorax (Diptera: Calliphoridae) in naturally infested dogs. Vet Parasitol Reg Stud Reports. 2021;24:100569. doi: 10.1016/j.vprsr.2021.100569. Epub 2021 Apr 3. PMID: 34024385. Tielemans E, Aouiche N, Saunders A, Besselaar JF, Beugnet F. Insecticidal efficacy of afoxolaner against Stomoxys calcitrans (Diptera: Muscidae) in dogs. Curr Res Parasitol Vector Borne Dis. 2021;1:100043. doi: 10.1016/j.crpvbd.2021.100043 . PMID: 35284852; PMCID: PMC8906105. Mihalca AD, Deak G, Panait LC, Rabei Ș, Beugnet F. Efficacy of afoxolaner (NexGard®) against natural infestations with Trichodectes canis in dogs under field conditions. Parasit Vectors. 2022;15(1):317. doi: 10.1186/s13071-022-05428-y . PMID: 36071527; PMCID: PMC9450229. Mihalca AD, Lolokote S, Moise A, Modrý D, Beugnet F. Efficacy of oral afoxolaner (NexGard®) against natural infestations with Heterodoxus spiniger (Phthiraptera: Boopiidae) in dogs under field conditions. Vet Parasitol Reg Stud Reports. 2025;63:101316. doi: 10.1016/j.vprsr.2025.101316 . Epub 2025 Jul 11. PMID: 40803800. Fankhauser R, Hamel D, Dorr P, Reinemeyer CR, Crafford D, Bowman DD, Ulrich M, Yoon S, Larsen DL. Efficacy of oral afoxolaner plus milbemycin oxime chewables against induced gastrointestinal nematode infections in dogs. Vet Parasitol. 2016;225:117 – 22. doi: 10.1016/j.vetpar.2016.06.003. Epub 2016 Jun 3. PMID: 27369586. Rehbein S, Knaus M, Mallouk Y, Breiltgens T, Brianti E, Capári B, Dantas-Torres F, Gau M, Joachim A, Kaulfuß KH, Kirkova Z, Lechner J, Mihalca AD, Mirabito R, Petkevičius S, Rapti D, Shukullari E, Sedeilhan M, Dollhofer D, Kley K, Lebon W, Visser M, Jeannin P. Efficacy against nematode infections and safety of afoxolaner plus milbemycin oxime chewable tablets in domestic dogs under field conditions in Europe. Parasitol Res. 2017;116(1):259–269. doi: 10.1007/s00436-016-5287-8 . Epub 2016 Oct 22. PMID: 27771803. Tielemans E, Lebon W, Dumont P, Taweethavonsawat P, Larsen D, Rehbein S. Efficacy of afoxolaner plus milbemycin oxime chewable tablets (NexGard Spectra®, Merial) against adult Ancylostoma ceylanicum hookworm, in dogs. Vet Parasitol. 2017;238:87–89. doi: 10.1016/j.vetpar.2017.03.028. Epub 2017 Apr 1. PMID: 28408213. Alvares CA, Stape JL, Sentelhas PC, Gonçalves JL de M, Sparovek G. Köppen's climate classification map for Brazil [Internet]. Meteorologische Zeitschrift. 2013; 22(6): 711–728. 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. Jacobs DE, Arakawa A, Courtney CH, Gemmell MA, McCall JW, Myers GH, Vanparijs O. 1994. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of anthelmintics for dogs and cats. Veterinary Parasitology 52, 179–202. Linardi PM, Guimarães LR. Siphonápteros do Brasil. São Paulo: Museu de Zoologia da Universidade de São Paulo; 2000. Aragão HB, Fonseca F. Notas de Ixodologia. VIII. Lista e chave para os representantes da fauna ixodológica brasileira. Mem Inst Oswaldo Cruz. 1961;59(2):115–129. Martins TF, Onofrio VC, Labruna MB, Guglielmone AA, Barros-Battesti DM. Família Ixodidae da região neotropical: características gerais, distribuição geográfica, hospedeiros e chaves de identificação das espécies que ocorrem no Brasil. In: Barros-Battesti DM, Machado RZ, André MR, editors. Ectoparasitofauna brasileira de importância veterinária: acarofauna de importância veterinária: Parasitiformes – Ixodida, Parte I. Jaboticabal: CBPV; 2024. p. 537. Werneck FL. Contribuição ao conhecimento dos Mallophagos encontrados nos mamíferos sul-americanos. Mem Inst Oswaldo Cruz. 1936;31(3):391–589. Werneck FL. De um estranho parasito do cão (Insecta, Mallophaga). Rev Bras Biol. 1941;1:47–55. Dryden MW, Canfield MS, Kalosy K, Smith A, Crevoiserat L, McGrady JC, Foley KM, Green K, Tebaldi C, Smith V, Bennett T, Heaney K, Math L, Royal C, Sun F. Evaluation of fluralaner and afoxolaner treatments to control flea populations, reduce pruritus and minimize dermatologic lesions in naturally infested dogs in private residences in west central Florida USA. Parasit Vectors. 2016;9(1):365. doi: 10.1186/s13071-016-1654-7 . PMID: 27352607; PMCID: PMC4924230. Dantas-Torres F. Canine vector-borne diseases in Brazil. Parasit Vectors. 2008;1(1):25. doi: 10.1186/1756-3305-1-25 . PMID: 18691408; PMCID: PMC2533296. Dantas-Torres F, de Sousa-Paula LC, Otranto D. The Rhipicephalus sanguineus group: updated list of species, geographical distribution, and vector competence. Parasit Vectors. 2024;17(1):540. doi: 10.1186/s13071-024-06572-3 . PMID: 39731169; PMCID: PMC11681662. Moore CO, André MR, Šlapeta J, Breitschwerdt EB. Vector biology of the cat flea Ctenocephalides felis. Trends Parasitol. 2024;40(4):324–337. doi: 10.1016/j.pt.2024.02.006 . Epub 2024 Mar 7. PMID: 38458883; PMCID: PMC11168582. Wang J, Gao L, Aksoy S. Microbiota in disease-transmitting vectors. Nat Rev Microbiol. 2023;21(9):604–618. doi: 10.1038/s41579-023-00901-6 . Epub 2023 May 22. PMID: 37217793; PMCID: PMC12397960. Traversa D, Frangipane di Regalbono A, Di Cesare A, La Torre F, Drake J, Pietrobelli M. Environmental contamination by canine geohelminths. Parasit Vectors. 