b Bayer Animal Health GmbH

Veterinary Therapeutics Vol. 9, No. 3, Fall 2008 Comparative Efficacy of Imidacloprid, Selamectin, Fipronil (S)-Methoprene, and Metaflumizone against Cats Experimentally Infested with Ctenocephalides felis* T. Schnieder, DVM, Dr. med. vet., DEVPC a S. Wolken, DVM, Dr. med. vet. a N. Mencke, DVM, Dr. med. vet., DEVPC b a Institute for Parasitology University of Veterinary Medicine Hannover Bünteweg 17 D-30559 Hannover Germany b Bayer Animal Health GmbH D-51368 Leverkusen Germany CLI NI CA L RE L E VA N C E Four active ingredients imidacloprid, selamectin, fipronil (S)-methoprene, and metaflumizone were tested to assess the speed of flea kill against existing flea infestations and subsequent reinfestations. Thirty flea-infested cats were allocated to four treatment groups and one untreated control group. Flea counts were performed 6, 18, and 48 hours after treatment (day 0) and 2, 4, and 24 hours after weekly flea reinfestations. Cats were also reinfested with fleas after the 6- and 18- hour counts on day 0 and after the 2- and 4-hour counts on subsequent count days. Imidacloprid provided significantly greater flea kill at diverse time points. At the 24-hour counts, all compounds showed expected and similar high efficacies. On study day 34, imidacloprid showed the highest efficacy at 24 hours after reinfestation, with 90.8% flea reduction compared with 55.7% to 67.4% in the other treatment groups. A single topical application of imidacloprid provided a high efficacy in the early elimination of adult fleas, limiting the risk of pathogen transmission and flea allergy dermatitis. INTRODUCTION Although substantial progress in flea control has been made during the past 15 years, fleas are still the predominant ectoparasite in cats and dogs, with cats being infested more often than dogs. During a 12-month observation period in Germany, about 5% of dogs and 14% of cats were found to be infested with fleas. 1 In Southern Italy, an average infestation rate of 18% was found in dogs during a similar 1-year *This study was funded by Bayer Animal Health GmbH, Leverkusen, Germany, and was originally presented at the Ninth International Parasite Control Symposium at the 2008 NAVC Conference. 176

T. Schnieder, S. Wolken, and N. Mencke observation period. 2 The cat flea, Ctenocephalides felis, is the most prevalent flea species infesting cats and dogs. 3 5 According to a survey conducted in Germany, 70% of about 1,700 small animal practitioners indicated that they frequently diagnose fleas on pets. 6 Despite the availability of a number of highly efficient ectoparasiticides, the prevalence of flea infestations has not been reduced considerably. On the contrary, because of the easy administration and high efficacy of the readily available insecticides, flea control is mainly done by pet owners and thus has escaped the supervision and care of veterinarians, sometimes resulting in unsuccessful treatments. The demand for flea control derives from pet owners concern for the health of their animal as well as their own well-being and that of other family members. However, there are additional reasons why flea control is important. Fleas are capable of causing or transmitting a number of diseases. They are intermediate hosts for Dipylidium caninum, a common tapeworm in dogs that can also infect humans. Furthermore, fleas are known to transmit several bacteria and rickettsiae with zoonotic potential, such as Bartonella henselae, the pathogen causing cat scratch fever, 7 11 and Rickettsia felis. 7,12 Recently, it was shown that cat fleas are carrying feline leukemia virus after feeding on an infected cat and probably are capable of transmitting the pathogen during their next blood meal. 13 Apart from transmitting infectious diseases, flea saliva injected during a blood meal can cause flea allergy dermatitis. 