- Email: [email protected]
Efficacy of milbemycin oxime in combination with spinosad in the treatment of larval and immature adult stages of Ancylostoma caninum and Toxocara canis in experimentally infected dogs
Veterinary Parasitology 205 (2014) 134–139
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Efficacy of milbemycin oxime in combination with spinosad in the treatment of larval and immature adult stages of Ancylostoma caninum and Toxocara canis in experimentally infected dogs Dwight D. Bowman a , Craig R. Reinemeyer b , Scott Wiseman c , Daniel E. Snyder d,∗ a b c d
College of Veterinary Medicine, Cornell University, Ithaca, NY, USA East Tennessee Clinical Research Inc., Rockwood, TN, USA Elanco Animal Health, Eli Lilly and Company Limited, Basingstoke, Hampshire, United Kingdom Elanco Animal Health Research and Development, a Division of Eli Lilly and Company, 2500 Innovation Way, Greenfield, IN, USA
a r t i c l e
i n f o
Article history: Received 15 May 2014 Received in revised form 9 July 2014 Accepted 20 July 2014 Keywords: Ancylostoma caninum Toxocara canis Spinosad Milbemycin oxime Larval Chemoprophylaxis
a b s t r a c t Ancylostoma caninum and Toxocara canis are two important zoonotic parasites of dogs. The primary objective of these studies were to confirm the oral effectiveness of milbemycin oxime (MO) and spinosad in dogs experimentally infected with immature (L4 and immature adult) stages of T. canis or A. caninum. Both trials were conducted as randomized, blinded, placebo-controlled dose confirmation studies. Treatments using the intended European commercial tablet formulation of Trifexis were administered in a timeframe relative to inoculation so that effectiveness could be assessed against specific immature stages of A. caninum or T. canis. In each study on Day 0, each of 32, 3–4 month old dogs were inoculated with 250 infective eggs of T. canis or 300 infective L3 of the hookworm, A. caninum. All dogs were weighed before their scheduled treatment, randomized to 1 of the 4 treatment groups in each study (8 dogs/group). All dogs were fed just prior to dosing. For T. canis, dogs were treated orally with an MO/spinosad tablet on Day 14 or Day 24. For A. caninum, dogs were treated orally with an MO/spinosad tablet on Day 7 or Day 11. Corresponding control groups in each study received a placebo tablet. Dogs were necropsied 5 or 6 days after their respective treatments. The digestive tract was removed and processed to recover, count, and identify all stages. The GM worm count for the MO/spinosad tablet on Day 14 (L4 T. canis) was 0.0, with efficacy calculated as 100%; however, only 3 of 8 control dogs had adequate infections. The GM worm count for the MO/spinosad tablet on Day 24 (immature adult stage) was 0.30; efficacy calculated at 96.15%. This is based on 5 of the 8 control dogs with adequate infections. In the two A. caninum studies, GM worm counts for the MO/spinosad tablets on Day 7 (L4 efficacy) was 2.37 and 0.8 with efficacy calculated as 98.92% and 99.25%, respectively. The GM count for the group treated with the MO/spinosad combination on Day 11 (immature adult) was 6.19 and 1.4; efficacy calculated at 97.77% and 98.58%, respectively. A minimum MO oral dose of 0.75 mg/kg was highly effective for the treatment of immature stages of T. canis and A. caninum infections in dogs. The ability to kill immature stages of these two parasites before they become patent will benefit dogs, their owners and family members due to reduced exposure to these potentially zoonotic parasites. © 2014 Elsevier B.V. All rights reserved.
∗ Corresponding author. Tel.: +1 317 277 4439; fax: +1 317 277 4167. E-mail address: snyder daniel [email protected] (D.E. Snyder). http://dx.doi.org/10.1016/j.vetpar.2014.07.023 0304-4017/© 2014 Elsevier B.V. All rights reserved.
D.D. Bowman et al. / Veterinary Parasitology 205 (2014) 134–139
1. Introduction Ancylostoma caninum and Toxocara canis are two of the most important intestinal nematode parasites of dogs, with infections reported from all parts of the world (Schnieder et al., 2011). Additionally, infected dogs can play an important role in the transmission of these two zoonotic nematodes by excreting eggs directly into the human environment. The veterinary and public health aspects of hookworm and Toxocara spp. infections in dogs are well-known and have been recently reviewed (Bowman et al., 2010; Deplazes et al., 2011; Lee et al., 2010; Overgaauw and van Knapen, 2013). Two hookworm species of significance in dogs are found in Europe, A. caninum and Uncinaria stenocephala. A. caninum is found predominantly in dogs located in central and southern Europe (ESCCAP, 2010).Chemoprophylactic strategies and different anthelmintic classes that can be used to control both larval and adult stages of A. caninum as well as T. canis have been reviewed based on the route of exposure or infection and the age of the dog (Epe, 2009). However, Blagburn et al., 1992 reported that milbemycin oxime (MO) at 0.5 mg/kg did not demonstrate acceptable (>90%) efficacy against larval (L4) or immature adult stages of A. caninum in dogs. They reported that 0.5 mg/kg had 49% efficacy against L3/L4 stages, 83% against L4 and 81% against early immature adult stages of A. caninum. Stansfield and Hepler (1991) reported that adult A. caninum was the dose-limiting parasite for MO, and required a minimum oral dose of 0.5 mg/kg. Bowman et al. (1988) demonstrated that oral MO at dose levels of 0.27–0.39 mg/kg had 100% efficacy against adult T. canis in experimentally induced infections. No currently labeled canine anthelmintics that contain MO and deliver a minimum dose of 0.5 mg/kg have label claims or indications for any immature stages of either T. canis or A. caninum. The efficacy of a combination of spinosad and MO in dogs naturally infected with different species of adult intestinal nematodes has been previously demonstrated (Schnitzler et al., 2012). More recently it was shown that a minimum dose of 0.75 mg/kg of MO in combination with spinosad will prevent the establishment of the adult stage of the French heartworm, Angiostrongylus vasorum (Böhm et al., 2014). Based on the improved efficacy against A. vasorum from higher dose levels of MO, it was decided to evaluate efficacy against immature stages of both A. caninum and T. canis. Thus, the primary objective of the dose confirmation studies described herein, using a higher minimum MO dose of 0.75 mg/kg, were to evaluate and confirm the effectiveness of a spinosad and milbemycin oxime (MO) combination tablet (Trifexis; Elanco Animal Health), administered orally at approximately the lower end (0.75–1.0 mg/kg MO) of the proposed MO tablet unit dose range (0.75–1.18 mg/kg MO), in dogs experimentally infected with immature (L4 and immature adult) stages of T. canis or A. caninum. 2. Materials and methods 2.1. Study design Three studies (one for T. canis; two for A. caninum) outlined below were conducted as randomized, blinded,
135
placebo-controlled dose confirmation studies according to Good Clinical Practice (GCP) standards based on VICH, Guideline 7 (2001), VICH, Guideline 9 (2001) and VICH, Guideline 19 (2002). Study protocols were approved by the study site Institutional Animal Care and Use Committee. In each of the studies, 32 purpose-bred laboratory research Beagles sourced from a USDA licensed vendor (mix of male/female; 3–4.1 months of age; 3.1–6.8 kg) were acclimated for a minimum of 14 days prior to inoculation with infective stages of T. canis or A. caninum on Day 0. All dogs were singly housed throughout the study, fed a balanced commercial dry dog food once daily and provided with water ad libitum. Dogs were individually identified by ear tattoo. Physical examinations were performed during the acclimation period to ensure dogs were healthy and were eligible to be enrolled into each study. After Day 0, all enrolled dogs were examined at least once daily for any abnormal clinical signs or adverse events. The primary inclusion criterion was body weight so that each selected dog fell into a defined weight range in order to ensure that dogs were dosed with whole tablet(s) to deliver between 0.75 and 1.0 mg/kg of MO, which is the approximate lower half of the tablet unit dose range. In each stand-alone study, on Day 0, each of 32 dogs was experimentally inoculated per os with 250 infective (larvated) eggs of the ascarid, T. canis or 300 third-stage infective larvae of the hookworm, A. caninum. All dogs were weighed using certified scales on the day before their scheduled treatment and then randomized to one of the four treatment groups. All dogs were fed a small portion of a moist canned food just prior to their scheduled dosing time point. For the T. canis study, 16 dogs from the pool of eligible candidates were allocated to Groups 1 (8 dogs) or 2 (8 dogs) on Day 13. From the pool of remaining dogs, 16 were then allocated to Groups 3 (8 dogs) or 4 (8 dogs) on Day 22 (2 days prior to scheduled dosing). Treatments using the intended European Union commercial solid oral dosage formulation (Trifexis; Elanco Animal Health) were administered in a timeframe relative to inoculation so that effectiveness could be assessed primarily against immature larval (L4) or immature adult stages of T. canis. In the T. canis study, dogs were treated with the MO/spinosad combination tablets on Day 14 (Group 2) or on Day 24 (Group 4). The corresponding control groups received a vehicle control tablet on Day 14 (Group 1) or Day 24 (Group 3). In each of the A. caninum studies, 16 dogs were allocated to Groups 1 and 2 and then Groups 3 and 4 (8 dogs/group). Treatments were administered in a time frame relative to inoculation so that effectiveness could be assessed primarily against immature larval (L4) or immature adult stages of A. caninum. Dogs were treated with the MO/spinosad combination tablets on Day 7 (Group 2) or on Day 11 (Group 4). The control groups received a vehicle control tablet on Day 7 (Group 1) or Day 11 (Group 3). The timing of dosing in relation to the time of inoculation of each parasite followed the methods as described for the AdvantageMulti/Advocate (Imidacloprid + moxidectin; Bayer) NADA (Anon, 2006; NADA 141–251) approved by the US FDA in 2006. All dogs in each study were humanely euthanized by lethal injection and necropsied 5 or 6 days after their respective treatments.
