Scholarly article on topic 'Nematode burdens of pastured cattle treated once at turnout with eprinomectin extended-release injection'

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Abstract of research paper on Biological sciences, author of scientific article — S. Rehbein, D.G. Baggott, E.G. Johnson, B.N. Kunkle, T.A. Yazwinski, et al.

Abstract The efficacy of eprinomectin in an extended-release injection (ERI) formulation was evaluated against infections with third-stage larvae or eggs of gastrointestinal and pulmonary nematodes in cattle under 120-day natural challenge conditions in a series of five studies conducted in the USA (three studies) and in Europe (two studies). For each study, 30 nematode-free (four studies) or 30 cattle harboring naturally acquired nematode infections (one study) were included. The cattle were of various breeds or crosses, weighed 107.5–273kg prior to treatment and aged approximately 4–11 months. For each study, animals were blocked based on pre-treatment bodyweight and then randomly allocated to treatment: ERI vehicle (control) at 1mL/50kg bodyweight or Eprinomectin 5% (w/v) ERI at 1mL/50kg bodyweight (1.0mg eprinomectin/kg) for a total of 15 and 15 animals in each group. Treatments were administered once on Day 0 by subcutaneous injection in front of the shoulder. In each study, all animals grazed one naturally contaminated pasture for 120 days. At regular intervals during the studies, fecal samples from all cattle were examined for nematode egg and larval counts. In four studies pairs of tracer cattle were used to monitor pasture infectivity at 28-day intervals before and/or during the grazing period. All calves were weighed before turnout onto pasture and at regular intervals until housing on Day 120. For parasite recovery, all study animals were humanely euthanized 27–30 days after removal from pasture. Cattle treated with Eprinomectin ERI had significantly (p <0.05) fewer strongylid eggs (≤1 egg per gram; egg count reduction≥94%) than the control cattle and zero lungworm larvae at each post-treatment time point. At euthanasia, cattle treated with Eprinomectin ERI had significantly (p <0.05) fewer of the following nematodes than the ERI vehicle-treated (control) cattle with overall reduction of nematode counts by >92%: Dictyocaulus viviparus (adults and fourth-stage larvae (L4), Bunostomum phlebotomum, Cooperia curticei, Cooperia oncophora, Cooperia punctata, Cooperia surnabada, Cooperia spp. inhibited L4, Haemonchus contortus, Haemonchus placei, Haemonchus spp. inhibited L4, Nematodirus helvetianus, Nematodirus spp. inhibited L4, Oesophagostomum radiatum, Oesophagostomum spp. inhibited L4, Ostertagia leptospicularis, Ostertagia lyrata, Ostertagia ostertagi, Ostertagia spp. inhibited L4, Trichostrongylus axei, Trichostrongylus colubriformis, Trichostrongylus spp. inhibited L4, Trichuris discolor, and Trichuris ovis. Over the 120-day grazing period, Eprinomectin ERI-treated cattle gained between 4.8kg and 31kg more weight than the controls. This weight gain advantage was significant (p <0.05) in three studies. All animals accepted the treatment well. No adverse reaction to treatment was observed in any animal in any study.

Academic research paper on topic "Nematode burdens of pastured cattle treated once at turnout with eprinomectin extended-release injection"

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Veterinary Parasitology

journal homepage: www.elsevier.com/locate/vetpar

Nematode burdens of pastured cattle treated once at turnout with eprinomectin extended-release injection

S. Rehbeina*, D.G. Baggottb, E.G. Johnsonc, B.N. Kunkled, T.A. Yazwinskie, S. Yoond, L.G. Cramerd, M.D. Solld

a Merial GmbH, Kathrinenhof Research Center, Walchenseestr. 8-12, 83101 Rohrdorf, Germany b Merial Animal Health Limited, Sandringham House, Harlow Business Park, Harlow, Essex CM19 5QA, United Kingdom c Johnson Research, L.L.C., 24007 Highway 20-26, Parma, ID 83660, USA d Merial Limited, 3239 Satellite Blvd., Duluth, GA 30096-4640, USA e Department of Animal Science, University of Arkansas, Fayetteville, AR 72701, USA