14 Therefore, adulticides used for flea control should remove adult fleas as quickly as possible to reduce the number of bites and blood meals and thus reduce the chance of transmitting pathogens and stop egg production by adult females at the earliest possible time. Because the importance of arthropod-transmitted diseases is increasingly being recog- TABLE 1. Study Protocol Explaining the Treatment and Ensuing Flea Counts and Flea Reinfestations Study Day Action 2 Flea infestation 1 Flea counts, group allocation, flea reinfestation 0 1 0 hr Treatment 6 hr Flea counts, flea reinfestation 18 hr (12 hr PI) Flea counts, flea reinfestation 42 hr (24 hr PI) Flea counts 6 7, 13 14, 20 21, 27 28, and 34 35 0 hr Flea reinfestation 2 hr PI Flea counts, flea reinfestation 4 hr PI Flea counts, flea reinfestation 24 hr PI Flea counts PI = postinfestation. TABLE 2. Geometric Mean Flea Counts of Untreated Control Cats 6, 18, and 42 Hours after Treatment and 2, 4, and 24 Hours after Subsequent Weekly Reinfestations Study Hours after Treatment Day 6 18 (12 hr PI) 42 (24 hr PI) 0 1 31.85 22.99 33.56 Hours Post-Reinfestation 2 4 24 6 7 44.25 55.17 32.92 13 14 37.14 40.86 40.09 20 21 24.99 18.48 22.87 27 28 35.03 29.33 30.64 34 35 34.18 40.98 40.50 PI = postinfestation. 177

Veterinary Therapeutics Vol. 9, No. 3, Fall 2008 nized, prevention schemes using insecticides and acaricides are recommended more frequently than in the past. However, the EMEA/CVMP guidelines 15 for labeling require that flea counts be performed 48 hours after infestation. Therefore, none of the existing comparative data for the various compounds address earlier efficacy, and none of the compounds has a label claim related to prevention against transmitted agents. This study was undertaken to investigate the speed of efficacy of some of the most important veterinary medicinal remedies against fleas at earlier times than defined in the EMEA/ CVMP guidelines to evaluate the suitability of these compounds for preventive use. The active ingredients imidacloprid, selamectin, and fipronil are widely used as ectoparasiticides on cats and dogs. Their efficacy has been demonstrated in numerous studies. 4,16 The active ingredient metaflumizone, a semicarbazone insecticide acting on the sodium channel of fleas, has only recently been introduced as a veterinary medicinal remedy, 17 and thus data on the speed of kill of fleas are not available. The aim of the study was to examine the speed of kill of fleas (C. felis) by imidacloprid (Advantage, Bayer Animal Health), selamectin (Stronghold [EU]/Revolution [USA], Pfizer Animal Health), fipronil (S)-methoprene (Frontline Combo [EU]/Frontline Plus [USA], Merial), and metaflumizone (ProMeris for cats, Fort Dodge Animal Health) after treatment and reinfestation. MATERIALS AND METHODS Study Design Thirty cats were acclimated to the study facilities for 1 week before each cat was experimentally infested with 50 fleas (C. felis) on study day 2. Flea counts were performed 24 hours after infestation (study day 1). Based on these counts, cats were allocated to five groups (four treatments and one untreated control) of six cats each. After randomization, cats were weighed and reinfested with 50 cat fleas each. On study day 0, cats were treated topically with the appropriate compound (imidacloprid, selamectin, fipronil (S)-methoprene, or metaflumizone) based on group assignment. The spot-on solutions were applied on the cat s neck at the base of the skull according to label instructions, and the cats were treated according to body weight using the recommended dosage. During the first week, the treatment success was monitored by flea counts performed 6 hours (±15 minutes) after treatment to allow the compound to be distributed on the cat s skin and fur. Cats were reinfested immediately after the counting procedure, and another flea count was performed 