136
D.D. Bowman et al. / Veterinary Parasitology 205 (2014) 134–139
2.2. Parasites (T. canis and A. caninum) The T. canis isolate used in this study was recently obtained from a naturally infected dog and was subsequently the source of infective eggs used to orally inoculate all dogs on Day 0. Two separate isolates of A. caninum, recently obtained from naturally infected dogs, were the source of infective L3 used to orally inoculate all dogs in each of these studies on Day 0. In order to obtain the A. caninum infective L3 and T. canis infective (larvated) eggs, fecal material from infected donor dogs were cultured using standard larval culture and ascarid egg embryonation methods. A qualitative fecal examination was conducted during acclimation to ensure dogs did not have a preexisting infection of any nematode species. Any dog with a pre-existing T. canis or A. caninum infection at the time of the fecal check would be excluded as per the study inclusion criteria. 2.3. Experimental inoculations On Day 0 of each study, 32 enrolled dogs were inoculated with 250 infective (larvated) eggs of T. canis or 300 third-stage infective larvae of A. caninum via oral gavage. All dogs within each study were inoculated orally using the same inoculation method. Any calculations needed to determine inoculum volume were recorded. Dogs were checked for vomiting at 1 h ± 30 min post-inoculation. 2.4. Necropsy/worm counts Food was removed for an overnight fast the day prior to each scheduled necropsy date. The entire digestive tract was removed and processed in accordance with the relevant standard operating procedures of each laboratory and standard parasitological procedures. Because immature stages of T. canis and A. caninum were being assessed, the intestinal contents were washed over fine mesh sieves. Additionally for A. caninum, the small intestine was soaked in warm tap water or saline to facilitate the emergence of immature stages from mucosal tissues. Materials recovered from soaked intestines were similarly sieved for worm recovery. The worms were preserved in 10% formalin prior to subsequently counting and the microscopic identification by species and stage.
difference (p < 0.05, two-sided) between the vehicle control group and the MO/spinosad treated groups was required to demonstrate effectiveness in the MO/spinosad treated groups against the immature L4 or immature adult stages of T. canis or A. caninum. As per VICH guidelines, a minimum of five T. canis or A. caninum specimens of any stage (total of immature L4, immature adult) was required in a minimum of six control dogs at the end of the study to meet adequacy of infection criteria for each parasite stage of interest. 2.5.2. Data analysis For each dog the total T. canis or A. caninum counts at necropsy were calculated as the sum of the immature L4, immature adult and adult counts. A logarithmic transformation (ln[count + 1]) was applied to the total post-treatment worm count for each individual animal to address the skewed nature of these data and also to allow zero counts. Back-transformed geometric means were calculated as ex¯ − 1, where x¯ is the arithmetic treatment mean of log-transformed counts at a given time point. The efficacy against each stage of T. canis or A. caninum was calculated using the following formula:
%Efficacy =
Geometric mean control − Geometric mean drug Geometric mean control
× 100
The transformed counts were analyzed with a general linear model with fixed effect Treatment and block as a random effect. In order to assess the effectiveness of the treatment against the immature L4 stage of T. canis or A. caninum, statistical contrasts were constructed comparing Group 1 (control) against Group 2 (treated) in each of the studies. Similarly in each study, Group 3 (control) and Group 4 (treated) were compared to determine the effectiveness of the treatment against the immature adult stage of T. canis or A. caninum. The residuals from the linear models were examined to assess the assumptions underlying the models. The Wilcoxon rank sum exact tests, analogous to the contrasts described above, were performed to validate the robustness of the parametric analyses, in case the normality assumption was not met. 3. Results 3.1. T. canis
2.5. Efficacy calculation/statistical analysis 2.5.1. Variable classification The primary variable evaluated was the total count of adult, immature L4 and immature adult stages of T. canis or A. caninum recovered at each post-treatment necropsy day. Treatment was administered in a time frame relative to inoculation so that effectiveness could be assessed primarily against immature L4 or immature adult stages of T. canis or A. caninum. Efficacy against experimentally induced T. canis or A. caninum populations was determined posttreatment for the MO/spinosad combination by comparing the total count with that in the corresponding vehicle control group. Efficacy ≥90% and a statistically significant
The geometric mean (GM) count at necropsy in the control groups was 7.71 for the group treated on Day 24 and necropsied on Day 29. A minimum of five T. canis were present in 5 of the 8 dogs in the control group necropsied on Day 29. The GM count for the group treated with the MO/spinosad combination tablet on Day 24 and necropsied on Day 29 (to assess effectiveness against the immature adult stage of T. canis) was 0.30, with efficacy calculated at 96.15%. This is based on 5 of the 8 control dogs with adequate infections (Table 1). Three of the 8 dogs in the control group treated on Day 14 and necropsied on Day 20 (to assess effectiveness against the L4 stage of T. canis) had a minimum of five T. canis present (GM = 1.37), therefore the infection was not
D.D. Bowman et al. / Veterinary Parasitology 205 (2014) 134–139
137
Table 1 Non-parametric analysis of post-treatment worm count data. Parasite stage
Statistic
Spinosad/milbemycin oxime combination
Placebo control
T. canis L4
Number dogs Geometric mean % Efficacy Treatment effect p-value
8 0.0
8 1.37
Number dogs Geometric mean % Efficacy Treatment effect p-value
8 0.3
Number dogs
8/8
8/8
Geometric mean % Efficacy Treatment effect p-value
2.37/0.79
220.58/105.56
Number dogs
8/8
8/8
Geometric mean % Efficacy Treatment effect p-value
6.19/1.39
277.96/98.06
T. canis immature adult
A. caninum L4 (study 1/study 2)
A. caninum immature adult (study 1/study 2)
Treatment comparison (0.75 mg/kg MO)
100 0.0769 8 7.71 96.15 0.0384
98.92/99.25 0.0002/0.0002
sufficient to draw definitive conclusions concerning the effectiveness against the L4 stage; however no worms were recovered in the treated group (efficacy = 100%). Examination of the residuals from the linear model and the Shapiro–Wilk test of normality indicated that the Normality assumption was not met, therefore, emphasis was placed on the non-parametric results; however, the nonparametric and parametric results were consistent. Using both model approaches, there was a statistically significant difference between the MO/spinosad and control groups necropsied on Day 29 (immature adult T. canis; Wilcoxon non-parametric model: p = 0.0384). There was no significant difference between the MO/spinosad treated group and its corresponding control group necropsied on Day 20 due to the low level of infection in the controls (L4 T. canis; Wilcoxon non-parametric model: p = 0.0769). There were no serious post-treatment adverse events in any treatment group. Non-serious adverse events (AEs) occurred in the following treatment groups: Vehicle Control: there were no non-serious AEs; MO/spinosad combination group, there was one non-serious AE in one dog consisting of a single episode of emesis. 3.2. A. caninum: study 1 The GM worm counts at necropsy in the control groups were 220.58 for the group treated on Day 7 and necropsied on Day 12, and 277.96 for the group treated on Day 11 and necropsied on Day 16. In each of the control groups at necropsy, all 8 dogs had 5 or more worms (range 121–352 worms per dog) thus demonstrating adequate infection for each stage of A. caninum that was assessed. The number of worms seen in each control group at each scheduled necropsy date confirmed the robust parasite challenge that was assessed in this study (Table 1). The GM count for
97.77/98.58 0.0002/0.0002
