Ninth International Parasite Control Symposium

Transcription

1 Ninth International Parasite Control Symposium Vol. 30, 6(B) June 2008 Proceedings of a symposium held at the 2008 NAVC Conference A Supplement to Compendium: Continuing Education for Veterinarians TM

2 Bayer was proud to sponsor this symposium on parasite control at the 2008 NAVC Conference. Bayer has an ongoing commitment to the veterinary profession, exemplified not only by its sponsorship of educational programs with up-to-date information on major topics of relevance but also by its continued commitment to research and development in the field of animal health. Bayer HealthCare AG Animal Health Leverkusen Germany Copyright 2008, Bayer HealthCare AG All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any electronic or mechanical means, by photocopying or recording, or otherwise without the prior permission of the copyright owner. Compilation and publication of this information constitute neither approval nor endorsement by the copyright holder or the publisher. The opinions expressed in this publication are those of the authors and do not necessarily reflect the points of view of the company or companies that manufacture and/or market any of the products mentioned. This manual is primarily intended as an overview of a scientific topic, as dosages are cited for several pharmacologic agents that may not be the same as those stated on their data sheets in a given country. In addition, dosages are included for pharmacologic products that may not be licensed for veterinary use in a given country. Veterinary practitioners should consult their local data sheet or equivalent for licensing/prescribing conditions in their particular country. Queries relating to the enclosed information should be addressed to the Bayer Animal Health Business Group in each individual country. Printed in USA. Designed and published by Veterinary Learning Systems, a division of MediMedia USA. Flea image 2008 Carolina K. Smith/Shutterstock.com This information has not been peer reviewed and does not necessarily reflect the opinions of, nor constitute or imply endorsement or recommendation by, the Publisher or Editorial Board. The Publisher is not responsible for any data, opinions, or statements provided herein.

3 Ninth International Parasite Control Symposium Vol. 30, 6(B) June 2008 Proceedings of a symposium held at the 2008 NAVC Conference A Supplement to Compendium: Continuing Education for Veterinarians TM

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5 Contents New Insights into Flea Resistance 4 Byron L. Blagburn, MS, PhD Michael W. Dryden, DVM, MS, PhD Patricia A. Payne, DVM, PhD Michael K. Rust, PhD Dennis E. Jacobs, DVM, PhD Ross Bond, DVM, PhD, DECVD Melanie J. Hutchinson, BSc, HND Ian Denholm, PhD Heinz Mehlhorn, PhD Imidacloprid/Moxidectin (Topical Solution) 7 to Prevent Heartworm (Dirofilaria immitis) Infection and Flea (Ctenocephalides felis) Infestation in Ferrets John McCall, PhD Ulf Wenzel, DVM, Dr. med. vet. Josef Heine, DVM, Dr. med. vet., DEVPC Robert G. Arther, DVM, PhD Sam D. Charles, BVSc, PhD, DACPV Heidrun Mengel, DVM, Dr. med. vet. Roland Schaper, DVM, Dr. med. vet. Demodex canis Control in Dogs 13 Ralf S. Mueller, DVM, Prof., DACVD, FACVSc, DECVD Comparative Speed of Efficacy of Imidacloprid against 18 Ctenocephalides felis in Experimentally Infested Cats Thomas Schnieder, DVM, Prof., DEVPC Sonja Wolken, DVM, Dr. med. vet. Norbert Mencke, DVM, Dr. med. vet., DEVPC Lice Management Options: Efficacy of Three 25 Imidacloprid-Containing Formulations against Natural Lice (Trichodectes canis) Infestations in Dogs Dorothee Stanneck, DVM, Dr. med. vet., DEVPC Jennifer Ketzis, PhD, BSc, MSc, MCert Proj Mngt Jeanie Doyle, BSc (Honors) Margret Fisher, DVM, DEVPC

6 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM New Insights into Flea Resistance Byron L. Blagburn, MS, PhD a, * Michael W. Dryden, DVM, MS, PhD b Patricia A. Payne, DVM, PhD b Michael K. Rust, PhD c Dennis E. Jacobs, DVM, PhD d Ross Bond, DVM, PhD, DECVD d Melanie J. Hutchinson, BSc, HND e Ian Denholm, PhD f Heinz Mehlhorn, PhD g a College of Veterinary Medicine, Auburn University, Auburn, Alabama, USA b College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA c College of Natural and Agricultural Sciences, University of California, Riverside, Riverside, California, USA d The Royal Veterinary College, University of London, Hatfield, Hertfordshire, UK e Shernacre Enterprise, Shernacre Cottage, Malvern, Worcester, UK f Rothamsted Research, Harpenden, Hertfordshire, UK g Institut für Zoomorphologie, Zellbiologie und Parasitologie, Heinrich-Heine Universität, Düsseldorf, Germany The preceding two decades have seen the introduction of revolutionary flea control products. Many would say that the outstanding efficacy of these products has virtually eliminated many of the problems associated with flea control. This is not so in some cases. To take full advantage of the new flea control products, we must use them wisely and understand how fleas behave. Failure to use these new agents properly or to appreciate the biological behavior of target parasites can result in real or perceived product failures. In this paper, we revisit important points about the biological behavior of fleas and compliance issues in flea control. We also discuss the necessity to monitor the performance of these products in the field as a safeguard for protecting their continued efficacy. FLEA BIOLOGY AND ITS EFFECTS ON PRODUCT EFFICACY Much information is now available on the biology and behavior of fleas. 1 Remember first that the vast majority of life stages (eggs, larvae, and pupae) of the flea remain off the host and inaccessible in many cases to our on-animal agents. This is particularly important for quiescent adults in the pupa (cocoon). Latent, unstimulated adults can emerge over extended developmental periods, leaving the unknowing pet owner with the false presumption that control efforts are *Corresponding author: B. Blagburn, Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA; phone, +1 (334) ; fax, +1 (334) ; , blagbbl@auburn.edu. Ectoparasiticide resistance is not restricted to a single chemical class of insecticides but has been demonstrated for many inorganic and synthetic organic compounds. having little effect. This is particularly true when these stages are in locations where the pet spends time. We must also remember that the flea life cycle is driven by abiotic factors such as temperature and relative humidity and that these conditions can affect the length of the life cycle, depending on geographic location. Cooler, drier climates result in longer life cycles, while warmer, humid climates support shorter cycles. Another feature of flea biology that should guide us in our control efforts is the demonstrated lack of host specificity shown by Ctenocephalides felis. The presence of cat fleas on many mammalian species, including some urban-adapted wildlife, can create sources of fleas in the pet s environment that are not immediately apparent to the pet owner. Finally, we should be reminded of the prolific egg-laying capabilities of the female flea. A small residual population of surviving fleas can produce many eggs in a short time. We now know that fleas survive even the most effective of our agents and that these fleas can serve as a continuing environmental source of reinfestation. If we can remember and communicate these points to pet owners, not only can we select and use products more effectively, we can also eliminate many client inquiries about perceived lack of efficacy. COMPLIANCE AND ECTOPARASITE CONTROL The dynamics and reasons for the recurrence of a flea problem may 4 Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

7 NAVC CONFERENCE, JANUARY 2008 TABLE 1. Differences between Product Failure due to Noncompliance and Insecticide Resistance Noncompliance Fleas tend to reappear more quickly when pet owners do not comply with product recommendations. If noncompliance is the issue, the flea problem improves when compliance behavior improves. If noncompliance is the issue, the flea problem may not improve when the client switches to a different product. Insecticide Resistance If resistance is the issue, fleas tend to reappear more slowly because of the slow resistance selection process. If resistance is the issue, the flea problem may not improve when compliance behavior improves. If resistance is the issue, the flea problem may improve if a different insecticide is used. Figure 1. Selecting flea eggs for placement in imidacloprid-treated media. Figure 2. Counting adult fleas on day 28. be different depending on whether compliance or resistance is the cause (Table 1). It is important to question the client regarding his or her observations to help determine why flea or tick control products may be failing. Surveys indicate that compliance represents an increasing threat to successful parasite control. 2 Efforts to encourage pet owner compliance must be intensified. Until we have achieved an acceptable level of pet owner responsibility, differentiating between product failure and noncompliance will be difficult. INTERNATIONAL IMIDACLOPRID SUSCEPTIBILITY MONITORING Ectoparasiticide resistance represents an increasingly prevalent problem facing industry and agriculture. 3 Resistance is not restricted to a single or even a few chemical classes of insecticides but has been demonstrated for many inorganic and synthetic organic compounds. 4 The majority of ectoparasites with demonstrated resistance to pesticides are crop pests. However, it was estimated that approximately 40% of resistant species are ectoparasites of humans or other animals. 5 The cat flea (C. felis) is the flea most commonly observed on pets and other animals in the United States and Europe. 1 According to the World Health Organization, C. felis is resistant to more insecticides in more chemical categories than has been reported for any other species of flea. 6 Despite the known capability of the cat flea to develop resistance to commonly applied insecticides, there existed no concerted effort or attempt to monitor the susceptibility of C. felis to currently marketed flea insecticides. In response to the need for such a monitoring initiative, a team of scientists, with support from Bayer Animal Health, developed an in vitro susceptibility monitoring assay using imidacloprid (Advantage Topical Solution) as the test compound and laboratory and field strains of C. felis. 7 9 The assay is conducted using flea eggs provided by participating veterinary clinics (Figure 1). Imidacloprid is solubilized in acetone and mixed at the target concentration in flearearing media. C. felis eggs are added to the laboratory dishes containing imidacloprid-treated media, and additional eggs are added to dishes containing media treated with acetone only. Dishes are incubated under conditions of temperature and relative humidity that promote optimal flea development (26 C to 28 C and 80% relative humidity) for 28 days after incubation with imidacloprid-treated media. Dishes are examined periodically for larval and pupal development. At day 28, the numbers of adult fleas present in the treated and control dishes are counted and compared (Figure 2). If fleas Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

