Evaluation of the Ovicidal Activity of Lufenuron and Spinosad on Fleas’ Eggs from Treated Dogs Michael W

Evaluation of the Ovicidal Activity of Lufenuron and Spinosad on Fleas’ Eggs from Treated Dogs Michael W. Drydena Patricia A. Paynea Smith Vickia Ritchie L. Debraa Allen Lynnb aDepartment of Diagnostic Medicine /Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS. 66503. bNovartis Animal Health US Inc., 3200 Northline Ave. Suite 300, Greensboro NC. 27408

Corresponding author: Michael W. Dryden phone: Department of Diagnostic Medicine /Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS. 66503. (785) 532-4613; FAX: (785) 532-4851; e-mail address: [email protected]

KEY WORDS: Ctenocephalides felis, fleas, into rearing media to determine adult flea KS1, dog, flea egg, lufenuron, spinosad, emergence. The percent adult flea emer- milbemycin oxime gence from eggs collected from untreated ABSTRACT control dogs ranged from 19.3% to 69.4% throughout the study. Eggs collected from This study was conducted to compare the dogs treated with spinosad had percent adult production and viability of flea eggs on emergence of 49.2%, 20.3%, and 37.9% on dogs treated with either the combination of collection days 30, 60, and 90, respectively. milbemycin oxime-lufenuron or spinosad. Even though eggs were collected on days Fifteen dogs were randomly distributed into 51, 72, and 79, those eggs were not viable. three treatment groups and housed individu- No adult fleas developed from eggs collect- ally. Dogs in group 1 served as untreated ed from dogs treated with lufenuron-milbe- controls, dogs in group 2 were treated with mycin oxime over the entire 90-day study. milbemycin oxime-lufenuron, and dogs in group 3 were treated with spinosad. Prod- INTRODUCTION ucts were administered orally according to The elimination of fleas on dogs and cats label directions on days 0, 30, and 60. All and in the surrounding environment can be dogs were infested with 250 cat fleas,Cteno - achieved by killing adult fleas on the host cephalides felis, KS1 strain, on days 14, 28, and eliminating environmental life stages. 42, 49, 58, 70, 77, and 88. Forty-eight hours Several residual insecticides such as dinote- after each infestation, dogs were placed furan, fipronil, imidacloprid, selamectin, and into egg collection cages for 3 hours. Flea spinosad do an excellent job of killing exist- eggs were collected, counted, and placed ing adult fleas on the pet.1-8 However, after 198 Vol. 10, No. 3, 2012 • Intern J Appl Res Vet Med. treatment, the pet still lives in an infested conducted in Alabama, North Carolina, and home environment with flea eggs, larvae, Ireland, using dogs 14 to 16 weeks of age, pupae, and emerging fleas. These remain- was given.15 The combined efficacy of the ing life stages provide an ongoing source of three trials on day 29 was remarkably only adult fleas that will continually reinfest pets 2% .15 It is unknown if this poor residual treated only with adulticidal compounds.9 efficacy was due to variability in flea strain Historically, repeated application of insec- or the age of the dogs. ticides and insect growth regulators formu- Then, in 2011, two studies were pub- lated as environmental sprays were used in lished using the KS1 flea strain.7 This pub- an attempt to eliminate the premises flea lication detailed two studies where the ef- life stages.9,10 However, client compliance ficacy of spinosad against the KS1 flea strain with environmental treatment protocols was in dogs was 22.1% and 32.5%, respectively, disappointing and often resulted in recurrent on day 29 post-treatment.7 In each of the infestations and client dis-satisfaction.. above reported studies, adulticidal efficacy New insecticides and insect growth began to wane at approximately day 15 post- regulators in convenient dosage forms treatment. Additionally, in a 2011 study (spot-ons, tablets, oral suspensions, and conducted in France, the residual efficacy of injectables),applied directly to the pet, pro- spinosad and fipronil against fleas infest- vide prolonged residual activity and have ing dogs at 24 and 48 hours, the efficacy of dramatically improved pet owner compli- spinosad was 85.0% and 89.0%, respectively ance, helped to eliminate recurrent infesta- on day 28 post-infestation was evaluated.16 tions, and improved client satisfaction.11-12 Because the adulticidal efficacy of Although residual flea adulticides spinosad has been shown to wane in several such as dinotefuran, fipronil, imidacloprid, of the aforementioned studies, we conducted selamectin, and spinosad provide prolonged this study to determine if fleas survived long adulticidal activity, efficacy may decrease enough on spinosad-treated dogs to feed, throughout the month following administra- mate, and produce viable eggs. Therefore, tion. 2,7,13-17 As the residual efficacy decreas- this study was conducted to compare the es, adult female fleas may live longer than production and viability of flea eggs on dogs 24 hours and start producing eggs.13,14,16,18 treated with either the combination of milbe- The production of viable eggs may extend mycin oxime/lufenuron or spinosad and the infestation within the home. infested with the KS1 flea strain. We used Providing ovicidal (lufenuron, me- lufenuron as a positive reference control for thoprene, pyriproxyfen, or selamectin) egg viability. activity as part of a flea treatment protocol MATERIALS AND METHODS is extremely beneficial because any eggs Fleas produced by adults surviving adulticide treatment will be rendered nonviable. 6,13,19-21 The KS-1 strain of Ctenocephalides felis was used in the study. The KS1 cat flea An orally administered residual adul- strain of Ctenocephalides felis has been ticide flea product containing spinosad has maintained as a closed colony at Kansas been approved for use in the U.S. market. In State University since 1990. The colony 2010 and 2011, studies indicated that under was originally established by removing certain conditions or with some flea strains, fleas from dogs and cats at a local animal the residual efficacy of spinosad decreases shelter in Riley county Kansas. In-vitro and markedly towards the end of the month.7,15 in-vivo evaluations have indicated that the In a presentation on spinosad residual ef- KS1 strain has some level of resistance or ficacy given at the 55th Annual Meeting reduced susceptibility to carbaryl, chlorpy- of the American Association of Veterinary riphos, fenthion, fipronil, imidacloprid, per- Parasitologists, results from three studies methrin, pyrethrins, and spinosad. 2,7,13,18, 22-26

