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Prevention of experimentally induced heartworm (Dirofilaria immitis) infections in dogs and cats with a single topical application of selamectin
Veterinary Parasitology 91 (2000) 259–268
Prevention of experimentally induced heartworm (Dirofilaria immitis) infections in dogs and cats with a single topical application of selamectin T.L. McTier a,∗ , D.J. Shanks b , P. Watson b,1 , J.W. McCall c , C. Genchi d , R.H. Six a , C.A. Thomas a,2 , S.K. Dickin b , G. Pengo e , T.G. Rowan b , A.D. Jernigan a a
b
Animal Health Clinical Affairs, Central Research Division, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA Animal Health Clinical Affairs, Central Research Division, Pfizer Limited, Sandwich, Kent CT13 9NJ, UK c Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA d Institute of General Veterinary Pathology, University of Milan, Milan, Italy e Via Marmara, 19/C Paderno Dugnano, Milan, Italy
Abstract In a series of six controlled studies (four in dogs, two in cats), heartworm-free dogs and cats were inoculated with Dirofilaria immitis larvae (L3 ) prior to topical treatment with the novel avermectin selamectin or a negative control containing inert formulation ingredients (vehicle). Selamectin and negative-control treatments were administered topically to the skin at the base of the neck in front of the scapulae. In dogs, selamectin was applied topically at dosages of 3 or 6 mg kg−1 at 30 days post-inoculation (PI), or of 3 or 6 mg kg−1 at 45 days PI, or of 6 mg kg−1 at 60 days PI. Cats were treated topically with unit doses providing a minimum dosage of 6 mg kg−1 selamectin at 30 days PI. Of the animals that were treated 30 days PI, some dogs were bathed with water or shampoo between 2 and 96 h after treatment, and some cats were bathed with shampoo at 24 h after treatment. Between 140 and 199 days PI, the animals were euthanized and examined for adult D. immitis. Adult heartworms developed in all control dogs (geometric mean count, 18.7 worms) and in 88% of control cats (geometric mean count, 2.1 worms). Selamectin was 100% effective in preventing heartworm development in dogs when administered as a single topical dose of 3 or 6 mg kg–1 at 30 days after infection, 3 or 6 mg kg−1 at 45 days after infection, or 6 mg kg−1 at 60 days after infection. Selamectin was 100% effective against heartworm infections in cats when administered as a
∗ Corresponding author. Tel.: +1-860-441-6454; Fax +1-860-441-5779. E-mail address: tom l [email protected] (T.L. McTier) 1 Present address: Royal Veterinary College, London, NW1 OUT, UK. 2 Present address: Animal Health Group, North American Region, Pfizer Inc., Exton, PA, USA.
0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 2 9 7 - 1
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single topical unit dose of 6 mg kg−1 . Bathing with water or shampoo between 2 and 96 h after treatment did not reduce the efficacy of selamectin as a heartworm prophylactic in dogs. Likewise, bathing with shampoo at 24 h after treatment did not reduce the efficacy of selamectin in cats. These studies demonstrated that, at the recommended dosage and treatment interval, a single topical administration of selamectin was 100% effective in preventing the development of D. immitis in dogs and cats. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Selamectin; RevolutionTM ; StrongholdTM ; Avermectin; Heartworm; Dirofilaria immitis; Canis familiaris; Felis catus; Dog; Cat
1. Introduction Infection with the filarial parasite Dirofilaria immitis (heartworm) is common among dogs living in warm temperate and tropical parts of the world where mosquito vectors are present. Surveys performed in the USA and Italy have demonstrated heartworm prevalence of up to 70% in dogs in some endemic areas (Genchi et al., 1991; Guerrero et al., 1992). In one experimental study in the southeastern region of the United States, 13 of 14 (93%) heartworm-naive dogs placed outdoors in heartworm endemic areas became infected in a one-year period by natural exposure (McTier et al., 1992a). In cats, global prevalence values of up to 23% in endemic areas are lower than in dogs in the same areas, but indicates the susceptibility of cats to naturally acquired heartworm infection (Courtney and Zeng, 1989; Genchi et al., 1992; Guerrero et al., 1992). In addition, other studies have shown that typically 60 to 80% of cats inoculated experimentally with D. immitis will develop adult heartworm infections (McTier et al., 1992b; Paul et al., 1992; Stewart et al., 1992; Mansour et al., 1995). In dogs, the severity and rate of development of heartworm disease are primarily related to the magnitude of the worm burden (Munnell et al., 1980; Thrall et al., 1980; Knight, 1983). Heavy infections commonly result in pulmonary hypertension and right-sided heart failure (Knight, 1983). In contrast, cats mount a more intense pathological response to the presence of the parasite than do dogs (Calvert et al., 1994), and show a wider variety of clinical signs (Holmes, 1993; Calvert et al., 1994; Dillon, 1998). As few as two adult heartworms have resulted in sudden death in cats (Calvert et al., 1994). Treatment of established heartworm infections carries serious risks for the host animal, as dead and dying heartworms can cause severe complications (Calvert et al., 1994; Miller, 1998) and chemoprophylaxis is highly recommended for animals living in heartworm-endemic areas (Calvert et al., 1994; Dillon, 1998). Heartworm chemoprophylaxis has been transformed within the last decade by the introduction of macrocyclic lactones that are administered as oral formulations at monthly intervals (e.g. ivermectin, milbemycin oxime). In addition, milbemycin oxime has activity against other endoparasites at the dose used for heartworm prevention. Initial studies demonstrated that selamectin (RevolutionTM /StrongholdTM , Pfizer, New York) was effective in preventing heartworm development when applied topically to the skin as a single dose within 30 days after larval inoculation in dogs and cats (McTier et al., 1998). Other studies have demonstrated the endectocidal activity of selamectin in dogs and
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cats against a variety of other internal and external parasites (Jernigan et al., 2000; McTier et al., 2000a,b,c; Shanks et al., 2000a,b). For dogs and cats, an endectocide product applied to the skin in a single spot provides the benefits of a convenient application together with a broad-spectrum of activity. The six studies reported in this paper investigated the efficacy of selamectin in preventing the development of heartworms in dogs and cats given experimental inoculations of D. immitis under conditions of simulated under-dosing, simulated missed or late treatment (treatment administered up to 60 days after larval inoculation), and bathing of animals 2, 6, 24, or 96 h after treatment.