12 hours (±15 minutes) later. Cats were again reinfested, and a third flea count was performed 24 hours (±1 hour) later. Subsequent reinfestations and flea counts were done in weekly intervals for 5 weeks after treatment. Starting with the second week, infestation and counting cycles were performed as follows (Table 1): Reinfestation Flea count and immediate reinfestation 2 hours (±15 minutes) later Flea count and immediate reinfestation 4 hours (±15 minutes) later Flea count 24 hours (±1 hour) later Artificial Infestations with C. felis On each artificial infestation, all cats were infested with approximately 50 new fleas originating from the Institute for Parasitology, University of Veterinary Medicine Hannover, Germany. For flea infestation, flea containers 178

T. Schnieder, S. Wolken, and N. Mencke TABLE 3. Efficacies (% Flea Reduction) of the Four Test Compounds Calculated from Geometric Mean Flea Counts on Treated and Control Cats 6, 18, and 42 Hours after Treatment and 2, 4, and 24 Hours after Subsequent Weekly Reinfestations Study Day 0 1 Hours after Treatment 6 18 42 (12 hr PI) (24 hr PI) Imidacloprid 61.1 a,b 100 b 100 Selamectin 47.4 99.5 100 Fipronil (S)-methoprene 18.8 92.8 100 Metaflumizone 8.3 0 73.9 6 7 Hours Post-Reinfestation 2 4 24 Imidacloprid 62.3 96.2 a,b,c 100 Selamectin 28.4 51.9 99.2 Fipronil (S)-methoprene 28.9 69.5 99.2 Metaflumizone 42.1 53.6 85.9 13 14 Imidacloprid 59.0 b 91.0 b,c 99.7 Selamectin 43.4 11.1 98.3 Fipronil (S)-methoprene 35.1 63.2 97.6 Metaflumizone 11.3 30.6 91.2 (cont. next column) Hours Post-Reinfestation Study Day 2 4 24 20 21 Imidacloprid 20.2 52.5 c 97.4 Selamectin 25.5 0 87.6 Fipronil (S)-methoprene 42.8 22.4 95.0 Metaflumizone 43.9 8.7 92.8 27 28 Imidacloprid 18.3 34.6 c 95.7 Selamectin 24.6 0 83.2 Fipronil (S)-methoprene 47.9 20.6 90.9 Metaflumizone 28.1 38.7 82.1 34 35 Imidacloprid 28.1 62.8 c 90.8 a,c Selamectin 30.7 13.3 55.7 Fipronil (S)-methoprene 60.1 58.9 57.8 Metaflumizone 59.2 52.4 67.4 a Significant difference compared with fipronil (S)-methoprene treated group; see Results for P value(s). b Significant difference compared with metaflumizonetreated group; see Results for P value(s). c Significant difference compared with selamectin-treated group; see Results for P value(s). PI = postinfestation. were opened in the animal s cage and fleas were allowed to disperse in the animal s haircoat. Fleas combed off the cats were counted and stored, and cats were reinfested for subsequent counts with new batches of fleas. Flea Counts The total body surface was examined in the following sequence by intensive combing with a fine-toothed flea comb until no further fleas were found: head, ears, neck, lateral areas, dorsal strip from shoulder blades to base of tail, tail and anal area, forelegs and shoulders, hind legs, abdominal area from chest to inside hind legs, feet. All fleas were removed, counted, and collected in a 50-ml falcon tube. Statistical Analysis Arithmetic and geometric means of flea counts were calculated. Efficacy was calculated 179

Veterinary Therapeutics Vol. 9, No. 3, Fall 2008 according to the EMEA/CVMP guidelines 15 using the formula: % Efficacy = (Geometric Mean Flea Counts Controls Geometric Mean Flea Counts Treated) Geometric Mean Flea Counts Controls 100 These calculations were performed for each treatment group. Statistical differences in flea counts between the different groups were compared using the Wilcoxon Mann Whitney U test. RESULTS Arithmetic means of the flea counts in the control group at the different time points varied between 24 and 58. Geometric means of the flea counts on the untreated control cats are listed in Table 2. At 4 hours after infestation on all but one occasion (day 27 + 4 hours), imidacloprid showed the lowest flea counts of all groups. Twenty-four hours after infestation, all treatments showed the expected and similar