Intern J Appl Res Vet Med • Vol. 10, No. 3, 2012. 199 Due to the hardiness of this flea strain, it has 14, 28, 42, 49, 58, 70, 77, and 88. This cor- been used extensively as an indicator of flea responds to 14 and 28 days following the product performance. In fact, almost every first treatment; 12, 19, and 28 days follow- major flea product has been tested against ing the second treatment; and 10, 17 and 28, this flea strain either before or shortly after days following the third dosing. introduction to the US market. Forty-eight hours after each flea infes- Animals and Housing tation, the dogs were removed from their This study used 15 purpose bred mongrel individual kennels and placed into stainless dogs between 6 and 12 months of age steel metabolic cages with expanded metal housed in individual kennels. The line of floors over stainless steel solid surface col- dogs is one approved by the FDA for pivotal lection pans for flea egg collections. Dogs studies; the age range is standard and usual were housed in the metabolic cages for 3 for conducting investigations. Dogs must be hours. After 3 hours, the dogs were brushed housed in individual kennels for individual to dislodge any remaining eggs. The dogs observation and to obtain accurate data. were then removed from the metabolic During the 14-day preconditioning phase, no cages, given one dose of Capstar® (niten- medications or pesticides were administered pyram) (minimum dose 1.0 mg/kg) orally, or used, and none of the dogs were bathed or and returned to their original kennels. shampooed. All animal care and procedures All flea eggs collected in the stainless conformed to the established guidelines of steel pans were counted. Up to 100 eggs the Kansas State University Institutional from each dog were placed in glass Petri Animal Care and Use Committee and those dishes containing growth media (sand, of Novartis Animal Health. ground dog chow, brewer’s yeast, and Animal Selection and Randomization dried blood), and held in a growth rearing On day 1, the dogs were weighed and chamber (I-30B, Percival Manufacturing randomly distributed into three groups, five Co, Boone, IA; 27-28° C, 70-80% relative dogs in each group. humidity, 24 hours dark). At 10 – 12 days after egg collection, pupae (and any larvae Treatments and Infestation that had not completed cocoon formation) Dogs allocated to group1 served as untreated were sifted from the media and placed into controls. Dogs in group 2 were treated with plastic vials with lids. Adult emergence was ® ® Sentinel Flavor Tabs (Novartis Animal determined by counting emerged adult fleas Health) milbemycin oxime - lufenuron at about 28 days after egg collection. Per- (minimum dosage 0.5mg/kg milbemycin ox- sonnel conducting egg counts, egg collec- ime, 10mg/kg lufenuron) per label directions tions, and viability assessments were blinded immediately after a meal. Dogs in group 3 to treatment group allocation. were treated with the appropriate size Com- Statistics fortis® (Elanco) spinosad tablet (minimum dosage 30 mg/kg) per label directions, with An analysis of variance (ANOVA, SAS a meal. PROC MIXED) was used to compare the percent of adult fleas that developed Dogs were observed to ensure they had from eggs recovered from each dog in the consumed a meal before treatment and ob- milbemycin oxime - lufenuron and spinosad served for 1 hour after tablet administration treatment groups. Data on the percent of to monitor for emesis. Dogs were treated on adult fleas was transformed using an (arcsine days 0, 30, and 60. (square root (percent flea hatch))) trans- Flea Infestations and Evaluation of Egg formation to normalize the variance. The production and Ovicidal Aactivity ANOVA was performed at all days post- All dogs were infested with 250 fleas, KS1 treatment. strain, 1 to 5 days post emergence, on days The following hypothesis was tested:

200 Vol. 10, No. 3, 2012 • Intern J Appl Res Vet Med. Table 1: Evaluation of the Ovicidal Activity of Lufenuron and Spinosad on Flea Eggs from Treated Dogs.

Collection Collection Collection days days days Treatment Day Day Treatment Day Day Day Treatment Day Day Day Day 0a 16b 30 Day 30 44 51 60 Day 60 72 79 90 Controls Total eggs 326 500 400 430 398 278 230 226 incubatedc Total adults 63 347 156 201 116 122 68 48 producedd % 19.3b 69.4b 39b 46.7b 29b 43.9b 29.6b 21.2b Developmente Lufenuron – milbemycin oxime Total eggs 500 500 500 500 500 500 317 ND* incubated Total adults 0 0 0 0 0 0 0 0 produced % 0a 0a 0a 0a 0a 0a 0a 0a Development Spinosad Total eggs 0 380 0 13 256 2 36 256 incubated Total adults 0 187 0 0 52 0 0 97 produced % 0na 49.2b 0na 0a 20.3b 0b 0b 37.9b Development a Dogs in group #2 were orally administered Sentinel® Flavor Tabs®(Novartis Animal Health) milbemycin oxime - lufenuron (minimum dosage 0.5mg/kg milbemycin oxime, 10mg/kg lufenuron) per label directions immediately after a meal. Dogs in group #3 were orally administered the appropriate size of Comfortis® (Elanco) spinosad (minimum dosage 30 mg/kg) per label directions, with a meal. b Dogs were infested with 250 C. felis 48 hours prior to each egg collection day. c. Five dogs in each treatment group and a maximum of 100 eggs/dog used for viability assessments. d Number of adult fleas emerging 28 days following egg collection. e Percent of adult fleas emerging from eggs collected. *ND – no data: no eggs were collected due to procedural error. Numbers in columns with unlike superscripts are significantly different at p < 0.05 a) Ho: Mean (Trans(Group X para- vs spinosad . sites +1)) = Mean (Trans(Group Y parasites Rejection of the null hypothesis (statis- +1)), vs Ha: Mean (Trans(Group X parasites tical significance) at a specific time point +1)) < Mean (Trans(Group Y parasites+1)) indicated that the product under test was Where: superior (lower number of adult fleas) to the Trans = (arcsine (square root (percent flea comparator product at that time point. hatch))), and, Parasites = percent of adult The data were grouped by month and fleas recovered from a random subset of the over all months, and categorized as either total eggs collected for group comparisons positive for adults developed from collected where X ≠ Y, where group comparisons eggs and assigned a score of ‘1,’ or negative were: milbemycin oxime - lufenuron vs for adults developed from collected eggs and untreated control, spinosad vs untreated assigned a score of ‘0.’ The percent of dogs control, and milbemycin oxime - lufenuron exhibiting inhibition was then calculated