2. Materials and methods A total of six controlled laboratory studies were conducted, five in the USA (one in cats, and four in dogs) and one (cats) in Italy. All investigations were conducted in compliance with the Food and Drug Administration Center for Veterinary Medicine Guidelines (CVM, 1992, 1997) and with the European guidelines (FEDESA, 1995). Animals were housed in appropriate accommodation that conformed to accepted guidelines for floor area/type, lighting, temperature, humidity and welfare (including environmental enrichment and social interaction), as required by local and national legislation. 2.1. Study design The two cat studies (A and B) were similar in design and evaluated the efficacy of a single topical unit dose providing a minimum dosage of 6 mg kg−1 selamectin administered 30 days post-inoculation (PI) in preventing heartworm development (Table 1). These studies also assessed the effect on efficacy of bathing of cats with a non-insecticidal shampoo (study A — Johnson’s pH 5.5 Shampoo® , Johnson and Johnson; study B — Allergroom® , Allerderm) at 24 h after selamectin treatment. The four dog studies (C–F) evaluated selamectin at dosages of 3 or 6 mg kg−1 with treatment at various times after inoculation of heartworms (30, 45, or 60 days PI; Tables 1–4). In addition, the impact on efficacy of soaking in water for a short time (approximately 1 min) 2 h after treatment and shampoo bathing with a non-insecticidal shampoo (Allergroom® , Allerderm) at 2, 6, 24, or 96 h after selamectin treatment was evaluated in dogs. 2.2. Animals Purpose-bred domestic short-haired cats were used in studies A and B, and purpose-bred Beagles were used in studies C–F. At the time of treatment, the dogs ranged in age from 6 to 13 months and in weight from 6.3 to 15.8 kg. Cats ranged in age from 3 to 5 months, and in weight from 1.5 to 2.9 kg. Each treatment had equal number of male and female animals. All animals were confirmed to be in satisfactory physical condition and free of D. immitis infection prior to the start of the study. In studies B–F, a blood sample obtained from each animal at 32 days prior to treatment was tested for the presence of circulating microfilariae and adult heartworm antigen. An additional set of tests was performed 90 days
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Table 1 Efficacy of a single topical unit dose of selamectin providing a minimum dosage of 6 mg kg−1 against Dirofilaria immitis in bathed and unbathed dogs and cats (studies A–C) Study
Treatment
Dosage
Days between Bathing larval inoculation and treatment
Time of Geometric mean bath after of heartworm treatment (h) counts (range)
Prevention rate (%)
A (cats)
Vehiclea 0.10 ml kg−1 30 Selamectin 6 mg kg−1 30 Selamectin 6 mg kg−1 30
None – None – Shampoo 24
1.1b (1–2) 0 0
NAc 100 100
B (cats)
Vehicle 0.10 ml kg−1 30 30 Selamectin 6 mg kg−1 Selamectin 6 mg kg−1 30
None – None – Shampoo 24
3.5d (0–13) 0 0
NA 100 100
C (dogs) Vehicle 0.05 ml kg−1 30 30 Selamectin 6 mg kg−1 Selamectin 6 mg kg−1 30
None – None – Shampoo 24
7.2 (1–16) 0 0
NA 100 100
a The negative-control treatment was the vehicle only (inert ingredients) for the commercial formulation of selamectin. Studies A and B were performed in cats (12 cats per treatment); study C was performed in dogs (eight dogs per treatment). All animals were inoculated with infective D. immitis larvae (L3 ) 30 days before treatment (50 larvae per animal in studies A and C, 100 larvae per animal in study B). Necropsy examinations were performed 199 days (study A), 169 days (study B), or 142 days (study C) after heartworm inoculation. b Includes two dead heartworms found in two cats (a single worm in each cat) which were present at the time of necropsy. c Not applicable. d Three cats were free of adult heartworms at necropsy.
after treatment in these studies, to detect infections that could have been present at the start of the study but which could not be detected by current diagnostic methods at that time. In study A, heartworm tests were performed 90 days after treatment only. None of the animals had been treated with an avermectin in the 60 days prior to inoculation with larvae. The animals were all housed in mosquito-proof accommodation for the duration of each study. In each study, the animals were allocated randomly within sex to treatments. For the duration of each study, the general health of the animals was observed twice daily. Clinical Table 2 Efficacy of a single topical unit dose of selamectin providing a minimum dosage of 6 mg kg−1 against D. immitis in water-bathed, shampoo-bathed, and unbathed dogs (study D)a Treatment
Dosage
Vehicle (control) 0.05 ml kg−1 Selamectin Selamectin Selamectin Selamectin
6 mg kg−1 6 mg kg−1 6 mg kg−1 6 mg kg−1
Days between larval inoculation and treatment
Bathing
Time of bath after treatment (h)
Geometric mean of heartworm counts (range)
Prevention rate (%)
30
None
–
29.0 (18–39)
NAb
30 30 30 30
None Water Shampoo Shampoo
– 2 2 6
0 0 0 0
100 100 100 100
a There were eight dogs in each treatment, all of which were inoculated with 50 D. immitis larvae (L ) 30 days 3 before treatment administration. Necropsy examinations were performed 140 days after heartworm inoculation. b Not applicable.
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Table 3 Efficacy of a single topical dose of 3 or 6 mg kg−1 selamectin against D. immitis when applied at 1 month after inoculation or at extended treatment intervals in dogs (study E)a Treatment
Dosage
Days between larval inoculation and treatment
Geometric mean of heartworm counts (range)
Prevention rate (%)
Vehicle (control)
0.05 ml kg−1
30, 45, 60
22.6 (10–29)
NAb
Selamectin Selamectin Selamectin Selamectin Selamectin
3 mg kg−1
30 30 45 45 60
6 mg kg−1 3 mg kg−1 6 mg kg−1 6 mg kg−1
0 0 0 0 0
100 100 100 100 100
a
There were six dogs in each treatment, all of which were inoculated with 50 D. immitis larvae (L3 ) 30, 45, or 60 days before treatment. The dogs were not bathed. Necropsy examinations were performed 140 days after heartworm inoculation. b Not applicable.