high efficacy, with slightly increasing flea counts toward the end of the study. The group differences have been calculated based on explorative two-sided Wilcoxon Mann Whitney U test (alpha 5%). The analysis showed the following statistically significant differences between imidacloprid and the comparative compounds. Six hours after treatment, flea counts in the imidacloprid group were significantly reduced compared with the fipronil (S)-methoprene (P =.0152) and metaflumizone (P =.0108) groups. There was still a significant difference (P =.0022) between imidacloprid- and metaflumizone-treated animals 12 hours after reinfestation. Four hours after infestation on study day 6, flea counts in the imidacloprid group were significantly different versus all the other groups (sel- amectin, P =.0022; fipronil (S)-methoprene, P =.0065; metaflumizone, P =.0022). On study day 13, the imidacloprid group showed significantly different flea counts at 2 hours compared with the metaflumizone groups (P =.0368) and at 4 hours compared with the selamectin (P =.0087) and metaflumizone (P =.0238) groups. Four hours after infestation on study days 20, 27, and 34, flea counts in the imidacloprid group were significantly different from the ones in the selamectin group (P =.0455,.0390, and.0022, respectively).the 24- hour insecticidal flea efficacy of imidacloprid on study day 35 was significantly different versus selamectin (P =.0173) and fipronil (S)- methoprene (P =.0368). The efficacies (% flea reduction) of the four tested compounds on study days 0, 6, 13, 20, 27, and 34 are shown in Table 3. DISCUSSION The aim of this study was to evaluate the speed of flea kill of four ectoparasiticides registered worldwide for flea control on cats. Although the EMEA/CVMP guidelines 15 request flea efficacy 48 hours after treatment and subsequent reinfestation to be above 95% for a label recommendation, it is the common understanding that this is too long a period to avoid cats being exposed to flea saliva or to reduce the likelihood of pathogen transmission by fleas. The study reported here confirmed that most tested compounds provided a high efficacy at 24 hours after infestation. It is well known that the efficacy of most compounds against fleas decreases with time and lasts for about 4 weeks. In the present study, efficacies 28 days after treatment were 96% for imidacloprid, 83% for selamectin, 91% for fipronil (S)-methoprene, and 82% for metaflumizone. Calculated efficacies are influenced by flea counts on the controls, biologic variations, and group sizes. In this trial, cats were 180

T. Schnieder, S. Wolken, and N. Mencke thoroughly combed for flea counts 18 times during the study, a process that likely removed some active compound from the fur of treated cats. While the frequent combing of the animals in the study may be in contrast to the fate of the active ingredients under field conditions, one should consider the impact of intensive grooming by cats on the pharmacokinetics of topically applied products. The frequent combing in this study may have contributed to the generally lower efficacies in all treatment groups toward the end of the study. Imidacloprid maintained more than 90% efficacy until 35 days after treatment. The chloronicotinyl imidacloprid is known for its fast onset of efficacy against a wide range of agricultural pests and fleas. 