8 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM TABLE 2. Flea Strains with 5% Adult Survival in the Imidacloprid Flea Larval Bioassay* Source/Year % Adult Flea Strain Acquired Emergence Comments R-003-FL-02 Florida/ % Pets in the household had been treated with imidacloprid. The last treatment was applied 6 to 12 months before collection. Further and extended analysis conducted with this isolate demonstrated that the LD 50 and LD 95 were no different than those from isolates collected and sampled from the field during the development of the assay. Results were interpreted as normal variations in susceptibility of individuals in this strain. B-006-CA-03 California/ % This animal was never treated for flea infestation. The assay was repeated several times in different laboratories with no survival in the subsequent studies. Results of the initial assay were interpreted as normal variations in susceptibility of individuals in this strain. D-015-LA-03 Louisiana/ % No available information on prior use of flea control products was available. A follow-up study showed no survival. Results of the initial assay were interpreted as normal variations in susceptibility of individuals in this strain. R-002-CA-03 California/ % Flea eggs were acquired from cats in a multiple-cat household. Cats had been treated with imidacloprid. When the last treatment was applied was unknown. The assay was repeated at the diagnostic dose of 3 ppm with no survivors. No further testing was performed. Results of the initial assay were interpreted as normal variations in susceptibility of individuals in this strain. R-003-CA-03 California/ % Pets had been treated with lufenuron. The last treatment for flea infestation was 2 to 6 months before collection. The laboratory was unsuccessful in rearing the next generation for additional testing. Results of the initial assay were interpreted as normal variations in susceptibility of individuals in this strain. R-002-CA-04 California/ % Flea eggs were collected from a multiple-cat household. Cats reportedly had been exposed to flea collars (active ingredient was unknown) but no other flea control products. A follow-up study showed no survivors. Results of the initial assay were interpreted as normal variations in susceptibility of individuals in this strain. *All strains were reevaluated and found to be fully susceptible to imidacloprid. survive in imidacloprid-treated media, they are propagated by placement on cats and subjected to further evaluation. From 2001 through 2006, 863 field flea isolates were collected and evaluated in laboratories in Germany, the United Kingdom, and the United States. Six strains in which survival of adult fleas equaled or exceeded 5% were identified (Table 2). These strains were reevaluated using either a diagnostic (single) dose or a full dose-response strategy and found to be fully susceptible to imidacloprid. 7 9 Our inability to identify resistant fleas thus far may not indicate definitively that resistance to imidacloprid does not exist in the pet population. However, it does imply that if fleas that are resistant to imidacloprid are present in pet populations, their numbers must be low. The efforts of the International Imidacloprid Flea Susceptibility Monitoring Study are ongoing. We will continue to sample flea populations throughout Europe, the United Kingdom, and the United States. If reduced susceptibility is confirmed in our in vitro assay using submitted field strains, the team will move forward to investigate metabolic and molecular bases for the reduced susceptibility. This novel effort to monitor susceptibility of flea populations to imidacloprid is a prudent step in ensuring successful flea control. ACKNOWLEDGMENTS The authors acknowledge additional current and past members of the imidacloprid susceptibility monitoring team: Dr. David Bledsoe, Dr. Terry Hopkins, Dr. Joe Hostetler, Dr. Norbert Mencke, Ms. Iris Schroeder, Dr. Michael Vaughn, and Dr. Sarah Weston. REFERENCES 1. Rust MK, Dryden MD. The biology, ecology, and management of the cat flea. Annu Rev Entomol 1997;42: American Animal Hospital Association (AAHA). The Path to High- Quality Care. Practical Tips for Improving Compliance. AAHA; Brown AW, Pal R. Insecticide Resistance in Arthropods. WHO Monograph Series No. 38. Geneva: World Health Organization; Bossard RL, Hinkle NC, Rust MK. Review of insecticide resistance in cat fleas (Siphonaptera: Pulicidae). J Med Entomol 1998;35: Roush RT. Occurrence, genetics and management of insecticide resistance. Parasitol Today 1993;9(5): Rust MK. Insecticide resistance in fleas. In: Knapp FW, ed. Proc Int Symp Ectoparasites Pets 1993: Blagburn BL, Dryden MW, Payne P, et al. New methods and strategies for monitoring susceptibility of fleas to current flea control products. Vet Ther 2006;7(2): Rust MK, Waggoner M, Hinkle NC, et al. Development of a larval bioassay for susceptibility of cat fleas (Siphonaptera: Pulicidae) to imidacloprid. J Med Entomol 2002;39: Rust MK, Denholm I, Dryden MW, et al. Determining a diagnostic dose for imidacloprid susceptibility testing of field-collected isolates of cat fleas (Siphonaptera: Pulicidae). J Med Entomol 2005;42: Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

9 NAVC CONFERENCE, JANUARY 2008 Imidacloprid/Moxidectin (Topical Solution) to Prevent Heartworm (Dirofilaria immitis) Infection and Flea (Ctenocephalides felis) Infestation in Ferrets John McCall, PhD a Ulf Wenzel, DVM, Dr. med. vet. b Josef Heine, DVM, Dr. med. vet., DEVPC c Robert G. Arther, DVM, PhD d Sam D. Charles, BVSc, PhD, DACPV d Heidrun Mengel, DVM, Dr. med. vet. e Roland Schaper, DVM, Dr. med. vet. c, * a TRS Labs Inc., Athens, Georgia, USA b Tierarztpraxis Dr. Wenzel, Leipzig, Germany c Bayer HealthCare AG, Animal Health, Leverkusen, Germany d Bayer HealthCare LLC, Shawnee Mission, Kansas, USA e KoVet, Leipzig, Germany The popularity of pet ferrets in heartworm-endemic and nonendemic areas is growing, with ferret ownership in the United States currently being estimated in the range of 8 to 10 million. 1 In Europe, although no published data are available, a recent market research study estimates the number of pet ferrets to be 115,000, 105,000, and 100,000 in Germany, Italy, and the United Kingdom, respectively. 2 The domestic ferret (Mustela putorius furo; Figure 1) is susceptible to naturally acquired and experimentally induced infections of Dirofilaria immitis (Figure 2). 3 There are relatively few reports on host parasite relationships between D. immitis and ferrets. 4,5 Laboratory studies have shown that ferrets are highly susceptible, with infection and recovery rates similar to those achieved in dogs and higher than those seen in cats. 6 Microfilaremia is usually of low concentration and transient in nature, similar to that seen in heartworm-infected cats. 6 A definitive diagnosis can be made from ELISA-based antigen tests, echocardiography, or angiography. Suggestive radiographic findings require additional supportive information to confirm a tentative diagnosis. 2 Prevention has been shown to be effective with oral ivermectin at a dosage of 3 µg/kg in experimental infections. 6 Currently, there is no approved compound available for the prevention or treatment of D. immitis in ferrets. *Corresponding author: R. Schaper, Bayer HealthCare AG, Animal Health, Global Veterinary Services, Leverkusen, Germany; phone, +49 (0) ; fax, +49 (0) ; , roland.schaper@ bayerhealthcare.com. A combination product containing 10% imidacloprid/ 1% moxidectin will protect ferrets against adult D. immitis infection and C. felis infestation. Flea infestations in ferrets are frequently reported. 7,8 The ubiquitous cat flea (Ctenocephalides felis; Figure 3) infests ferrets as it does other host species. 9 It is unknown how satisfactorily ferrets host cat fleas. Experimental infestations of ferrets with cat fleas have clearly demonstrated the capability of C. felis to maintain an infestation on ferrets. 10 In a household contaminated with C. felis by other flea hosts such as cats and dogs, pet ferrets are at risk for becoming infested with fleas from that environment, similar to any other potential host. Further, ferrets may suffer from flea-derived dermatologic conditions caused by hypersensitivity to flea saliva, such as papulocrustous dermatitis and self-inflicted alopecia. 11 The efficacy of imidacloprid against experimental infestations of cat fleas on ferrets has previously been reported. 10,12 The flea study reported here is the first report to evaluate the efficacy of imidacloprid in combination with moxidectin against C. felis in ferrets. MATERIALS AND METHODS Heartworm Study This study was conducted according to the Guideline on Good Clinical Practice, EMEA/CVMP/VICH/595/98-final, July 4, Twelve male ferrets were placed on acclimatization at the study facility for 7 days. During the acclimatization period (day 37 to day 31), daily general health observations were performed. All ferrets selected were free of D. immitis microfilariae and adult D. immitis antigen before the artificial infection, as determined by microfilariae and antigen blood examinations conducted from blood samples collected on day 36. Diagnosis for the microfilarial examination was based on Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

10 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM TABLE 1. Details of Ferrets Included in the Heartworm Study 2008 Dmitry Grivenko/Shutterstock.com Figure 1. Domestic ferret (Mustela putorius furo). Weight at Approximate Age Animal No. Sex Day 1 (kg) at Day 0 (months) Treatment Group 321A M A M A M A M A M A M Average Control Group 225A M A M A M A M A M A M Average Figure 2. Ferret heart with heartworms (Dirofilaria immitis). the modified Knott s test. The Dirocheck Antigen Test kit (Synbiotics Corporation, San Diego, CA, USA) was used for the adult D. immitis antigen screening. Each ferret underwent a physical examination on day 35. On day 30, the 12 ferrets were artificially infected with 25 infective third-stage larvae of D. immitis subcutaneously. On day 1, 12 ferrets were randomized and allocated to two groups, each containing six ferrets (Table 1). On study day 0, animals assigned to group 1 were treated with 10% imidacloprid/1% moxidectin at a dose of 0.4 ml by personnel who were blinded to treatments (Table 2). Animals assigned to group 2 served as untreated controls. On day 124, the ferrets in groups 1 and 2 were euthanized and necropsies were performed to determine the worm burden (Table 3). At necropsy, the pleural and peritoneal cavities were examined for worms before removal of the heart and lungs. The heart and lungs from each ferret were dissected and examined for heartworms. The heartworms recovered were identified as to genus, species, sex, and stage of development; they were counted and preserved in 10% buffered formalin. The response to treatment was evaluated by comparison of worm counts in ferrets treated with 10% imidacloprid/1% moxidectin with those in the untreated control group. Efficacy was calculated according to the recommendations for controlled tests described in Guideline VlCH GL7: % Effectiveness (Reduction) = (N1 N2) / N1 100 Figure 3. Cat flea (Ctenocephalides felis). where N1 equals the geometric mean nematode count for the control group and N2 equals the geometric mean nematode count for the group treated with test article. 8 Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