observations were made on all animals by a veterinarian before treatment, on at least three occasions in the 8 h following treatment, and 24 h after treatment. The veterinarians and other personnel who assessed the animals’ health were not aware of treatment allocations. 2.3. Inoculation with D. immitis larvae The animals were inoculated with D. immitis at 30, 45, or 60 days before treatment. Each inoculum consisted of 50 (studies A, C–F) or 100 (study B: cats ) infective D. immitis larvae (L3 ) in Hank’s balanced salt solution. The animals were infected by subcutaneous inoculation in the inguinal area (studies B–F) or by subcutaneous injection of the inoculum into the neck (study A: cats). Third-stage larvae (L3 ) were reared and collected as described by McCall et al. (1980). The heartworm larvae used were from a laboratory strain maintained by Dr. John McCall (University of Georgia, Athens, GA). 2.4. Selamectin and negative-control treatments Selamectin was formulated at concentrations of 60 and 120 mg ml−1 in an excipient (vehicle) consisting of glycol ether, isopropyl alcohol, and butylhydroxytoluene. In studies E and F, the exact dose of selamectin was administered by using a 1- or 3-ml syringe. In all other studies (A–D), selamectin was supplied in the commercial formulation in unit dose tubes. Selamectin unit doses were designed to deliver selamectin at a minimum recommended dosage of 6 mg kg−1 (range, 6–12 mg kg−1 for dogs and 6–17.3 mg kg−1 for cats). In all studies, the vehicle for the commercial formulation of selamectin was used as a negative-control treatment at a dosage of 0.05 ml kg−1 in dogs, and 0.10 ml kg−1 in cats. Unit doses for both the negative-control and selamectin treatments were selected according to body weight ranges. All animals were weighed within 3 days before treatment for calculation of the appropriate dose. In all studies, selamectin and the vehicle were applied in a similar manner topically to the skin at the base of the neck in front of the scapulae on day zero.
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2.5. Necropsy procedure Each animal received an intravenous injection with heparin and an approved euthanasia solution. Necropsy for heartworm counts was performed between 140 and 142 days PI for dogs and between 169 and 199 days PI for cats (Tables 1–4). Necropsies were conducted within approximately 2 h of euthanasia. In dogs, the postmortem procedure involved clamping of the anterior and posterior vena cava, removal of the heart and lungs, dissection and examination of the anterior vena cava, right atrium, right ventricle, pulmonary arteries, and exploration of the pleural and peritoneal cavities. In cats, in addition to the above procedures, the liver was removed and the cardiac–hepatic portion of the posterior vena cava was examined. Any adult D. immitis identified were recorded as being dead or alive according to the procedure of Holmes et al. (1986). Within each study, the order in which the necropsies were performed was randomized across treatments. The personnel who performed the examinations did not know to which treatment the animals had been allocated. 2.6. Data analysis Heartworm counts from postmortem examinations were summarized for each treatment in each study and geometric mean heartworm counts were calculated. The heartworm prevention rate, defined as the percentage of selamectin-treated animals free of adult heartworms at necropsy, compared with that for controls, was estimated for each treatment. Treatments with prevention rates of 100% were considered efficacious. Statistical analysis of the data was not performed. 3. Results 3.1. Prophylactic efficacy When applied topically to the skin at a dosage of 3 or 6 mg kg−1 , 30 days PI, selamectin prevented heartworm infections in all 140 treated animals (92 dogs, 48 cats; Tables 1–4). When the effects of a simulated 15-day delay in treatment were investigated (treatment 45 days PI), dosages of 3 or 6 mg kg−1 remained 100% effective against heartworms (Tables 3 and 4). When treatment was delayed until 60 days PI in dogs, simulating a missed monthly dose, a single application of 6 mg kg−1 selamectin was completely effective in preventing the development of heartworms (Tables 3 and 4). The effect of bathing after treatment was investigated in a total of 72 animals (48 dogs, 24 cats), all of which had been treated with 6 mg kg−1 selamectin 30 days PI. When dogs were bathed with water 2 h after treatment, or with a non-insecticidal shampoo 2, 6, 24, or 96 h after treatment, the efficacy of selamectin remained at 100% (Tables 1, 2 and 4). Similarly, bathing cats at 24 h after treatment with a non-insecticidal shampoo did not reduce the efficacy of selamectin (Table 1). 3.2. Clinical observations There were no mortalities among the animals during any of these studies, and the abnormalities that occurred in association with treatment were only minor and temporary.
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Stiffening of the hairs, hairs adhering together, and the presence of a slight white powder on the hairs were observed at the treatment site in some selamectin- and vehicle-treated animals for a period of 24 h after treatment.
4. Discussion When administered at the recommended dosage (6 mg kg−1 ), selamectin was 100% effective in preventing heartworm development in all of the 126 animals tested (78 dogs, 48 cats). This prevention rate was achieved in the face of larval challenges that resulted in the development of adult heartworms in 100% of control dogs (n=30) and 21 of 24 control cats (88%). In dogs, when administered 30 days after larval inoculation, selamectin remained 100% effective at half the recommended dosage (3 mg kg−1 ; n=14). In the studies that simulated delayed treatment (administration 45 days after larval inoculation), a single unit dose of selamectin at 3 or 6 mg kg−1 was 100% effective in all dogs tested (n=14 at both dose levels). Also, when administered at 6 mg kg−1 at 60 days PI, simulating a missed monthly treatment, selamectin remained completely effective in preventing heartworm maturation in dogs (n=14). The complete efficacy of a single topical unit dose of 6 mg kg−1 selamectin administered 45 days PI compares favorably with other macrocyclic lactones approved for heartworm prevention in dogs. Both milbemycin oxime and ivermectin are known to be effective in preventing heartworm development when administered as a single treatment at the recommended dosages at 45 days PI, providing a “grace period” if monthly treatment is delayed (Grieve et al., 1991; Paul et al., 1986, 1991). The current studies have shown that selamectin not only provides a “grace period” if treatment is delayed in dogs, but the “grace period” is maintained if a monthly treatment is inadvertently missed. Moxidectin also maintains a grace period similar to that for selamectin in dogs (McTier et al., 1992c). In dogs, these studies have shown that the 100% efficacy of a single unit dose providing a minimum dosage of 6 mg kg−1 selamectin in preventing heartworm maturation is not reduced by bathing the animal, even when the haircoat is soaked with water or bathed with shampoo as early as 2 h after treatment. A total of 48 dogs were bathed, either with water 2 h after treatment (n=8) or with shampoo 2 h (n=8), 6 h (n=8), 24 h (n=16), or 96 h (n=8) after treatment. Adult heartworms did not develop in any of these dogs. Also, bathing cats with shampoo 24 h after treatment (n=24) with a topical unit dose of 6 mg kg−1 did not reduce the efficacy of selamectin. Bathing of dogs at 2, 6, or 24 h after treatment and bathing of cats at 24 h after treatment did not reduce the efficacy of a single unit dose of selamectin against weekly experimental flea infestations for 1 month (McTier et al., 2000a). At the time of necropsy, heartworms were present in all control dogs (n=30) and in 88% of control cats (n=24). The mean heartworm counts from control animals were 18.7 for all four dog studies (C–F) combined and 2.1 for the two cats studies (A and B) combined. These numbers are similar to those achieved by other investigators in similar studies in dogs (McCall et al., 1980; Paul et al., 1986; McTier et al., 1992c; Blagburn et al., 1995) and in cats (McTier et al., 1992b; Paul et al., 1992; Stewart et al., 1992) and are sufficient to allow thorough assessment of the efficacy of selamectin as a heartworm preventive. Cats are naturally more resistant to the development of adult heartworm infection than are dogs,
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and this is reflected in the results of these studies. Inoculation of cats with 50 D. immitis larvae resulted in mean worm counts of 1.1 for cats (study A), and inoculation of cats with 100 larvae resulted in a mean worm count of 3.5 (study B).