2014;7:67. doi: 10.1186/1756-3305-7-67 . PMID: 24524656; PMCID: PMC3929561. Traversa D. 2012. Pet roundworms and hookworms: a continuing need for global worming. Parasites & Vectors, 5, 91–110. Baker CF, McCall JW, McCall SD, Drag MD, Mitchell EB, Chester ST, Larsen D. Ability of an oral formulation of afoxolaner to protect dogs from Borrelia burgdorferi infection transmitted by wild Ixodes scapularis ticks. Comp Immunol Microbiol Infect Dis. 2016;49:65–69. doi: 10.1016/j.cimid.2016.09.004. Epub 2016 Sep 29. PMID: 27865266. Beugnet F, Halos L, Larsen D, Labuschagné M, Erasmus H, Fourie J. The ability of an oral formulation of afoxolaner to block the transmission of Babesia canis by Dermacentor reticulatus ticks to dogs. Parasit Vectors. 2014;7:283. doi: 10.1186/1756-3305-7-283 . PMID: 24957215; PMCID: PMC4078974. Beugnet F, Lebon W, de Vos C. Prevention of the transmission of Babesia rossi by Haemaphysalis elliptica in dogs treated with Nexgard®. Parasite. 2019;26:49. doi: 10.1051/parasite/2019051 . Epub 2019 Aug 21. PMID: 31432778; PMCID: PMC6702822. Tielemans E, Rautenbach C, Viljoen A, Beugnet F. Efficacy of an oral combination of afoxolaner and milbemycin oxime for the prevention of transmission of Babesia canis by Dermacentor reticulatus ticks to dogs. Parasit Vectors. 2025;18(1):142. doi: 10.1186/s13071-025-06787-y . PMID: 40234985; PMCID: PMC12001589. Dantas-Torres F, Otranto D. 2014. Dogs, cats, parasites, and humans in Brazil: opening the black box. Parasites and Vectors, 7:22. Additional Declarations Competing interest reported. A.C, L.P and E.T. are employees of Boehringer Ingelheim, manufacturer of the investigated products (NexGard® and NexGard Spectra®) and sponsor of this study. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 13 May, 2026 Reviewers agreed at journal 11 May, 2026 Reviewers agreed at journal 09 May, 2026 Reviewers invited by journal 07 May, 2026 Submission checks completed at journal 07 May, 2026 First submitted to journal 30 Apr, 2026 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9294152","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":639970502,"identity":"f44ce859-3e7a-4945-a748-b372713bcaa2","order_by":0,"name":"Andre A. 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R.","lastName":"Pelissoni","suffix":""},{"id":639970504,"identity":"d3ba01dc-ec80-4d26-a7fa-9f105e955d5c","order_by":2,"name":"Tassia Lopes Vale","email":"","orcid":"","institution":"Federal University of Maranhão (UFMA)","correspondingAuthor":false,"prefix":"","firstName":"Tassia","middleName":"Lopes","lastName":"Vale","suffix":""},{"id":639970505,"identity":"599608dd-1718-4b78-bc09-710c9df50081","order_by":3,"name":"Livio Martins Costa-Junior","email":"","orcid":"","institution":"Federal University of Maranhão (UFMA)","correspondingAuthor":false,"prefix":"","firstName":"Livio","middleName":"Martins","lastName":"Costa-Junior","suffix":""},{"id":639970506,"identity":"2312994c-6c6c-412f-bd96-10fe33090893","order_by":4,"name":"Isabella Chaves Sousa","email":"","orcid":"","institution":"Federal University of Maranhão (UFMA)","correspondingAuthor":false,"prefix":"","firstName":"Isabella","middleName":"Chaves","lastName":"Sousa","suffix":""},{"id":639970507,"identity":"a1690011-a5da-4eda-99a7-fabb99f4813a","order_by":5,"name":"Naylene Carvalho Sales Silva","email":"","orcid":"","institution":"Federal University of Maranhão (UFMA)","correspondingAuthor":false,"prefix":"","firstName":"Naylene","middleName":"Carvalho Sales","lastName":"Silva","suffix":""},{"id":639970508,"identity":"c9202e75-ac0f-4271-bd2a-a50d22d7a06a","order_by":6,"name":"Cristiano Grisi do Nascimento","email":"","orcid":"","institution":"Convolution Divisão de Antiparasitários","correspondingAuthor":false,"prefix":"","firstName":"Cristiano","middleName":"Grisi do","lastName":"Nascimento","suffix":""},{"id":639970509,"identity":"9b096299-a66a-4960-a064-b324dd6cfe66","order_by":7,"name":"Maycon Junior Heidmann","email":"","orcid":"","institution":"Universidade Federal de Mato Grosso","correspondingAuthor":false,"prefix":"","firstName":"Maycon","middleName":"Junior","lastName":"Heidmann","suffix":""},{"id":639970511,"identity":"1d80337e-591e-42a9-9b33-58067695d466","order_by":8,"name":"Bruno Gomes de Castro","email":"","orcid":"","institution":"Universidade Federal de Mato Grosso","correspondingAuthor":false,"prefix":"","firstName":"Bruno","middleName":"Gomes","lastName":"de Castro","suffix":""},{"id":639970516,"identity":"5250580a-95de-47e7-be66-1fb48e73cc9f","order_by":9,"name":"Eric TIELEMANS","email":"data:image/png;base64,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","orcid":"","institution":"Boehringer Ingelheim (France)","correspondingAuthor":true,"prefix":"","firstName":"Eric","middleName":"","lastName":"TIELEMANS","suffix":""}],"badges":[],"createdAt":"2026-04-01 15:55:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9294152/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9294152/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109760022,"identity":"929f5433-c24f-4eaf-b3f3-67f10d241ce1","added_by":"auto","created_at":"2026-05-22 07:28:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":270384,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9294152/v1/28e2d27f-b724-4f49-bb4c-cc03c3822f20.pdf"}],"financialInterests":"Competing interest reported. A.C, L.P and E.T. are employees of Boehringer Ingelheim, manufacturer\nof the investigated products (NexGard® and NexGard Spectra®) and sponsor of this\nstudy.","formattedTitle":"Efficacy of afoxolaner or a combination of afoxolaner and milbemycin