ARTICLE INFO ABSTRACT

The efficacy of eprinomectin in an extended-release injection (ERI) formulation was evaluated against infections with third-stage larvae or eggs of gastrointestinal and pulmonary nematodes in cattle under 120-day natural challenge conditions in a series of five studies conducted in the USA (three studies) and in Europe (two studies). For each study, 30 nematode-free (four studies) or 30 cattle harboring naturally acquired nematode infections (one study) were included. The cattle were of various breeds or crosses, weighed 107.5-273 kg prior to treatment and aged approximately 4-11 months. For each study, animals were blocked based on pre-treatment bodyweight and then randomly allocated to treatment: ERI vehicle (control) at 1 mL/50 kg bodyweight or Eprinomectin 5% (w/v) ERI at 1 mL/50 kg bodyweight (1.0 mg eprinomectin/kg) for a total of 15 and 15 animals in each group. Treatments were administered once on Day 0 by subcutaneous injection in front of the shoulder. In each study, all animals grazed one naturally contaminated pasture for 120 days. At regular intervals during the studies, fecal samples from all cattle were examined for nematode egg and larval counts. In four studies pairs of tracer cattle were used to monitor pasture infectivity at 28-day intervals before and/or during the grazing period. All calves were weighed before turnout onto pasture and at regular intervals until housing on Day 120. For parasite recovery, all study animals were humanely euthanized 27-30 days after removal from pasture.

Cattle treated with Eprinomectin ERI had significantly (p <0.05) fewer strongylid eggs (<1 egg per gram; egg count reduction >94%) than the control cattle and zero lungworm larvae at each post-treatment time point. At euthanasia, cattle treated with Eprinomectin ERI had significantly (p <0.05) fewer of the following nematodes than the ERI vehicle-treated (control) cattle with overall reduction of nematode counts by >92%: Dictyocaulus viviparus (adults and fourth-stage larvae (L4), Bunostomum phlebotomum, Cooperia cur-ticei, Cooperia oncophora, Cooperia punctata, Cooperia surnabada, Cooperia spp. inhibited L4, Haemonchus contortus, Haemonchus placei, Haemonchus spp. inhibited L4, Nematodirus helvetianus, Nematodirus spp. inhibited L4, Oesophagostomum radiatum, Oesophagostomum spp. inhibited L4, Ostertagia leptospicularis, Ostertagia lyrata, Ostertagia ostertagi, Osterta-gia spp. inhibited L4, Trichostrongylus axei, Trichostrongylus colubriformis, Trichostrongylus

Keywords: Eprinomectin Extended-release injection Efficacy

Chemoprophylaxis

Nematodes

Cattle

* Corresponding author. Tel.: +49 8032 70750. E-mail address: steffen.rehbein@merial.com (S. Rehbein).

0304-4017/$ - see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.11.038

spp. inhibited L4, Trichuris discolor, and Trichuris ovis. Over the 120-day grazing period, Eprinomectin ERI-treated cattle gained between 4.8 kg and 31 kg more weight than the controls. This weight gain advantage was significant (p < 0.05) in three studies. All animals accepted the treatment well. No adverse reaction to treatment was observed in any animal in any study.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Collectively, gastrointestinal nematodes and lungworms are still considered the most commonly occurring and most economically important parasites of grazing cattle in temperate regions worldwide.

Although parasitic infections in the majority of the cattle are often subclinical in current management systems where antiparasitics are used for prophylactic control, infection with those parasites can cause severe parasitic gastroenteritis and parasitic bronchitis, especially in young stock which are most susceptible in their first grazing season. Knowledge of the epidemiology of parasitic nema-todes has led to the creation of parasite control programs which aim to suppress the fecal egg output of grazing cattle by the use of early season anthelmintic prophylaxis and thus reduce the accumulation of infective larvae in the environment later in the season. The repeated strategic (prophylactic) administration of anthelmintics has proven to be highly effective in the prevention of clinical disease and/or production losses. The superior efficacy and extended duration of activity of the macrocyclic lactones in injectable and pour-on formulations led to an important extension of the intervals between treatments in these programs. High labor and equipment costs resulting from frequent gathering of cattle for treatment administration resulted in the development of various forms of intrarumi-nal boli (controlled-release devices) that were formulated for delivery of anthelmintics for up to 4 months following a single administration, either by continuous low-level release or by pulse-release of the drug.