5. Conclusions This series of studies demonstrated that selamectin was completely effective in preventing the development of D. immitis when applied topically to the skin of dogs and cats at a dosage of 6 mg kg−1 at 30 days PI. Under-dosing (3 mg kg−1 ) or delay of treatment (6 mg kg−1 ) by up to 1 month did not reduce the efficacy. In addition, soaking or shampooing dogs 2 h after treatment or shampooing cats 24 h after treatment did not alter selamectin’s efficacy. Selamectin provides robust protection against heartworms in a convenient topical formulation. References Blagburn, B.L., Hendrix, C.M., Vaughan, J.L., Lindsay, D.S., Tebbit, G.L., 1995. Efficacy of a chewable formulation of milbemycin oxime against preadult Dirofilaria immitis and adult Ancylostoma caninum in naturally and experimentally infected dogs. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium’95. American Heartworm Society, Washington, DC, pp. 171–175. Calvert, C.A., Rawlings, C.A., McCall, J.W., 1994. Feline heartworm disease. In: Sherding, R.G. (Ed.), The Cat: Diseases and Clinical Management, 2nd Edition. Churchill Livingstone, New York, pp. 623–645. CVM, 1992. Conduct of clinical investigations: responsibilities of clinical investigators and monitors for investigational new animal drug studies. Technical Report, October. Center for Veterinary Medicine, Rockville, MD. CVM, 1997. Good target animal study practices: clinical investigators and monitors. Technical Report, May. Center for Veterinary Medicine, Rockville, MD. Courtney, C.H., Zeng, Q.Y., 1989. The structure of heartworm populations in dogs and cats in Florida. In: Otto, G.F. (Ed.), Proceedings of the Heartworm Symposium’89. American Heartworm Society, Washington, DC, pp. 1–6. Dillon, R., 1998. Clinical significance of feline heartworm disease. Vet. Clin. N. Am.: Small Anim. Pract. 28 (6), 1547–1565. FEDESA, 1995. Good clinical practice for the conduct of clinical trials for veterinary medicinal products. Fédération Européenne de la Santé Animale, CVMP, Brussels, Belgium. Genchi, C., Vezzoni, A., Di Sacco, B., Baroni, G., 1991. Dog heartworm infection: epidemiology and prophylaxis. Veterinaria. 5, 83–90. Genchi, C., Guerro, J., Di Sacco, B.F., 1992. Prevalence of Dirofilaria immitis in Italian cats. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium’92. American Heartworm Society, Batavia, IL, pp. 97–102. Grieve, R.B., Frank, G.R., Stewart, V.A., Parsons, J.C., Belasco, D.L., Hepler, D.I., 1991. Chemoprophylactic effects of milbemycin oxime against larvae of Dirofilaria immitis during prepatent development. Am. J. Vet. Res. 52, 2040–2042. Guerrero, J., McCall, J.W., Dzimianski, M.T., McTier, T.L., Holmes, R.A., Newcomb, K.H., 1992. Prevalence of Dirofilaria immitis infection in cats from the southeastern United States. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium’92. American Heartworm Society, Batavia, IL, pp. 91–95. Holmes, R.A., 1993. Feline dirofilariasis. Vet. Clin. N. Am.: Small Anim. Pract. 23 (1), 125–138. Holmes, R.A., McCall, J.W., Prasse, K.W., 1986. Thiacetarsamide in dogs with Dirofilaria immitis: influence of decreased liver function on drug efficacy. Am. J. Vet. Res. 47, 1341–1344. Jernigan, A.D., McTier, T.L., Chieffo, C., Thomas, C.A., Krautmann, M.J., Hair, J.A., Young, D.R., Wang, C., Rowan, T.G., 2000. Efficacy of selamectin against experimentally induced tick (Rhipicephalus sanguineus and Dermacentor variabilis) infestations on dogs. Vet. Parasitol. 91, 359–375.
