Controlled FECRT were conducted during 2006–2009 in 14 trials, to evaluate the anthelmintic efficacy of the proposed commercial formulation of derquantel-abamectin (Startect; Pfizer New Zealand Ltd, Auckland, NZ) when administered orally to sheep. They were conducted against naturally acquired, mixed infestations of gastrointestinal nematodes, on farms that were considered representative of sheep enterprises in the region, and managed as a commercial operation. Trial sites were selected to capture a range of geographic locations, climatic conditions, farming operations, breeds, sexes and ages, and to ensure representation of the economically important species of gastrointestinal nematodes. All trials were approved by either the AgResearch Grasslands Animal Ethics Committee, Palmerston North, New Zealand, or the Kaiawhina Animal Ethics Committee, Palmerston North, New Zealand.
The design of the study and technical procedures were similar across all farms, and consistent with the recommendations of the World Association for the Advancement of Veterinary Parasitology (WAAVP) for evaluating the efficacy of anthelmintics in ruminants (Wood et al. 1995
). For each type of nematode egg (strongyle and/or Nematodirus
sp.), efficacy of the product under investigation and each reference anthelmintic was given by the percentage reduction in geometric mean FEC compared with a negative control group. Percentage reductions in arithmetic mean FEC were also determined for completeness. In all trials, the interval between treatment (Day 0) and collection of faecal samples post-treatment was 14 (± 1) days. Pooled larval cultures were assessed pre- and post-treatment, to identify the strongyle genera present.
A summary of the location, animal details, pre-treatment strongyle FEC, group size and reference anthelmintics used in each trial is presented in . Each was a single-site, negatively controlled efficacy trial, using a randomised block design, with the individual animal as the experimental unit. Negative control animals either received tap water as a placebo or remained untreated.
Table 1. Details of individual trials conducted to evaluate the field effiicacy of the novel combination anthelmintic derquantel-abamectin in sheep, with range of pre-treatment strongyle faecal nematode egg counts (FEC), target group size (n), and reference anthelmintics (more ...)
Trials 1–5 were conducted to support registration of the derquantel-abamectin combination product, and did not include reference anthelmintics; a prior history of anthelmintic resistance was not a requirement for selection of these farms. Trials 6–14 were conducted to generate additional efficacy data on sheep farms with a history of resistance to single-active and/or combination anthelmintics available commercially. The selection of reference anthelmintics for these trials was guided by the results of previous anthelmintic-resistance tests on each farm, and were selected from albendazole (Albendazole Sheep; Ancare New Zealand Ltd, Auckland, NZ), levamisole (Levicare; Ancare New Zealand Ltd), combined albendazole-levamisole (Arrest; Ancare New Zealand Ltd), ivermectin (Ivomec Liquid for Sheep and Goats; Merial New Zealand Ltd, Manukau City, NZ), abamectin (Genesis Oral Drench; Ancare New Zealand Ltd), and moxidectin (Vetdectin Oral Drench for Sheep; Fort Dodge Animal Health Ltd, Auckland, NZ).
The sheep used were aged 2–12 months, weighed 18.5–53.0 kg at the time of treatment, and represented a range of breeds. A single sex (female) was used in nine trials, and mixed sex (female/castrated male) in the other five (see ). A total of 838 animals were enrolled across the 14 individual trials.
On each trial site, individual mobs were screened in the weeks leading up to commencement of the trial, to determine the parasite burden and range of species present. Source flocks with a mean strongyle FEC of ≥400 epg, and with at least two nematode genera present, were required. On some farms the study animals had significantly higher nematode burdens than this, with several genera represented. Potential source flocks were only considered on the basis that they had not been treated in the previous 60 days with a persistent macrocyclic lactone, or the previous 150 days with a sustained-release anthelmintic.
Faecal samples were collected 2–5 days prior to treatment, for the determination of individual FEC, as well as for differentiation of larvae from pooled coproculture. In each trial, up to 50% more animals than the target number were sampled, to ensure that all enrolled animals had an established nematode burden. Individual animals were included on the basis of good general health and a strongyle FEC of ≥ 100 epg; the animals with the highest egg counts were selected for each trial. In certain trials, animals with a very high FEC were excluded on welfare grounds, due to the potential for clinical parasitism in the event that those animals were allocated to the negative control group.
Allocation to experimental groups
The selected animals were sorted and blocked by pre-treatment strongyle FEC and, when possible, Nematodirus sp. FEC, and randomly allocated to experimental groups within each block. As a result of this procedure, each group had a similar mean and range of FEC prior to treatment.
Administration of test and reference anthelmintics
Animals were weighed for calculation of the dose on Day 0 (the day of treatment), or up to 5 days prior. In Trials 1–5, animals were treated with either derquantel-abamectin or tap water placebo; the dose for each animal was calculated on individual bodyweight at the rate of 1 ml/5 kg (nominal dose rates of 2 mg/ kg derquantel and 0.2 mg/kg abamectin). In Trials 6–14, the doses of derquantel-abamectin and each reference anthelmintic were based on the heaviest animal enrolled in the trial; in Trial 7, the animals were split into two lines in order to avoid excessive overdosing, and as such the doses in this trial were based on the heaviest animal in each line. Based on the stated concentration(s) and recommended label dose for each reference anthelmintic, the nominal (minimum) dose rate of each active drug was 4.75 mg/kg albendazole, 7.5 mg/kg levamisole, 0.2 mg/kg ivermectin, 0.2 mg/kg abamectin, and 0.2 mg/kg moxidectin. The negative control animals remained untreated in Trials 6–14. In all trials, individual doses were administered using a 10-ml or 20-ml disposable plastic syringe. Except in Trial 1, the presence or absence of coughing immediately following treatment was assessed and recorded.