the group treated with the combination tablet on Day 7 and necropsied on Day 12 (to assess effectiveness against the L4 stage of hookworm) was 2.37 with efficacy calculated as 98.92%. The GM count for the group treated with the combination tablet on Day 11 and necropsied on Day 16 (to assess effectiveness against the immature adult stage of hookworm) was 6.19 with efficacy calculated at 97.77%. Examination of the residuals from the linear model and the Shapiro–Wilk test of normality indicated that the Normality assumption was not met, therefore emphasis was placed on the non-parametric results; however, the non-parametric and parametric results were consistent. The MO/spinosad groups necropsied on Days 12 and 16 were both significantly different (Wilcoxon nonparametric model: p = 0.0002) from their corresponding placebo control groups. There were no post-treatment serious AEs in any treatment group. Non-serious AEs occurred in the following treatment groups: Vehicle Control, there was one dog with bloody diarrhea which is a known clinical sign associated with this blood-sucking hookworm species; Spinosad/MO combination, there were six dogs with non-serious, mild transient signs of emesis that occurred on the day of/day following dosing and therefore are possibly related to the administration of spinosad. None of these dogs were retreated. 3.3. A. caninum: study 2 The GM worm counts at necropsy in the control groups were 105.56 for the group treated on Day 7 and necropsied on Day 12, and 98.06 for the group treated on Day 11 and necropsied on Day 16. In each of the control groups at necropsy, all 8 dogs had five or more worms thus demonstrating adequate infection for each stage of A. caninum
138
D.D. Bowman et al. / Veterinary Parasitology 205 (2014) 134–139
that was assessed. The number of worms seen in each control group at each scheduled necropsy date confirmed the robust parasite challenge that was assessed in this study (Table 1). The GM count for the group treated with the combination tablet on Day 7 and necropsied on Day 12 (to assess effectiveness against the L4 stage of hookworm) was 0.79 with efficacy calculated as 99.25%. The GM count for the group treated with the combination tablet on Day 11 and necropsied on Day 16 (to assess effectiveness against the immature adult stage of hookworm) was 1.39 with efficacy calculated at 98.58%. Examination of the residuals from the linear model and the Shapiro–Wilk test of normality indicated that the Normality assumption was not met, therefore emphasis was placed on the non-parametric results; however, the non-parametric and parametric results were consistent. The MO/spinosad groups necropsied on Days 12 and 16 were both significantly different (Wilcoxon non-parametric model: p = 0.0002) from their corresponding placebo control groups. One dog in Group 1 (control) at necropsy had a single small (ca. 2 cm) male Toxascaris leonina. Based on the worm’s immature size, the length of the pre-patent period for this ascarid species and when the dog arrived at the study site, the dog was most likely already infected upon arrival and the infection was obtained while at the dog vendor’s location and could not be confirmed (no eggs being shed) when the pre-treatment qualitative fecal egg counts were conducted. The presence of this single worm in this single control dog did not impact the experimentally induced A. caninum infections or the interpretation of the results against the L4 stage of A. caninum. There were no post-treatment serious AEs in any treatment group. Post-treatment non-serious AEs occurred in the following treatment groups: Vehicle Control: one control dog (group 1) had an AE consisting of a single non-serious event of emesis that occurred on the day of dosing approximately 1 h post-dosing; Spinosad/MO combination,there were no non-serious AEs in these treated groups. 4. Discussion As described in Section 1, the zoonotic potential and prevalence of T. canis has been well established in the scientific literature, including Europe. Human exposure to T. canis is exemplified by seroprevalence data generated in different regions of the world where the age adjusted Toxocara seroprevalence was 13.9% (Won et al., 2008; Manini et al., 2012). The most common control measures for T. canis include regular and frequent anthelmintic treatment of dogs starting at an early age, education and enforcement of laws for the disposal of canine feces, dog legislation and personal hygiene (Macpherson, 2013). Reducing environmental contamination is important since it is known that T. canis eggs are very resistant and survive well over most winters in temperate climates, surviving for 6–12 months. Some eggs may be able to survive in moist, cool conditions for 2–4 years or longer (Macpherson, 2013). Since T. canis is also a serious zoonotic parasite in Europe and other regions of the world and can be routinely found in both
juvenile and mature dogs (Fahrion et al., 2008), a higher minimum effective point dose (e.g. −0.75 mg/kg) of MO will more effectively and consistently kill immature adult T. canis as demonstrated in this study and will reduce or eliminate shedding of these highly resilient eggs prior to female worms becoming patent. This environmental contamination potential is further exemplified by reports that have demonstrated an association between contamination of public areas and seropositivity for Toxocara spp. in children. The role of pet dogs and cats in the transmission of helminthic zoonoses, including T. canis, in Europe and other regions of the world has been documented (Macpherson, 2013). Schnieder et al. (2011) thoroughly reviewed the larval development of T. canis including the current knowledge of infection routes and the subsequent development of larvae within the canine host. They additionally reviewed information about the clinical, pathological, enzymatic, hematological and histopathological changes in dogs when they are infected with T. canis. When preadults have matured, eggs are first shed approximately 4–5 weeks post-inoculation (p.i.) in experimentally infected puppies whereas in older hosts prepatency seems to be extended to 40 up to 56 days p.i. These variations in the pre-patency period of T. canis highlight the importance of routine deworming treatments of dogs of various ages, in particular, when infected dogs are in close association with higher risk groups such as children, the elderly and immunocompromised pet owners. The zoonotic potential of A. caninum also has been well established in the scientific literature (Bowman et al., 2010). The prevalence of this hookworm species has been shown in prevalence surveys in various regions of the world and confirms that this parasite is commonly found in both young and adult dogs at all times of the year (Nolan and Smith, 1995; Little et al., 2009). Chemoprophylactic strategies to control A. caninum as well as T. canis have been reviewed based on the route of exposure or infection and the age of the dog (Epe, 2009). However, it is critical that the mg/kg dose of MO and other anthelmintics are administered accurately and just small variations can impact the overall efficacy against different stages of these zoonotic parasites. The effectiveness of the combination MO/spinosad tablet at a minimum dosage level of 0.75 mg/kg MO against both immature larval L4 and immature adult stages of A. caninum was clearly demonstrated in both dose confirmation studies summarized above. In contrast, Blagburn et al., 1992 reported that slightly lower doses of MO at 0.5 mg/kg did not demonstrate acceptable (>90%) efficacy against larval (L4) or immature adult stages of this pathogenic hookworm species of dogs. They reported that 0.5 mg/kg had 49% efficacy against L3/L4 stages, 83% against L4 and 81% against early (immature) stages of A. caninum. Stansfield and Hepler (1991) reported that adult A. caninum is the dose-limiting parasite for MO (which requires a minimum oral dose of 0.5 mg/kg). Results from the studies presented here, in combination with previously conducted studies at lower dose levels below 0.75 mg/kg, indicate that the larval stages of this parasite should now represent the dose-limiting intestinal nematode species (stage). A higher minimum effective
D.D. Bowman et al. / Veterinary Parasitology 205 (2014) 134–139
