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Onset of efficacy and residual speed of kill over one month of a topical dinotefuran-permethrin-pyriproxyfen combination (Vectra®3D) against the adult cat flea (Ctenocephalides felis felis) on dogs
Veterinary Parasitology 211 (2015) 89–92
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Onset of efficacy and residual speed of kill over one month of a topical dinotefuran-permethrin-pyriproxyfen combination (Vectra® 3D) against the adult cat flea (Ctenocephalides felis felis) on dogs Marie Varloud a,∗ , Josephus J. Fourie b a b
Ceva Santé Animale S. A., 10 Avenue de la Ballastière, 33500 Libourne, France ClinVet International (Pty) Ltd., P.O. Box 11186, Universitas 9321, South Africa
a r t i c l e
i n f o
Article history: Received 8 December 2014 Received in revised form 27 March 2015 Accepted 7 May 2015 Keywords: Flea adulticidal efficacy Insecticide Canis familiaris Control Prevention
a b s t r a c t This study was designed to assess the onset of therapeutic and residual insecticidal efficacy of a topical ectoparasiticide (Vectra® 3D, DPP) on dogs over one month against the cat flea, Ctenocephalides felis felis. Adult dogs (n = 32, 11.0–18.7 kg) were infested with 100 adult fleas on days −6, −2, 7, 14, 21 and 28. Based on flea retention, the dogs were allocated in two groups and were treated topically on day 0 with a control solution (CS) or DPP. Each group was divided in two subgroups in which live fleas were counted by combing the dogs 2 or 6 h after treatment and at each re-infestation. The insecticidal efficacy was calculated using arithmetic and geometric means at each time point. The flea retention rate ranged from 64.0 to 86.5% in the CS group throughout the study. Based on arithmetic means, the therapeutic efficacy was 96.4% 6 h post-treatment and residual efficacies ranged from 96.8 to 99.9% 2 h after each re-infestation. The residual speed of killing effectiveness, >96% at 2 h, persisted for one month after treatment. DPP administration was well tolerated. This study confirms that DPP starts killing fleas within 2 h after treatment and reaches efficacy levels >95% at 6 h. We have shown that DPP kills >96.8% of fleas for one month after treatment within 2 h after infestation. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Fleas are common hematophagous ectoparasites of dogs. The cat flea (Ctenocephalides felis felis) is the most frequent species and represents a threat to both animal and human health because of its bites and the risk of disease transmission. Fleas start their first blood meal shortly after invading the host (Varloud et al., 2014) and perform numerous blood meals daily on the same host but can accidentally infest humans. On human skin, each probing induces visible lesions and pruritic wheals that can occur in clusters (Bitam et al., 2010). In dogs, flea bites can induce hypersensitivity reactions associated with typical dermatological conditions (Bruet et al., 2012). C. felis is considered to be a vector of several pathogens: viruses such as calicivirus (Mencke et al., 2009), bacteria such as Rickettsia felis (Capelli et al., 2009) and Bartonella (Bouhsira et al., 2012), and helminths such as Dipylidium caninum (Hinaidy, 1991). Some of these organisms are zoonotic. Transmission of these pathogens can occur through ingestion of the infective flea, through
∗ Corresponding author. Tel.: +33 557556349; fax: +33 57554246. E-mail address: [email protected] (M. Varloud).
blood feeding, and also by infective feces that can be ingested or can contaminate a breach in the skin barrier. After the blood meal, digestion starts immediately and the fleas excrete their first feces approximately 30 min after infestation (Wang et al., 2012). When fleas are infected with Bartonella, they can produce infective feces for several days (Bouhsira et al., 2013; Kernif et al., 2014). Due to the risks associated with flea infestations, various ectoparasiticides are available for the control and prevention of flea infestations in dogs. While the efficacy of such products has been generally evaluated 48 h after treatment of infestation, they are expected to act much faster to relieve dogs from their fleas, as well as to reduce the risks of disease transmission. This study was therefore designed to assess the onset and residual performance 2 and 6 h post-treatment and after re-infestations over one month using a topical dinotefuranpermethrin-pyriproxyfen combination (Vectra® 3D) against adult cat fleas (C. felis) on dogs. 2. Materials and methods The animals were not treated by any individual involved in performing the post-treatment assessments and observations. Furthermore, the study groups were coded to blind the assessors.
http://dx.doi.org/10.1016/j.vetpar.2015.05.005 0304-4017/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
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M. Varloud, J.J. Fourie / Veterinary Parasitology 211 (2015) 89–92
Table 1 Body weight and hair length of dogs.
