Veterinary Therapeutics Vol. 4, No. 3, Fall 2003 Persistent Efficacy of Moxidectin Canine Sustained- Release Injectable Against Experimental Infections of Ancylostoma caninum and Uncinaria stenocephala in Dogs* Dwight D. Bowman, MS, PhD a Tracey Rock, DVM b Kathleen Heaney, DVM b Norwood R. Neumann, DVM, PhD a Michael Ulrich, BS a Deborah Amodie, BS b a CHK-R&D 17190 Polk Road Stanwood, MI 49346 b Fort Dodge Animal Health PO Box 5366 Princeton, NJ 08543-5366 ABSTRACT The efficacy of moxidectin canine sustainedrelease injectable administered at fixed intervals before administration of an oral hookworm challenge was evaluated in 40 laboratory dogs. Groups of eight dogs were treated with the moxidectin sustained-release formulation by SC injection approximately 3, 4, 5, or 6 months before being given oral inoculations with 300 Ancylostoma caninum larvae on Day 0 and 400 Uncinaria stenocephala larvae one day later. Dogs were euthanized 21 days after parasite inoculations. Fecal samples and the intestinal contents collected at necropsy from each dog were examined for hookworms. Fecal egg count reductions based on geometric means relative to controls were 99.2% (3 months) to 81.2% (6 months). The reduction in A. can- *This study was sponsored by Fort Dodge Animal Health, Overland Park, KS. inum worm recoveries at 3, 4, 5, and 6 months based on geometric means were 94.7%, 90.3%, 82.0%, and 60.2%, respectively. The mean reductions in U. stenocephala worm counts at these intervals were 94.6%, 85.3%, 71.6%, and 48.2%, respectively. INTRODUCTION Moxidectin is derived from modification of nemadectin, a fermentation product of Streptomyces cyaneogriseus subsp noncyanogenus. Moxidectin is a pentacyclic 16-membered lactone macrolide that has activity resulting in paralysis and death of affected parasites. Given by SC injection at 0.17 mg/kg, moxidectin is effective against the tissue larval stage of Dirofilaria immitis 1 as well as against larval and adult stages of the canine hookworms Ancylostoma caninum and Uncinaria stenocephala. Peak blood levels are observed approximately 7 to 14 days after treat- 228
D. D. Bowman, T. Rock, K. Heaney, N. R. Neumann, M. Ulrich, and D. Amodie ment. ProHeart 6 (Fort Dodge Animal Health), the commercial formulation of sustained-release (SR) injectable moxidectin, is administered to dogs every 6 months for prevention of infection by D. immitis and canine hookworms. Moxidectin has previously been shown to have activity against larval hookworms. 2,3 Three studies examined the efficacy against hookworm larvae (one with a combination infection with both A. caninum and U. stenocephala, one with A. caninum only, and one with U. stenocephala only). 2 In those studies, treatments were administered 6 days after dogs were infected with A. caninum and 5 days after infection with U. stenocephala (so all larvae were in the fourth stage). The dogs were necropsied 42 days after infections. Efficacy against A. caninum larvae was 100% in both studies in which this species was evaluated. Efficacy against U. stenocephala larvae was 100% when animals were infected with both species and 99.5% when given as a single-species infection. In a later study using a similar treatment/infection interval, 3 moxidectin SR injection was 99% to 100% effective against both species for dogs necropsied 7 or 14 days after infection. In another study, a single injection of moxidectin (Cydectin 1% aqueous injectable solution formulated for sheep) at 1 mg/kg given to bitches at the time of conception and again on Day 55 of pregnancy was completely effective in prevention of transmammary infection of puppies with A. caninum. 