4 This fast onset of action against pest insects and fleas includes the antifeeding effect reported for imidacloprid 18 were infested with 100 adult cat fleas each and flea counts were performed 3 and 8 hours later. The efficacies reported at the 3-hour count were 26.9% for imidacloprid and 24.3% for fipronil; at 8 hours, efficacies were 82.8% (imidacloprid) and 62.6% (fipronil). Because the study was performed to compare topically applied insecticides with the orally administered nitenpyram, which is eliminated and excreted in cats within 3 days, the evaluations were done only at 3 and 8 hours after treatment. The other recorded speed of flea kill study conducted in cats 23 compared selamectin with imidacloprid and fipronil (S)-methoprene using fleas from the special Kansas 1 colony (KS1) established and maintained as a closed colony at Kansas State University since 1990. After infestation with 100 adult cat fleas, comb counts were performed at 6, 12, 24, and 48 Six hours after treatment, flea counts in the imidacloprid group were already reduced by more than 60%. and the ability to quickly eliminate fleas from an infested host to prevent the fleas from biting. Studies evaluating the speed of flea kill of insecticides registered as veterinary medicinal remedies have been recorded previously. Spray and spot-on formulations of fipronil were evaluated in comparison with imidacloprid by Marchiondo and colleagues 19 and Cruthers and associates. 20 In these studies, efficacy was evaluated 8 hours after treatment and reinfestations or at 6 and 12 hours, respectively. In another study, the speed of flea kill was recorded for selamectin and imidacloprid at 6, 12, and 24 hours after treatment and reinfestation. 21 All of these studies, however, were conducted in dogs. Studies evaluating the speed of flea kill on cats are limited. In a study by Schenker et al, 22 cats hours after treatment (day 0) and after reinfestation (days 7, 14, 21, 28). On day 0, the onset of efficacy was fastest for imidacloprid at 86.7% versus 0% for selamectin and 28.6% for fipronil (S)-methoprene. At 12 hours, imidacloprid achieved 99.3% efficacy versus 59.7% for selamectin and 89.6% for fipronil (S)-methoprene. The study presented here confirmed that imidacloprid provides high and early efficacy. Six hours after treatment, flea counts in the imidacloprid group were already reduced by more than 60%. During the first 2 weeks after treatment, efficacy of imidacloprid was about 60% at 2 hours and exceeded 90% at 4 hours after reinfestation. The flea reduction at the 4- hour flea counts was significantly higher in the imidacloprid-treated group than in most of the 181

Veterinary Therapeutics Vol. 9, No. 3, Fall 2008 other groups during the first 2 weeks following treatment (see Results for P values). Mean flea counts in the control group showed considerable variations. Newly developed pupae were weighed, and an equivalent of 50 fleas was used for each infestation and reinfestation, resulting in flea counts ranging from 24 to 58. It is well known that individual grooming behavior, especially in cats, leads to different recovery rates. According to the guidelines, 40% to 75% 24 or 50% 15 of the infestation rate should be found on the controls. With a mean infestation of 37 fleas per cat, these requirements were met. The lowest infestation rate in the controls, 48% (arithmetic means, 24 fleas), was noted on study day 20 at 4 hours after reinfestation. CONCLUSION This study demonstrated that a single topical application of imidacloprid on cats at the recommended dosage provided a high level of efficacy in the early elimination of adult cat fleas. With this rapid onset of flea efficacy, imidacloprid is likely able to reduce flea bites and thus limit the risk of flea-induced or flea-transmitted diseases. REFERENCES 1. Beck W, Boch K, Mackensen H, et al. Qualitative and quantitative observations on the flea population dynamics of dogs and cats in several areas of Germany. Vet Parasitol 2006;137:130-136. 2. Rinaldi L, Spera G, Musella V, et al. A survey of fleas on dogs in southern Italy. Vet Parasitol 2007;148:375-378. 3. Dryden MW, Rust MK. The cat flea: biology, ecology and control. Vet Parasitol 1994;52:1-19. 4. Kraemer F, Mencke N. Flea Biology and Control. The Biology of the Cat Flea Control and Prevention with Imidacloprid in Small Animals. Heidelberg, Germany: Springer; 2001:192. 