Intern J Appl Res Vet Med • Vol. 10, No. 3, 2012. 201 as the number of dogs exhibiting complete ratory maintained flea strains for various control (ie, no adults developed) divided by insecticides,2,7,13,18, 22-26 numerous field col- the total number of dogs in the group. lected cat flea strains have been identified 26-28 RESULTS that were even less susceptible. Several field collected cat flea strains were found Eggs collected from untreated control dogs to be less susceptible to organophosphates had percent adult emergence ranging from a and pyrethroids than the KS1 strain.26 In low of 19.3% to a high of 69.4% (Table 1). 2001, a strain designated as R6 from a Viability of eggs in some of the collections veterinary practice in Lakeland Florida was was likely reduced due to urine and feces significantly less susceptible to fipronil than contamination of egg collection pans. Urine the KS1 strain.27 Additionally, a number and feces contamination occurred sporadi- of field collected flea strains were found to cally in all treatment groups. be less susceptible to imidacloprid than the Eggs collected from dogs treated with KS1 strain.28 spinosad had percent adult emergence of Lufenuron provided 100% ovicidal 49.2%, 20.3%, and 37.9% on collection days activity against the KS1 flea strain through- 30, 60, and 90, respectively. Even though out this study; no viable eggs were collected eggs were collected on days 51, 72 and 79, from dogs treated with lufenuron-milbemy- those eggs were not viable (Table 1). At no cin oxime. collection period over the 90 day study did Flea infestations can be dramatically any adult fleas develop from eggs collected reduced or eliminated using topical and/or from dogs treated with lufenuron-milbemy- systemic flea adulticides if flea death occurs cin oxime (Table 1). prior to initiating reproduction or if repro- DISCUSSION duction is directly inhibited. One previous While a previously published study had study provided evidence of remarkable shown a remarkably high level of residual residual adulticidal efficacy (100% at 40mg/ activity of spinosad,8 several other investi- kg) of spinosad at day 30 post-treatment.8 gations demonstrated a significant reduction However, another study demonstrated in adulticide efficacy after Day 15 against reduced adulticidal efficacy (89% at day a variety of flea strains.7,15,16 This current 30) with limited production of flea eggs study revealed that egg production from from dogs 30 days following treatment with spinosad treated dogs infested with the spinosad.16 KS1 flea strain began within 12 to 19 days Data from the current study shows that following treatment, and that by day 30 viable eggs were produced on spinosad post-treatment for each dosing period, many treated dogs infested with the KS1 strain of of those eggs were viable. cat fleas between monthly doses. Therefore, When conducting efficacy trials with variability in susceptibility to spinosad may laboratory maintained flea strains, it is often also exist in other flea populations. difficult to assess how relevant the data is Acknowledgements, Funding, and to real world “in-clinic” situations. How Conflict of Interest Statements: the data generated in individual studies This study was funded in part by Novartis relates to clinical practice is often difficult Animal Health US, Inc. Dr Dryden per- to determine. Efficacy results are specific forms consulting work for Novartis Animal to the conditions of the individual study Health and other pharmaceutical companies. and the susceptibility of the laboratory flea Drs Dryden and Payne have been sponsored strain used in those studies. The KS1 flea to present lectures by Novartis Animal strain has been extensively evaluated over Health and other pharmaceutical companies. the past 2 decades, and while the KS1 flea Vicki Smith and Debra Ritchie are research strain is one of the least susceptible labo- technicians working in laboratories super-

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