The endectocidal effectiveness of eprinomectin, the most recent commercialized member of the macrocyclic lactone class of parasiticides as used in a 0.5% pour-on formulation at 0.5 mg eprinomectin/kg bodyweight, has been extensively documented (Barth et al., 1997; Gogolewski et al., 1997a,b; Holste et al., 1997, 1998; Pitt et al., 1997; Williams et al., 1997; Yazwinski et al., 1997; Campbell et al., 2001; Davey and George, 2002; Rehbein et al., 2005). Dose titration studies have demonstrated eprinomectin represents a threefold greater potency against ruminant gastrointestinal nematodes than ivermectin (Shoop et al., 1996; Shoop and Soll, 2002). The commercialized epri-nomectin pour-on formulation has considerable persistent activity against a range of important nematodes (Cramer et al., 2000; Holste et al., 2002) and has been used successfully in prophylactic treatment programmes (Batty et al., 1999; Epe et al., 1999; Dorny et al., 2000).

In order to extend the persistency of activity of eprinomectin against endoparasites, an injectable formulation has been developed which is not only effective in removing existing nematode infections (Hunter et al., 2013; Rehbein

et al., 2013) but also releases the active ingredient in concentrations to provide effective control of nematode infections in cattle for up to 150 days after treatment as demonstrated in single point challenge studies (Soll et al., 2013). In this extended-release formulation, eprinomectin is released from a matrix formed with poly(D,L-lactide-co-glycolic)acid (PLGA). PLGA is known as a safe and effective biodegradable material which has been used as a drug delivery system for extended release applications of various pharmaceutical compounds including ivermectin (Lewis, 1990; Miller et al., 1999; Clark et al., 2004; Winzenburg et al., 2004).

The studies reported here were designed to confirm the efficacy of eprinomectin extended-release injection (ERI) over the entire duration of activity of the product,

1.e., nematode-free or naturally infected calves received a single injection of eprinomectin ERI at turnout and were subsequently and continuously exposed to nematode challenge by grazing permanent pasture for 120 days.

2. Materials and methods

Atotal of five controlled studies were conducted according to similar protocols, three in the USA and one each in Germany and in the UK. In four studies (Studies 1-4), nematode-free calves were used, while one study utilized calves harboring naturally acquired nematode infections (Study 5). The studies were designed and conducted to comply with the regulatory requirements of both the FDA/CVM and the European Medicines Agency/Committee for Medicinal Products for Veterinary Use, and according to relevant guidelines for Good Clinical Practices (GCPs) and for establishing the efficacy of cattle anthelmintics.

The studies were performed as blinded studies, i.e., all personnel involved in collecting data were masked to the treatment assignment of the animals.

2.1. Experimental animals

A total of 150 (67 male castrate, 83 female) healthy, ruminating Holstein, Limousin, Pinzgauer and cross-bred beef cattle, weighing 107.5-273 kg prior to treatment (Days -3, -1 or 0), and aged approximately 4-11 months were included in five studies conducted in the USA (Studies 3, 4, and 5 - Arkansas, Idaho, Missouri), in Germany (Study 2, Upper Bavaria), and in the UK (Study 1, Hertfordshire). The animal descriptions and details are presented in Table 1. Animals had not been previously treated with an aver-mectin or milbemycin product.

Animals enrolled in Studies 1-4 were worm-free (as determined by fecal examination prior to study start, i.e., Days -15 or -14), with the exception of one animal in

Table l

Animal description and details.

Study#a Treatmentb/animals per treatment Breed Sex ~Age (months) Pre-treatment bodyweight (kg)

1 Control, n = 15 EpERI, n = 15 Limousin, Limousin cross, Salers cross Female 4-б 107.5-199

2 Control, n = 15 EpERI, n = 15 Pinzgauer Male castrate б 117-178

3 Control, n = 15 EpERI, n = 15 Mixed stocker breeds Female б 143.8-182.3

4 Control, n = 15 EpERI, n = 15 Holstein Male castrate 5 192.3-240.4

5 Control, n = 15 EpERI, n = 15 Angus, Angus cross, Charolais, Charolais cross 6 Male castrate, 24 Female 9-11 190-27б

a Study sites: Study 1 - Hertfordshire, UK; Study 2 - Upper Bavaria, Germany; Study 3 - Arkansas, USA; Study 4 - Idaho, USA; Study 5 - Missouri, USA. b Control, ERI vehicle-treated; EpERI, Eprinomectin ERI.