Clinical observations and bodyweight
Clinical observations were performed by a veterinarian, who was blinded to allocation to treatment groups, from the commencement of treatment until at least 30 minutes after the final animal was treated. In Trials 1–5, additional clinical observations were made at 2 and 6 hours following treatment. After completion of clinical observations on Day 0, the study animals were returned to pasture and run as a single group until Day 14 (± 1 day). During this period, the animals were observed in the paddock by the farm manager on the day following treatment, then at least three times a week. The animals were weighed on the final day of the trial, except in Trial 7.
Faecal samples were collected per rectum pre- and post-treatment, and transported to a commercial veterinary laboratory (Gribbles Veterinary Pathology, Palmerston North or Dunedin), for individual FEC and pooled larval culture. In several trials it was not possible to obtain a faecal sample on the final day of the trial from every enrolled animal; this resulted in no more than one animal from any experimental group being excluded from the efficacy calculations. FEC were performed according to standard laboratory procedures, using a modified McMaster technique, and reported as epg; pre-treatment samples were counted at a sensitivity of 1:100 epg, while post-treatment samples were counted at either 1:50 epg (eight trials) or 1:100 epg (six trials). To reduce observational bias, post-treatment faecal samples were not sorted into their respective treatment groups prior to counting, and laboratory personnel were blinded to the allocation to treatment groups. In Trial 5, a repeat FEC was performed due to a suspected mix-up or identification error of samples; this repeat count was performed 6 days after collection of the samples, using new sub-samples from stored faeces, and laboratory personnel remained blinded to the allocation to treatment groups.
Once FEC were completed, faecal samples collected post-treatment were pooled by treatment group for larval culture and identification, according to standard laboratory procedures. Differentiation of larvae for strongyle genera is reported as the percentage of each genus identified. In line with standard practice, Chabertia and Oesophagostomum spp. larvae were not differentiated, thus percentages of larvae of those genera are reported as a single combined figure.
Statistical analysis and efficacy calculations
As per WAAVP guidelines (Wood et al. 1995
), the primary outcome measure was the percentage reduction in the geometric mean of individual FEC, compared with a negative control group, for each type of nematode egg identified, i.e. strongyle or Nematodirus
sp. A log-transformation [ln(x+1)] was applied to the FEC data prior to analysis, and the transformed values were analysed using a GLM including the fixed effect of treatment group and the random effect of block. Geometric means were obtained using back transformation, and treatment differences were assessed at the 5% level of significance (two-tailed). Arithmetic means for each treatment group were also determined using a corresponding analysis of untransformed data.
Provided there was overall evidence of a treatment effect (p<0.05), each treated group was compared with the negative control group, to determine the statistical significance of treatment differences, with no further adjustments for multiple comparisons, and to estimate treatment efficacy. Statistical comparisons between the derquantel-abamectin group and the reference groups have not been presented, as the reference anthelmintics were used in these trials solely to establish or confirm the resistance profile of the nematode population present on each farm.
Geometric and arithmetic means were used to estimate efficacy for each of the treated groups (derquantel-abamectin, and each reference anthelmintic where relevant), using the following formula:
where T01 represents the negative control group, and T0X the treated group of interest
In the case of Nematodirus sp., efficacy calculations were not performed where the number of egg-positive animals in the negative control group at Day 14 (± 1 day) was fewer than six.
In Trials 6–14, larval differentiation figures post-treatment were used to estimate the percentage efficacy of derquantel-abamectin, as well as the reference anthelmintics, against each strongyle genus identified. The arithmetic mean count for each genus was determined by multiplying the arithmetic mean strongyle FEC by the larval culture percentage, for each experimental group. Efficacy was then calculated using the arithmetic mean genus counts using the formula above; geometric means were not calculated due to larval differentiation being based on a pooled culture rather than culture of individual faecal samples. Where the derived arithmetic mean in the negative control group was <50 epg, efficacy calculations were not considered a valid estimate of the true efficacy against that genus (McKenna 1996
; Miller et al. 2006
), and are therefore not reported.
The presence of resistance to any one of the broad-spectrum reference anthelmintics used was defined as <95% reduction in mean FEC (Presidente 1985
; Coles et al. 2006
For those trials where the animals were weighed twice (all except Trial 7), the change in bodyweight was analysed using a GLM, with the fixed effect of treatment group, the random effect of block, and the bodyweight pre-treatment fitted as a covariate. LSM changes in bodyweight are reported for each experimental group. Where there was overall evidence of a treatment effect (p<0.05), pair-wise differences significant at the 5% level are reported. For trials with more than two experimental groups, Tukey's method was used to adjust for multiple comparisons.
Statistical analyses were performed using SAS for Windows v9.1 (SAS Institute Inc, Cary NC, USA).