4 Based on these results, it was hypothesized that the levels of moxidectin present in the dogs treated with the SR formulation would be effective against the larval stages of any infections introduced to the dogs for 6 months following treatment. There are no data available, however, regarding the actual duration of activity of this formulation against hookworms. The objective of the present study was to determine the duration of activity of moxidectin SR injectable when administered SC to dogs at the recommended dosage of 0.17 mg/kg body weight. Targeted parasites for this study were A. caninum and U. stenocephala (hookworms). The study examined the ability of the commercial formulation of moxidectin SR injectable to protect dogs against these two hookworm species when administered 3, 4, 5, and 6 months prior to challenge with infective-stage larvae. MATERIALS AND METHODS Animals Forty beagles, including 20 males and 20 females approximately 7 months of age, were obtained from USDA-approved sources that routinely supply research animals to the facility. Three fecal examinations were performed for each dog during the acclimation period to verify that all dogs were initially negative for hookworm infection. The dogs were individually housed in cages or runs from the time of arrival at the facility and throughout the study. Housing, handling, and routine care conformed to standards established at the facility in compliance with federal regulatory requirements. Each animal was identified by an ear tattoo with a unique identification number, and each cage was marked with the animal s identification number. Animals were fed a commercial diet (Purina HiPro, Nestlé Purina PetCare Company) and allowed free access to tap water. Allocation and Treatment The study was scheduled such that the day of inoculation with the infective-stage larvae of A. caninum would be Day 0. The 40 dogs were weighed on Day 173 and were ranked by sex in descending order according to this weight. Within sex and according to this ranking order, dogs were sequentially stratified into five groups of four. Treatments were randomly allocated to the five dogs within each stratum thus formed, as follows: 229
Veterinary Therapeutics Vol. 4, No. 3, Fall 2003 Group 1: Moxidectin canine SR injectable on Day 173 (6 months) Group 2: Moxidectin canine SR injectable on Day 145 (5 months) Group 3: Moxidectin canine SR injectable on Day 117 (4 months) Group 4: Moxidectin canine SR injectable on Day 89 (3 months) Group 5: Saline injection on Day 89. All moxidectin and saline treatments were given by SC injection. Doses of moxidectin for individual dogs were calculated to deliver 0.17 mg moxidectin/kg, and all dogs were weighed before treatment. Hookworm Larval Inoculations Ancylostoma caninum and U. stenocephala infective-stage larvae were obtained in separate cultures from recent isolates from naturally infected dogs. On the day of inoculation (designated Day 0), each dog was given approximately 150 A. caninum infective-stage larvae PO in the morning and 150 larvae in the evening. The dogs were observed for vomiting for approximately 30 minutes after each dosing, and any dog that vomited within this period was reinoculated. Soft food (Pedigree Puppy Canned Food for Puppies Chicken and Beef Dinner, Mars) was given to each dog 1 hour after each inoculation. The following day (Day 1), each dog was inoculated PO with approximately 200 U. stenocephala infective-stage larvae both in the morning and in the evening. The dogs were observed for vomiting for approximately 30 minutes after each inoculation, and any dog that vomited within this time was reinoculated. As was done on Day 0, canned food was given to each dog 1 hour after each inoculation. Necropsies The trial was terminated 21 days after inoc- ulation with A. caninum larvae. All dogs were euthanized the same day. A fecal sample was collected from each dog at necropsy and the number of hookworm eggs per gram of feces (EPG) was calculated using a modified centrifugal sugar flotation procedure. There was no attempt to distinguish between the eggs of the two different hookworms in the fecal samples. At necropsy, the intestinal tract was opened and the contents of the lumen and scrapings from the mucosa were processed for hookworm recovery by passing the contents of the intestinal tract through an 80-mesh US Standard Mesh sieve. All worms recovered from this process were counted and identified according to sex and species. Efficacy Calculations Egg and worm counts were transformed to the log 10 (count + 1) before analysis to stabilize the variance and normalize the data. Geometric means for these two variables were calculated for each group, and the percent efficacy for each treatment/infection interval