2.4. Flea infestations
Body weight (kg) Group CS DPP
Hair length (mm)
Mean
Min
Max
Mean
Min
Max
14.7 13.6
11.2 11.0
18.7 17.5
29.0 21.8
17.0 14.3
63.3 28.5
2.1. Dogs Thirty-two healthy mongrel dogs (>6 months old, 1:1 sex ratio) were enrolled in the study and completed an acclimation period of at least 7 days. The dogs had not been treated with an acaricide or insecticide for 12 weeks prior to day 0 of the study. All dogs were identified with electronic transponders and were dewormed at the beginning of the study. Their body weight (BW) ranged from 11.0 to 18.7 kg and their hair length ranged from 14 to 63 mm (Table 1). The dogs were housed individually in an indoor animal unit that was temperature controlled and set for a period of 12 h light and 12 h darkness. The dogs were fed commercial dog food once daily with water available ad libitum. No contact between dogs was possible during the study. All dogs were observed for general health status and adverse reactions to treatment once per day, from day −6 until day 30, except on day 0, when a general examination, including local tolerance assessments, was performed within 2 h before and 1, 2 and 8 h (±30 min) post-treatment. This protocol was approved by an independent animal ethics committee.
A laboratory-bred strain of C. felis felis was used for the experimental infestation. Fleas were routinely fed on cats. Each dog was infested with 100 adult fleas on days −6, −2, 7, 14, 21 and 28. The fleas were unfed and of mixed sex. Flea counts were conducted by combing and removing the parasites on the dogs at 2 ± 0.5 h in the first subgroup (a) and at 6 ± 0.5 h post-infestation or treatment in the second subgroup (b). 2.5. Statistical analysis The calculations were based on the geometric means of the flea (count +1) data. One (1) was subsequently subtracted from the result to obtain a meaningful value for the geometric mean of the study groups. Arithmetic means and efficacies based on the arithmetic means were also calculated. The percent efficacy for each subgroup (a, 2 h or b, 6 h) on each day was calculated as: Efficacy (%) = 100 ×
(MC − MT) MC
where MC is the mean (geometric or arithmetic) of live fleas in the CS group and MT is the mean (geometric or arithmetic) of the live fleas in the treated group. The groups were compared using analysis of variance (ANOVA) with a treatment effect after a logarithmic transformation of the (flea count +1) data. Statistical significance was declared at a twosided p-value of 0.05.
2.2. Allocation The study followed a randomized block design. The 32 dogs included were ranked, within gender, in descending order of individual flea counts on day −5. Within each gender, the animals were assigned to blocks of four dogs each. Within each block, the dogs were randomly allocated to groups 1 and 2, and in each group the dogs were furthermore randomly assigned to one of two subgroups: a and b. 2.3. Treatment Each dog was treated with the allocated treatment on day 0. Dogs in group 1 received the placebo control solution (CS) containing the vehicle of the commercial formulation Vectra® 3D (Ceva Animal Health, Lenexa, KS, USA) with no active ingredient. Dogs in group 2 were treated with the commercial formulation Vectra® 3D (DPP) containing the active ingredients dinotefuran (4.95%), pyriproxifen (0.44%) and permethrin (36.08%). Both treatments were administered topically, as spot-on, according to the manufacturer’s label directions, at a rate of 3.6 mL per dog applied equally (1.2 mL per site) in three spots at the shoulder blades, the mid-back and the base of the tail (Table 2).
2.6. Guidelines The study was conducted in accordance with the current and appropriate guidelines (CVMP, 2000, 2007). 3. Results The study was run in October and November; this time period corresponds to the end of spring and the beginning of summer in South Africa. The temperature inside the housing unit remained between 17.0 and 26.4 ◦ C and the recorded relative humidity ranged from 19.9 to 79.0%. No adverse effects were observed in any of the treated dogs. Cosmetic effects, described as the “spiking with a wet paint brush effect”, were noticed on all dogs 1–8 h after administration of the treatment. In the CS group, the average flea retention rate in the subgroups collected 2 and 6 h post-treatment was 64.8% and 64.0%, respectively (Table 3). There were from 28 to 94 live fleas on each dog in the CS group. Two hours after the administration of DPP, one dog was free of fleas. The insecticidal efficacy based on arithmetic means was 23.0%. The number of live fleas found on dogs ranged
Table 2 Concentrations of active ingredients delivered individually in each subgroup on day 0. mg/kg BWa
mg/m2 BSAb
Subgroup
Active ingredient
Mean
Min
Max
Mean
Min
Max
2h
Dinotefuran Pyriproxyfen Permethrin
14.9 1.3 108.3
11.2 1.0 81.5
17.9 1.6 130.1
313.4 27.9 2284.5
291.1 25.9 2121.8
345.5 30.7 2518.2
6h
Dinotefuran Pyriproxyfen Permethrin
14.5 1.3 105.6
12.7 1.1 92.9
17.8 1.6 129.7
320.6 28.5 2336.8
266.2 23.7 1940.4
375.8 33.4 2739.5
a b
BW: body weight. BSA: body surface area.