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(Ed.), Recent Advances in Heartworm Disease: Symposium’98. American Heartworm Society, Batavia, IL, pp. 187–192. McTier, T.L., Jones, R.L., Holbert, M.S., Murphy, M.G., Watson, P., Sun, F., Smith, D.G., Rowan, T.G., Jernigan, A.D., 2000a. Efficacy of selamectin against adult flea infestations (Ctenocephalides felis felis and Ctenocephalides canis) on dogs and cats. Vet. Parasitol. 91, 187–199. McTier, T.L., Shanks, D.J., Jernigan, A.D., Rowan, T.G., Jones, R.L., Murphy, M.G., Wang, C., Smith, D.G., Holbert, M.S., Blagburn, B.L., 2000b. Evaluation of the effects of selamectin against adult and immature stages of fleas (Ctenocephalides felis felis) on dogs and cats. Vet. Parasitol. 91, 201–312. McTier, T.L., Shanks, D.J., Wren, J.A., Six, R.H., Bowman, D.D., McCall, J.W., Pengo, G., Genchi, C., Smothers, C.D., Rowan, T.G., Jernigan, A.D., 2000c. Efficacy of selamectin against experimentally induced and naturally acquired infections of Toxocara cati and Ancylostoma tubaeforme in cats. Vet. Parasitol. 91, 311–319. Miller, M.W., 1998. Canine heartworm disease. Clin. Tech. Small Anim. Pract. 13, 113–118. Munnell, J.F., Weldon, J.S., Lewis, R.E., Thrall, D.E., McCall, J.W., 1980. Intimal lesions of the pulmonary artery in dogs with experimental dirofilariasis. Am. J. Vet. Res. 41, 1108–1112. Paul, A.J., McCall, J.W., Todd Jr., K.S., 1986. Efficacy of ivermectin against Dirofilaria immitis larvae in dogs forty-five days after induced infection. In: Otto, G.F. (Ed.), Proceedings of the Heartworm Symposium’86. American Heartworm Society, Washington, DC, pp. 33–35. Paul, A.J., Todd, K.S., Acre, K.E., Plue, R.E., Wallace, D.H., French, R.A., Wallig, M.A., 1991. Efficacy of ivermectin chewable tablets and two new ivermectin tablet formulations against Dirofilaria immitis larvae in dogs. Am. J. Vet. Res. 52, 1922–1923. Paul, A.J., Acre, K.E., Todd, K.S., Wallace, D.H., Jernigan, A.D., Wallig, M.A., 1992. Efficacy of ivermectin against Dirofilaria immitis in cats 30 and 45 days post-infection. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium’92. American Heartworm Society, Batavia, IL, pp. 117–119. Shanks, D.J., McTier, T.L., Rowan, T.G., Watson, P., Thomas, C.A., Bowman, D.D., Hair, J.A., Pengo, G., Genchi, C., Smothers, C.A., Smith, D.G., Jernigan, A.D., 2000a. The efficacy of selamectin in the treatment of naturally acquired aural infestations of Otodectes cynotis on dogs and cats. Vet. Parasitol. 91, 283–290. Shanks, D.J., McTier, T.L., Behan, S., Pengo, G., Genchi, C., Bowman, D.D., Holbert, M.S., Smith, D.G., Jernigan, A.D., Rowan, T.G., 2000b. The efficacy of selamectin in the treatment of naturally acquired infestations of Sarcoptes scabiei on dogs. Vet. Parasitol. 91, 269–281. Stewart, V.A., Blagburn, B.L., Hendrix, C.M., Hepler, D.I., Grieve, R.B., 1992. Milbemycin oxime as an effective preventative of heartworm (Dirofilaria immitis) infection in cats. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium’92. American Heartworm Society, Batavia, IL, pp. 127–131. Thrall, D.E., Badertscher, R.R., Lewis, R.E., McCall, J.W., Lonsonsky, J.M., 1980. Radiographic changes associated with developing dirofilariasis in experimentally infected dogs. Am. J. Vet. Res. 41, 81–90.
Prevention of experimentally induced heartworm (Dirofilaria immitis) infections in dogs and cats with a single topical application of selamectin T.L. McTier a,∗ , D.J. Shanks b , P. Watson b,1 , J.W. McCall c , C. Genchi d , R.H. Six a , C.A. Thomas a,2 , S.K. Dickin b , G. Pengo e , T.G. Rowan b , A.D. Jernigan a a
b
Animal Health Clinical Affairs, Central Research Division, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA Animal Health Clinical Affairs, Central Research Division, Pfizer Limited, Sandwich, Kent CT13 9NJ, UK c Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA d Institute of General Veterinary Pathology, University of Milan, Milan, Italy e Via Marmara, 19/C Paderno Dugnano, Milan, Italy
Abstract In a series of six controlled studies (four in dogs, two in cats), heartworm-free dogs and cats were inoculated with Dirofilaria immitis larvae (L3 ) prior to topical treatment with the novel avermectin selamectin or a negative control containing inert formulation ingredients (vehicle). Selamectin and negative-control treatments were administered topically to the skin at the base of the neck in front of the scapulae. In dogs, selamectin was applied topically at dosages of 3 or 6 mg kg−1 at 30 days post-inoculation (PI), or of 3 or 6 mg kg−1 at 45 days PI, or of 6 mg kg−1 at 60 days PI. Cats were treated topically with unit doses providing a minimum dosage of 6 mg kg−1 selamectin at 30 days PI. Of the animals that were treated 30 days PI, some dogs were bathed with water or shampoo between 2 and 96 h after treatment, and some cats were bathed with shampoo at 24 h after treatment. Between 140 and 199 days PI, the animals were euthanized and examined for adult D. immitis. Adult heartworms developed in all control dogs (geometric mean count, 18.7 worms) and in 88% of control cats (geometric mean count, 2.1 worms). Selamectin was 100% effective in preventing heartworm development in dogs when administered as a single topical dose of 3 or 6 mg kg–1 at 30 days after infection, 3 or 6 mg kg−1 at 45 days after infection, or 6 mg kg−1 at 60 days after infection. Selamectin was 100% effective against heartworm infections in cats when administered as a
∗ Corresponding author. Tel.: +1-860-441-6454; Fax +1-860-441-5779. E-mail address: tom l [email protected] (T.L. McTier) 1 Present address: Royal Veterinary College, London, NW1 OUT, UK. 2 Present address: Animal Health Group, North American Region, Pfizer Inc., Exton, PA, USA.
0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 2 9 7 - 1
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single topical unit dose of 6 mg kg−1 . Bathing with water or shampoo between 2 and 96 h after treatment did not reduce the efficacy of selamectin as a heartworm prophylactic in dogs. Likewise, bathing with shampoo at 24 h after treatment did not reduce the efficacy of selamectin in cats. These studies demonstrated that, at the recommended dosage and treatment interval, a single topical administration of selamectin was 100% effective in preventing the development of D. immitis in dogs and cats. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Selamectin; RevolutionTM ; StrongholdTM ; Avermectin; Heartworm; Dirofilaria immitis; Canis familiaris; Felis catus; Dog; Cat
1. Introduction Infection with the filarial parasite Dirofilaria immitis (heartworm) is common among dogs living in warm temperate and tropical parts of the world where mosquito vectors are present. Surveys performed in the USA and Italy have demonstrated heartworm prevalence of up to 70% in dogs in some endemic areas (Genchi et al., 1991; Guerrero et al., 1992). In one experimental study in the southeastern region of the United States, 13 of 14 (93%) heartworm-naive dogs placed outdoors in heartworm endemic areas became infected in a one-year period by natural exposure (McTier et al., 1992a). In cats, global prevalence values of up to 23% in endemic areas are lower than in dogs in the same areas, but indicates the susceptibility of cats to naturally acquired heartworm infection (Courtney and Zeng, 1989; Genchi et al., 1992; Guerrero et al., 1992). In addition, other studies have shown that typically 60 to 80% of cats inoculated experimentally with D. immitis will develop adult heartworm infections (McTier et al., 1992b; Paul et al., 1992; Stewart et al., 1992; Mansour et al., 1995). In dogs, the severity and rate of development of heartworm disease are primarily related to the magnitude of the worm burden (Munnell et al., 1980; Thrall et al., 1980; Knight, 1983). Heavy infections commonly result in pulmonary hypertension and right-sided heart failure (Knight, 1983). In contrast, cats mount a more intense pathological response to the presence of the parasite than do dogs (Calvert et al., 1994), and show a wider variety of clinical signs (Holmes, 1993; Calvert et al., 1994; Dillon, 1998). As few as two adult heartworms have resulted in sudden death in cats (Calvert et al., 1994). Treatment of established heartworm infections carries serious risks for the host animal, as dead and dying heartworms can cause severe complications (Calvert et al., 1994; Miller, 1998) and chemoprophylaxis is highly recommended for animals living in heartworm-endemic areas (Calvert et al., 1994; Dillon, 1998). Heartworm chemoprophylaxis has been transformed within the last decade by the introduction of macrocyclic lactones that are administered as oral formulations at monthly intervals (e.g. ivermectin, milbemycin oxime). In addition, milbemycin oxime has activity against other endoparasites at the dose used for heartworm prevention. Initial studies demonstrated that selamectin (RevolutionTM /StrongholdTM , Pfizer, New York) was effective in preventing heartworm development when applied topically to the skin as a single dose within 30 days after larval inoculation in dogs and cats (McTier et al., 1998). Other studies have demonstrated the endectocidal activity of selamectin in dogs and
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cats against a variety of other internal and external parasites (Jernigan et al., 2000; McTier et al., 2000a,b,c; Shanks et al., 2000a,b). For dogs and cats, an endectocide product applied to the skin in a single spot provides the benefits of a convenient application together with a broad-spectrum of activity. The six studies reported in this paper investigated the efficacy of selamectin in preventing the development of heartworms in dogs and cats given experimental inoculations of D. immitis under conditions of simulated under-dosing, simulated missed or late treatment (treatment administered up to 60 days after larval inoculation), and bathing of animals 2, 6, 24, or 96 h after treatment.