point dose of MO at 0.75 mg/kg will more effectively and consistently kill these immature stages of A. caninum and will reduce or eliminate shedding of infective eggs prior to female worms becoming patent. Additionally, both immature and adult stages of A. caninum are voracious blood suckers and can be very pathogenic in young dogs. Killing these blood-sucking hookworms as early in the life cycle of this parasite as possible will provide significant health benefits to infected dogs. The routine administration of broad spectrum endectocides that effectively kill both adult and immature intestinal nematode stages will help to address recommendations made to veterinarians (Epe, 2009) and expert groups such as ESCCAP (2010). These entities recommend routine treatment and control of T. canis and A. caninum, which are consistently found in dogs of all ages and throughout the year in prevalence studies conducted in different areas of the world, including Europe (MartinezMoreno et al., 2007; Riggio et al., 2013; Neves et al., 2014). Since re-infection is common in dogs with T. canis and A. caninum, the ability to kill immature adult stages of T. canis and L4/immature stages of A. caninum before they even start shedding eggs into its local environment will help to minimize and prevent environmental contamination with these zoonotic parasites. This improved parasite spectrum against immature stages of these two zoonotic intestinal nematode species of dogs using a minimum effective dose of 0.75 mg/kg of MO as reported herein will be a benefit for both dogs that includes reduced pathogenicity, re-infections, and killing immature female worms before they start to lay eggs, while their owners and family members will have less exposure to these potentially zoonotic parasites. This appears to be the first report demonstrating that a higher dose of MO using a minimum oral dose of 0.75 mg/kg can effectively kill immature intestinal stages of two very important nematodes of dogs, T. canis and A. caninum. Conflict of interest statement The studies as reported herein were funded by Elanco Animal Health. The authors from Cornell University and East Tennessee Clinical Research were contracted to perform these studies; the remaining authors are current employees of Elanco Animal Health and assisted with the study design, study conduct, data analysis, and review of the manuscript; however, there were no conflicting interests that may have biased the work reported in this paper. References Anon, 2006. NADA 141-251. United States Food and Drug Administration. Freedom of Information Summary. ADVANTAGE MULTI for Dogs http://www.fda.gov/downloads/ (Imidacloprid+Moxidectin), AnimalVeterinary/Products/ApprovedAnimalDrugProducts/ FOIADrugSummaries/UCM051438.pdf Blagburn, B.L., Hendrix, C.M., Lindsay, D.S., Vaughan, J.L., Hepler, D.I., Wright, J.C., 1992. Efficacy of milbemycin oxime against naturally
139
acquired or experimentally induced Ancylostoma spp. and Trichuris vulpis infections in dogs. Am. J. Vet. Res. 53, 513–516. Böhm, C., Schnyder, M., Thamsborg, S.M., Thompson, C.M., Trout, C., Wolken, S., Schnitzler, B., 2014. Assessment of the combination of spinosad and milbemycin oxime in preventing the development of canine Angiostrongylus vasorum infections. Vet. Parasitol. 199, 272–277. Bowman, D.D., Parsons, J.C., Grieve, R.B., Hepler, D.I., 1988. Effects of milbemycin on adult Toxocara canis in dogs with experimentally induced infection. Am. J. Vet. Res. 49, 1986–1989. Bowman, D.D., Montgomery, S.P., Zajac, A.M., Eberhard, M.L., Kazacos, K.R., 2010. Hookworms of dogs and cats as agents of cutaneous larva migrans. Trends Parasitol. 26, 162–167. Deplazes, P., van Knapen, F., Schweiger, A., Overgaauw, P.A., 2011. Role of pet dogs and cats in the transmission of helminthic zoonoses in Europe, with a focus on echinococcosis and toxocarosis. Vet. Parasitol. 182, 41–53. Epe, C., 2009. Intestinal nematodes: biology and control. Vet. Clin. Small Anim. 39, 1091–1107. ESCCAP, 2010. Worm Control in Dogs and Cats. Guideline 01, second ed., pp. 1–28, http://www.esccap.org Fahrion, A.S., Staebler, S., Deplazes, P., 2008. Patent Toxocara canis infections in previously exposed and in helminth-free dogs after infection with low numbers of embryonated eggs. Vet. Parasitol. 152, 108–115. Lee, A.C.Y., Schantz, P.M., Kazacos, K.R., Montgomery, S.P., Bowman, D.D., 2010. Epidemiologic and zoonotic aspects of ascarid infections in dogs and cats. Trends Parasitol. 26, 155–161. Little, S.E., Johnson, E.M., Lewis, D., Jaklitsch, R.P., Payton, M.E., Blagburn, B.L., Bowman, D.D., Moroff, S., Tams, T., Rich, L., Aucoin, D., 2009. Prevalence of intestinal parasites in pet dogs in the United States. Vet. Parasitol. 166, 144–152. Manini, M.P., Marchioro, A.A., Colli, C.M., Nishi, L., Falavigna-Guilherme, A.L., 2012. Association between contamination of public squares and seropositivity for Toxocara spp. in children. Vet. Parasitol. 188, 48–52. Martinez-Moreno, F.J., Hernandez, S., Lopez-Cobos, E., Becerra, C., Acosta, I., Martinez-Moreno, A., 2007. Estimation of canine intestinal parasites in Cordoba (Spain) and their risk to public health. Vet. Parasitol. 143, 7–13. Macpherson, C.N.L., 2013. The epidemiology and public health importance of toxocariasis: a zoonosis of global importance. Int. J. Parasitol. 43, 999–1008. Neves, D., Lobo, L., Brilhante Simoes, P., Cardoso, L., 2014. Frequency of intestinal parasites in pet dogs from an urban area (Greater Oporto northern Portugal). Vet. Parasitol. 200, 295–298. Nolan, T.J., Smith, G., 1995. Time series analysis of the prevalence of endoparasitic infections in cats and dogs presented to a veterinary teaching hospital. Vet. Parasitol. 59 (2), 87–96. Overgaauw, P.A.M., van Knapen, F., 2013. Veterinary and public health aspects of Toxocara spp. Vet. Parasitol. 193, 398–403. Riggio, F., Mannella, R., Ariti, G., Perrucci, S., 2013. Intestinal and lung parasites in owned dogs and cats from central Italy. Vet. Parasitol. 193, 78–84. Schnieder, T., Laabs, E.-M., Welz, C., 2011. Larval development of Toxocara canis in dogs. Vet. Parasitol. 175, 193–206. Schnitzler, B., Hayes, B., Wiseman, S., Snyder, D.E., 2012. Confirmation of the efficacy of a combination tablet of spinosad and milbemycin oxime against naturally acquired infections of canine intestinal nematode parasites. Vet. Parasitol. 184, 279–283. Stansfield, D.G., Hepler, D.I., 1991. Safety and efficacy of milbemycin oxime for parasite control. Canine Pract. 16, 11–16. VICH, Guideline 7, 2001a. Efficacy requirements for anthelmintics: overall guidelines. Veterinary International Cooperation on Harmonization. European Agency for the Evaluation of Medicinal Products, London. VICH, Guideline 9, 2001b. Good Clinical Practice. Veterinary International Cooperation on Harmonization. European Agency for the Evaluation of Medicinal Products, London. VICH, Guideline 19, 2002. Efficacy of Anthelmintics: Specific Recommendations for Canine. Veterinary International Cooperation on Harmonization. European Agency for the Evaluation of Medicinal Products, London. Won, K.Y., Kruszon-Moran, D., Schantz, P.M., Jones, J.L., 2008. National seroprevalence and risk factors for zoonotic Toxocara spp. infection. Am. J. Trop. Med. Hyg. 79, 552–557.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Efficacy of milbemycin oxime in combination with spinosad in the treatment of larval and immature adult stages of Ancylostoma caninum and Toxocara canis in experimentally infected dogs Dwight D. Bowman a , Craig R. Reinemeyer b , Scott Wiseman c , Daniel E. Snyder d,∗ a b c d
College of Veterinary Medicine, Cornell University, Ithaca, NY, USA East Tennessee Clinical Research Inc., Rockwood, TN, USA Elanco Animal Health, Eli Lilly and Company Limited, Basingstoke, Hampshire, United Kingdom Elanco Animal Health Research and Development, a Division of Eli Lilly and Company, 2500 Innovation Way, Greenfield, IN, USA
a r t i c l e
i n f o
Article history: Received 15 May 2014 Received in revised form 9 July 2014 Accepted 20 July 2014 Keywords: Ancylostoma caninum Toxocara canis Spinosad Milbemycin oxime Larval Chemoprophylaxis