M. Varloud, J.J. Fourie / Veterinary Parasitology 211 (2015) 89–92
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Table 3 Flea counts and efficacy 2 and 6 h. after treatment and weekly (days −2, 7, 14, 21 and 28) infestations with adult C. felis on dogs treated with a control (CS) or a topical ectoparasiticide (DPP) on day 0. Days
0
Mean
AMb
a
64.8 49.9 23.0 1/8 64.0 2.3 96.4 2/8
CS 2 h DPP 2 h Efficacy (%) FF 2 h (n) CS 6 h DPP 6 h Efficacy (%) FF 6 h (n)
7 GM 62.0 26.6 57.1 60.7 1.7 97.2
AM 85.9 0.4 99.5 5/8 68.0 0.0 100 8/8
14 GM 85.6 0.3* 99.7 65.3 0.0* 100
AM 76.9 0.4 99.5 6/8 64.1 0.0 100 8/8
21 GM 76.4 0.3* 99.7 62.8 0.0* 100
AM 78.1 0.1 99.9 7/8 67.9 0.0 100 8/8
28 GM 77.2 0.1* 99.9 66.1 0.0* 100
AM 86.5 2.8 96.8 2/8 73.0 1.0 98.6 6/8
GM 86.2 2.0* 97.7 70.4 0.5* 99.3
a
CS: control solution; DPP: dinotefuran, pyriproxyfen, permethrin; FF: number of dogs free of live fleas. AM: arithmetic mean; GM: geometric mean. *Group differed (p < 0.05) within each time point. b
between 0 and 93. Six hours after the administration of DPP, two dogs were free of fleas. The number of live fleas found on the dogs ranged from 0 to 5. The insecticidal efficacy based on arithmetic means was 96.5%. In the CS group, the average flea retention rate observed in weekly re-infestations ranged from 64.1 to 86.5% (Table 3). In the first DPP-treated subgroup, there were from 0 to 6 live fleas found on the dogs 2 h after infestation. The insecticidal efficacy based on arithmetic means was above 99% for 3 weeks post-treatment and was 96.8% during the fourth week. In the second subgroup, there were from 0 to 5 live fleas found on DPP-treated dogs 6 h after infestation. The insecticidal efficacy based on arithmetic means was 100% for 3 weeks post-treatment and was 98.6% during the fourth week. All DPP-treated dogs from subgroup b were free of fleas at every time point, except for 2 dogs during the fourth week. 4. Discussion As demonstrated in the CS group, which was characterized by an average flea retention rate of 72.2%, the flea challenges were vigorous during the entire duration of the study. 4.1. Therapeutic efficacy In this study, the therapeutic efficacy was measured 2 and 6 h after the administration of the products on dogs already infested with fleas for 2 days. Although the active ingredients needed time to spread over the body surface and coat to reach the most distal parts of the animal where fleas could hide, 1 out of the 8 dogs was already flea-free 2 h after treatment in subgroup a and the insecticidal efficacy reached 96.4% 6 h after treatment in subgroup b. These results confirmed previous evaluations of the therapeutic efficacy of DPP against flea infestations in dogs that were also measured 6 h after treatment (97.2%) but using a different strain of fleas (Dryden et al., 2011). Although the therapeutic speed of kill is often considered to be a marker of the parasiticide performance, it has less clinical relevance compared with the residual speed of kill. The therapeutic speed of kill occurs only on the day of treatment for a dog already infested for an undetermined length of time while the residual speed of kill is linked to the duration of protection expected from the product. 4.2. Residual efficacy From days 7 to 28, based on arithmetic means, DPP killed >96.8 % of fleas in 2 h after each infestation. There were no fleas found on dogs 6 h after infestation from days 7 to 21 and efficacy on day 28 was 98.6%. Such results confirm the residual speed of kill performance of DPP against fleas that was already evaluated 6 and 24 h