2. Materials and methods A total of six controlled laboratory studies were conducted, five in the USA (one in cats, and four in dogs) and one (cats) in Italy. All investigations were conducted in compliance with the Food and Drug Administration Center for Veterinary Medicine Guidelines (CVM, 1992, 1997) and with the European guidelines (FEDESA, 1995). Animals were housed in appropriate accommodation that conformed to accepted guidelines for floor area/type, lighting, temperature, humidity and welfare (including environmental enrichment and social interaction), as required by local and national legislation. 2.1. Study design The two cat studies (A and B) were similar in design and evaluated the efficacy of a single topical unit dose providing a minimum dosage of 6 mg kg−1 selamectin administered 30 days post-inoculation (PI) in preventing heartworm development (Table 1). These studies also assessed the effect on efficacy of bathing of cats with a non-insecticidal shampoo (study A — Johnson’s pH 5.5 Shampoo® , Johnson and Johnson; study B — Allergroom® , Allerderm) at 24 h after selamectin treatment. The four dog studies (C–F) evaluated selamectin at dosages of 3 or 6 mg kg−1 with treatment at various times after inoculation of heartworms (30, 45, or 60 days PI; Tables 1–4). In addition, the impact on efficacy of soaking in water for a short time (approximately 1 min) 2 h after treatment and shampoo bathing with a non-insecticidal shampoo (Allergroom® , Allerderm) at 2, 6, 24, or 96 h after selamectin treatment was evaluated in dogs. 2.2. Animals Purpose-bred domestic short-haired cats were used in studies A and B, and purpose-bred Beagles were used in studies C–F. At the time of treatment, the dogs ranged in age from 6 to 13 months and in weight from 6.3 to 15.8 kg. Cats ranged in age from 3 to 5 months, and in weight from 1.5 to 2.9 kg. Each treatment had equal number of male and female animals. All animals were confirmed to be in satisfactory physical condition and free of D. immitis infection prior to the start of the study. In studies B–F, a blood sample obtained from each animal at 32 days prior to treatment was tested for the presence of circulating microfilariae and adult heartworm antigen. An additional set of tests was performed 90 days
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Table 1 Efficacy of a single topical unit dose of selamectin providing a minimum dosage of 6 mg kg−1 against Dirofilaria immitis in bathed and unbathed dogs and cats (studies A–C) Study
Treatment
Dosage
Days between Bathing larval inoculation and treatment
Time of Geometric mean bath after of heartworm treatment (h) counts (range)
Prevention rate (%)
A (cats)
Vehiclea 0.10 ml kg−1 30 Selamectin 6 mg kg−1 30 Selamectin 6 mg kg−1 30
None – None – Shampoo 24
1.1b (1–2) 0 0
NAc 100 100
B (cats)
Vehicle 0.10 ml kg−1 30 30 Selamectin 6 mg kg−1 Selamectin 6 mg kg−1 30
None – None – Shampoo 24
3.5d (0–13) 0 0
NA 100 100
C (dogs) Vehicle 0.05 ml kg−1 30 30 Selamectin 6 mg kg−1 Selamectin 6 mg kg−1 30
None – None – Shampoo 24
7.2 (1–16) 0 0
NA 100 100
a The negative-control treatment was the vehicle only (inert ingredients) for the commercial formulation of selamectin. Studies A and B were performed in cats (12 cats per treatment); study C was performed in dogs (eight dogs per treatment). All animals were inoculated with infective D. immitis larvae (L3 ) 30 days before treatment (50 larvae per animal in studies A and C, 100 larvae per animal in study B). Necropsy examinations were performed 199 days (study A), 169 days (study B), or 142 days (study C) after heartworm inoculation. b Includes two dead heartworms found in two cats (a single worm in each cat) which were present at the time of necropsy. c Not applicable. d Three cats were free of adult heartworms at necropsy.
after treatment in these studies, to detect infections that could have been present at the start of the study but which could not be detected by current diagnostic methods at that time. In study A, heartworm tests were performed 90 days after treatment only. None of the animals had been treated with an avermectin in the 60 days prior to inoculation with larvae. The animals were all housed in mosquito-proof accommodation for the duration of each study. In each study, the animals were allocated randomly within sex to treatments. For the duration of each study, the general health of the animals was observed twice daily. Clinical Table 2 Efficacy of a single topical unit dose of selamectin providing a minimum dosage of 6 mg kg−1 against D. immitis in water-bathed, shampoo-bathed, and unbathed dogs (study D)a Treatment
Dosage
Vehicle (control) 0.05 ml kg−1 Selamectin Selamectin Selamectin Selamectin
6 mg kg−1 6 mg kg−1 6 mg kg−1 6 mg kg−1
Days between larval inoculation and treatment
Bathing
Time of bath after treatment (h)
Geometric mean of heartworm counts (range)
Prevention rate (%)
30
None
–
29.0 (18–39)
NAb
30 30 30 30
None Water Shampoo Shampoo
– 2 2 6
0 0 0 0
100 100 100 100
a There were eight dogs in each treatment, all of which were inoculated with 50 D. immitis larvae (L ) 30 days 3 before treatment administration. Necropsy examinations were performed 140 days after heartworm inoculation. b Not applicable.