was calculated using the following formula: Geometric Geometric mean (control) mean (treated) 100 Geometric mean (control) Using the PROC MIXED procedure (SAS version 8.2, SAS Institute), transformed total counts were analyzed by two-way analysis of variance with a model that considered treatment as a fixed effect and block as a random effect. Treatment was tested against the residual error at the 5% level of significance (two-sided test). Least squares means were calculated for each group, and the least squares mean for each moxidectin-treated group was compared with that of the control group by a one-sided Student s t-test at the 5% level of significance. No attempt was made to make comparisons among moxidectin-treated groups. 230
D. D. Bowman, T. Rock, K. Heaney, N. R. Neumann, M. Ulrich, and D. Amodie TABLE 1. Fecal Egg Counts and Recovery of Hookworms at Necropsy from Dogs Treated with Moxidectin Sustained-Release Injectable 3, 4, 5, or 6 Months Before Inoculation (PO) with Infective-Stage Larvae Fecal Egg Animal Count Ancylostoma caninum Uncinaria stenocephala ID (eggs/gram) Male Female Total Male Female Total Control Geometric mean 1,948.7 26.6 21.9 48.9 130.9 129.8 260.9 Range (770 5,760) (13 61) (17 45) (30 106) (52 230) (48 233) (100 463) Moxidectin 3 mo before infection Geometric mean 16.2* 1.6 1.2 2.6* 8.3 6.7 14.0* Range (0 836) (0 10) (0 10) (0 20) (0 43) (0 49) (0 92) Efficacy 99.2% 94.7% 94.6% Moxidectin 4 mo before infection Geometric mean 59.1* 2.6 2.1 4.7* 17.4 20.3 38.5* Range (3 850) (0 20) (0 11) (0 31) (0 126) (2 156) (2 282) Efficacy 97.0% 90.3% 85.3% Moxidectin 5 mo before infection Geometric mean 295.5* 5.6 3.3 8.8* 35.3 38.6 74.0* Range (97 2,440) (1 17) (0 11) (1 28) (5 149) (11 156) (16 305) Efficacy 84.8% 82.0% 71.6% Moxidectin 6 mo before infection Geometric mean 366.6* 10.4 8.1 19.5* 71.2 65.3 135.1 Range (0 3,240) (1 44) (0 36) (4 80) (4 167) (2 158) (6 308) Efficacy 81.2% 60.2% 48.2% *Significant reduction in egg or worm count versus controls (P <.05). Percent efficacy = [Geometric mean (control) geometric mean (treated)/geometric mean (control)] 100. RESULTS Fecal Egg Counts The geometric mean egg count for the control group at necropsy was 1,949 EPG (Table 1). Dogs treated with moxidectin (at any time) had significantly (P <.05) reduced egg counts compared with counts for control dogs. Egg counts were reduced by 99.2% for dogs treated with moxidectin 3 months before infection, 97.0% for dogs treated at 4 months, 84.8% for dogs treated at 5 months, and 81.2% for dogs treated at 6 months before infection (Table 1). Hookworm Recoveries Worms were recovered from all control dogs at necropsy (Table 1). A mean of 48.9 A. caninum (range = 30 to 106) were recovered from controls. Dogs receiving moxidectin (at any time) had significantly (P <.05) reduced A. caninum present at necropsy compared with dogs in the control group. Efficacy of moxidectin SR injectable against A. caninum, based on reductions in the number of worms recovered at necropsy, was 94.7% (3 months), 90.3% (4 months), 82.0% (5 months), and 60.2% (6 months) (Table 1). The frequency of male and female worms was relatively similar within all groups. A mean of 260.9 U. stenocephala (range = 100 to 463) was recovered at necropsy from 231
Veterinary Therapeutics Vol. 4, No. 3, Fall 2003 saline-treated control dogs. All dogs treated with moxidectin, with the exception of the group treated 6 months before infection, had a significant (P <.05) reduction in U. stenocephala counts compared with populations recovered from dogs in the control group. Based on geometric means, efficacy of moxidectin SR injection against U. stenocephala was 94.6% at 3 months, 85.3% at 4 months, 71.6% at 5 months, and 48.2% at 6 months. DISCUSSION There was a significant (P <.05) impact on both the number of adult worms and hookworm egg counts in dogs infected 3 months (89 days), 4 months (117 days), and 5 months (145 days) after treatment with moxidectin SR injectable. For infections given 3 months after treatment, population reductions were essentially equivalent for both species (approximately 95%), and egg counts were reduced by 99.2%. For infections given 4 months after treatment, moxidectin