oxime against fleas, ticks, lice and/or intestinal nematodes in dogs naturally infested in Brazil","fulltext":[{"header":"Background","content":"\u003cp\u003eFleas, ticks, lice and intestinal nematodes are commonly identified in domestic dogs and have a significant veterinary and public health importance [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These parasites can have a direct pathogenic effect on the host, associated to the immune reactions caused by their presence [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], their life cycle, and their feeding on host tissue [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Many ectoparasites have an indirect pathogenic effect, being vector of filarial nematode, protozoan, bacterial and viral diseases. Importantly, several canine parasites and several vector-borne agents are zoonotic [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfoxolaner is an insecticide and acaricide isoxazoline compound formulated as an oral chewable ectoparasiticide tablet for dogs (NexGard\u0026reg;, Boehringer Ingelheim). Milbemycin oxime (MO) is a macrocyclic lactone nematicide compound formulated in combination with afoxolaner as an oral chewable endectoparasiticide tablet for dogs (NexGard Spectra\u0026reg;, Boehringer Ingelheim). Isoxazolines antagonize the GABA-gated chloride ion channels [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], while macrocyclic lactones, mainly agonize the Glutamate-gated chloride ion channel [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] of the parasites. Afoxolaner, alone or in combination with MO has been reported efficacious several times against fleas [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], ticks [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], mites [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], flies [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and lice [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], in laboratory or in field conditions. Milbemycin oxime, in combination with afoxolaner has also been reported efficacious several times against intestinal nematodes [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis manuscript describes a field study conducted in two cities in different Brazilian states, Mato Grosso and Maranh\u0026atilde;o, located respectively in the Center-West and Northeast administrative regions of Brazil, for the investigation of the efficacy and tolerance of NexGard and NexGard Spectra in dogs naturally infested with fleas, ticks and/or intestinal helminths. The objectives of this study were to verify the absence of local effects on sensitivity to the parasiticide molecules in the field and to evaluate the parasitism of domestic dogs in these regions.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis field trial was conducted during the period October 2018 - February 2019, in urban areas, in two states in Brazil, Mato Grosso (Sinop and Ipiranga do Norte cities) and Maranh\u0026atilde;o (metropolitan region of S\u0026atilde;o Lu\u0026iacute;s). Although both S\u0026atilde;o Lu\u0026iacute;s and Sinop have tropical climates with high temperatures throughout the year, their environmental conditions differ markedly due to their geographic separation (~\u0026thinsp;1,600 km) and distinct climatic influences. S\u0026atilde;o Lu\u0026iacute;s experiences a humid tropical coastal climate characterized by high relative humidity and a short dry winter (Aw, K\u0026ouml;ppen classification), whereas Sinop, located inland in the Amazon\u0026ndash;Cerrado transition zone, presents a tropical monsoon climate influenced by the South American monsoon system, with a pronounced dry season (Am) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThis study was conducted in accordance with the relevant WAAVP (World Association for the Advancement of Veterinary Parasitology) guidelines for the evaluation of efficacy of parasiticides on dogs and cats against fleas and ticks [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and anthelmintics [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]; the relevant VICH (Veterinary Internation Conference in Harmonization) International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products guidelines: VICH GL7, \u0026ldquo;Efficacy of Anthelmintics: General Requirements\u0026rdquo;, VICH GL19 \u0026ldquo;Efficacy of Anthelmintics: Specific Recommendations for Canines\u0026rdquo;, and VICH GL9 \u0026ldquo;Good Clinical Practices\u0026rdquo;.\u003c/p\u003e \u003cp\u003eThis study was designed to test the efficacy of NexGard\u0026reg; and NexGard Spectra\u0026reg; against natural infestations with fleas, ticks and intestinal nematodes (NexGard Spectra\u0026reg; only) in the field in two regions of Brazil, and to confirm their safety.