a b s t r a c t Ancylostoma caninum and Toxocara canis are two important zoonotic parasites of dogs. The primary objective of these studies were to confirm the oral effectiveness of milbemycin oxime (MO) and spinosad in dogs experimentally infected with immature (L4 and immature adult) stages of T. canis or A. caninum. Both trials were conducted as randomized, blinded, placebo-controlled dose confirmation studies. Treatments using the intended European commercial tablet formulation of Trifexis were administered in a timeframe relative to inoculation so that effectiveness could be assessed against specific immature stages of A. caninum or T. canis. In each study on Day 0, each of 32, 3–4 month old dogs were inoculated with 250 infective eggs of T. canis or 300 infective L3 of the hookworm, A. caninum. All dogs were weighed before their scheduled treatment, randomized to 1 of the 4 treatment groups in each study (8 dogs/group). All dogs were fed just prior to dosing. For T. canis, dogs were treated orally with an MO/spinosad tablet on Day 14 or Day 24. For A. caninum, dogs were treated orally with an MO/spinosad tablet on Day 7 or Day 11. Corresponding control groups in each study received a placebo tablet. Dogs were necropsied 5 or 6 days after their respective treatments. The digestive tract was removed and processed to recover, count, and identify all stages. The GM worm count for the MO/spinosad tablet on Day 14 (L4 T. canis) was 0.0, with efficacy calculated as 100%; however, only 3 of 8 control dogs had adequate infections. The GM worm count for the MO/spinosad tablet on Day 24 (immature adult stage) was 0.30; efficacy calculated at 96.15%. This is based on 5 of the 8 control dogs with adequate infections. In the two A. caninum studies, GM worm counts for the MO/spinosad tablets on Day 7 (L4 efficacy) was 2.37 and 0.8 with efficacy calculated as 98.92% and 99.25%, respectively. The GM count for the group treated with the MO/spinosad combination on Day 11 (immature adult) was 6.19 and 1.4; efficacy calculated at 97.77% and 98.58%, respectively. A minimum MO oral dose of 0.75 mg/kg was highly effective for the treatment of immature stages of T. canis and A. caninum infections in dogs. The ability to kill immature stages of these two parasites before they become patent will benefit dogs, their owners and family members due to reduced exposure to these potentially zoonotic parasites. © 2014 Elsevier B.V. All rights reserved.
∗ Corresponding author. Tel.: +1 317 277 4439; fax: +1 317 277 4167. E-mail address: snyder daniel [email protected] (D.E. Snyder). http://dx.doi.org/10.1016/j.vetpar.2014.07.023 0304-4017/© 2014 Elsevier B.V. All rights reserved.
D.D. Bowman et al. / Veterinary Parasitology 205 (2014) 134–139
1. Introduction Ancylostoma caninum and Toxocara canis are two of the most important intestinal nematode parasites of dogs, with infections reported from all parts of the world (Schnieder et al., 2011). Additionally, infected dogs can play an important role in the transmission of these two zoonotic nematodes by excreting eggs directly into the human environment. The veterinary and public health aspects of hookworm and Toxocara spp. infections in dogs are well-known and have been recently reviewed (Bowman et al., 2010; Deplazes et al., 2011; Lee et al., 2010; Overgaauw and van Knapen, 2013). Two hookworm species of significance in dogs are found in Europe, A. caninum and Uncinaria stenocephala. A. caninum is found predominantly in dogs located in central and southern Europe (ESCCAP, 2010).Chemoprophylactic strategies and different anthelmintic classes that can be used to control both larval and adult stages of A. caninum as well as T. canis have been reviewed based on the route of exposure or infection and the age of the dog (Epe, 2009). However, Blagburn et al., 1992 reported that milbemycin oxime (MO) at 0.5 mg/kg did not demonstrate acceptable (>90%) efficacy against larval (L4) or immature adult stages of A. caninum in dogs. They reported that 0.5 mg/kg had 49% efficacy against L3/L4 stages, 83% against L4 and 81% against early immature adult stages of A. caninum. Stansfield and Hepler (1991) reported that adult A. caninum was the dose-limiting parasite for MO, and required a minimum oral dose of 0.5 mg/kg. Bowman et al. (1988) demonstrated that oral MO at dose levels of 0.27–0.39 mg/kg had 100% efficacy against adult T. canis in experimentally induced infections. No currently labeled canine anthelmintics that contain MO and deliver a minimum dose of 0.5 mg/kg have label claims or indications for any immature stages of either T. canis or A. caninum. The efficacy of a combination of spinosad and MO in dogs naturally infected with different species of adult intestinal nematodes has been previously demonstrated (Schnitzler et al., 2012). More recently it was shown that a minimum dose of 0.75 mg/kg of MO in combination with spinosad will prevent the establishment of the adult stage of the French heartworm, Angiostrongylus vasorum (Böhm et al., 2014). Based on the improved efficacy against A. vasorum from higher dose levels of MO, it was decided to evaluate efficacy against immature stages of both A. caninum and T. canis. Thus, the primary objective of the dose confirmation studies described herein, using a higher minimum MO dose of 0.75 mg/kg, were to evaluate and confirm the effectiveness of a spinosad and milbemycin oxime (MO) combination tablet (Trifexis; Elanco Animal Health), administered orally at approximately the lower end (0.75–1.0 mg/kg MO) of the proposed MO tablet unit dose range (0.75–1.18 mg/kg MO), in dogs experimentally infected with immature (L4 and immature adult) stages of T. canis or A. caninum. 2. Materials and methods 2.1. Study design Three studies (one for T. canis; two for A. caninum) outlined below were conducted as randomized, blinded,
135
placebo-controlled dose confirmation studies according to Good Clinical Practice (GCP) standards based on VICH, Guideline 7 (2001), VICH, Guideline 9 (2001) and VICH, Guideline 19 (2002). Study protocols were approved by the study site Institutional Animal Care and Use Committee. In each of the studies, 32 purpose-bred laboratory research Beagles sourced from a USDA licensed vendor (mix of male/female; 3–4.1 months of age; 3.1–6.8 kg) were acclimated for a minimum of 14 days prior to inoculation with infective stages of T. canis or A. caninum on Day 0. All dogs were singly housed throughout the study, fed a balanced commercial dry dog food once daily and provided with water ad libitum. Dogs were individually identified by ear tattoo. Physical examinations were performed during the acclimation period to ensure dogs were healthy and were eligible to be enrolled into each study. After Day 0, all enrolled dogs were examined at least once daily for any abnormal clinical signs or adverse events. The primary inclusion criterion was body weight so that each selected dog fell into a defined weight range in order to ensure that dogs were dosed with whole tablet(s) to deliver between 0.75 and 1.0 mg/kg of MO, which is the approximate lower half of the tablet unit dose range. In each stand-alone study, on Day 0, each of 32 dogs was experimentally inoculated per os with 250 infective (larvated) eggs of the ascarid, T. canis or 300 third-stage infective larvae of the hookworm, A. caninum. All dogs were weighed using certified scales on the day before their scheduled treatment and then randomized to one of the four treatment groups. All dogs were fed a small portion of a moist canned food just prior to their scheduled dosing time point. For the T. canis study, 16 dogs from the pool of eligible candidates were allocated to Groups 1 (8 dogs) or 2 (8 dogs) on Day 13. From the pool of remaining dogs, 16 were then allocated to Groups 3 (8 dogs) or 4 (8 dogs) on Day 22 (2 days prior to scheduled dosing). Treatments using the intended European Union commercial solid oral dosage formulation (Trifexis; Elanco Animal Health) were administered in a timeframe relative to inoculation so that effectiveness could be assessed primarily against immature larval (L4) or immature adult stages of T. canis. In the T. canis study, dogs were treated with the MO/spinosad combination tablets on Day 14 (Group 2) or on Day 24 (Group 4). The corresponding control groups received a vehicle control tablet on Day 14 (Group 1) or Day 24 (Group 3). In each of the A. caninum studies, 16 dogs were allocated to Groups 1 and 2 and then Groups 3 and 4 (8 dogs/group). Treatments were administered in a time frame relative to inoculation so that effectiveness could be assessed primarily against immature larval (L4) or immature adult stages of A. caninum. Dogs were treated with the MO/spinosad combination tablets on Day 7 (Group 2) or on Day 11 (Group 4). The control groups received a vehicle control tablet on Day 7 (Group 1) or Day 11 (Group 3). The timing of dosing in relation to the time of inoculation of each parasite followed the methods as described for the AdvantageMulti/Advocate (Imidacloprid + moxidectin; Bayer) NADA (Anon, 2006; NADA 141–251) approved by the US FDA in 2006. All dogs in each study were humanely euthanized by lethal injection and necropsied 5 or 6 days after their respective treatments.