11 NAVC CONFERENCE, JANUARY 2008 TABLE 2. Imidacloprid/Moxidectin Dose Applied to Treated Ferrets Included in the Heartworm Study Dose of Dosage of Weight at Imidacloprid/ Imidacloprid/ Day of Moxidectin Applied Moxidectin Applied Animal Treatment per Animal per Animal No. (kg) (ml) (mg/kg body weight) 321A / A / A / A / A / A /3.33 Flea Study The study was conducted according to the Guideline on Good Clinical Practice, EMEA/CVMP/VICH/595/98-final, July 4, 2000, and Guidelines 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, EMEA/CVMP/005/2000- Rev-2, October 12, Sixteen adult ferrets (eight males and eight females ranging from 10 to 24 months of age) were included in the study. The ferrets were kept during the acclimatization phase and during the study in individual metal wire cages with an attached wooden sleeping box. The ferrets were weighed at study start on day 7 and at the end of the study on day 30. All ferrets were infested with 50 fleas each on days 7, 1, 7, 14, 21, and 28. The fleas used in the study were nonfed adult cat fleas (C. felis). Based on flea counts at day 6 and ferret body weight, the ferrets were randomly allocated to two groups (Table 4). On study day 0, each ferret in group 1 was treated with one pipette containing 0.4 ml of imidacloprid 10%/moxidectin 1% spot-on (Table 5). The second group was left untreated (control group). The therapeutic efficacy of the 10% imidacloprid/1% moxidectin spot-on was determined by performing a flea count on study day 1 (24 hours + 1 hour after). The preventive efficacy was determined by flea counts on each animal on days 8, 15, 23, and 30 (Table 6). The number of living fleas on each ferret was determined by combing with a flea comb through the ferret s coat following its lay. The majority of ferrets did not tolerate the counting procedure and were sedated with ketamine hydrochloride SC (0.1 ml/kg) in combination with xylazine hydrochloride SC (0.1 ml/kg). Efficacy was defined as group mean reduction of fleas at all time points following treatment compared with the control group. The number of counted fleas was recorded; the physical condition of the TABLE 3. Schedule of Events in the Heartworm Study Study Day Activity* 37 Start acclimatization 36 Heartworm testing (microfilaria, antigen) 35 Clinical examination 30 Experimental infection 3 Clinical examination 1 Weighing Randomization 0 Treatment Clinical assessment Weighing 90 Heartworm testing (microfilaria, antigen) Weighing 124 Euthanasia and necropsy Weighing Worm counts *In addition, there were daily general health observations from day 37 to study end at day 124. fleas, when deviating from normal, was documented. Dead fleas and fleas that were visually affected and damaged were recorded separately. The latter involved fleas lying on their side and/or ones that had uncontrolled leg movements. The percentage efficacy for treated groups at each considered time point was calculated as follows: % Efficacy = 100 (C T) / C where C is the geometric mean of fleas in the control group and T is the geometric mean of fleas in the treatment group. These calculations were performed for each treatment group. Statistical analyses included the test on efficacy as described in the EMEA s Guidelines 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 and tests on superiority of the IVP treatment versus untreated control for parameters of flea reduction. General health observations were performed daily from day 7 until the end of the study except on days with clinical assessments. Clinical assessments, including dermal safety aspects, were carried out on study days 7, 1, 1, and 28. On days 7, 14, and 21, only the dermal safety aspects were assessed. RESULTS Heartworm Study The treatment was well tolerated in all animals. No adverse events were noted after treatment or during daily health observations. None of the ferrets (0/6) in the treatment group had any Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

12 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM TABLE 4. Details of Ferrets Included in the Flea Study Weight at day Flea Count at Animal No. Sex 7 (kg) Day 6 Treatment Group 1 M F F M M M F F Average Control Group 3 F M M M M F F F Average TABLE 5. Imidacloprid/Moxidectin Dosage Applied to Treated Ferrets Included in the Flea Study Dose of Dosage of Weight at Imidacloprid/ Imidacloprid/ Day of Moxidectin Applied Moxidectin Applied Animal Treatment per Animal per Animal No. (kg) (ml) (mg/kg body weight) / / / / / / / /4 adult D. immitis recovered at necropsy, while 6 of 6 ferrets in the untreated control group had 13 or more adult D. immitis worms recovered at necropsy, showing adequacy of infection (Table 7). TABLE 6. Schedule of Events in the Flea Study Study Day Activity* 7 Experimental flea infestation Clinical examination Weighing 6 Counting of fleas Randomization 1 Experimental flea infestation Clinical examination 0 Treatment 1 Counting of fleas Clinical examination 7 Experimental flea infestation Clinical examination, restricted to dermal safety aspects 8 Counting of fleas 14 Experimental flea infestation Clinical examination, restricted to dermal safety aspects 15 Counting of fleas 21 Experimental flea infestation Clinical examination, restricted to dermal safety aspects 23 Counting of fleas 28 Experimental flea infestation Clinical examination 30 Counting of fleas Weighing *In addition, there were daily general health observations from study start at day 7 to study end at day 30 on all days without clinical examination. The efficacy of the combination of 10% imidacloprid/1% moxi - dectin, at a dose of 0.4 ml, against artificial infections with D. immitis third-stage larvae in ferrets was 100% when administered once 30 days after initial infection. Flea Study The treatment was well tolerated in all animals. No adverse events were noted after treatment or during daily health observations. The infestation rates for individual ferrets as well as the geometric mean per group at the respective combing times of 24 or 48 hours following infestation showed consistently high flea counts in the control group, which demonstrated an establishment rate of 57% (Table 8). In the treatment group, no fleas were found on days 1, 8, or 15, and low numbers were found on days 23 and 30 (Table 8). Also for the treatment group, the therapeutic efficacy was 100% at day 1 (Table 9). The preventive efficacy was 100% at days 8 and 15 and greater than 97% and 90% at days 23 and 30, respectively (Table 9). The one-sided Mann- Whitney U test of superiority showed highly significant superiority of the treatment group for all time points (P <.001) after treatment. 10 Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

13 NAVC CONFERENCE, JANUARY 2008 TABLE 7. Worm Recovery of Dirofilaria immitis in Ferrets Untreated or Treated with 10% Imidacloprid/1% Moxidectin at a Topical Dose of 0.4 ml per Ferret Animal Adult Female Adult Male Total No. D. immitis D. immitis D. immitis Treatment Group 321A A A A A A GM = 0 Control Group 225A A A A A A GM = 17.2 GM = geometric mean. DISCUSSION Heartworm Study Ferrets in heartworm-endemic areas are at risk for D. immitis infection. They are as susceptible as dogs but develop disease more similar to cats, characterized by low worm burdens with severe clinical signs. Currently, there is no approved compound for the prevention of D. immitis infection in ferrets. In an experimental infection with low worm numbers, 13 treatment with melarsomine dihydrochloride, which is approved for the treatment of D. immitis infection in dogs, achieved cure rates from 80% to 83% using a similar regimen as recommended in dogs. On the other hand, under clinical conditions, with natural infections, melarsomine dihydrochloride showed disappointing results and that survival rate with the treatment was low. 5 This provides strong evidence that prevention of the development of adult D. immitis is the preferable alternative in ferrets. Successful prevention has been previously demonstrated with monthly oral dosing of ivermectin at 3 µg/kg in experimental infections. 6 In the study outlined in this paper, a combination of 10% imidacloprid/1% moxidectin topical solution, given at a dose rate of 0.4 ml (the volume designed for a cat up to 4 kg), prevented the development of adult D. immitis following treatment 30 days after exper- TABLE 8. Individual Flea Count Results of Living Fleas at Different Counting Dates Animal No. Sex SD 6 SD 1 SD 8 SD 15 SD 23 SD 30 Treatment Group (GM = 1) 1 M F F M M M F F Control Group (GM = 2) 3 F M M M M F F F Average all SD: 28.3 Establishment rate: 57% GM = geometric mean; SD = study day. imental infection. The treatment was well tolerated, was easy to apply, and would be a viable option to prevent heartworm disease in ferrets when applied at monthly intervals. Flea Study Ferrets are at risk of acquiring flea infestations when living in households with other flea hosts such as dogs or cats. The cat flea (C. felis) is the predominant flea species in dogs and cats. 11 Members of the family Mustelidae such as the mink (Mustela vison) in a more natural environment usually seem to be infested with the squirrel flea. 14 However, ferrets are also susceptible to infections with the cat flea, as previous reports have shown. 7,9,15 Successful experimental infestation of ferrets with cat fleas has been demonstrated in a study by Hutchinson and colleagues. 10 This finding was confirmed in the study reported here, although the high flea establishment levels of more than 80% in the study by Hutchinson and colleagues were not achieved in this investigation. Here, an average establishment rate of 57% was achieved, which may be a result of the different laboratory-bred flea strains used in the studies. Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

14 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM TABLE 9. Geometric Mean Flea Infestation at Different Study Days and Efficacy of Treatment Study Day* Control Group Treatment Group* % Efficacy** *Geometric mean of living fleas at flea count procedure. **The Mann-Whitney U test showed highly significant superiority of the treatment group for all time points (P <.001) after treatment. It is suggested that in pet ferrets kept in households with other pets, C. felis may be the dominating flea species. Ferrets may play an important role as a source for reinfestation of other pet household members (or potentially even humans) if treatment is omitted. 15 Successful treatment and prevention are possible with imidacloprid, as earlier studies have shown. 10,12 The study reported here confirmed the efficacy of imidacloprid in the combination of imidacloprid and moxidectin against existing flea infestations. Efficacy of 100% against an existing flea infestation was demonstrated. A preventive effect could be demonstrated yielding 100% efficacy at 1 and 2 weeks following treatment and an efficacy greater than 97% at 3 weeks and greater than 90% at 4 weeks following administration. This is a considerably longer protection against reinfestation with fleas as compared with the results of the other experimental study, 10 in which a high preventive efficacy was seen for only 1 week. In that study, the dosage of imidacloprid was the minimum recommended dosage of 10 mg/kg. In the study reported here, the dosage range was between 20 and 50 mg/kg, which resulted in considerably longer prevention against reinfestation with fleas. CONCLUSION A combination product containing 10% imidacloprid/1% moxi - dectin applied at a dose of 0.4 ml will protect ferrets against the potentially life-threatening adult heartworm, D. immitis. At the same time, it will treat existing adult flea infestations and provide protection against flea infestation with C. felis for up to 4 weeks. The treatment is well tolerated without any adverse reactions. REFERENCES 1. Personal communication, P. Hendrix, President, American Ferret Association, 2/10/ World Animal Health Markets [Animal Pharm Report]. London: Informa Healthcare; 2006: McCall JW. Dirofilariasis in the domestic ferret. Clin Tech Small Anim Pract 1998;13(2): Kemmer D. Heartworm disease in the domestic ferret. Proc Heartworm Symp : Antinoff N. Clinical observations in ferret with naturally occurring heartworm disease, and preliminary evaluation of treatment with ivermectin with and without melarsomine. Proc Heartworm Symp : Supakorndej P, McCall JW, Lewis RE. Biology, diagnosis, and prevention of heartworm infection in ferrets. In: Soll MD, ed. Proc Heartworm Symp : Timm KL. Pruritus in rabbits, rodents and ferrets. Vet Clin North Am Small Anim Pract 1988;18: Oxenhamm M. Flea control in ferrets. Vet Rec 1996;138(15): Visser M, Rehbein D, Wiedemann C. Species of flea (Siphonaptera) infesting pets and hedgehogs in Germany. J Vet Med B Infect Dis Vet Public Health 2001;48(3): Hutchinson MJ, Jacobs DE, Mencke N. Establishment of the cat flea (Ctenocephalides felis felis) on the ferret (Mustela putorius furo) and its control with imidacloprid. Med Vet Entomol 2001;15: Krämer F, Mencke N. Flea Biology and Control: The Biology of the Cat Flea Control and Prevention with Imidacloprid in Small Animals. Berlin: Springer; 2001: Larsen KS, Siggurdson H, Mencke N. Efficacy of imidacloprid, imidacloprid/permethrin and phoxim for flea control in the Mustelidae (ferret, mink). Parasitol Res 2005;97(suppl 1): Supakorndej P, McCall JW, Supakorndej N, et al. Evaluation of melarsomine dihydrochloride as a heartworm adulticide for ferrets. In: Soll MD, ed. Proc Heartworm Symp : Larsen KS. A study of the squirrel flea (Ceratophyllus sciurorum) related to its occurrence on farmed mink [PhD thesis]. Denmark: University of Aarhus; Beck W. Ectoparasites, endoparasites, and heartworm control in small animals. Compend Contin Educ Vet 2007;29(5 suppl): Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