\u003c/p\u003e \u003cp\u003eThe study was fully conducted in households. The households were recruited on voluntary basis, without other veterinary-related purposes. To qualify for the study, a dog was naturally infested with fleas, ticks or intestinal nematode. A maximum of three dogs per household were included for efficacy assessment, and a maximum of five dogs per household were acceptable (households with dogs and cats were excluded). The same treatment was administered to all dogs in a household. Dogs infected with intestinal nematodes were assigned to the NexGard Spectra\u0026reg; group. Dogs only infested with fleas and/or ticks were assigned to the NexGard\u0026reg; group (except for dogs within a household of a dog assigned to NexGard Spectra\u0026reg;, in which case treated with the same treatment). In a household, dogs that were not included for efficacy evaluation received the same treatment and were evaluated for safety. No masking procedure was implemented during this study.\u003c/p\u003e \u003cp\u003eFollowing treatment, dogs were assessed for ectoparasites and/or intestinal nematodes based on their diagnosed parasitism at inclusion day.\u003c/p\u003e \u003cp\u003eNo parasiticide compound with remanent activity within the three previous months of inclusion had been used on dogs or in their environment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eThree-hundred and forty-five (345) domestic dogs (177 males, 168 females) of various age (2 months to 15 years), bodyweight (2 to 46 kg) and breed (85% mongrels) were included for efficacy assessment. These dogs belonged to 84 households in Mato Grosso, and 130 households in Maranh\u0026atilde;o. All dogs were infested with at least six live fleas and/or four live ticks and/or were infected with intestinal nematode (hookworm, roundworm, whipworm) through identification of eggs in feces. Dogs were maintained at home during the study in their usual routine, environmental and husbandry conditions.\u003c/p\u003e\n\u003ch3\u003eTreatment\u003c/h3\u003e\n\u003cp\u003eOn Day 0, dogs were treated with NexGard\u0026reg; (when they had been included on the basis of ectoparasite infestation, only) or with NexGard Spectra\u0026reg; (when they or any other dog from their household had been included on the basis of endoparasite infestation, namely). The products were administered per label recommendations (orally and with a tablet strength corresponding to their bodyweight, delivering 2.5 to 7.1 mg/kg afoxolaner and 0.5 to 1.1 mg/kg milbemycin oxime in the combined product). In Mato Grosso, 86 and 84 dogs and in Maranh\u0026atilde;o, 63 and 112 dogs were treated with NexGard\u0026reg; and NexGard Spectra\u0026reg;, respectively (exclusive of the 21 dogs that were not evaluated for efficacy).\u003c/p\u003e\n\u003ch3\u003eParasite evaluations\u003c/h3\u003e\n\u003cp\u003eLive flea counts were performed by combing all parts of the animal\u0026rsquo;s haircoat using a fine-tooth comb (11 to 13 teeth per centimeter) for at least 5 minutes after the encounter of the last ectoparasite on the animal\u0026rsquo;s skin.\u003c/p\u003e \u003cp\u003eLive ticks found during the flea combing process or haircoat search were counted and removed manually with fingertips or forceps. The interdigital and pad areas, internal face of ears, pubic, perineal and axillary areas were individually checked.\u003c/p\u003e \u003cp\u003eSamples of removed ectoparasites for each dog were collected and kept stored in ethanol 70% solution for species identification based on morphological characteristics. Fleas and ticks were examined under stereomicroscope and identified using dichotomous keys according to Linardi and Guimar\u0026atilde;es [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], as well as taxonomic references described by Arag\u0026atilde;o and Fonseca [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] subsequently updated by Martins et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Lice specimens were mounted on glass slides using a drop of lactophenol solution and identified following the criteria established by Werneck [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIntestinal nematodes, i.e. hookworms (\u003cem\u003eAncylostoma\u003c/em\u003e sp.), roundworms (\u003cem\u003eToxocara\u003c/em\u003e sp.) and whipworms (\u003cem\u003eTrichuris\u003c/em\u003e sp.) were evaluated from fecal samples by copromicroscopy using Centrifugal Flotation and McMaster techniques, for identification at genus level per morphological characteristics and calculation of eggs per gram (EPG). The species of roundworm and whipworm were probably \u003cem\u003eT. canis\u003c/em\u003e and \u003cem\u003eT. vulpis\u003c/em\u003e, but were not identified at molecular level, which is a limitation of this study. Fecal material was not obtained from all the dogs that were included for ectoparasite efficacy evaluation due to lack of feces available from few animals, by the time of inclusion visit.\u003c/p\u003e \u003cp\u003eBefore treatment, dogs were evaluated for ectoparasites and intestinal nematodes. After treatment, dogs were evaluated for the parasites for which they had been included, i.e. fleas and/or ticks and/or intestinal nematodes. For ectoparasites the post-treatment evaluations were performed on Days 14 (\u0026plusmn;\u0026thinsp;4) and 30 (\u0026plusmn;\u0026thinsp;4), for intestinal nematodes on Days 14 (\u0026plusmn;\u0026thinsp;4).