136
D.D. Bowman et al. / Veterinary Parasitology 205 (2014) 134–139
2.2. Parasites (T. canis and A. caninum) The T. canis isolate used in this study was recently obtained from a naturally infected dog and was subsequently the source of infective eggs used to orally inoculate all dogs on Day 0. Two separate isolates of A. caninum, recently obtained from naturally infected dogs, were the source of infective L3 used to orally inoculate all dogs in each of these studies on Day 0. In order to obtain the A. caninum infective L3 and T. canis infective (larvated) eggs, fecal material from infected donor dogs were cultured using standard larval culture and ascarid egg embryonation methods. A qualitative fecal examination was conducted during acclimation to ensure dogs did not have a preexisting infection of any nematode species. Any dog with a pre-existing T. canis or A. caninum infection at the time of the fecal check would be excluded as per the study inclusion criteria. 2.3. Experimental inoculations On Day 0 of each study, 32 enrolled dogs were inoculated with 250 infective (larvated) eggs of T. canis or 300 third-stage infective larvae of A. caninum via oral gavage. All dogs within each study were inoculated orally using the same inoculation method. Any calculations needed to determine inoculum volume were recorded. Dogs were checked for vomiting at 1 h ± 30 min post-inoculation. 2.4. Necropsy/worm counts Food was removed for an overnight fast the day prior to each scheduled necropsy date. The entire digestive tract was removed and processed in accordance with the relevant standard operating procedures of each laboratory and standard parasitological procedures. Because immature stages of T. canis and A. caninum were being assessed, the intestinal contents were washed over fine mesh sieves. Additionally for A. caninum, the small intestine was soaked in warm tap water or saline to facilitate the emergence of immature stages from mucosal tissues. Materials recovered from soaked intestines were similarly sieved for worm recovery. The worms were preserved in 10% formalin prior to subsequently counting and the microscopic identification by species and stage.
difference (p < 0.05, two-sided) between the vehicle control group and the MO/spinosad treated groups was required to demonstrate effectiveness in the MO/spinosad treated groups against the immature L4 or immature adult stages of T. canis or A. caninum. As per VICH guidelines, a minimum of five T. canis or A. caninum specimens of any stage (total of immature L4, immature adult) was required in a minimum of six control dogs at the end of the study to meet adequacy of infection criteria for each parasite stage of interest. 2.5.2. Data analysis For each dog the total T. canis or A. caninum counts at necropsy were calculated as the sum of the immature L4, immature adult and adult counts. A logarithmic transformation (ln[count + 1]) was applied to the total post-treatment worm count for each individual animal to address the skewed nature of these data and also to allow zero counts. Back-transformed geometric means were calculated as ex¯ − 1, where x¯ is the arithmetic treatment mean of log-transformed counts at a given time point. The efficacy against each stage of T. canis or A. caninum was calculated using the following formula:
%Efficacy =
Geometric mean control − Geometric mean drug Geometric mean control
× 100
The transformed counts were analyzed with a general linear model with fixed effect Treatment and block as a random effect. In order to assess the effectiveness of the treatment against the immature L4 stage of T. canis or A. caninum, statistical contrasts were constructed comparing Group 1 (control) against Group 2 (treated) in each of the studies. Similarly in each study, Group 3 (control) and Group 4 (treated) were compared to determine the effectiveness of the treatment against the immature adult stage of T. canis or A. caninum. The residuals from the linear models were examined to assess the assumptions underlying the models. The Wilcoxon rank sum exact tests, analogous to the contrasts described above, were performed to validate the robustness of the parametric analyses, in case the normality assumption was not met. 3. Results 3.1. T. canis
2.5. Efficacy calculation/statistical analysis 2.5.1. Variable classification The primary variable evaluated was the total count of adult, immature L4 and immature adult stages of T. canis or A. caninum recovered at each post-treatment necropsy day. Treatment was administered in a time frame relative to inoculation so that effectiveness could be assessed primarily against immature L4 or immature adult stages of T. canis or A. caninum. Efficacy against experimentally induced T. canis or A. caninum populations was determined posttreatment for the MO/spinosad combination by comparing the total count with that in the corresponding vehicle control group. Efficacy ≥90% and a statistically significant
The geometric mean (GM) count at necropsy in the control groups was 7.71 for the group treated on Day 24 and necropsied on Day 29. A minimum of five T. canis were present in 5 of the 8 dogs in the control group necropsied on Day 29. The GM count for the group treated with the MO/spinosad combination tablet on Day 24 and necropsied on Day 29 (to assess effectiveness against the immature adult stage of T. canis) was 0.30, with efficacy calculated at 96.15%. This is based on 5 of the 8 control dogs with adequate infections (Table 1). Three of the 8 dogs in the control group treated on Day 14 and necropsied on Day 20 (to assess effectiveness against the L4 stage of T. canis) had a minimum of five T. canis present (GM = 1.37), therefore the infection was not
D.D. Bowman et al. / Veterinary Parasitology 205 (2014) 134–139
137
Table 1 Non-parametric analysis of post-treatment worm count data. Parasite stage
Statistic
Spinosad/milbemycin oxime combination
Placebo control
T. canis L4
Number dogs Geometric mean % Efficacy Treatment effect p-value
8 0.0
8 1.37
Number dogs Geometric mean % Efficacy Treatment effect p-value
8 0.3
Number dogs
8/8
8/8
Geometric mean % Efficacy Treatment effect p-value
2.37/0.79
220.58/105.56
Number dogs
8/8
8/8
Geometric mean % Efficacy Treatment effect p-value
6.19/1.39
277.96/98.06
T. canis immature adult
A. caninum L4 (study 1/study 2)
A. caninum immature adult (study 1/study 2)
Treatment comparison (0.75 mg/kg MO)
100 0.0769 8 7.71 96.15 0.0384
98.92/99.25 0.0002/0.0002
sufficient to draw definitive conclusions concerning the effectiveness against the L4 stage; however no worms were recovered in the treated group (efficacy = 100%). Examination of the residuals from the linear model and the Shapiro–Wilk test of normality indicated that the Normality assumption was not met, therefore, emphasis was placed on the non-parametric results; however, the nonparametric and parametric results were consistent. Using both model approaches, there was a statistically significant difference between the MO/spinosad and control groups necropsied on Day 29 (immature adult T. canis; Wilcoxon non-parametric model: p = 0.0384). There was no significant difference between the MO/spinosad treated group and its corresponding control group necropsied on Day 20 due to the low level of infection in the controls (L4 T. canis; Wilcoxon non-parametric model: p = 0.0769). There were no serious post-treatment adverse events in any treatment group. Non-serious adverse events (AEs) occurred in the following treatment groups: Vehicle Control: there were no non-serious AEs; MO/spinosad combination group, there was one non-serious AE in one dog consisting of a single episode of emesis. 3.2. A. caninum: study 1 The GM worm counts at necropsy in the control groups were 220.58 for the group treated on Day 7 and necropsied on Day 12, and 277.96 for the group treated on Day 11 and necropsied on Day 16. In each of the control groups at necropsy, all 8 dogs had 5 or more worms (range 121–352 worms per dog) thus demonstrating adequate infection for each stage of A. caninum that was assessed. The number of worms seen in each control group at each scheduled necropsy date confirmed the robust parasite challenge that was assessed in this study (Table 1). The GM count for