after weekly re-infestations (Dryden et al., 2011). The residual efficacy was assessed weekly in situations in which the parasites must enter the coat of the dogs and move on each animal’s skin up to their location for feeding, increasing both their level of metabolism and their exposure to already-treated skin and hair. However, in such circumstances, the concentration of active ingredients in the animals often follows a decay-curve pattern. This fact was observed with topical administrations of permethrin (Lüssenhop et al., 2011), imidacloprid (Chopade et al., 2010) and fipronil (Bigelow Dyk et al., 2012), and with oral administrations of spinosad (Holmstrom et al., 2011) and afoxolaner (Letendre et al., 2014). The decrease in concentration of active ingredients can be due to different factors such as formulation (Endris et al., 2003), metabolism, hair loss and exposure to sunlight or water. The efficacy level can be maintained at the required threshold until the next administration when the assessments are performed 48 h after re-infestations while it is less common for the speed of kill. Indeed, for example with spinosad, the efficacy measured at 4 h against C. canis can decrease from 81% on the day of treatment to 42% on day 28 after the treatment (Franc and Bouhsira 2009). On the other hand, the DPP formulation provides a high and residual speed of kill, effective against new flea infestations for one month after administration. These aspects of DPP should be explored to preventively protect against flea bites and flea-borne diseases. 4. Conclusion This experiment has demonstrated that DPP not only starts killing a significant number of fleas within 2 h after treatment but also relieves dogs of almost all fleas in 6 h. DPP also provides protection against flea infestations that lasts for a month, as demonstrated by the high (>96.8%) residual insecticidal efficacy within 2 h after infestation at every time point for one month. Although further investigations should be performed to determine the performance of DPP over shorter time intervals after flea infestations, this combination should be considered as a reliable veterinary prevention tool to protect dogs against infestations by the cat flea C. felis. Competing interests This study was funded by Ceva Animal Health, where Marie Varloud is an employee. Josephus J. Fourie works in ClinVet an independent Contract Development Organization that was contracted to perform the study. Authors’ contributions JJF designed, drafted the protocol and conducted the study. MV drafted the manuscript. All authors approved the final manuscript.
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Acknowledgements The authors are grateful to Dr. Cathy A. Ball and Mr Reiner A. Zwiegers who contributed to the study and to the ClinVet staff who took part in the study. References Bigelow Dyk, M., Liu, Y., Chen, Z., Vega, H., Krieger, R.I., 2012. Fate and distribution of fipronil on companion animals and in their indoor residences following spot-on flea treatments. J. Environ. Sci. Health 47, 913–924. Bitam, I., Dittmar, K., Parola, P., Whiting, M.F., Raoult, D., 2010. Fleas and flea-borne diseases. Int. J. Infect. Dis. 14, 667–676. Bouhsira, E., Ferrandez, Y., Liu, M., Franc, M., Boulouis, H.J., Biville, F., 2012. Ctenocephalides felis: an in vitro potential vector for five Bartonella species. Comp. Immunol. Microbiol. Infect. Dis. 36, 105–111. Bouhsira, E., Franc, M., Boulouis, H.J., Jacquiet, P., Raymond-Letron, I., Liénard, E., 2013. Assessment of persistence of Bartonella henselae in Ctenocephalides felis. Appl. Environ. Microbiol. 79, 7439–7444. Bruet, V., Bourdeau, P.J., Roussel, A., Imparato, L., Desfontis, J.C., 2012. Characterization of pruritus in canine atopic dermatitis: flea bite hypersensitivity and flea infestation and its role in diagnosis. Vet. Dermatol. 23, 487–e93. Capelli, G., Montarsi, F., Porcellato, E., Maioli, G., Furnari, C., Rinaldi, L., Oliva, G., Otranto, D., 2009. Occurrence of Rickettsia felis in dog and cat fleas (Ctenocephalides felis) from Italy. Parasit. Vectors 2 (Suppl. 1), S8. Chopade, H., Eigenberg, D., Solon, E., Strzemienski, P., Hostetler, J., McNamara, T., 2010. Skin distribution of imidacloprid by microautoradiography after topical administration to beagle dogs. Vet. Ther. 11, 1–10. CVMP, 2000. VICH GL9 guideline on good clinical practice under animal ethics committee approval.. CVMP, 2007. Guideline for the testing and evaluation of the efficacy of anti-parasitic substances for the treatment and prevention of tick and flea infestations in dogs and cats. .