\u003c/p\u003e\n\u003ch3\u003eTolerance evaluations\u003c/h3\u003e\n\u003cp\u003eOwners were requested to observe their dog(s) daily and for two hours after each treatment, and report any abnormality to the veterinarian in charge. At the scheduled visits (on Days 14 (\u0026plusmn;\u0026thinsp;4) and 30 (\u0026plusmn;\u0026thinsp;4) when applicable), a veterinarian performed a physical examination, which together with any adverse reaction reported by owners (resulting or not in an unplanned veterinary consultation, veterinary care, concurrent medication) was considered for an evaluation of the tolerance. Relatedness to treatment for all adverse reactions and abnormalities were evaluated by the Investigator. This involved the 345 dogs included for efficacy assessment and another 21 dogs from multiple dog households that were treated but not assessed for efficacy, as not diagnosed positive for a parasite at inclusion or being a 4th or 5th dog of the household.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003e No untreated control group was used in this study, each dog served as its own control, the parasite count obtained before treatment at inclusion served as control value. For each dog, the number of live fleas, live attached ticks and the EPG by nematode genus were transformed to the natural logarithm of (count\u0026thinsp;+\u0026thinsp;1) for analysis and calculation of the geometric means. The percent efficacy was calculated using the formula 100 x [(B - T) / B], where B\u0026thinsp;=\u0026thinsp;geometric mean of the Day 0 (baseline) visit count and T\u0026thinsp;=\u0026thinsp;geometric mean of the appropriate visit day count. The geometric mean was calculated by taking the anti-logarithm of the average of the log-counts and then subtracting 1, or was computed by taking the anti-logarithm of the least square mean minus 1 from the analysis model. The flea and tick efficacies were also calculated with arithmetic means.\u003c/p\u003e \u003cp\u003eNormality was first assessed using the D\u0026rsquo;Agostino\u0026ndash;Pearson test, which indicated non-normal distribution of parasite count data; therefore, pre- and post-treatment comparisons were performed using the Wilcoxon signed-rank test for paired samples with statistical significance set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eParasitism at inclusion\u003c/h2\u003e \u003cp\u003eThe parasites detected at inclusion are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and the types of (co)-infestations and are described in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParasitism per individual dog at inclusion by infestation pattern and state.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInfections\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eBrazilian State\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA (n\u0026thinsp;=\u0026thinsp;175)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMT (n\u0026thinsp;=\u0026thinsp;170)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eSingle Infections\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e181 (52.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAncylostoma\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToxocara\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eDual Infections\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100 (29.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAncylostoma\u003c/em\u003e sp., \u003cem\u003eToxocara\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAncylostoma\u003c/em\u003e sp., \u003cem\u003eTrichuris\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAncylostoma\u003c/em\u003e sp., \u003cem\u003eToxocara\u003c/em\u003e sp., \u003cem\u003eTrichuris\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlea, \u003cem\u003eAncylostoma\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlea, Lice\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTick, \u003cem\u003eAncylostoma\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTick, Flea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTick, \u003cem\u003eToxocara\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTick, Lice\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eMultiple infections (\u0026ge;\u0026thinsp;3 parasites)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64 (18.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlea\u0026thinsp;+\u0026thinsp;Intestinal nematodes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTick\u0026thinsp;+\u0026thinsp;Intestinal nematodes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlea, Tick\u0026thinsp;+\u0026thinsp;Intestinal nematodes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlea, Lice, \u003cem\u003eAncylostoma\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTick, Flea, Lice\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eMT\u0026thinsp;=\u0026thinsp;Mato Grosso state; MA\u0026thinsp;=\u0026thinsp;Maranh\u0026atilde;o state.To qualify for inclusion, a dog was infested with at least six live fleas and/or four live ticks and/or was diagnosed infected with intestinal nematode (\u003cem\u003eAncylostoma\u003c/em\u003e sp., \u003cem\u003eToxocara\u003c/em\u003e sp., \u003cem\u003eTrichuris\u003c/em\u003e sp.). It was not possible to evaluate feces from all included animals during inclusion, so the co-infection with helminths by dogs harboring ectoparasites is underestimated\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOverall distribution of parasitism categories at inclusion (n\u0026thinsp;=\u0026thinsp;345).