15 NAVC CONFERENCE, JANUARY 2008 Demodex canis Control in Dogs Ralf S. Mueller, DVM, Prof., DACVD, FACVSc, DECVD * Faculty of Veterinary Medicine, Ludwig Maximilian University, Munich, Germany LOCALIZED DEMODICOSIS Localized demodicosis is a mild disease that usually heals spontaneously in 6 to 8 weeks but may wax and wane in a localized area for months (Figure 1). Anecdotally, there is no difference between treated and untreated cases. If therapeutic intervention is desired, mupirocin or benzoyl peroxide gel can be massaged gently into the alopecic area once daily. Thus, treating localized demodicosis is in most cases not a problem. Because any stress on the immune system such as endoparasites or concurrent systemic disease may predispose dogs to demodicosis, possible endoparasites should be treated and the patient s general health status checked very carefully. Although most dogs with localized demodicosis will go into remission, in some dogs the disease will develop into the generalized form. Therefore, patients with localized demodicosis should be reevaluated after a few weeks to months. During return visits, the veterinarian can determine whether there are any indications of generalized demodicosis. 1 I do not treat the localized form at all with ectoparasiticides. Abstinence from miticidal treatment allows identification of those patients with progression to generalized disease. The reason to differentiate dogs with localized disease from those with the generalized form is that young dogs with generalized demodicosis have a strong genetic predisposition for the disease. Breeding with such dogs is strongly discouraged, as they would pass on this genetic trait and their offspring may be more likely to get the disease. I advise all owners of dogs with generalized demodicosis to neuter their pets. GENERALIZED DEMODICOSIS Although the prognosis for dogs with demodicosis has improved dramatically in recent decades, successful treatment may still be a *Corresponding author: R. Mueller, Faculty of Veterinary Medicine, Ludwig Maximilian University, Veterinärstrasse 13, Munich, Germany; phone, +49 (0) ; fax, +49 (0) ; , ralf.mueller@ med.vetmed.uni-muenchen.de. Dogs with generalized demodicosis need to be reevaluated typically every 2 to 4 weeks, and skin scrapings need to be obtained to monitor success of therapy. challenge in some cases. About 90% of patients can be cured, but it may take up to 12 months of treatment. 2 However, the average time to clinical and microscopic remission in reported studies is approximately 2 to 4 months. 2 The most common reason for treatment failure is premature cessation of therapy. 1 Dogs with juvenile-onset disease (Figure 2) may not always need miticidal therapy. However, there are no good studies evaluating how many young dogs with generalized demodicosis recover spontaneously, and anecdotal estimates vary widely, from 0% to 50%. In intact females, ovariohysterectomy is strongly recommended because the dogs may deteriorate or the disease may recur. 3 In dogs older than 1 to 2 years of age or those with adult-onset generalized demodicosis (Figure 3), systemic disorders can be the cause of demodicosis and the reason for an unfavorable treatment response. Some dogs may recover without miticidal therapy if the underlying disease is treated successfully, although for ethical reasons I am usually not bold enough to withhold anti-mite therapy for several weeks or months in dogs with adultonset demodicosis while treating the underlying disease. Dogs with generalized demodicosis need to be reevaluated typically every 2 to 4 weeks, and skin scrapings need to be obtained to monitor success of therapy. 2 To determine treatment efficacy, skin scrapings should always be taken from the same sites. If there is no clinical improvement or if skin scrapings show the same high number of mites especially immature stages such as nymphs, larvae, or eggs a change in treatment should be considered. Up to 70% of dogs that do not respond well to one treatment will improve and go into remission once the mode of therapy is changed. 2 Secondary pyoderma is almost always present in dogs with demodicosis. 1 The most common bacterial isolate is Staphylococcus intermedius. Empirical therapy for 3 to 8 weeks is usually appropriate. The most common gram-negative bacteria found in canine demodicosis are Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis. If rods predominate, bacterial culture and sensitivity is rec- Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

16 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM Figure 1. One-year-old female German shepherd dog with periocular dermatitis caused by Demodex mites. Figure 2. One-year-old female German shepherd mix with generalized demodicosis. ommended, as the resistance pattern of these organisms is more unpredictable. While waiting for results of the bacterial culture and sensitivity, the infection is treated symptomatically with an antibiotic that is frequently effective for such infections. Fluoroquinolones such as enrofloxacin may be chosen initially. I dispense a small amount sufficient for 7 to 10 days of treatment and then additional medication depending on the results of the sensitivity testing. In addition to systemic antibiotics, antibacterial shampoo therapy is frequently used to remove crusts and treat bacteria topically. Benzoyl peroxide shampoo is most commonly recommended because of its presumed follicular flushing activity. 4 At this time, the cure rate of demodicosis is around 90%. Commonly used therapies include amitraz and macrocyclic lactones such as ivermectin, moxidectin, and milbemycin oxime. 2 COMPOUNDS USED TO TREAT DEMODICOSIS Amitraz Amitraz is licensed in Europe and Australia as a concentration to prepare a therapeutic wash application for the treatment of generalized demodicosis. Amitraz is a monoaminooxidase inhibitor and α 2 - adrenergic agonist that inhibits prostaglandin synthesis. It is available as Mitaban in the United States and Canada and as Ectodex in Europe. Amitraz is also available in a 9% anti-tick collar; unfortunately, this collar is ineffective for the treatment of generalized demodicosis. 1 Some studies using amitraz in various formulations show clinical cure rates up to 90% with special protocols. 5,6 Dogs with medium or long coats should be clipped to allow the solution to come in contact with the skin and better penetrate the hair follicle. All crusts should be removed. Before using amitraz, the dog should be thoroughly washed with a medicated shampoo designed to kill bacteria and remove scales. Amitraz is then applied by wetting and sponging. The solution must be applied to the entire body. Adverse effects such as respiratory problems have been observed in humans, 1,7 so the washing procedure should be performed outside or in a wellventilated area and protective gloves should be worn. In addition, if the dog is placed under anesthesia, it is important to remember that amitraz is an α 2 -adrenergic agonist and that synergistic toxicity with α 2 -adrenergic sedatives such as benzodiazepine or xylazine is possible. The recommended treatment protocol in the United States is 0.025% amitraz in a wash every 2 weeks. Higher concentrations and/or frequencies seem to be associated with a higher success rate. Daily therapy with 0.125% amitraz on alternating halves of the body was reported to be efficacious for 73% of 32 dogs with generalized demodicosis resistant to conventional therapy. 8 Recently, amitraz at 1.25% weekly was used successfully to treat generalized demodicosis in eight dogs that had failed to respond to the drug at lower concentrations. 9 These dogs were premedicated with atipamezole at 0.1 mg/kg IM once and yohimbine 0.1 mg/kg/day PO for a further 3 days in an attempt to counteract the α 2 -adrenergic activity of amitraz, which results in bradycardia, a decrease in rectal temperature, and hyperglycemia. In some of these patients, a transient erythematous rash was observed after rinsing. To maximize the efficacy of amitraz, it is important that rinsed patients do not get wet between rinses. This is particularly difficult in rainy climates and in dogs whose paws are affected. In patients with pododemodicosis or demodectic otitis externa, a mixture of amitraz and paraffin or mineral oil has been recommended topically daily to every 3 days. 2 Adult-onset demodicosis may respond less favorably to therapy. In a recent analysis of studies evaluating treatments for demodicosis, the success rate in adult-onset disease was approximately 30% Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

17 NAVC CONFERENCE, JANUARY 2008 cure rates varied from 15% to 92%, depending on the dosage used and the age at onset of disease. Several studies have shown a better success rate with higher doses. 11,13 Dogs with adult-onset disease respond more poorly to treatment than dogs with juvenile-onset disease. Some rare adverse effects have been reported; these include ataxia, stupor, and trembling in two dogs given a dosage of 3.8 mg/kg/day; lethargy; and transient vomiting. Figure 3. Twelve-year-old spayed Boston terrier with generalized demodicosis. Adverse effects with amitraz therapy were ataxia, depression, diarrhea, polyphagia/polydipsia, sleepiness, and vomiting. With 1.25% amitraz, generalized erythema, scaling, and an unpleasant odor were noted. 9 Macrocyclic Lactones Milbemycins (e.g., milbemycin oxime, moxidectin) and avermectins (e.g., ivermectin, doramectin) are antiparasitic agents produced by the fermentation of various actinomycetes. They bind selectively to glutamate-gated chloride channels, resulting in increased cell permeability for chloride ions, and cause neuromuscular blocking, resulting in paralysis and death of the parasite. They also interact with γ- aminobutyric acid (GABA) sites. These compounds have a wide margin of safety in mammals because the mammalian peripheral nervous system doesn t have glutamate-gated chloride channels. In addition, GABA is a central nervous system neurotransmitter in mammals, and these compounds cross the blood brain barrier at a very low level. 10 Milbemycin Milbemycin oxime, a fermentation product of Streptomyces hygroscopicus subsp aureolacrimosus, is used as a heartworm preventive and endoparasitic agent. In Europe, milbemycin is registered as an oral formulation against demodicosis and is licensed for use as a monthly heartworm and intestinal parasite preventive in dogs at an oral dosage of 0.5 mg/kg. A variety of dogs of all breeds, including those that are sensitive to high doses of ivermectin, and various ages at onset of both diseases and therapy were treated with milbemycin at dosages varying from 0.5 to 3.8 mg/kg/day. 7,11 14 The mean duration of treatment needed to achieve negative skin scrapings was 8 to 26 weeks; mean treatment duration was 12 to 30 weeks. 2 Clinical Moxidectin Moxidectin was used in three studies at oral dosages ranging from 0.2 to 0.4 mg/kg/day In these studies, 52 dogs with generalized demodicosis, 41 dogs with juvenile-onset disease, and 11 dogs with adult-onset disease were treated with oral moxidectin. Thirty-one (76%) of the dogs with juvenile-onset demodicosis were in remission after 8 to 14 weeks, and seven were lost to follow-up. Of the 11 dogs with adult-onset demodicosis, nine were in remission after 8 to 16 weeks. All dogs were cured, but details on long-term follow-up were unavailable. Transient side effects included anorexia, ataxia, lethargy, stupor, and vomiting. 26 More studies with longer follow-up periods are needed to identify potential benefits and disadvantages of the oral use of moxidectin. Moxidectin as part of a combination with imidacloprid spot-on approved for the treatment of many endo- and ectoparasites including demodicosis has recently become available in many countries. In one multicenter study, this product administered monthly showed a success rate comparable to that of oral milbemycin. 29 The efficacy and suitable protocols of this spot-on for treatment of canine generalized demodicosis are currently under further investigation in another multicenter study with dermatologists in four European countries. Preliminary results of this study show that remission was achieved using moxidectin in this spot-on every 2 weeks in approximately two-thirds of the cases with less than 20 mites per scraping. At Ludwig Maxi - milian University, Munich, Germany, we currently use this spot-on weekly for our patients with generalized demodicosis. Ivermectin Ivermectin is a fermentation product of Streptomyces avermitilis. In the United States and Europe, it is only licensed in small animals for the prevention of dirofilariasis at an oral dosage of mg/kg once monthly. To my knowledge, ivermectin is not licensed for treating demodicosis in any country. It was first reported as a treatment for generalized demodicosis in the mid-1980s. Oral dosages usually vary from 0.3 to 0.6 mg/kg/day, and treatment courses range from 35 to 210 days. Published data on the efficacy of ivermectin for canine demodicosis are more limited than that for amitraz, with approximately 100 cases available for review. 3,15 19 The rate of clinical cure varied from 83% to 100% in individual studies. 2 An early protocol, treating with ivermectin at 0.4 mg/kg SC once weekly, did not demonstrate much efficacy. 5 Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