Endris, R.G., Hair, J.A., Anderson, G., Rose, W.B., Disch, D., Meyer, J.A., 2003. Efficacy of two 65% permethrin spot-on formulations against induced infestations of Ctenocephalides felis (Insecta: Siphonaptera) and Amblyomma americanum (Acari: Ixodidae) on beagles. Vet. Ther. 4, 47–55. Franc, M., Bouhsira, E., 2009. Evaluation of speed and duration of efficacy of spinosad tablets for treatment and control of Ctenocephalides canis (Siphonaptera: Pulicidae) infestations in dogs. Parasite 16, 125–128. Dryden, M.W., Payne, P.A., Smith, V., Kobuszewski, D., 2011. Efficacy of topically applied dinotefuran formulations and orally administered Spinosad tablets against the KS1 flea strain infesting dogs. Intern. J. Appl. Res. Vet. Med. 9, 124–129. Hinaidy, H.K., 1991. A contribution on the biology of Dipylidium caninum Part II. J. Vet. Med. 38, 329–336. Holmstrom, S.D., Totten, M.L., Newhall, K.B., Qiao, M., Riggs, K.L., 2011. Pharmacokinetics of spinosad and milbemycin oxime administered in combination and separately per os to dogs. J. Vet. Pharmacol. Ther. 35, 351–364. Kernif, T., Leulmi, H., Socolovschi, C., Berenger, J.M., Lepidi, H., Bitam, I., Rolain, J.M., Raoult, D., Parola, P., 2014. Acquisition and excretion of Bartonella quintana by the cat flea, Ctenocephalides felis felis. Mol. Ecol. 23, 1204–1212. Letendre, L., Huang, R., Kvaternick, V., Harriman, J., Drag, M., Soll, M., 2014. The intravenous and oral pharmacokinetics of afoxolaner used as a monthly chewable antiparasitic for dogs. Vet. Parasitol. 201, 190–197. Lüssenhop, J., Stahl, J., Wolken, S., Schnieder, T., Kietzmann, M., Bäumer, W., 2011. Distribution of permethrin in hair and stratum corneum after topical administration of four different formulations in dogs. J. Vet. Pharmacol. Ther. 35, 206–208. Mencke, N., Vobis, M., Mehlhorn, H., D’Haese, J., Rehagen, M., Mangold-Gehring, S., Truyen, U., 2009. Transmission of feline calicivirus via the cat flea (Ctenocephalides felis). Parasitol. Res. 105, 185–189. Varloud, 2014 Varloud, M., Warin, S., Fourie, J.J., et al., 2014. Inhibition of Flea Feeding in Dogs Treated with a Dinotefuran-permethrin-pyriproxyfen Spot-on and Weekly Challenged with Adult Fleas (Ctenocephalides felis), 528. BSAVA Congress. Wang, C., Mount, J., Butler, J., Gao, D., Jung, E., Blagburn, B.L., Kaltenboeck, B., 2012. Real-time PCR of the mammalian hydroxymethylbilane synthase (HMBS) gene for analysis of flea (Ctenocephalides felis) feeding patterns on dogs. Parasit. Vectors. 5, 4.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Onset of efficacy and residual speed of kill over one month of a topical dinotefuran-permethrin-pyriproxyfen combination (Vectra® 3D) against the adult cat flea (Ctenocephalides felis felis) on dogs Marie Varloud a,∗ , Josephus J. Fourie b a b
Ceva Santé Animale S. A., 10 Avenue de la Ballastière, 33500 Libourne, France ClinVet International (Pty) Ltd., P.O. Box 11186, Universitas 9321, South Africa
a r t i c l e
i n f o
Article history: Received 8 December 2014 Received in revised form 27 March 2015 Accepted 7 May 2015 Keywords: Flea adulticidal efficacy Insecticide Canis familiaris Control Prevention
a b s t r a c t This study was designed to assess the onset of therapeutic and residual insecticidal efficacy of a topical ectoparasiticide (Vectra® 3D, DPP) on dogs over one month against the cat flea, Ctenocephalides felis felis. Adult dogs (n = 32, 11.0–18.7 kg) were infested with 100 adult fleas on days −6, −2, 7, 14, 21 and 28. Based on flea retention, the dogs were allocated in two groups and were treated topically on day 0 with a control solution (CS) or DPP. Each group was divided in two subgroups in which live fleas were counted by combing the dogs 2 or 6 h after treatment and at each re-infestation. The insecticidal efficacy was calculated using arithmetic and geometric means at each time point. The flea retention rate ranged from 64.0 to 86.5% in the CS group throughout the study. Based on arithmetic means, the therapeutic efficacy was 96.4% 6 h post-treatment and residual efficacies ranged from 96.8 to 99.9% 2 h after each re-infestation. The residual speed of killing effectiveness, >96% at 2 h, persisted for one month after treatment. DPP administration was well tolerated. This study confirms that DPP starts killing fleas within 2 h after treatment and reaches efficacy levels >95% at 6 h. We have shown that DPP kills >96.8% of fleas for one month after treatment within 2 h after infestation. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Fleas are common hematophagous ectoparasites of dogs. The cat flea (Ctenocephalides felis felis) is the most frequent species and represents a threat to both animal and human health because of its bites and the risk of disease transmission. Fleas start their first blood meal shortly after invading the host (Varloud et al., 2014) and perform numerous blood meals daily on the same host but can accidentally infest humans. On human skin, each probing induces visible lesions and pruritic wheals that can occur in clusters (Bitam et al., 2010). In dogs, flea bites can induce hypersensitivity reactions associated with typical dermatological conditions (Bruet et al., 2012). C. felis is considered to be a vector of several pathogens: viruses such as calicivirus (Mencke et al., 2009), bacteria such as Rickettsia felis (Capelli et al., 2009) and Bartonella (Bouhsira et al., 2012), and helminths such as Dipylidium caninum (Hinaidy, 1991). Some of these organisms are zoonotic. Transmission of these pathogens can occur through ingestion of the infective flea, through