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEctoparasite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e311\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90.1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndoparasite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEctoparasite only\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e61.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndoparasite only\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEcto and endoparasite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e195\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e59.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlea and tick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e* percentage relative to the 345 included dogs. It was not possible to evaluate feces from all included animals during inclusion, so occurrence of intestinal helminths in dogs harboring ectoparasites and co-infection by helminths is underestimated\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAmong the 311 dogs infested with ectoparasites, 66% were infested with ticks and 63% with fleas. All the ticks were determined to be \u003cem\u003eRhipicephalus linnaei\u003c/em\u003e and the fleas \u003cem\u003eCtenocephalides felis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAmong the 133 dogs harboring endoparasites, 110 (83%), 45 (34%) and 21 (19%) were shedding hookworm, roundworm and whipworm eggs, respectively.\u003c/p\u003e \u003cp\u003eInterestingly, 11 dogs belonging to nine different households in the Maranh\u0026atilde;o state were also infested with lice identified as \u003cem\u003eHeterodoxus spiniger.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eParasiticide efficacy\u003c/h2\u003e \u003cp\u003eAll the results described below are inclusive of all dogs in both regions and of both treatments for ectoparasites. No regional or treatment effect were observed.\u003c/p\u003e \u003cp\u003eFor the 204 dogs evaluated for ticks, the efficacy ranged from 96.9% to 100% on Day 14, and from 97.7% to 100% on Day 30.\u003c/p\u003e \u003cp\u003eFor the 196 dogs evaluated for fleas, the efficacy ranged from 98.4% to 100% on Day 14, and from 98.8% to 100% on Day 30.\u003c/p\u003e \u003cp\u003eIn Maranh\u0026atilde;o, \u003cem\u003eHeterodoxus spiniger\u003c/em\u003e lice were found on 11 dogs from 9 households. Seven were treated with NexGard and four with NexGard Spectra. The efficacy was 100% on Days 14 and 30.\u003c/p\u003e \u003cp\u003eFor the 110 dogs were evaluated for \u003cem\u003ehookworm\u003c/em\u003e the efficacy was \u0026ge;\u0026thinsp;97.8%, for the 44 dogs evaluated for roundworm the efficacy was \u0026ge;\u0026thinsp;94.7%, and for the 23 dogs evaluated for whipworm, the efficacy was \u0026ge;\u0026thinsp;97.7%.\u003c/p\u003e \u003cp\u003eAll efficacy evaluations were significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSafety\u003c/h2\u003e \u003cp\u003eThere was no adverse event related to treatment reported by the owners, post-treatment or during the study. No significant abnormality deemed related to treatment was observed during this study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study allowed the confirmation that, in two regions in Brazil and in field conditions, NexGard\u0026reg; and NexGard Spectra\u0026reg; were efficacious against \u003cem\u003eC. felis\u003c/em\u003e and \u003cem\u003eR. linnaei\u003c/em\u003e natural infestations, and that NexGard Spectra\u0026reg; is also highly effective against \u003cem\u003eAncylostoma, Toxocara\u003c/em\u003e, and \u003cem\u003eTrichuris\u003c/em\u003e infestations. A high efficacy of both products against the chewing louse \u003cem\u003eH. spiniger\u003c/em\u003e, with a total elimination of the parasite two weeks after the treatment, was also demonstrated and confirms a previous report of efficacy of afoxolaner in Europe [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll the parasites diagnosed and eliminated in this study have a significant veterinary importance. The ectoparasites, have a direct pathogenic effect on their host with a level of severity correlated to their number, duration of infestation and inherent host conditions (e.g. age, immune status). Ticks, fleas and lice may provoke mild to severe inflammatory cutaneous reactions in relation to their bite wounds that may also cause allergic reactions, such as the Flea Allergic Dermatitis, often leading to pruritus-related damages and secondary infections [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Systemic effects such as anemia may occur because of spoliation caused by the hematophagous actions of fleas and ticks in high number. Importantly ectoparasites have a significant indirect effect on their vertebrate host, through their ability of vector borne disease (VBD) transmission. This is especially true for ticks but also a reality with fleas and lice [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. \u003cem\u003eRhipicehalus linnaei\u003c/em\u003e previously identified as \u003cem\u003eRhipicephalus sanguineus\u003c/em\u003e s.l. is a recognized vector of the protozoan \u003cem\u003eBabesia vogeli\u003c/em\u003e, the bacteria \u003cem\u003eRickettsia rickettsii\u003c/em\u003e (an agent of spotted fever), \u003cem\u003eAnaplasma platys, Ehrlichia canis\u003c/em\u003e (the agent of the canine monocytic ehrichiosis), \u003cem\u003eMycoplasma