18 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM Daily oral treatment of ivermectin was shown in seven studies that included a total of 120 patients to have a good success rate. The dosages used ranged from 0.3 to 0.6 mg/kg. The mean time to achieve negative skin scrapings was 6.5 to 28 weeks; the mean treatment duration was 10 to 33 weeks. Higher dosing does not seem to influence the time to first negative skin scrapings: An alternate-day protocol, in which dogs received 0.45 to 0.6 mg/kg, seemed equally efficacious as 0.3 mg/kg/day. 20 However, the pour-on formulation of ivermectin, which is very effective in the treatment of nonfollicular mites in dogs and cats, performed very poorly in dogs with generalized demodicosis when it was applied three times weekly at a dosage of 1.5 mg/kg. Only 2 of 12 treated dogs achieved parasitologic cure. 21 Side effects of ivermectin include ataxia, edematous wheals, lethargy, and mydriasis. These develop as late as 10 weeks into treatment. Collies are particularly sensitive. 2 One report recommended gradually increasing the dosage of ivermectin from 0.05 to 0.1, 0.2, and 0.3 mg/kg during the first days of treatment to identify sensitive animals. 22 Because of ivermectin s relatively long half-life, serum concentrations of ivermectin administered daily continue to increase for weeks before reaching equilibrium at much higher levels than with weekly therapy. Thus, chronic toxicity due to cumulative therapy may develop with prolonged daily ivermectin treatment. Today, some veterinary laboratories offer a gene test to recognize dogs with a defect of the MDR-1 gene encoding for the multidrug resistant transport P-glycoprotein. 23 This gene was reported to be associated with ivermectin toxicity in collies. 24 The MDR-1 defect is autosomal recessive. However, a study presented at the North American Veterinary Dermatology Forum in 2007 indicated that most dogs showing chronic toxicity have a normal MDR-1 gene. 25 These dogs did not develop the severe acute toxicity at low doses that is seen in collies but typically showed subchronic toxicity and clinical signs after 4 days to 5 weeks of therapy. Doramectin In one study, 23 dogs were injected weekly with 0.6 mg/kg doramectin SC for 5 to 23 weeks. 30 Ten of the dogs were cured, seven relapsed after 1 to 24 months (two of which responded to repeat doramectin treatment), and six were lost to follow-up. More studies are needed to evaluate the efficacy and optimal dosing of doramectin for the treatment of canine demodicosis. OTHER TREATMENT OPTIONS A number of other drugs have been evaluated as treatment for canine demodicosis, including amitraz collars, 31 closantel, 32 deltamethrin, 33 herbal 34 and homeopathic preparations, 35 muramyl dipeptide, 36 phoxime, 37 vitamin E, 38,39 pour-on ivermectin, 21 levamisole, 40,41 lufenuron, 42 and ronnel, 40,43 but insufficient evidence exists regarding the efficacy of these products. Typically, a follow-up of 12 months is recommended after remission of a dog with generalized demodicosis, as recurrences after cessation of therapy are possible. In a recent meta-analysis, it was concluded that if a recurrence of generalized demodicosis is treated again either with the original medication or a different type of drug, two of three dogs will go into long-term remission and can be cured after the second treatment course. 2 Thus, treatment of recurrent demodicosis should be recommended and does not necessarily indicate a poor prognosis. However, some dogs will never go into microscopic remission (clinically, these patients appear healthy, but skin scrapings always identify a few mites) and may show frequent recurrence of clinical signs. In these dogs, life-long monthly amitraz rinses or weekly treatment with macrocyclic lactones (the one chosen differs depending on the case) may provide relief and a normal life without clinical skin disease. REFERENCES 1. Scott DW, Miller WH, Griffin CE. Small Animal Dermatology. 6th ed. Philadelphia: WB Saunders; Mueller RS. Treatment protocols for demodicosis: an evidence-based review. Vet Derm 2004;15: Mueller RS, Hastie K, Bettenay SV. Daily oral ivermectin for the treatment of generalised demodicosis in 23 dogs. Aust Vet Pract 1999;29: Mueller RS. Topical dermatological therapy. In: Maddison JE, Page SW, Church D, eds. Small Animal Clinical Pharmacology. London: WB Saunders; 2002: Scott DW, Walton DK. Experiences with the use of amitraz and ivermectin for the treatment of generalized demodicosis in dogs. JAAHA 1985;21: Muller GH. Amitraz treatment of demodicosis. JAAHA 1983;19: Mueller RS, Bettenay SV. Milbemycin oxime in the treatment of canine demodicosis. Aust Vet Pract 1995;25: Medleau L, Willemse T. Efficacy of daily amitraz therapy for refractory, generalized demodicosis in dogs: two independent studies. JAAHA 1995;31: Hugnet C, Bruchon-Hugnet C, Royer H, et al. Efficacy of 1.25% amitraz solution in the treatment of generalized demodicosis (eight cases) and sarcoptic mange (five cases) in dogs. Vet Dermatol 2001;12: Campbell WC, Fisher MH, Stapley EQ. Ivermectin a potent new antiparasitic agent. Science 1983;221: Garfield RA, Reedy LM. The use of oral milbemycin oxime (Interceptor) in the treatment of chronic generalized canine demodicosis. Vet Dermatol 1992;3: Holm B. Efficacy of milbemycin oxime in the treatment of canine generalized demodicosis: a retrospective study of 99 dogs ( ). Vet Dermatol 2004;15: Miller WH, Scott DW, Cayatte SM, et al. Clinical efficacy of increased dosages of milbemycin oxime for treatment of generalized demodicosis in adult dogs. JAAHA 1995;207: Miller WH, Scott DW, Wellington JR, et al. Clinical efficacy of milbemycin oxime in the treatment of generalized demodicosis in adult dogs. JAAHA 1993;203: Medleau L, Ristic Z, McElveen DR. Daily ivermectin for treatment of generalized demodicosis in dogs. Vet Dermatol 1996;7: Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

19 NAVC CONFERENCE, JANUARY Fondati A. Efficacy of daily oral ivermectin in the treatment of 10 cases of generalized demodicosis in adult dogs. Vet Dermatol 1996;7: Ristic Z, Medleau L, Paradis M, et al. Ivermectin for treatment of generalized demodicosis in dogs. JAAHA 1995;207: Paradis M, Laperriere E. Efficacy of daily ivermectin treatment in a dog with amitraz-resistant, generalized demodicosis. Vet Dermatol 1992;3: Guaguere E. Efficacy of daily oral ivermectin treatment in 38 dogs with generalized demodicosis: a study of relapse rates. In: Kwochka KW, Willemse T, Von Tscharner C, eds. Advances in Veterinary Dermatology. Oxford: Butterworth-Heinemann; 1998: Tapp T, Muse R, Rosenkrantz WS. Efficacy of alternate day oral ivermectin in the treatment of generalized demodicosis. Annu Meet Am Acad Vet Derm Am Coll Vet Dermatol 1998: Paradis M, Page N. Topical (pour-on) ivermectin in the treatment of chronic generalized demodicosis in dogs. Vet Dermatol 1998;9: Mueller RS, Bettenay SV. A proposed new therapeutic protocol for the treatment of canine mange with ivermectin. JAAHA 1999;35: Geyer J, Doring B, Godoy JR, et al. Development of a PCR-based diagnostic test detecting a nt230(del4) MDR1 mutation in dogs: verification in a moxidectin-sensitive Australian shepherd. J Vet Pharmacol Ther 2005;28: Neff MW, Robertson KR, Wong AK, et al. Breed distribution and history of canine mdr1-1delta, a pharmacogenetic mutation that marks the emergence of breeds from the collie lineage. Proc Natl Acad Sci USA 2004: Bissonnette S, Paradis M, Daneau I, et al. Survey of the MDR1 gene for heterozygous mutations in dogs displaying subchronic toxicity signs to systemic macrocyclic lactones following treatment regimen for a generalized demodicosis. North Am Vet Forum 2007: Wagner R, Wendlberger U. Field efficacy of moxidectin in dogs and rabbits naturally infested with Sarcoptes spp., Demodex spp. and Psoroptes spp. mites. Vet Parasitol 2000;93: Bensignor E, Carlotti D. Moxidectin in the treatment of generalized demodicosis in dogs: a pilot study: 8 cases. In: Kwochka KW, Willemse T, Von Tscharner C, eds. Advances in Veterinary Dermatology. Oxford: Butterworth-Heinemann; 1998: Burrows A. Evaluation of the clinical efficacy of two different doses of moxidectin in the treatment of generalized demodicosis in the dog. Sci Meet Aust Coll Vet Sci Heine J, Krieger K, Dumont P, et al. Evaluation of the efficacy and safety of imidacloprid 10% plus moxidectin 2.5% spot-on in the treatment of generalized demodicosis in dogs: results of a European field study. Parasitol Res 2005;97(suppl 1): Johnstone IP. Doramectin as a treatment for canine and feline demodicosis. Aust Vet Pract 2002;32: Franc M, Soubeyroux H. Le traitement de la demodecie du chien par un collier a 9% d amitraz. Rev Med Vet (Toulouse) 1986;137: Losson B, Benakhla A. Efficacite du closantel dans le traitement de la gale demodectique du chien. Ann Med Vet 1980;124: Kamboj DS, Singh KB, Avtar S, et al. Studies on the therapeutic efficacy of amitraz, deltamethrin and ivermectin on canine demodicosis. Indian Vet J 1993;70: Das SS. Efficacy of Maggacite, an indigenous preparation against demodicosis in dogs and its comparison with deltamethrin. Indian Vet J 1998;75: Nayak DC, Tripathy SB, Dey PC, et al. Therapeutic efficacy of some homeopathic preparations against experimentally produced demodicosis in canines. Indian Vet J 1998;75: Kraiss A, Gothe R. Demodikosetherapie mit Muramyl dipeptide und Amitraz. Kleintierpraxis 1983;28: , Sikovec J. Demodicosis and sarcoptic mange in the dog: therapy trials with phoxim (Sebacil). Vet Med Rev 1983;2: Gilbert PA, Griffin CE, Rosenkrantz WS. Serum vitamin E levels in dogs with pyoderma and generalized demodicosis. JAAHA 1992;28: Figueiredo C, Viana JA, Curi PR. Clinical evaluation of the effect of vitamin E in the treatment of generalized canine demodicosis. In: Ihrke PJ, Mason IS, White SD, eds. Advances in Veterinary Dermatology. Oxford: Pergamon Press; 1993: Scott DW, Schultz RD, Baker EB. Further studies on the therapeutic and immunologic aspects of generalized demodectic mange in the dog. JAAHA 1976;12: Mojzisova J, Paulik S, Bajova V, et al. The immunomodulatory effect of levamisole with the use of amitraz in dogs with uncomplicated generalized demodicosis. Vet Med Czech 1997;42: Schwassmann M, Kunkle GA, Hepler DI, et al. Use of lufenuron for treatment of generalized demodicosis in dogs. Vet Dermatol 1997;8: Baker BB, Stannard AA, Yaskulski SG, et al. Evaluation of topical application of ronnel solution for generalized demodicosis in dogs. JAVMA 1976;168: Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