Study 3, which shed 2 strongylid eggs per gram on Day 0 (=day of treatment). Calves included in Studies 1 and 2 were nematode-naive while calves used in Studies 3 and 4 were treated with fenbendazole (10 mg/kg bodyweight per os) on Day -21 before pre-treatment fecal examination on Day -14.

Animals used in Study 5 harbored naturally acquired nematode infections and were shedding 1.33-321 strongylid eggs per gram of feces at examination on Day -7.

For proof of pasture infectivity (pasture contamination), eight to ten tracer cattle were included in Studies 1-4 (see Tables 3-6). The tracer cattle were similar to the study animals and were demonstrated free of patent nema-tode infection as confirmed by fecal examination prior to turnout onto pasture.

All animals were handled with due regard to their welfare and in compliance with Merial Institutional Animal Care and Use Committee (1ACUC) approvals, any applicable local regulations, and requirements of any local 1ACUC.

2.2. Study design

Studies 1-4 were conducted under the same protocol design utilizing a randomized block design based on pre-treatment (Day -1 or 0) bodyweights such that 15 replicates of two cattle each were formed sequentially. Within replicates, animals were randomly allocated to treatment: one to the control (ERI vehicle-treated) group and one to the Eprinomectin ER1 group, for a total of 15 and 15 animals in each group. For Study 5, a total of 75 cattle were ranked by pre-treatment (Day -3) bodyweight grouped into replicates of five animals each, and randomly allocated to one of five treatment groups within each replicate: control (ERI vehicle-treated) group, Eprinomectin ERI group, and three animals to one of three other additional treatment groups (data not shown), for a total of 15 animals per treatment group. All studies were initiated in June.

Treatments, ERI vehicle (formulation that consisted of the excipients of the Eprinomectin ERI) as well as Eprinomectin ERI (eprinomectin 5% in a PLGA-based formulation), were administered at 1 mL/50kg (1 mL/110lb) bodyweight once on Day 0 by subcutaneous injection in

front of the shoulder using commercial syringes and needles. Dose volumes were rounded to the next 0.1 mL above the calculated dose volume if the bodyweight was between increments.

All cattle were observed hourly for 4h post-treatment and thereafter once daily throughout the studies for health problems or adverse drug events.

All study animals were weighed prior to treatment (Day -3 or -1 or 0) for allocation and dose calculation, and on Days 28, 56, 84, and 120.

To provide an indication of the level of contamination of the pasture with helminth larvae/eggs, pairs of worm-free tracer calves were placed in the same pasture as the study animals (principals) for approximately 28-day intervals in Studies 1-4. The tracer calves were humanely euthanized immediately after removal from pasture or after housing for up to three weeks under conditions which were designed to preclude further nematode infections (for details see Tables 3-6).

In all studies, control (ERI vehicle-treated) and Eprinomectin ERI-treated cattle grazed the same naturally contaminated pasture to ensure continuous nematode exposure. After 120 days, the animals were housed under conditions designed to preclude further nematode exposure. All study animals were humanely euthanized 27-30 days later for nematode recovery.

2.3. Parasitological techniques

2.3.1. Fecal examinations

In Studies 1-4, fecal samples were collected from all study animals on Day 0 before treatment and on Days 28, 56, 84, and 120 after treatment for fecal egg and lung-worm larval count determination. For fecal egg counting either a modified McMaster method (flotation using saturated NaCl solution; count sensitivity 1 egg = 50 eggs per gram (EPG) in Study 1 or 1 egg = 10 EPG in Study 2) or a quantitative direct centrifugation/flotation technique (Study 3: flotation using saturated MgSO4 solution, count sensitivity 1 egg = 2 EPG; Study 4: flotation using saturated NaCl solution, count sensitivity 1 egg = 10 EPG) was used. For lungworm (Dictyocaulus) larval recovery, the Baermann technique was used based on 10-g fecal samples.