haemocanis\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. \u003cem\u003eCtenocephalides felis\u003c/em\u003e and \u003cem\u003eH. spiniger\u003c/em\u003e namely carry and transmit \u003cem\u003eRickettsia, Bartonella, Dipylidium\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Identically, the intestinal nematodes identified in this study provoke pathogenic effects by spoliation of nutrients (roundworms and whipworms) and hematophagous action (hookworms), enteritis through irritation or damage of intestinal mucosa, and very importantly tissue damage associated to their migratory cycle (hookworms and roundworms), namely in the liver or in lungs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral of these vector-borne agents have a high public health significance, namely \u003cem\u003eR. rickettsii Bartonella, D. caninum. Ancylostoma\u003c/em\u003e have a high zoonotic potential through cutaneous \u003cem\u003elarva migrans\u003c/em\u003e properties and \u003cem\u003eToxocara\u003c/em\u003e have a severe zoonotic potential namely through visceral, cerebellar or ocular tissue \u003cem\u003elarva migrans\u003c/em\u003e properties [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is thus important in a One-Health perspective to control ecto- and endoparasites, both for veterinary and public health purposes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The parasiticide effect of afoxolaner, a systemic molecule, requires a blood meal from the arthropod vector. Thus, the direct effect of afoxolaner for blocking of transmission is related to the speed of transmission of the VBD agent. Afoxolaner has been demonstrated efficacious for the prevention of slowly transmitted VBD agents such as \u003cem\u003eBorrelia\u003c/em\u003e, and \u003cem\u003eBabesia\u003c/em\u003e [\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The efficacy of afoxolaner to block agents transmitted more quickly (e.g. \u003cem\u003eAnaplasma, Rickettsia, Ehrlichia\u003c/em\u003e) is unclear, nevertheless the hypothesis that its use in endemic areas may decrease the local biomass of these VBD agents, through a decrease of their vectors is sound and supports a beneficial effect for a decrease of these VBD presence and transmission.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrated a significant level of parasitism in domestic dogs in two regions of Brazil, and a high efficacy of afoxolaner formulated alone or combined with milbemycin oxime, against ectoparasites \u003cem\u003e(R. linnaei, C. felis\u003c/em\u003e and \u003cem\u003eH. spiniger).\u003c/em\u003e Comparably, a high efficacy of milbemycin oxime against the intestinal hookworms, roundworm and whipworms was demonstrated. The veterinary and public health importance of these parasites [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] advocate consistent treatment of the domestic canine population with these products.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eAll dog owners were duly informed about study protocol before inclusion, agreeing by writing with all procedures performed after signing the informed consent. The study was approved by Universidade Federal de Mato Grosso (23108.954157/2018-91) and Universidade Federal do Maranhao Institutional (CIAEP: 01.0341.2014) Animal Care and Use Committees (IACUC) with the concurrence of Boehringer Ingelheim.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors have reviewed this document and consent to publish the data.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interest\u003c/strong\u003e \u003cp\u003eThe work reported herein was funded by Boehringer-Ingelheim. AC, LP, ET are current employees or contractors of Boehringer-Ingelheim. 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).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eA.C, L.P and E.T. are employees of Boehringer Ingelheim, manufacturerof the investigated products (NexGard\u0026reg; and NexGard Spectra\u0026reg;) and sponsor of thisstudy.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe work reported herein was funded by Boehringer Ingelheim Animal Health GA, USA.\u003c/p\u003e \u003cp\u003eAvailability of data and materials\u003c/p\u003e \u003cp\u003eAll relevant data are provided within the paper.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAndre A. Cutolo: study conceptualization, study protocol, study monitoring, funding, writing and language editing, general supervisionLuis. G. R. Pelissoni: study protocol, funding, writing and language editingT\u0026aacute;ssia L. Vale: data gathering Livio M. Costa-Junior: investigation, validation, data analysis, writing and language editingIsabella C. Sousa: data gatheringNaylene C. S. Silva: data gatheringCristiano Grisi do Nascimento: study coordinationMaycon Junior Heidmann: data gatheringBruno Gomes de Castro: investigation, validation, data analysis, writing and language editingEric Tielemans: Manuscript drafting\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe gratefully acknowledge the National Institute of Science and Technology in Innovation in Animal Parasitology (INCT-IPA) sponsored by Brazil\u0026rsquo;s National Council for Scientific and Technological Development (CNPq), grant no. 408812/2024-9. To all R\u0026amp;D Boehringer Ingelheim Animal Health Brazil staff for the support on the logistics and study data review.