∗ Corresponding author. Tel.: +33 557556349; fax: +33 57554246. E-mail address: [email protected] (M. Varloud).
blood feeding, and also by infective feces that can be ingested or can contaminate a breach in the skin barrier. After the blood meal, digestion starts immediately and the fleas excrete their first feces approximately 30 min after infestation (Wang et al., 2012). When fleas are infected with Bartonella, they can produce infective feces for several days (Bouhsira et al., 2013; Kernif et al., 2014). Due to the risks associated with flea infestations, various ectoparasiticides are available for the control and prevention of flea infestations in dogs. While the efficacy of such products has been generally evaluated 48 h after treatment of infestation, they are expected to act much faster to relieve dogs from their fleas, as well as to reduce the risks of disease transmission. This study was therefore designed to assess the onset and residual performance 2 and 6 h post-treatment and after re-infestations over one month using a topical dinotefuranpermethrin-pyriproxyfen combination (Vectra® 3D) against adult cat fleas (C. felis) on dogs. 2. Materials and methods The animals were not treated by any individual involved in performing the post-treatment assessments and observations. Furthermore, the study groups were coded to blind the assessors.
http://dx.doi.org/10.1016/j.vetpar.2015.05.005 0304-4017/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
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M. Varloud, J.J. Fourie / Veterinary Parasitology 211 (2015) 89–92
Table 1 Body weight and hair length of dogs.
2.4. Flea infestations
Body weight (kg) Group CS DPP
Hair length (mm)
Mean
Min
Max
Mean
Min
Max
14.7 13.6
11.2 11.0
18.7 17.5
29.0 21.8
17.0 14.3
63.3 28.5
2.1. Dogs Thirty-two healthy mongrel dogs (>6 months old, 1:1 sex ratio) were enrolled in the study and completed an acclimation period of at least 7 days. The dogs had not been treated with an acaricide or insecticide for 12 weeks prior to day 0 of the study. All dogs were identified with electronic transponders and were dewormed at the beginning of the study. Their body weight (BW) ranged from 11.0 to 18.7 kg and their hair length ranged from 14 to 63 mm (Table 1). The dogs were housed individually in an indoor animal unit that was temperature controlled and set for a period of 12 h light and 12 h darkness. The dogs were fed commercial dog food once daily with water available ad libitum. No contact between dogs was possible during the study. All dogs were observed for general health status and adverse reactions to treatment once per day, from day −6 until day 30, except on day 0, when a general examination, including local tolerance assessments, was performed within 2 h before and 1, 2 and 8 h (±30 min) post-treatment. This protocol was approved by an independent animal ethics committee.
A laboratory-bred strain of C. felis felis was used for the experimental infestation. Fleas were routinely fed on cats. Each dog was infested with 100 adult fleas on days −6, −2, 7, 14, 21 and 28. The fleas were unfed and of mixed sex. Flea counts were conducted by combing and removing the parasites on the dogs at 2 ± 0.5 h in the first subgroup (a) and at 6 ± 0.5 h post-infestation or treatment in the second subgroup (b). 2.5. Statistical analysis The calculations were based on the geometric means of the flea (count +1) data. One (1) was subsequently subtracted from the result to obtain a meaningful value for the geometric mean of the study groups. Arithmetic means and efficacies based on the arithmetic means were also calculated. The percent efficacy for each subgroup (a, 2 h or b, 6 h) on each day was calculated as: Efficacy (%) = 100 ×
(MC − MT) MC
where MC is the mean (geometric or arithmetic) of live fleas in the CS group and MT is the mean (geometric or arithmetic) of the live fleas in the treated group. The groups were compared using analysis of variance (ANOVA) with a treatment effect after a logarithmic transformation of the (flea count +1) data. Statistical significance was declared at a twosided p-value of 0.05.