20 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM Comparative Speed of Efficacy of Imidacloprid against Ctenocephalides felis in Experimentally Infested Cats Thomas Schnieder, DVM, Prof., DEVPC a, * Sonja Wolken, DVM, Dr. med. vet. a Norbert Mencke, DVM, Dr. med. vet., DEVPC b a University of Veterinary Medicine Hannover, Hannover, Germany b Bayer HealthCare AG, Animal Health, Leverkusen, Germany 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 cat infestation occurring more frequently than dog infestation. 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 observation period. 2 The cat flea, Ctenocephalides felis, is the most prevalent species of flea infesting cats and dogs. 3 5 According to a survey conducted in Germany, 70% of about 1,700 small animal practitioners answered 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 to a considerable degree. On the contrary, due to easy administration and high efficacy of the readily available insecticides, flea control is mainly done by pet owners and 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 that causes cat scratch fever, 7 11 and Rickettsia felis. 7,12 *Corresponding author: T. Schnieder, University of Veterinary Medicine Hannover, Buenteweg 17, D Hannover, Germany; phone, +49 (0) ; fax, +49 (0) ; , thomas.schnieder@ tiho-hannover.de. The chloronicotinyl imidacloprid is known for its fast onset of efficacy against a wide range of agricultural pests and fleas. Recently, it was shown that cat fleas carry FeLV after feeding on an infected cat and 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 (FAD). 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 the egg production of adult female fleas at the earliest possible time. Because the importance of arthropod-transmitted diseases is increasingly being recognized, prevention schemes using insecticides and acaricides are recommended more often than in the past. However, the EMEA/CVMP guidelines 15 for labeling require that flea counts only be performed 48 hours after infestation. Therefore, none of the existing compounds comparative data concerning earlier efficacy are available, and none of the compounds have a label claim related to prevention against transmitted agents. In this study, the speed of efficacy of some of the most important veterinary medicinal remedies against fleas is investigated at earlier time points than defined in the guideline requirements to evaluate the suitability of these compounds for preventive use. The active ingredients imidacloprid, sela mectin, and fipronil are widely used as ectoparasiticides on both cats and dogs. Their efficacy has been recorded in numerous studies. 4,16 The active 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 flea kill are not available. The aim of the study was to examine the speed of flea kill caused by imidacloprid (Advantage, Bayer Animal Health), selamectin 18 Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

21 NAVC CONFERENCE, JANUARY 2008 TABLE 1. Study Groups and Active Ingredients Applied Group Treatment Application a Untreated b Imidacloprid Spot-on c Selamectin Spot-on d Fipronil/(S)-methoprene Spot-on e Metaflumizone Spot-on (Stronghold /Revolution, Pfizer Animal Health), fipronil/(s)- methoprene (Frontline Combo /Frontline Plus, Merial), and metaflumizone (ProMeris for cats, Fort Dodge Animal Health) against C. felis after treatment and reinfestation. MATERIALS AND METHODS Study Design Thirty cats were acclimatized to the study facilities for 1 week before each cat was experimentally infested with 50 fleas (C. felis) on study day 2. Twenty-four hours after infestation (study day 1), flea counts were performed. Based on these flea counts, cats were then allocated to five groups (four treatments and one untreated control) of six cats each. After randomization, cats were weighed and reinfested with 50 fleas per cat. On study day 0, cats were treated topically with the different ectoparasiticides (Table 1) according to label instructions. The spoton solutions were applied on the neck at the base of the skull. The cats were treated according to body weight using the recommended dosage. During the first week, treatment success was monitored by flea counts 6 hours (±15 minutes) after treatment to initially allow a distribution of the compounds in the cats fur. Immediately following the counting procedure, cats were reinfested; another flea count was performed 12 hours (±15 minutes) later. Thereafter, the cats were again reinfested, and a third flea count was performed 24 hours (±1 hour) later. Subsequent reinfestation and counting were done in weekly intervals for 5 weeks after treatment. Starting with the second week, infestation and counting cycles were performed as follows (Table 2): Reinfestation Flea counts and immediate reinfestation 2 hours (±15 minutes) later Flea counts and immediate reinfestation 4 hours (±15 minutes) later Flea counts 24 hours (±1 hour) later Artificial Infestations with C. felis On each artificial infestation, all cats were infested with approximately 50 new fleas (determined by weighing pupae) of approximately balanced sex proportion originating from the Institute for Parasitology, University of Veterinary Medicine Hannover, Germany. For flea infestation, flea containers 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, and feet. All fleas were removed, counted, and collected in a 50 ml falcon tube. TABLE 2. Experimental Design of the Comparative Speed of Flea Kill Study Study Days 0/1 Study Day 2 Study Day 1 0 hr + 6 hr + 12 hr + 24 hr Flea infestation Flea counts Treatment Flea counts Flea counts Flea counts Allocation to groups Flea reinfestation Flea reinfestation Flea reinfestation Study Days 6/7, 13/14, 20/21, 27/28, 34/35 0 h + 2 hr + 4 hr + 24 hr Flea reinfestation Flea counts Flea counts Flea counts Flea reinfestation Flea reinfestation Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

22 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM 40 Flea Count b 6 d e e b Study Day 0: Hours after Treatment and Infestation Control Imidacloprid Selamectin Fipronil/(S )-methoprene Metaflumizone Figure 1. Geometric means of flea counts at 6 hours after treatment and 12 and 24 hours after reinfestation. Significant differences in flea counts were noted between group b and groups d and e. Flea Count b c Study Day 6: Hours after Infestation d e Control Imidacloprid Selamectin Fipronil/(S )-methoprene Metaflumizone Figure 2. Geometric means of flea counts at 2, 4, and 24 hours after reinfestation. Significant differences in flea counts were noted between group b and groups c, d, and e. Statistical Analysis Arithmetic and geometric means of flea counts were calculated. The percentage efficacy was calculated according to the EMEA/CVMP guidelines 15 using the formula % Efficacy = 100 (C T) / C where C is the geometric mean of fleas in the control group and T is the geometric mean of fleas in the treatment group. These calculations were performed for each treatment group. Statistical differences of flea counts in the different groups were compared using the Wilcoxon-Mann-Whitney U test. RESULTS Geometric means of all groups on all study days are shown in Figures 1 to 6. The 4-hour geometric mean flea count (6-hour count on day 0) for imidacloprid was lower than all other groups at five of the six evaluation points (days 0, 6, 13, 20, and 34). Twenty-four hours after infestation, all treatments showed the similar and expected high efficacy (Figure 1), with a slight drop-off toward the end of the study (Figure 6). The group differences shown above have been calculated based on the explorative two-sided Mann-Whitney U test (alpha 5%). The analysis showed statistically significant differences between imi - 20 Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

23 NAVC CONFERENCE, JANUARY Flea Count b e c e 10 0 b Study Day 13: Hours after Infestation Control Imidacloprid Selamectin Fipronil/(S )-methoprene Metaflumizone Figure 3. Geometric means of flea counts at 2, 4, and 24 hours after reinfestation. Significant differences in flea counts were noted between group b and groups c and e Flea Count Study Day 20: Hours after Infestation Control Imidacloprid Selamectin Fipronil/(S )-methoprene Metaflumizone Figure 4. Geometric means of flea counts at 2, 4, and 24 hours after reinfestation. dacloprid and comparative compounds at the days/times as shown in Table 3. On study day 13, imidacloprid-treated animals showed significant differences in flea counts at 2 hours compared to metaflumizone and at 4 hours compared to selamectin and metaflumizone (Figure 3). The 24-hour insecticidal flea efficacy of imidacloprid on study day 34 appeared better than the comparative compounds. The efficacies of the four tested compounds (given in % flea reduction) on study days 0, 6, 13, 20, 27, and 34 are given in Table 4. DISCUSSION The aim of the study was to evaluate the speed of flea kill of four ectoparasiticides registered worldwide for flea control on cats. While the EMEA/CVMP guidelines 15 require 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 of time to avoid exposure of cats to flea saliva or to reduce the likelihood of pathogen transmission. The study reported here confirmed that all tested compounds showed a similar efficacy at 24 hours after infestation, as required for registration. 18 It is well known that the efficacy of many compounds against fleas decreases with time and generally lasts for about 4 weeks. In the present study, efficacies at 28 days posttreatment were 96% for imidacloprid, 83% for selamectin, 91% for fipronil, and 82% for metaflumizone. Calculated efficacies are influenced by the flea count of the controls, biologic variations, and group sizes. In this trial, conditions were unique in that cats were combed 18 times Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