97.77/98.58 0.0002/0.0002
the group treated with the combination tablet on Day 7 and necropsied on Day 12 (to assess effectiveness against the L4 stage of hookworm) was 2.37 with efficacy calculated as 98.92%. The GM count for the group treated with the combination tablet on Day 11 and necropsied on Day 16 (to assess effectiveness against the immature adult stage of hookworm) was 6.19 with efficacy calculated at 97.77%. Examination of the residuals from the linear model and the Shapiro–Wilk test of normality indicated that the Normality assumption was not met, therefore emphasis was placed on the non-parametric results; however, the non-parametric and parametric results were consistent. The MO/spinosad groups necropsied on Days 12 and 16 were both significantly different (Wilcoxon nonparametric model: p = 0.0002) from their corresponding placebo control groups. There were no post-treatment serious AEs in any treatment group. Non-serious AEs occurred in the following treatment groups: Vehicle Control, there was one dog with bloody diarrhea which is a known clinical sign associated with this blood-sucking hookworm species; Spinosad/MO combination, there were six dogs with non-serious, mild transient signs of emesis that occurred on the day of/day following dosing and therefore are possibly related to the administration of spinosad. None of these dogs were retreated. 3.3. A. caninum: study 2 The GM worm counts at necropsy in the control groups were 105.56 for the group treated on Day 7 and necropsied on Day 12, and 98.06 for the group treated on Day 11 and necropsied on Day 16. In each of the control groups at necropsy, all 8 dogs had five or more worms thus demonstrating adequate infection for each stage of A. caninum
138
D.D. Bowman et al. / Veterinary Parasitology 205 (2014) 134–139
that was assessed. The number of worms seen in each control group at each scheduled necropsy date confirmed the robust parasite challenge that was assessed in this study (Table 1). The GM count for the group treated with the combination tablet on Day 7 and necropsied on Day 12 (to assess effectiveness against the L4 stage of hookworm) was 0.79 with efficacy calculated as 99.25%. The GM count for the group treated with the combination tablet on Day 11 and necropsied on Day 16 (to assess effectiveness against the immature adult stage of hookworm) was 1.39 with efficacy calculated at 98.58%. Examination of the residuals from the linear model and the Shapiro–Wilk test of normality indicated that the Normality assumption was not met, therefore emphasis was placed on the non-parametric results; however, the non-parametric and parametric results were consistent. The MO/spinosad groups necropsied on Days 12 and 16 were both significantly different (Wilcoxon non-parametric model: p = 0.0002) from their corresponding placebo control groups. One dog in Group 1 (control) at necropsy had a single small (ca. 2 cm) male Toxascaris leonina. Based on the worm’s immature size, the length of the pre-patent period for this ascarid species and when the dog arrived at the study site, the dog was most likely already infected upon arrival and the infection was obtained while at the dog vendor’s location and could not be confirmed (no eggs being shed) when the pre-treatment qualitative fecal egg counts were conducted. The presence of this single worm in this single control dog did not impact the experimentally induced A. caninum infections or the interpretation of the results against the L4 stage of A. caninum. There were no post-treatment serious AEs in any treatment group. Post-treatment non-serious AEs occurred in the following treatment groups: Vehicle Control: one control dog (group 1) had an AE consisting of a single non-serious event of emesis that occurred on the day of dosing approximately 1 h post-dosing; Spinosad/MO combination,there were no non-serious AEs in these treated groups. 4. Discussion As described in Section 1, the zoonotic potential and prevalence of T. canis has been well established in the scientific literature, including Europe. Human exposure to T. canis is exemplified by seroprevalence data generated in different regions of the world where the age adjusted Toxocara seroprevalence was 13.9% (Won et al., 2008; Manini et al., 2012). The most common control measures for T. canis include regular and frequent anthelmintic treatment of dogs starting at an early age, education and enforcement of laws for the disposal of canine feces, dog legislation and personal hygiene (Macpherson, 2013). Reducing environmental contamination is important since it is known that T. canis eggs are very resistant and survive well over most winters in temperate climates, surviving for 6–12 months. Some eggs may be able to survive in moist, cool conditions for 2–4 years or longer (Macpherson, 2013). Since T. canis is also a serious zoonotic parasite in Europe and other regions of the world and can be routinely found in both
juvenile and mature dogs (Fahrion et al., 2008), a higher minimum effective point dose (e.g. −0.75 mg/kg) of MO will more effectively and consistently kill immature adult T. canis as demonstrated in this study and will reduce or eliminate shedding of these highly resilient eggs prior to female worms becoming patent. This environmental contamination potential is further exemplified by reports that have demonstrated an association between contamination of public areas and seropositivity for Toxocara spp. in children. The role of pet dogs and cats in the transmission of helminthic zoonoses, including T. canis, in Europe and other regions of the world has been documented (Macpherson, 2013). Schnieder et al. (2011) thoroughly reviewed the larval development of T. canis including the current knowledge of infection routes and the subsequent development of larvae within the canine host. They additionally reviewed information about the clinical, pathological, enzymatic, hematological and histopathological changes in dogs when they are infected with T. canis. When preadults have matured, eggs are first shed approximately 4–5 weeks post-inoculation (p.i.) in experimentally infected puppies whereas in older hosts prepatency seems to be extended to 40 up to 56 days p.i. These variations in the pre-patency period of T. canis highlight the importance of routine deworming treatments of dogs of various ages, in particular, when infected dogs are in close association with higher risk groups such as children, the elderly and immunocompromised pet owners. The zoonotic potential of A. caninum also has been well established in the scientific literature (Bowman et al., 2010). The prevalence of this hookworm species has been shown in prevalence surveys in various regions of the world and confirms that this parasite is commonly found in both young and adult dogs at all times of the year (Nolan and Smith, 1995; Little et al., 2009). Chemoprophylactic strategies to control A. caninum as well as T. canis have been reviewed based on the route of exposure or infection and the age of the dog (Epe, 2009). However, it is critical that the mg/kg dose of MO and other anthelmintics are administered accurately and just small variations can impact the overall efficacy against different stages of these zoonotic parasites. The effectiveness of the combination MO/spinosad tablet at a minimum dosage level of 0.75 mg/kg MO against both immature larval L4 and immature adult stages of A. caninum was clearly demonstrated in both dose confirmation studies summarized above. In contrast, Blagburn et al., 1992 reported that slightly lower doses of MO at 0.5 mg/kg did not demonstrate acceptable (>90%) efficacy against larval (L4) or immature adult stages of this pathogenic hookworm species of dogs. They reported that 0.5 mg/kg had 49% efficacy against L3/L4 stages, 83% against L4 and 81% against early (immature) stages of A. caninum. Stansfield and Hepler (1991) reported that adult A. caninum is the dose-limiting parasite for MO (which requires a minimum oral dose of 0.5 mg/kg). Results from the studies presented here, in combination with previously conducted studies at lower dose levels below 0.75 mg/kg, indicate that the larval stages of this parasite should now represent the dose-limiting intestinal nematode species (stage). A higher minimum effective