When present, eggs were referred to as strongylid (nematode genera including Bunostomum, Cooperia, Haemonchus, Oesophagostomum, Ostertagia, and Trichostrongylus), Nematodirus, Strongyloides, Capillaria, and/or Trichuris eggs.

In Study 5, fecal samples were collected from all study animals on Day -7 (prior to treatment) and on Days 28, 56, 84, and 120 after treatment for fecal egg count determination using a double centrifuga-tion/flotation technique (flotation using Sheather's sucrose solution; count sensitivity 1 egg = 0.33 EPG). Fecal lung-worm (Dictyocaulus) larval counts using the Baermann technique based on 50-g fecal samples were conducted only on Day -7. When present, eggs were referred to as strongylid (nematode genera including Bunostomum, Cooperia, Haemonchus, Nematodirus, Oesophagostomum, Ostertagia, and Trichostrongylus), Strongyloides, Capillaria, and/or Trichuris eggs.

2.3.2. Pepsinogen determination

Plasma derived from blood samples collected on Days -1, 28, 56,84, and 120 from Study 2 animals was analyzed for pepsinogen concentration using the method of Berghen et al. (1987).

2.3.3. Parasite counts

All study animals were humanely euthanized for nematode counting 27-30 days after removal from pasture. At necropsy, the lungs, abomasum, small intestine, and large intestine were removed. Lungs were examined completely for lungworms either by lengthwise opening of all accessible air passages and soaking of the dissected lungs (Studies 2 and 3) or by pulmonary tract perfusion (Oakley, 1980; Studies 1, 4 and 5). The contents of the abomasum, small and large intestines were collected separately and diluted with water. Abomasum and small intestine were incubated (saline soak) to recover mucosal stages of the parasites for identification and counting. The entire contents or a known percentage of the contents of each organ (abomasum and small intestine contents: 5% aliquots; abomasum and small intestine soaks: 2%, 5% or 10% aliquots; large intestine content: 5%, 20%, 25% aliquots or total count) were examined and the appropriate dilution factor applied to calculate the final count. To facilitate isolation and counting of nema-todes, organ contents and soaks were screened over sieves of appropriate mesh sizes to remove the debris.

Counts of each parasite species for each animal were calculated by multiplying the number of worms actually counted from each organ by the aliquot factor and summing over all organs.

At necropsy, nematodes were identified to species or genus level and stage of development according to recognized methods and procedures. Adult female nematodes and/or fourth-stage larvae were not identified always to species level. For some genera of nematodes (e.g., Coope-ria, Ostertagia, and Trichostrongylus), the total counts were estimated by proportionally assigning adult females to each species based on the adult male counts. When adult females within genera were counted in the presence of no males, the counts were either proportioned equally or assigned based on the species present in other animals. For Trichostrongylus spp. in some studies, non-speciated

nematodes were assigned to species based on the location in which the nematodes were found.

Necropsies ofthe tracer calves followed the same principles as described for the study animals; however, the sizes of examined aliquots were different in some cases.

2.4. Statistical methods

Fecal eggs per gram counts or Dictyocaulus larval counts per gram for each animal at each time point were transformed to the natural logarithm of (count + 1) for calculation of geometric means. Exclusion criterion for individual analysis for Nematodirus, Strongyloides, Capillaria, Trichuris, and/or Dictyocaulus egg or larval counts was based on an infection rate (prevalence) of <40% in the control (ERI vehicle-treated) animals at the post-treatment time points.

Efficacy was calculated as percent reduction in egg/larval counts at each time point as 100[(C-T)/C], where C is the geometric mean of the control group and T is the geometric mean of the Eprinomectin ERI-treated group.

All nematode counts were subject to the methods described above regarding transformation and calculation of geometric means and efficacy. Treatment groups were compared using the Wilcoxon rank sum test. A two-sided test was used at a = 0.05.

Pepsinogen concentrations were analyzed with a repeated-measures analysis of variance (ANOVA) method in a randomized block design.