24 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM Flea Count Study Day 27: Hours after Infestation Control Imidacloprid Selamectin Fipronil/(S )-methoprene Metaflumizone Figure 5. Geometric means of flea counts at 2, 4, and 24 hours after reinfestation Flea Count Study Day 34: Hours after Infestation Control Imidacloprid Selamectin Fipronil/(S )-methoprene Metaflumizone Figure 6. Geometric means of flea counts at 2, 4, and 24 hours after reinfestation. throughout the study for flea counts; thus, there is the likelihood that active compound was removed from the fur of the treated cats. While this frequent combing of the animals in the study may be in contrast to the fate of the actives under field conditions, one should consider the impact of intensive grooming of cats on the pharmacokinetics of topically applied actives. The frequent combing may explain 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 effects includes the antifeeding effect reported for imidacloprid 19 and the ability to quickly eliminate fleas from an infested host to prevent the fleas from biting. Studies evaluating the speed of flea kill by insecticides registered as veterinary medicinal remedies have been recorded. Fipronil in a spray and spot-on formulation was evaluated in comparison to imidacloprid by Marchiondo and colleagues 20 and Cruthers and colleagues. 21 In these studies, efficacy was evaluated at 8 hours after treatment and reinfestations, or 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. 22 However, all of these studies were conducted on dogs. Studies evaluating the speed of flea kill on cats are limited. In a study conducted by Schenker and colleagues, 23 cats were infested with 100 adult cat fleas per animal and flea counts were performed 3 and 8 hours after treatment. The efficacies reported at the 3-hour count were 26.9% for imidacloprid and 24.3% for fipronil; at 8 22 Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

25 NAVC CONFERENCE, JANUARY 2008 hours, the results were 82.8% for imidacloprid and 62.6% for fipronil. Because the study aimed to compare topically applied insecticides with the orally applied nitenpyram that is eliminated Days/Time Points with Statistically Significant Differences and excreted in cats within 3 days, the evaluations 3 and 8 hours in Flea Counts between Imidacloprid and Other Tested Compounds after treatment were the only ones carried out. The other recorded speed of flea kill study conducted in cats 24 Study Study Study compared selamectin with imidacloprid and fipronil/(s)-methoprene Day hr Day hr Day hr using the special KS-1 flea strain. After infestation with Selamectin X X 100 adult cat fleas, comb counts were performed at 6, 12, 24, and Fipronil/(S)-methoprene X X 48 hours after treatment (day 0) and after reinfestation (days 7, Metaflumizone X X X 14, 21, and 28). At day 0, the onset of flea efficacies was the fastest for imidacloprid, with 86.7% reduction compared with 0% X denotes statistical significance. for selamectin and 28.6% for fipronil. At 12 hours, imidacloprid achieved 99.3% reduction TABLE 4. compared with 59.7% for selamectin and 89.6% for fipronil. Flea Efficacies (in % Flea Reduction)* The study presented here confirmed that imidacloprid shows high and early efficacy. Six hours after treatment, flea count in the imidacloprid Study Days 0/1 Imidacloprid Day hr 61.1 Day hr 100 Day hr 100 group was already reduced by more Selamectin Fipronil/(S)-methoprene than 60%. During the first 2 weeks after treatment, Metaflumizone efficacy of imidacloprid was already about 60% at 2 hours and more than 90% at 4 Study Days 6/7 Day hr Day hr Day hr hours after reinfestation. The flea reduction at Imidacloprid Selamectin hour flea counts was significantly higher in Fipronil/(S)-methoprene the imidacloprid-treated group than in most of the other groups during the first 2 weeks following treatment. Metaflumizone Study Days 13/ Day hr 53.6 Day hr 85.9 Day hr Imidacloprid Mean flea counts in the control group Selamectin showed considerable variations. Newly developed Fipronil/(S)-methoprene pupae were weighed, and an equivalent of Metaflumizone fleas taken for every infestation and reinfestation resulted in flea counts between 24 and Study Days 20/21 Day hr Day hr Day hr Imidacloprid It is well known that individual grooming Selamectin behavior, especially in cats, leads to different recovery rates. According to the guidelines, Fipronil/(S)-methoprene Metaflumizone % to 75% 25 or 50% 15 of the infestation rate Study Days 27/28 Day hr Day hr Day hr should be found on the controls. With a mean Imidacloprid infestation of 50 fleas per cat per infestation, Selamectin these requirements were met, with the lowest infestation rate of 48% on study day 20 at 4 hours postinfestation. Fipronil/(S)-methoprene Metaflumizone Study Days 34/ Day hr Day hr Day hr CONCLUSION This study demonstrated that a single topical application of imidacloprid at the recommended dosage provided a high efficacy in the early elimination of adult cat fleas. With this fast onset of TABLE 3. Imidacloprid Selamectin Fipronil/(S)-methoprene Metaflumizone *Calculated from geometric means of flea counts on the treated and control cats at 6 hours after treatment and 12 and 24 hours after reinfestation (study days 0/1) and 2, 4, and 24 hours after subsequent weekly reinfestations. Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

26 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM flea efficacy, it is likely that imidacloprid is 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, Wiegand B, Pfister K. Qualitative and quantitative observations on the flea population dynamics of dogs and cats in several areas of Germany. Vet Parasitol 2006;137: Rinaldi L, Spera G, Musella V, et al. A survey of fleas on dogs in southern Italy. Vet Parasitol 2007;148: Dryden MW, Rust MK. The cat flea: biology, ecology and control. Vet Parasitol 1994;52: 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-Verlag; 2001: Rust MK, Dryden MW. The biology, ecology, and management of the cat flea. Annu Rev Entomol 1997;42: 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: Breitschwerdt ED, Levine JF, Radulovic S, et al. Bartonella henselae and Rickettsia seroreactivity in a sick cat population from North Carolina. Int J Appl Res Vet Med 2005;3: 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: Chomel BB, Kasten RW, Floyd-Hawkins K, et al. Experimental transmission of Bartonella henselae by the cat flea. Eur J Clin Microbiol 1996;34: Kordick DL, Brown TT, Shin KO, et al. Clinical and pathological evaluation of chronic Bartonella henselae or Bartonella clarridgeiae infection in cats. Eur J Clin Microbiol 1999;33: 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: Rolain JM, Franc M, Davoust B, et al. 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: Vobis M, D Haese J, Mehlhorn H, et al. Evidence of horizontal transmission of feline leukemia virus by the cat flea (Ctenocephalides felis). Parasitol Res 2003;91: Halliwell REW. Flea allergy dermatitis. In: Kirk RW, ed. Current Veterinary Therapy VIII. Philadelphia: WB Saunders; 1983: Committee for Medicinal Products for Veterinary Use. 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 [Doc. Ref. EMEA/CVMP/EWP/005/2000-Rev.2] Gaulliard JM. Fipronil: a broad spectrum insecticide. Phytoma 1996; 488: 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: Franc M, Yao KP. Comparison of the activity of selamectin, imidacloprid and fipronil for the treatment of cats infested experimentally with Ctenocephalides felis felis and Ctenocephalides felis strongylus. Vet Parasitol 2007;143: 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): Marchiondo AA, Green SE, Plue RE, et al. Comparative speed of kill of Frontline spray, Frontline Top Spot and Advantage against adult cat fleas (Ctenocephalides felis) on dogs. Proc 5th Int Symp Ectopara Pets 1999: Cruthers L, Guerrero J, Robertson-Plouch C. Evaluation of the speed of kill of fleas and ticks with fipronil or imidacloprid. Proc 5th Int Symp Ectopara Pets 1999: Everett R, Cunningham J, Arther B, et al. Comparative evaluation of the speed of flea kill of imidacloprid and selamectin on dogs. Vet Ther 2000;1: Schenker R, Tinembart O, Humbert-Droz E, et al. Comparative speed of kill between nitenpyram, fipronil, imidacloprid, selamectin and cythioate against Ctenocephalides felis (Bouche) on cats and dogs. Vet Parasitol 2003;112: Dryden MW, Smith V, Payne PA, et al. Comparative speed of kill of selamectin, imidacloprid, and fipronil-(s)-methoprene spot-on formulations against fleas on cats. Vet Ther 2005;6: 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: Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

27 NAVC CONFERENCE, JANUARY 2008 Lice Management Options: Efficacy of Three Imidacloprid- Containing Formulations against Natural Lice (Trichodectes canis) Infestations in Dogs Dorothee Stanneck, DVM, Dr. med. vet., DEVPC a * Jennifer Ketzis, PhD, BSc, MSc, MCert Proj Mngt b Jeanie Doyle, BSc (Honors) b Margret Fisher, DVM, DEVPC c a Bayer HealthCare AG, Animal Health, Clinical Development Parasiticides, Leverkusen, Germany b Charles River Laboratories, BioLabs Europe, Ballina, County Mayo, Ireland c Shernacre Enterprise, Shernacre Cottage, Malvern, Worcester, UK Biting lice (Trichodectes canis) are small, dorsoventrally flattened, wingless insects (Figure 1). Like other species of louse, T. canis is very host specific, and this species parasitizes dogs. They have mouthparts adapted to rasping the surface of the skin and eating the debris. T. canis spend their entire life on the host: Female lice attach their eggs to hair shafts, and immature lice (which appear similar to adult lice but are smaller in size; Figure 2) hatch after 1 to 2 weeks and mature to adults about 3 to 5 weeks after the eggs are laid. Lice can be found on dogs throughout the world, but they appear to be particularly common in Scandinavia. 1 In this northerly region, lice are often the main ectoparasite because the climate does not support the flea or tick life cycle. In other geographic areas, lice infestation occurs sporadically, usually as concurrent parasites on dogs kept under robust, outdoor housing conditions. Lice populations can become very large, especially in young, old, or un healthy dogs, perhaps because grooming is not as effective. Small numbers of lice may be well tolerated, but an established population can cause intense pruritus associated with self-trauma, as demonstrated by a study conducted in Sweden in which sucking lice (Linognathus setosus) appeared to be the underlying cause in 23% of cases of pyotraumatic dermatitis (hot spots) in 44 dogs. 2 In addition to the distress caused by the lice themselves, T. canis can also act as an effective *Corresponding author: D. Stanneck, Bayer HealthCare AG, Animal Health, Clinical Development Parasiticides, BHC-AH-RD-CRD-Para, Building 6700, Leverkusen, Germany; phone, +49 (0) ; fax, +49 (0) ; , dorothee.stanneck@bayerhealthcare.com. The active ingredient imidacloprid has also been successfully combined with permethrin and with moxidectin to provide products with broader spectra of activity. intermediate host for the tapeworm Dipylidium caninum. 3,4 Traditional treatments have tended to be ineffective against stages within the egg, so two treatments were often necessary. 5,6 However, because the entire louse life cycle occurs on the host, as long as the treatment can penetrate the egg or remain active for about 2 weeks and affect immature lice as they hatch, a single treatment is all that is necessary. This has been achieved in recent years with long-lasting active ingredients whose efficacy exceeds the development time of the louse life cycle. 1,7 10 One of these active ingredients is imidacloprid, a chloronicotinyl nitroguanidine that has been marketed as a flea control product for almost a decade. The active ingredient imidacloprid has also been successfully combined with permethrin and with moxidectin to provide products with broader spectra of activity. Imidacloprid has been recognized for some time as having insecticidal activity against lice, 1 but so far the proof of these observations by formal, controlled studies has been lacking. MATERIALS AND METHODS Three consecutive studies were conducted using an identical design to prove the efficacy of three imidacloprid-containing formulations against lice. All the studies were conducted under the principles of Good Clinical Practice (GCP), as given in the Guideline VICH GL9, and followed the recommendations of the Guideline 7AE17a (Notice to Applicants VOL 7A, 1993): Demonstration of Efficacy of Ectoparasiticides (III/3682/92), and the Guideline for the Testing and Evalua- Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