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Table 3 Efficacy of a single topical dose of 3 or 6 mg kg−1 selamectin against D. immitis when applied at 1 month after inoculation or at extended treatment intervals in dogs (study E)a Treatment
Dosage
Days between larval inoculation and treatment
Geometric mean of heartworm counts (range)
Prevention rate (%)
Vehicle (control)
0.05 ml kg−1
30, 45, 60
22.6 (10–29)
NAb
Selamectin Selamectin Selamectin Selamectin Selamectin
3 mg kg−1
30 30 45 45 60
6 mg kg−1 3 mg kg−1 6 mg kg−1 6 mg kg−1
0 0 0 0 0
100 100 100 100 100
a
There were six dogs in each treatment, all of which were inoculated with 50 D. immitis larvae (L3 ) 30, 45, or 60 days before treatment. The dogs were not bathed. Necropsy examinations were performed 140 days after heartworm inoculation. b Not applicable.
observations were made on all animals by a veterinarian before treatment, on at least three occasions in the 8 h following treatment, and 24 h after treatment. The veterinarians and other personnel who assessed the animals’ health were not aware of treatment allocations. 2.3. Inoculation with D. immitis larvae The animals were inoculated with D. immitis at 30, 45, or 60 days before treatment. Each inoculum consisted of 50 (studies A, C–F) or 100 (study B: cats ) infective D. immitis larvae (L3 ) in Hank’s balanced salt solution. The animals were infected by subcutaneous inoculation in the inguinal area (studies B–F) or by subcutaneous injection of the inoculum into the neck (study A: cats). Third-stage larvae (L3 ) were reared and collected as described by McCall et al. (1980). The heartworm larvae used were from a laboratory strain maintained by Dr. John McCall (University of Georgia, Athens, GA). 2.4. Selamectin and negative-control treatments Selamectin was formulated at concentrations of 60 and 120 mg ml−1 in an excipient (vehicle) consisting of glycol ether, isopropyl alcohol, and butylhydroxytoluene. In studies E and F, the exact dose of selamectin was administered by using a 1- or 3-ml syringe. In all other studies (A–D), selamectin was supplied in the commercial formulation in unit dose tubes. Selamectin unit doses were designed to deliver selamectin at a minimum recommended dosage of 6 mg kg−1 (range, 6–12 mg kg−1 for dogs and 6–17.3 mg kg−1 for cats). In all studies, the vehicle for the commercial formulation of selamectin was used as a negative-control treatment at a dosage of 0.05 ml kg−1 in dogs, and 0.10 ml kg−1 in cats. Unit doses for both the negative-control and selamectin treatments were selected according to body weight ranges. All animals were weighed within 3 days before treatment for calculation of the appropriate dose. In all studies, selamectin and the vehicle were applied in a similar manner topically to the skin at the base of the neck in front of the scapulae on day zero.
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2.5. Necropsy procedure Each animal received an intravenous injection with heparin and an approved euthanasia solution. Necropsy for heartworm counts was performed between 140 and 142 days PI for dogs and between 169 and 199 days PI for cats (Tables 1–4). Necropsies were conducted within approximately 2 h of euthanasia. In dogs, the postmortem procedure involved clamping of the anterior and posterior vena cava, removal of the heart and lungs, dissection and examination of the anterior vena cava, right atrium, right ventricle, pulmonary arteries, and exploration of the pleural and peritoneal cavities. In cats, in addition to the above procedures, the liver was removed and the cardiac–hepatic portion of the posterior vena cava was examined. Any adult D. immitis identified were recorded as being dead or alive according to the procedure of Holmes et al. (1986). Within each study, the order in which the necropsies were performed was randomized across treatments. The personnel who performed the examinations did not know to which treatment the animals had been allocated. 2.6. Data analysis Heartworm counts from postmortem examinations were summarized for each treatment in each study and geometric mean heartworm counts were calculated. The heartworm prevention rate, defined as the percentage of selamectin-treated animals free of adult heartworms at necropsy, compared with that for controls, was estimated for each treatment. Treatments with prevention rates of 100% were considered efficacious. Statistical analysis of the data was not performed. 3. Results 3.1. Prophylactic efficacy When applied topically to the skin at a dosage of 3 or 6 mg kg−1 , 30 days PI, selamectin prevented heartworm infections in all 140 treated animals (92 dogs, 48 cats; Tables 1–4). When the effects of a simulated 15-day delay in treatment were investigated (treatment 45 days PI), dosages of 3 or 6 mg kg−1 remained 100% effective against heartworms (Tables 3 and 4). When treatment was delayed until 60 days PI in dogs, simulating a missed monthly dose, a single application of 6 mg kg−1 selamectin was completely effective in preventing the development of heartworms (Tables 3 and 4). The effect of bathing after treatment was investigated in a total of 72 animals (48 dogs, 24 cats), all of which had been treated with 6 mg kg−1 selamectin 30 days PI. When dogs were bathed with water 2 h after treatment, or with a non-insecticidal shampoo 2, 6, 24, or 96 h after treatment, the efficacy of selamectin remained at 100% (Tables 1, 2 and 4). Similarly, bathing cats at 24 h after treatment with a non-insecticidal shampoo did not reduce the efficacy of selamectin (Table 1). 3.2. Clinical observations There were no mortalities among the animals during any of these studies, and the abnormalities that occurred in association with treatment were only minor and temporary.
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Stiffening of the hairs, hairs adhering together, and the presence of a slight white powder on the hairs were observed at the treatment site in some selamectin- and vehicle-treated animals for a period of 24 h after treatment.