D.D. Bowman et al. / Veterinary Parasitology 205 (2014) 134–139
point dose of MO at 0.75 mg/kg will more effectively and consistently kill these immature stages of A. caninum and will reduce or eliminate shedding of infective eggs prior to female worms becoming patent. Additionally, both immature and adult stages of A. caninum are voracious blood suckers and can be very pathogenic in young dogs. Killing these blood-sucking hookworms as early in the life cycle of this parasite as possible will provide significant health benefits to infected dogs. The routine administration of broad spectrum endectocides that effectively kill both adult and immature intestinal nematode stages will help to address recommendations made to veterinarians (Epe, 2009) and expert groups such as ESCCAP (2010). These entities recommend routine treatment and control of T. canis and A. caninum, which are consistently found in dogs of all ages and throughout the year in prevalence studies conducted in different areas of the world, including Europe (MartinezMoreno et al., 2007; Riggio et al., 2013; Neves et al., 2014). Since re-infection is common in dogs with T. canis and A. caninum, the ability to kill immature adult stages of T. canis and L4/immature stages of A. caninum before they even start shedding eggs into its local environment will help to minimize and prevent environmental contamination with these zoonotic parasites. This improved parasite spectrum against immature stages of these two zoonotic intestinal nematode species of dogs using a minimum effective dose of 0.75 mg/kg of MO as reported herein will be a benefit for both dogs that includes reduced pathogenicity, re-infections, and killing immature female worms before they start to lay eggs, while their owners and family members will have less exposure to these potentially zoonotic parasites. This appears to be the first report demonstrating that a higher dose of MO using a minimum oral dose of 0.75 mg/kg can effectively kill immature intestinal stages of two very important nematodes of dogs, T. canis and A. caninum. Conflict of interest statement The studies as reported herein were funded by Elanco Animal Health. The authors from Cornell University and East Tennessee Clinical Research were contracted to perform these studies; the remaining authors are current employees of Elanco Animal Health and assisted with the study design, study conduct, data analysis, and review of the manuscript; however, there were no conflicting interests that may have biased the work reported in this paper. References Anon, 2006. NADA 141-251. United States Food and Drug Administration. Freedom of Information Summary. ADVANTAGE MULTI for Dogs http://www.fda.gov/downloads/ (Imidacloprid+Moxidectin), AnimalVeterinary/Products/ApprovedAnimalDrugProducts/ FOIADrugSummaries/UCM051438.pdf Blagburn, B.L., Hendrix, C.M., Lindsay, D.S., Vaughan, J.L., Hepler, D.I., Wright, J.C., 1992. Efficacy of milbemycin oxime against naturally
139
acquired or experimentally induced Ancylostoma spp. and Trichuris vulpis infections in dogs. Am. J. Vet. Res. 53, 513–516. Böhm, C., Schnyder, M., Thamsborg, S.M., Thompson, C.M., Trout, C., Wolken, S., Schnitzler, B., 2014. Assessment of the combination of spinosad and milbemycin oxime in preventing the development of canine Angiostrongylus vasorum infections. Vet. Parasitol. 199, 272–277. Bowman, D.D., Parsons, J.C., Grieve, R.B., Hepler, D.I., 1988. Effects of milbemycin on adult Toxocara canis in dogs with experimentally induced infection. Am. J. Vet. Res. 49, 1986–1989. Bowman, D.D., Montgomery, S.P., Zajac, A.M., Eberhard, M.L., Kazacos, K.R., 2010. Hookworms of dogs and cats as agents of cutaneous larva migrans. Trends Parasitol. 26, 162–167. Deplazes, P., van Knapen, F., Schweiger, A., Overgaauw, P.A., 2011. Role of pet dogs and cats in the transmission of helminthic zoonoses in Europe, with a focus on echinococcosis and toxocarosis. Vet. Parasitol. 182, 41–53. Epe, C., 2009. Intestinal nematodes: biology and control. Vet. Clin. Small Anim. 39, 1091–1107. ESCCAP, 2010. Worm Control in Dogs and Cats. Guideline 01, second ed., pp. 1–28, http://www.esccap.org Fahrion, A.S., Staebler, S., Deplazes, P., 2008. Patent Toxocara canis infections in previously exposed and in helminth-free dogs after infection with low numbers of embryonated eggs. Vet. Parasitol. 152, 108–115. Lee, A.C.Y., Schantz, P.M., Kazacos, K.R., Montgomery, S.P., Bowman, D.D., 2010. Epidemiologic and zoonotic aspects of ascarid infections in dogs and cats. Trends Parasitol. 26, 155–161. Little, S.E., Johnson, E.M., Lewis, D., Jaklitsch, R.P., Payton, M.E., Blagburn, B.L., Bowman, D.D., Moroff, S., Tams, T., Rich, L., Aucoin, D., 2009. Prevalence of intestinal parasites in pet dogs in the United States. Vet. Parasitol. 166, 144–152. Manini, M.P., Marchioro, A.A., Colli, C.M., Nishi, L., Falavigna-Guilherme, A.L., 2012. Association between contamination of public squares and seropositivity for Toxocara spp. in children. Vet. Parasitol. 188, 48–52. Martinez-Moreno, F.J., Hernandez, S., Lopez-Cobos, E., Becerra, C., Acosta, I., Martinez-Moreno, A., 2007. Estimation of canine intestinal parasites in Cordoba (Spain) and their risk to public health. Vet. Parasitol. 143, 7–13. Macpherson, C.N.L., 2013. The epidemiology and public health importance of toxocariasis: a zoonosis of global importance. Int. J. Parasitol. 43, 999–1008. Neves, D., Lobo, L., Brilhante Simoes, P., Cardoso, L., 2014. Frequency of intestinal parasites in pet dogs from an urban area (Greater Oporto northern Portugal). Vet. Parasitol. 200, 295–298. Nolan, T.J., Smith, G., 1995. Time series analysis of the prevalence of endoparasitic infections in cats and dogs presented to a veterinary teaching hospital. Vet. Parasitol. 59 (2), 87–96. Overgaauw, P.A.M., van Knapen, F., 2013. Veterinary and public health aspects of Toxocara spp. Vet. Parasitol. 193, 398–403. Riggio, F., Mannella, R., Ariti, G., Perrucci, S., 2013. Intestinal and lung parasites in owned dogs and cats from central Italy. Vet. Parasitol. 193, 78–84. Schnieder, T., Laabs, E.-M., Welz, C., 2011. Larval development of Toxocara canis in dogs. Vet. Parasitol. 175, 193–206. Schnitzler, B., Hayes, B., Wiseman, S., Snyder, D.E., 2012. Confirmation of the efficacy of a combination tablet of spinosad and milbemycin oxime against naturally acquired infections of canine intestinal nematode parasites. Vet. Parasitol. 184, 279–283. Stansfield, D.G., Hepler, D.I., 1991. Safety and efficacy of milbemycin oxime for parasite control. Canine Pract. 16, 11–16. VICH, Guideline 7, 2001a. Efficacy requirements for anthelmintics: overall guidelines. Veterinary International Cooperation on Harmonization. European Agency for the Evaluation of Medicinal Products, London. VICH, Guideline 9, 2001b. Good Clinical Practice. Veterinary International Cooperation on Harmonization. European Agency for the Evaluation of Medicinal Products, London. VICH, Guideline 19, 2002. Efficacy of Anthelmintics: Specific Recommendations for Canine. Veterinary International Cooperation on Harmonization. European Agency for the Evaluation of Medicinal Products, London. Won, K.Y., Kruszon-Moran, D., Schantz, P.M., Jones, J.L., 2008. National seroprevalence and risk factors for zoonotic Toxocara spp. infection. Am. J. Trop. Med. Hyg. 79, 552–557.