5. Rust MK, Dryden MW. The biology, ecology and management of the cat flea. Ann Rev Entomol 1997;42:451-473. 6. Beck W, Pfister K. Erhebungen zu Vorkommen und Epidemiologie von Flöhen bei Hunden und Katzen in Deutschland Ein Fragebogen-Survey. Berl Munch Tierarztl Wochenschr 2006;119:355-359. 7. Breitschwerdt ED, Levine JF, Radulovic S, et al. Bartonella henselae and Rickettsia seroreactivity in a sick cat population from North Carolina. Intl J Appl Res Vet Med 2005;3:287-302. 8. Breitschwerdt ED, Maggi RG, Duncan AW, et al. Bartonella species in blood of immunocompetent persons with animal and arthropod contact. Emerg Infect Dis 2007;13:938-941. 9. Chomel BB, Kasten RW, Floyd-Hawkins K, et al. Experimental transmission of Bartonella henselae by the cat flea. J Clin Microbiol 1996;34:1952-1956. 10. Kordick DL, Brown TT, Shin KO, Breitschwerdt EB. Clinical and pathological evaluation of chronic Bartonella henselae or Bartonella clarridgeiae infection in cats. J Clin Microbiol 1999;33:1536-1547. 11. Lappin MR, Griffin B, Brunt J, et al. Prevalence of Bartonella species, haemoplasma species, Ehrlichia species, Anaplasma phagocytophilum, and Neorickettsia risticii DNA in the blood of cats and their fleas in the United States. J Feline Med Surg 2006;8:85-90. 12. Rolain JM, Franc M, Davoust B, Raoult D. Molecular detection of Bartonella quintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, and Wolbachia pipientis in cat fleas in France. Emerg Infect Dis 2003;9: 338-342. 13. Vobis M, D Haese J, Mehlhorn H, Mencke N. Evidence of horizontal transmission of feline leukemia virus by the cat flea (Ctenocephalides felis). Parasitol Res 2003;91:467-470. 14. Halliwell REW. Flea allergy dermatitis. In: Kirk RW, ed. Current Veterinary Therapy VIII. Philadelphia: WB Saunders; 1983:496-499. 15. Committee for Medicinal Products for Veterinary Use (CVMP). Guideline for the Testing and Evaluation of the Efficacy of Antiparasitic Substances for the Treatment and Prevention of Tick and Flea Infestation in Dogs and Cats. London: European Medicines Agency; 2007. Accessed August 2008 at www.emea.europa.eu/pdfs/ vet/ewp/000500-rev.2.pdf. 16. Gaulliard JM. Fipronil: a broad spectrum insecticide. Phytoma 1996;488:59-61. 17. Holzmer S, Hair JA, Dryden MW, et al. Efficacy of a novel formulation of metaflumizone for the control of fleas (Ctenocephalides felis) on cats. Vet Parasitol 2007; 150:219-224. 18. Rust MK, Hinkle NC, Waggoner M, et al. The influence of imidacloprid on adult cat flea feeding. Comp Cont Educ Vet Pract 2001;23(suppl):18-21. 19. Marchiondo AA, Green SE, Plue RE, et al. Compara- 182

T. Schnieder, S. Wolken, and N. Mencke tive speed of kill of Frontline spray, Frontline Top Spot and Advantage against adult cat fleas (Ctenocephalides felis) on dogs [no. 30]. Proc. 5th Int Symp Ectopara Pets 1999. 20. Cruthers L, Guerrero J, Robertson-Plouch C. Evaluation of the speed of kill of fleas and ticks with fipronil or imidacloprid [no. 27]. Proc. 5th Int Symp Ectopara Pets 1999. 21. Everett R, Cunnigham J, Arther B, et al. Comparative evaluation of the speed of flea kill of imidacloprid and selamectin on dogs. Vet Ther 2000;1:229-234. 22. Schenker R, Tinembart O, Humbert-Droz E, et al. Comparative speed of kill between nitenpyram, fipronil, imidacloprid, selamectin and cythioate against Ctenocephalides felis (Bouché) on cats and dogs. Vet Parasitol 2003;112:249-254. 23. Dryden MW, Smith V, Payne PA, McTier TL. Comparative speed of kill of selamectin, imidacloprid, and fipronil-(s)-methoprene spot-on formulations against fleas on cats. Vet Ther 2005;6:228-236. 24. Marchiondo AA, Holdsworth PA, Green P, et al. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of parasiticides for the treatment, prevention and control of flea and tick infestation on dogs and cats. Vet Parasitol 2007;145:332-344. 183