2.2. Allocation The study followed a randomized block design. The 32 dogs included were ranked, within gender, in descending order of individual flea counts on day −5. Within each gender, the animals were assigned to blocks of four dogs each. Within each block, the dogs were randomly allocated to groups 1 and 2, and in each group the dogs were furthermore randomly assigned to one of two subgroups: a and b. 2.3. Treatment Each dog was treated with the allocated treatment on day 0. Dogs in group 1 received the placebo control solution (CS) containing the vehicle of the commercial formulation Vectra® 3D (Ceva Animal Health, Lenexa, KS, USA) with no active ingredient. Dogs in group 2 were treated with the commercial formulation Vectra® 3D (DPP) containing the active ingredients dinotefuran (4.95%), pyriproxifen (0.44%) and permethrin (36.08%). Both treatments were administered topically, as spot-on, according to the manufacturer’s label directions, at a rate of 3.6 mL per dog applied equally (1.2 mL per site) in three spots at the shoulder blades, the mid-back and the base of the tail (Table 2).
2.6. Guidelines The study was conducted in accordance with the current and appropriate guidelines (CVMP, 2000, 2007). 3. Results The study was run in October and November; this time period corresponds to the end of spring and the beginning of summer in South Africa. The temperature inside the housing unit remained between 17.0 and 26.4 ◦ C and the recorded relative humidity ranged from 19.9 to 79.0%. No adverse effects were observed in any of the treated dogs. Cosmetic effects, described as the “spiking with a wet paint brush effect”, were noticed on all dogs 1–8 h after administration of the treatment. In the CS group, the average flea retention rate in the subgroups collected 2 and 6 h post-treatment was 64.8% and 64.0%, respectively (Table 3). There were from 28 to 94 live fleas on each dog in the CS group. Two hours after the administration of DPP, one dog was free of fleas. The insecticidal efficacy based on arithmetic means was 23.0%. The number of live fleas found on dogs ranged
Table 2 Concentrations of active ingredients delivered individually in each subgroup on day 0. mg/kg BWa
mg/m2 BSAb
Subgroup
Active ingredient
Mean
Min
Max
Mean
Min
Max
2h
Dinotefuran Pyriproxyfen Permethrin
14.9 1.3 108.3
11.2 1.0 81.5
17.9 1.6 130.1
313.4 27.9 2284.5
291.1 25.9 2121.8
345.5 30.7 2518.2
6h
Dinotefuran Pyriproxyfen Permethrin
14.5 1.3 105.6
12.7 1.1 92.9
17.8 1.6 129.7
320.6 28.5 2336.8
266.2 23.7 1940.4
375.8 33.4 2739.5
a b
BW: body weight. BSA: body surface area.