4. Discussion When administered at the recommended dosage (6 mg kg−1 ), selamectin was 100% effective in preventing heartworm development in all of the 126 animals tested (78 dogs, 48 cats). This prevention rate was achieved in the face of larval challenges that resulted in the development of adult heartworms in 100% of control dogs (n=30) and 21 of 24 control cats (88%). In dogs, when administered 30 days after larval inoculation, selamectin remained 100% effective at half the recommended dosage (3 mg kg−1 ; n=14). In the studies that simulated delayed treatment (administration 45 days after larval inoculation), a single unit dose of selamectin at 3 or 6 mg kg−1 was 100% effective in all dogs tested (n=14 at both dose levels). Also, when administered at 6 mg kg−1 at 60 days PI, simulating a missed monthly treatment, selamectin remained completely effective in preventing heartworm maturation in dogs (n=14). The complete efficacy of a single topical unit dose of 6 mg kg−1 selamectin administered 45 days PI compares favorably with other macrocyclic lactones approved for heartworm prevention in dogs. Both milbemycin oxime and ivermectin are known to be effective in preventing heartworm development when administered as a single treatment at the recommended dosages at 45 days PI, providing a “grace period” if monthly treatment is delayed (Grieve et al., 1991; Paul et al., 1986, 1991). The current studies have shown that selamectin not only provides a “grace period” if treatment is delayed in dogs, but the “grace period” is maintained if a monthly treatment is inadvertently missed. Moxidectin also maintains a grace period similar to that for selamectin in dogs (McTier et al., 1992c). In dogs, these studies have shown that the 100% efficacy of a single unit dose providing a minimum dosage of 6 mg kg−1 selamectin in preventing heartworm maturation is not reduced by bathing the animal, even when the haircoat is soaked with water or bathed with shampoo as early as 2 h after treatment. A total of 48 dogs were bathed, either with water 2 h after treatment (n=8) or with shampoo 2 h (n=8), 6 h (n=8), 24 h (n=16), or 96 h (n=8) after treatment. Adult heartworms did not develop in any of these dogs. Also, bathing cats with shampoo 24 h after treatment (n=24) with a topical unit dose of 6 mg kg−1 did not reduce the efficacy of selamectin. Bathing of dogs at 2, 6, or 24 h after treatment and bathing of cats at 24 h after treatment did not reduce the efficacy of a single unit dose of selamectin against weekly experimental flea infestations for 1 month (McTier et al., 2000a). At the time of necropsy, heartworms were present in all control dogs (n=30) and in 88% of control cats (n=24). The mean heartworm counts from control animals were 18.7 for all four dog studies (C–F) combined and 2.1 for the two cats studies (A and B) combined. These numbers are similar to those achieved by other investigators in similar studies in dogs (McCall et al., 1980; Paul et al., 1986; McTier et al., 1992c; Blagburn et al., 1995) and in cats (McTier et al., 1992b; Paul et al., 1992; Stewart et al., 1992) and are sufficient to allow thorough assessment of the efficacy of selamectin as a heartworm preventive. Cats are naturally more resistant to the development of adult heartworm infection than are dogs,
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and this is reflected in the results of these studies. Inoculation of cats with 50 D. immitis larvae resulted in mean worm counts of 1.1 for cats (study A), and inoculation of cats with 100 larvae resulted in a mean worm count of 3.5 (study B).
5. Conclusions This series of studies demonstrated that selamectin was completely effective in preventing the development of D. immitis when applied topically to the skin of dogs and cats at a dosage of 6 mg kg−1 at 30 days PI. Under-dosing (3 mg kg−1 ) or delay of treatment (6 mg kg−1 ) by up to 1 month did not reduce the efficacy. In addition, soaking or shampooing dogs 2 h after treatment or shampooing cats 24 h after treatment did not alter selamectin’s efficacy. Selamectin provides robust protection against heartworms in a convenient topical formulation. References Blagburn, B.L., Hendrix, C.M., Vaughan, J.L., Lindsay, D.S., Tebbit, G.L., 1995. Efficacy of a chewable formulation of milbemycin oxime against preadult Dirofilaria immitis and adult Ancylostoma caninum in naturally and experimentally infected dogs. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium’95. American Heartworm Society, Washington, DC, pp. 171–175. Calvert, C.A., Rawlings, C.A., McCall, J.W., 1994. Feline heartworm disease. In: Sherding, R.G. (Ed.), The Cat: Diseases and Clinical Management, 2nd Edition. Churchill Livingstone, New York, pp. 623–645. CVM, 1992. Conduct of clinical investigations: responsibilities of clinical investigators and monitors for investigational new animal drug studies. Technical Report, October. Center for Veterinary Medicine, Rockville, MD. CVM, 1997. Good target animal study practices: clinical investigators and monitors. Technical Report, May. Center for Veterinary Medicine, Rockville, MD. Courtney, C.H., Zeng, Q.Y., 1989. The structure of heartworm populations in dogs and cats in Florida. In: Otto, G.F. (Ed.), Proceedings of the Heartworm Symposium’89. American Heartworm Society, Washington, DC, pp. 1–6. Dillon, R., 1998. Clinical significance of feline heartworm disease. Vet. Clin. N. Am.: Small Anim. Pract. 28 (6), 1547–1565. FEDESA, 1995. Good clinical practice for the conduct of clinical trials for veterinary medicinal products. Fédération Européenne de la Santé Animale, CVMP, Brussels, Belgium. Genchi, C., Vezzoni, A., Di Sacco, B., Baroni, G., 1991. Dog heartworm infection: epidemiology and prophylaxis. Veterinaria. 5, 83–90. Genchi, C., Guerro, J., Di Sacco, B.F., 1992. Prevalence of Dirofilaria immitis in Italian cats. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium’92. American Heartworm Society, Batavia, IL, pp. 97–102. Grieve, R.B., Frank, G.R., Stewart, V.A., Parsons, J.C., Belasco, D.L., Hepler, D.I., 1991. Chemoprophylactic effects of milbemycin oxime against larvae of Dirofilaria immitis during prepatent development. Am. J. Vet. Res. 52, 2040–2042. Guerrero, J., McCall, J.W., Dzimianski, M.T., McTier, T.L., Holmes, R.A., Newcomb, K.H., 1992. Prevalence of Dirofilaria immitis infection in cats from the southeastern United States. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium’92. American Heartworm Society, Batavia, IL, pp. 91–95. Holmes, R.A., 1993. Feline dirofilariasis. Vet. Clin. N. Am.: Small Anim. Pract. 23 (1), 125–138. Holmes, R.A., McCall, J.W., Prasse, K.W., 1986. Thiacetarsamide in dogs with Dirofilaria immitis: influence of decreased liver function on drug efficacy. Am. J. Vet. Res. 47, 1341–1344. Jernigan, A.D., McTier, T.L., Chieffo, C., Thomas, C.A., Krautmann, M.J., Hair, J.A., Young, D.R., Wang, C., Rowan, T.G., 2000. Efficacy of selamectin against experimentally induced tick (Rhipicephalus sanguineus and Dermacentor variabilis) infestations on dogs. Vet. Parasitol. 91, 359–375.
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