28 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM Figure 1. The dog biting louse, Trichodectes canis. tion of the Efficacy of Antiparasitic Substances for the Treatment and Prevention of Tick and Flea Infestation in Dog and Cats, CVMP/005/00, January In each of the studies, dogs with natural biting louse infestations were ranked by their louse counts and then paired, with one of each pair randomly allocated to one of two groups. Thus, two groups of 10 dogs each were created per study. Dogs in one group received a spot-on treatment on day 0, while those in the second group served as untreated controls. Treatments for the first, second, and third study were the minimum therapeutic dosage of 0.1 ml/kg 10% imidacloprid (Bayer Advantage ), 10% imidacloprid/2.5% moxidectin (Bayer Advocate /Advantage Multi ), or 10% imidacloprid/50% permethrin (Bayer Advantix /K9 Advantix ), respectively. The product was administered as a topical spot-on between the shoulder blades or along the back topline of the dogs, according to the label instructions. Dogs were individually housed throughout the study, and cages were color-coded to ensure that treatment was not accidentally transferred into control pens. Lice were counted before treatment (the counts used for allocation) and on days 1, 2, 7, 14, 21, 28, and 35 or 36. On each occasion, the animals were sedated and the numbers of lice were counted in defined areas: ears (right and left), forehead, side of thorax (left and right), abdomen, base of tail, and a strip (approximately 8 cm long 2 cm wide) along the midline of the back. For the sides of the thorax and the abdomen, an area of approximately 6 7 cm was searched. Louse counts were performed on each animal for a minimum of 5 minutes. When the total louse count recorded for an animal was zero, the following areas were also searched: the underside (ventral surface) of the neck, the hind leg rump area, and the caudal aspect of each limb. Again, when a total louse count of zero was recorded for these areas, a general sweep of each side of the animal s body was performed. At each of the specified count locations, the hair of the animal was gently separated and back-combed (using a fine-toothed comb, forceps/tweezers, or finger), allowing the operator to count any lice present. For animal welfare reasons, when counts were found to be greater than 100, enough lice were removed from the animal to achieve a remaining population of 10 to 100 lice (except in the second study, in which four animals in group 1 were combed on day 1 until their Figure 2. Adults and one juvenile stage of Trichodectes canis. remaining louse counts equaled 100). The total number of lice counted on each animal at each examination time point was transformed to the natural logarithm (count + 1) for calculation of geometric means. Percent efficacy was calculated according to the recommendations for controlled tests described in Guideline 7AE17a using Abbott s formula: % Effectiveness (reduction) = 100 (C T) / C where C is the geometric mean count of T. canis for the untreated control group (group 1) and T is the geometric mean count of T. canis for the treated group (group 2). Louse counts were formally analyzed using analysis of variance. Factors in the model were the treatment (treated or untreated). On day 2, louse counts were analyzed using a (nonparametric) Mann Whitney test because transformations could not be appropriately performed. Louse counts on days 7, 14, 21, 28, and 35 were analyzed using (nonparametric) Fisher s exact test because analysis of variance and transformations were not appropriate. The adequacy of infection was assessed on day 1 using the lower 95% CI of geometric louse counts in the untreated group. The lower 95% CI was above 10% of the appropriate central tendency calculation in the control group. All statistical tests were two-tailed, with a level of significance of 5%. RESULTS In all three studies, there was no difference between louse counts in the two groups before treatment. 26 Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

29 NAVC CONFERENCE, JANUARY 2008 TABLE 1. Summary of the Efficacy of 10% Imidacloprid against Trichodectes canis in Naturally Infected Dogs Control Imidacloprid Range of Geometric Mean No. of Range of Geometric Mean No. of Day Louse Counts Louse Count Dogs with Lice Louse Counts Louse Count Dogs with Lice % Efficacy * * * * * * * *Count significantly different from control. TABLE 2. Summary of the Efficacy of 10% Imidacloprid/2.5% Moxidectin against Trichodectes canis in Naturally Infected Dogs Control Imidacloprid/Moxidectin Range of Geometric Mean No. of Range of Geometric Mean No. of Day Louse Counts Louse Count Dogs with Lice Louse Counts Louse Count Dogs with Lice % Efficacy * * * * * * * *Count significantly different from control. In the imidacloprid study, by day 1 (24 hours after treatment) counts had been reduced from a range of 29 to 111 lice to a range of 1 to 68 lice. By day 7, no lice remained on the treated dogs, and apart from two lice found on one dog on day 14, dogs remained louse free until the end of the study on day 36. In contrast, all except two dogs in the control group maintained a louse count of 16 to 280. Thus, imidacloprid at the standard minimum dosage was 100% effective in removing an established biting louse population (Table 1). In the imidacloprid/moxidectin study, louse counts were markedly reduced on all dogs by 1 day after treatment; however, the count on one dog that had 719 lice on day 3 was reduced to only 201 on day 1. In this dog (75533) and one other dog (90016), in which the count was 96 on day 1, imidacloprid/moxidectin had an overall efficacy of 89.9% on day 1. Counts in all dogs continued to diminish, and all dogs had zero counts by day 28, except dog 75533, which had started with the highest count. Louse numbers continued to decline on that dog, but one louse was found on day 35 at the conclusion of the study. Therefore, overall efficacy of the treatment reached 99.9% (Table 2). In the imidacloprid/permethrin study, louse counts were markedly reduced on all dogs by 1 day after treatment; on one dog (05336), however, single lice could be found up to day 28. Counts in all other dogs had diminished to and remained at zero from day 7 (Table 3), except dog 84098, which showed two lice on day 3. No health problems related to treatment were observed in the studies. Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June

30 NINTH INTERNATIONAL PARASITE CONTROL SYMPOSIUM TABLE 3. Summary of the Efficacy of 10% Imidacloprid/50% Permethrin against Trichodectes canis in Naturally Infected Dogs Control Imidacloprid/Permethrin Range of Geometric Mean No. of Range of Geometric Mean No. of Day Louse Counts Louse Count Dogs with Lice Louse Counts Louse Count Dogs with Lice % Efficacy * * * * * * * *Count significantly different from control. DISCUSSION In all three laboratory studies, 95% efficacy was obtained by 2 days after treatment and louse infestations were either 100% eliminated with a single treatment (imidacloprid study) or more than 99% eliminated (imidacloprid/moxidectin study and imidacloprid/permethrin study) within 7 days. At the end of the treatment interval, there were only single dogs with single louse specimens. Reasons for this could be an extremely high louse burden at the time of treatment, resulting in a heavy reinfection pressure due to the number of nits (eggs) already present in the dog s coat at the time of treatment (imidacloprid/ moxidectin study); a diseased, underweight dog with a much higher susceptibility to the louse infestation (imidacloprid/ permethrin study); or a possible delayed hatching of eggs present on the dog from the prior infestation. However, because numbers of lice found were always in the single digits despite intensive combing of the whole dog, it is unlikely that these single lice on a dog would be able to reestablish an infestation. Consistently high efficacy values were obtained after the application of a single treatment of imidacloprid on dogs representing a range of breeds naturally infested with T. canis under laboratory conditions. Therefore, the results support the statement that a single treatment at the established dosage will effectively treat infestations of the biting louse T. canis. Needing only a single treatment is particularly useful. In the past, two treatments with nonpersistent products were often needed to eliminate an infection 11 because unhatched lice are protected within the nits from the insecticide and reinfestation occurs with newly hatched lice 1 to 2 weeks after treatment. The choice of biting lice for this study can be judged as a particularly critical test for long-term efficacy against both dog louse species: The egg-to-egg development period of T. canis is 3 to 5 weeks, 12 whereas L. setosus develops in just 8 to 10 days. 12 Therefore, the treatment success in the slowly developing biting lice is a strong hint for the same long-term efficacy in the fast-developing sucking lice. This is in line with the data from a field study 1 that demonstrated good efficacy of imidacloprid against both biting and sucking lice for 6 weeks. REFERENCES 1. Hanssen I, Mencke N, Asskildt H, et al. Field study on the insecticidal efficacy of Advantage against natural infestations of dogs with lice. Parasitol Res 1999;85: Holm BR, Rest JR, Seewald W. A prospective study of the clinical findings, treatment and histopathology of 44 cases of pyotraumatic dermatitis. Vet Derm 2004;15: Jueco NL. Parasitic zoonoses. In: Steele JH, ed. Handbook Series in Zoonoses. Boca Raton, FL: CRC Press; 1982: Wall R, Shearer D, eds. Veterinary Entomology. London: Chapman Hall; 1997: O Dair H. Differential diagnosis of pruritus in cats. In Pract 1992; 14: Scott DW, Miller WH, Griffen CE. Parasitic skin diseases. In: Scott DW, Miller WH, Griffen CE, eds. Muller and Kirk s Small Animal Dermatology. 5th ed. Philadelphia: WB Saunders; 1995: Cooper PR, Penaliggon J. Use of fipronil to eliminate recurrent infestation by Trichodectes canis in a pack of bloodhounds. Vet Rec 1996;139: Endris RG, Reuter VE, Nelson J, Nelson JA. Efficacy of a topical spoton containing 65% permethrin against the dog louse, Trichodectes canis (Mallophaga:Tichodectidae). Vet Ther 2001;2(2): Gautier P, Allan J, Pengo G, et al. The efficacy of the novel avermectin, selamectin, against biting lice on dogs and cats. Proc 6th Int Symp Ectopara Pets 2001: Pollmeier M, Pengo G, Jeannin P, Soll M. Evaluation of the efficacy of fipronil formulations in the treatment and control of biting lice, Trichodectes canis, on dogs. Vet Parasitol 2002;107: Bowman DD, ed. Orders Anoplura, bloodsucking lice, and Mallophaga, chewing lice. In: Georgi s Parasitology for Veterinarians. 7th ed. Philadelphia: WB Saunders; 1999: Zlotorzycka J, Eichler W, Ludwig HW, eds. Taxonomie und Biologie der Mallophagen und Läuse mitteleuropäischer Haus- und Nutztiere. Vol st ed. Jena: VEB Gustav Fischer Verlag; 1974: Supplement to Compendium: Continuing Education for Veterinarians Vol. 30, No. 6(B), June 2008

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