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll relevant data are provided within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAraes-Santos AI, Moraes-Filho J, Peixoto RM, Spolidorio MG, Azevedo SS, Costa MM, Labruna MB, Horta MC. Ectoparasite Infestations and Canine Infection by Rickettsiae and Ehrlichiae in a Semi-Arid Region of Northeastern Brazil. Vector Borne Zoonotic Dis. 2015;15(11):645\u0026ndash;51. doi: 10.1089/vbz.2015.1786. PMID: 26565771; PMCID: PMC4652196.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRostami A, Riahi SM, Hofmann A, Ma G, Wang T, Behniafar H, Taghipour A, Fakhri Y, Spotin A, Chang BCH, Macpherson CNL, Hotez PJ, Gasser RB. Global prevalence of Toxocara infection in dogs. 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Parasites and Vectors, 7:22.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"Dog, efficacy, afoxolaner, milbemycin oxime, Ctenocephalides felis, Rhipicephalus linnaei, Heterodoxus spiniger, Ancylostoma, Toxocara, Trichuris","lastPublishedDoi":"10.21203/rs.3.rs-9294152/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9294152/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eInfestations with fleas, ticks and intestinal nematodes are commonly identified in domestic dogs and have significant veterinary and public health importances. The study described in this manuscript was conducted to verify the natural parasitism of domestic dogs in two Brazilian regions, and to confirm the efficacy of the isoxazoline afoxolaner, formulated alone (NexGard\u0026reg;) or in combination with the macrocyclic lactone milbemycin oxime (NexGard Spectra\u0026reg;), for the control of fleas and ticks, and to confirm the efficacy of the milbemycin oxime combination for the control of intestinal nematodes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe trial was conducted in Mato Grosso (MT) and Maranh\u0026atilde;o (MA) states, Center-West and Northeast region. Three hundred and forty-five (345) domestic dogs, 170 in MT and 175 in MA, infested with at least one of these parasites were studied. Dogs were treated once and evaluated approximately two and four weeks later for ectoparasites, and two weeks later for intestinal nematodes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eApproximately half (47.5%) of the dogs were infested with more than one parasite, more than one third (38.6%) with intestinal nematodes. All fleas were identified as \u003cem\u003eCtenocephalides felis\u003c/em\u003e, ticks as \u003cem\u003eRhipicephalus linnaei\u003c/em\u003e and intestinal nematodes eggs as \u003cem\u003eAncylostoma, Toxocara\u003c/em\u003e or \u003cem\u003eTrichuris\u003c/em\u003e. Eleven dogs were also diagnosed infested with the chewing louse \u003cem\u003eHeterodoxus spiniger.\u003c/em\u003e Both products had an identical efficacy against fleas (98.4% to 100%), ticks (96.9% to 100%) and lice (100%). The MO formulation had an efficacy of \u0026ge;\u0026thinsp;97.8% against \u003cem\u003eAncylostoma\u003c/em\u003e, \u0026ge;\u0026thinsp;94.7% against \u003cem\u003eToxocara\u003c/em\u003e and \u0026ge;\u0026thinsp;97.7% against \u003cem\u003eTrichuris.\u003c/em\u003e\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study demonstrated a significant parasitic abundance in domestic dogs in Brazil, and confirmed a high efficacy of afoxolaner against fleas, ticks and lice and a high efficacy of MO against intestinal nematodes.\u003c/p\u003e","manuscriptTitle":"Efficacy of afoxolaner or a combination of afoxolaner and milbemycin oxime against fleas, ticks, lice and/or intestinal nematodes in dogs naturally infested in Brazil","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 18:09:55","doi":"10.21203/rs.3.rs-9294152/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"286940975150846072913624263686021185189","date":"2026-05-13T23:46:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315544726465462277323939086077177241661","date":"2026-05-11T14:19:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230134934640547842239513706150754402168","date":"2026-05-09T14:03:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-07T10:39:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-07T06:22:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2026-04-30T16:07:03+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":"55d444d4-278e-4ec5-ab37-8db04e8710a3","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"286940975150846072913624263686021185189","date":"2026-05-13T23:46:52+00:00","index":23,"fulltext":""},{"type":"reviewerAgreed","content":"315544726465462277323939086077177241661","date":"2026-05-11T14:19:06+00:00","index":21,"fulltext":""},{"type":"reviewerAgreed","content":"230134934640547842239513706150754402168","date":"2026-05-09T14:03:23+00:00","index":15,"fulltext":""},{"type":"reviewersInvited","content":"8","date":"2026-05-07T10:39:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-07T06:22:46+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T18:09:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 18:09:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9294152","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9294152","identity":"rs-9294152","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}