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Table 3 Flea counts and efficacy 2 and 6 h. after treatment and weekly (days −2, 7, 14, 21 and 28) infestations with adult C. felis on dogs treated with a control (CS) or a topical ectoparasiticide (DPP) on day 0. Days
0
Mean
AMb
a
64.8 49.9 23.0 1/8 64.0 2.3 96.4 2/8
CS 2 h DPP 2 h Efficacy (%) FF 2 h (n) CS 6 h DPP 6 h Efficacy (%) FF 6 h (n)
7 GM 62.0 26.6 57.1 60.7 1.7 97.2
AM 85.9 0.4 99.5 5/8 68.0 0.0 100 8/8
14 GM 85.6 0.3* 99.7 65.3 0.0* 100
AM 76.9 0.4 99.5 6/8 64.1 0.0 100 8/8
21 GM 76.4 0.3* 99.7 62.8 0.0* 100
AM 78.1 0.1 99.9 7/8 67.9 0.0 100 8/8
28 GM 77.2 0.1* 99.9 66.1 0.0* 100
AM 86.5 2.8 96.8 2/8 73.0 1.0 98.6 6/8
GM 86.2 2.0* 97.7 70.4 0.5* 99.3
a
CS: control solution; DPP: dinotefuran, pyriproxyfen, permethrin; FF: number of dogs free of live fleas. AM: arithmetic mean; GM: geometric mean. *Group differed (p < 0.05) within each time point. b
between 0 and 93. Six hours after the administration of DPP, two dogs were free of fleas. The number of live fleas found on the dogs ranged from 0 to 5. The insecticidal efficacy based on arithmetic means was 96.5%. In the CS group, the average flea retention rate observed in weekly re-infestations ranged from 64.1 to 86.5% (Table 3). In the first DPP-treated subgroup, there were from 0 to 6 live fleas found on the dogs 2 h after infestation. The insecticidal efficacy based on arithmetic means was above 99% for 3 weeks post-treatment and was 96.8% during the fourth week. In the second subgroup, there were from 0 to 5 live fleas found on DPP-treated dogs 6 h after infestation. The insecticidal efficacy based on arithmetic means was 100% for 3 weeks post-treatment and was 98.6% during the fourth week. All DPP-treated dogs from subgroup b were free of fleas at every time point, except for 2 dogs during the fourth week. 4. Discussion As demonstrated in the CS group, which was characterized by an average flea retention rate of 72.2%, the flea challenges were vigorous during the entire duration of the study. 4.1. Therapeutic efficacy In this study, the therapeutic efficacy was measured 2 and 6 h after the administration of the products on dogs already infested with fleas for 2 days. Although the active ingredients needed time to spread over the body surface and coat to reach the most distal parts of the animal where fleas could hide, 1 out of the 8 dogs was already flea-free 2 h after treatment in subgroup a and the insecticidal efficacy reached 96.4% 6 h after treatment in subgroup b. These results confirmed previous evaluations of the therapeutic efficacy of DPP against flea infestations in dogs that were also measured 6 h after treatment (97.2%) but using a different strain of fleas (Dryden et al., 2011). Although the therapeutic speed of kill is often considered to be a marker of the parasiticide performance, it has less clinical relevance compared with the residual speed of kill. The therapeutic speed of kill occurs only on the day of treatment for a dog already infested for an undetermined length of time while the residual speed of kill is linked to the duration of protection expected from the product. 4.2. Residual efficacy From days 7 to 28, based on arithmetic means, DPP killed >96.8 % of fleas in 2 h after each infestation. There were no fleas found on dogs 6 h after infestation from days 7 to 21 and efficacy on day 28 was 98.6%. Such results confirm the residual speed of kill performance of DPP against fleas that was already evaluated 6 and 24 h
after weekly re-infestations (Dryden et al., 2011). The residual efficacy was assessed weekly in situations in which the parasites must enter the coat of the dogs and move on each animal’s skin up to their location for feeding, increasing both their level of metabolism and their exposure to already-treated skin and hair. However, in such circumstances, the concentration of active ingredients in the animals often follows a decay-curve pattern. This fact was observed with topical administrations of permethrin (Lüssenhop et al., 2011), imidacloprid (Chopade et al., 2010) and fipronil (Bigelow Dyk et al., 2012), and with oral administrations of spinosad (Holmstrom et al., 2011) and afoxolaner (Letendre et al., 2014). The decrease in concentration of active ingredients can be due to different factors such as formulation (Endris et al., 2003), metabolism, hair loss and exposure to sunlight or water. The efficacy level can be maintained at the required threshold until the next administration when the assessments are performed 48 h after re-infestations while it is less common for the speed of kill. Indeed, for example with spinosad, the efficacy measured at 4 h against C. canis can decrease from 81% on the day of treatment to 42% on day 28 after the treatment (Franc and Bouhsira 2009). On the other hand, the DPP formulation provides a high and residual speed of kill, effective against new flea infestations for one month after administration. These aspects of DPP should be explored to preventively protect against flea bites and flea-borne diseases. 4. Conclusion This experiment has demonstrated that DPP not only starts killing a significant number of fleas within 2 h after treatment but also relieves dogs of almost all fleas in 6 h. DPP also provides protection against flea infestations that lasts for a month, as demonstrated by the high (>96.8%) residual insecticidal efficacy within 2 h after infestation at every time point for one month. Although further investigations should be performed to determine the performance of DPP over shorter time intervals after flea infestations, this combination should be considered as a reliable veterinary prevention tool to protect dogs against infestations by the cat flea C. felis. Competing interests This study was funded by Ceva Animal Health, where Marie Varloud is an employee. Josephus J. Fourie works in ClinVet an independent Contract Development Organization that was contracted to perform the study. Authors’ contributions JJF designed, drafted the protocol and conducted the study. MV drafted the manuscript. All authors approved the final manuscript.
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