was still at least 90% effective in reducing A. caninum populations and hookworm egg counts, whereas efficacy against U. stenocephala was slightly below 90% (85.3%) at that interval. Although geometric mean worm burdens in dogs infected 5 months after moxidectin treatment were significantly (P <.05) less than those in controls, efficacy was considered marginal (82.0% for A. caninum and 71.6% for U. stenocephala) for this treatment/infection interval. Mean hookworm egg counts were reduced by at least 81% for all moxidectin-treated groups for 6 months in this study. All larval infections in this study were administered orally. When A. caninum larvae are acquired by this route of infection, they typically do not undergo a liver lung migration that occurs with percutaneous infection. Instead, they develop within the gastric glands or the glands of Lieberkühn in the small intestine for a few days and then migrate back into the lumen of the intestine, where they molt to the fourth stage and eventually become adults. 5 The prepatent period following oral inoculation can be as short as 14 days. If larvae penetrate the skin, they migrate through the lungs, and the prepatent period will be extended by approximately 2 weeks. 5 Thus, larvae acquired by the percutaneous route would seem to have a better chance of being destroyed by any product present systemically within the body of dogs. It is unclear what percentage of infections with A. caninum occurs through the percutaneous route versus the oral route. Another hookworm species, Ancylostoma braziliense, which was not evaluated in this study, seems to be the most ardent skin penetrator of the canine species, but again, it is not known to what extent one mode of infection occurs more commonly than the other. In the case of U. stenocephala, it is suspected that the majority of infections are obtained orally, but these larvae are also capable of skin penetration. It could be speculated, however, that efficacy against hookworm larvae obtained by skin penetration could be higher than that observed in the groups of dogs infected orally in this study by virtue of having prolonged contact with moxidectin. The persistent reductions noted in hookworm egg shedding following administration of moxidectin SR injectable could result in diminished environmental contamination, ultimately decreasing public health risks to hookworm exposure. CONCLUSION Results of this study indicate that induced populations of A. caninum and U. stenocephala were significantly (P <.05) reduced for 5 months after treatment with moxidectin SR injectable (ProHeart 6) compared with populations present in untreated controls. Moxidectin 232
D. D. Bowman, T. Rock, K. Heaney, N. R. Neumann, M. Ulrich, and D. Amodie was at least 90% effective against A. caninum larvae administered PO 4 months after treatment, whereas efficacy against U. stenocephala fell slightly below 90% after 3 months. At 5 months after dosing, efficacy against both species of hookworm was less than 90%. Fecal hookworm egg counts were reduced by at least 90% for 4 months and by at least 80% for 6 months following administration of moxidectin SR injectable. REFERENCES 1. Lok JB, Knight DH, Wang GT, et al: Activity of an injectable, SR formulation of moxidectin administered prophylactically to mixed-breed dogs to prevent infection with Dirofilaria immitis. Am J Vet Res 62(11):1721 1726, 2001. 2. Bowman DD, McCall JW, Supakorndej P, et al: Efficacy of moxidectin canine SR (slow-release) injectable against experimental hookworm (Ancylostoma caninum and Uncinaria stenocephala) infection in dogs [abstract]. Proc 45 th Annu Meet AAVP, 2000. 3. Ranjan S, Heaney K, Simkins K: Efficacy of moxidectin canine sustained release injectable against larval/immature stages of T. vulpis, A. caninum, and U. stenocephala infection in dogs [abstract]. Proc 45 th Annu Meet AAVP, 2002. 4. Rosler K: Zur Wirkung von Moxidectin auf reaktivierte somatische Larven von Ancylostoma caninum Ercolani 1859 (Ancylostomatidae) in der graviden Hndin [thesis]. Tierarztliche Hochschule Hannover, Hannover; Germany; 1998. 5. Reinecke RK: Veterinary Helminthology. Woburn, MA, Butterworth Publishers, 1983, pp 163 166. 233