Pre-treatment bodyweight, Day 120 bodyweight, and weight gain until removal of animals from pasture (Day 120) were analyzed using ANOVA for a randomized block design.

3. Results

All animals accepted treatment, Eprinomectin ERI or ERI vehicle (control), well, as they were reported as normal during hourly observations for four hours post-treatment. There were no drug-related health problems or adverse drug events observed at any time during the studies. The Eprinomectin ERI-treated cattle were not reported as showing clinical signs of parasitic disease, but control animals in Studies 1 and 2 became sufficiently parasitized to compromise their well being with one animal dying from dictyocaulosis and parasitic gastroenteritis in Study 2. Therefore, salvage treatments with fenbendazole (PANACUR® 10% suspension; Intervet, B.V.) according to the manufacturer's recommendation were administered orally to all controls on Days 58, 85 and 101 in Study 1, and on Day 56 in Study 2.

As summarized in Table 2, cattle treated with Epri-nomectin ERI had significantly (p <0.05) fewer strongylid eggs than the vehicle-treated (control) cattle at each post-treatment time point, if present. Eprinomectin ERI treatment suppressed nematode egg production consistently through all study sites, and efficacy was >94% for all post-treatment time points. Nematodirus eggs were observed infrequently at fecal examinations so meaningful analysis of that parasite was possible only on data

Table 2

Summary of fecal egg and larval count data.

Study #A TreatmentB Eggs or larvae per gram counts (GMC)

Efficacy (%)D

Days before treatment Days after treatment

-15,-14 or-7 0 28 56 84 120

Strongylid eggs 1

Control EpERI

Control EpERI

Control EpERI

Control EpERI

Control EpERI

0 0a naE

0 0a na

nsF ns na

194 0b 100%

45 0b 100%

<1b >99%

<1b 98%

390 <1b >99%

634 0b

<1b >99%

<1b 96%

<1b >99%

493 <1b >99%

192 1b

3 <1b 94%

<1b >99%

212 <1b >99%

221 1b

<1b 99%

<1b 99%

Nematodirus spp. eggs

1 Control EpERI

2 Control EpERI

3 Control EpERI

4 Control EpERI

0 0 na

0 0 na

0 0 na

0 0 na

0 0 na

0 0 na

0 0 na

0 0 na

Dictyocaulus spp. larvae 2 Control

0 0 na

0 0 na

A Study sites: Study 1 - Hertfordshire, UK; Study 2 - Upper Bavaria, Germany; Study 3 - Arkansas, USA; Study 4 - Idaho, USA; Study 5 - Missouri, USA. B Control, ERI vehicle-treated; EpERI, Eprinomectin ERI.

C Geometric mean strongylid and Nematodirus egg and Dictyocaulus larval counts (based on transformation to the natural logarithm of [count +1]). D Efficacy = 100 [(geometric mean control - geometric mean Eprinomectin ERI)/geometric mean control]. E Not applicable. F Not sampled.

G Not analyzed due to a prevalence of <40% in the control group. a Probability from the Wilcoxon rank sum test: not significant at a = 0.05. b Probability from the Wilcoxon rank sum test: p < 0.05.

collected at some time points; cattle treated with Epri-nomectin ERI had significantly (p <0.05) fewer Nematodirus eggs than the control cattle at Days 56 (Studies 1 and 2), 84 (Study 3), and 120 (Study 4) with 100% reduction in egg counts. Cattle treated with Eprinomectin ERI had significantly (p <0.05) fewer Dictyocaulus larvae than the control cattle at each post-treatment time point in Study 2 with 100% efficacy (p<0.05) for all post-treatment time points. No lungworm larvae were recovered from any fecal sample collected from Eprinomectin ERI-treated animals following treatment. Strongyloides, Capillaria, and/or Trichuris spp. eggs were found in some animals in some studies; however, there were insufficient numbers of animals present

with those eggs for a reliable assessment. The summary of the fecal strongylid and Nematodirus egg and Dictyocaulus larval count data for all studies is shown in Table 2.

The pepsinogen concentration as measured in Study 2 animals was significantly (p <0.05) higher in the ERI vehicle-treated (control) cattle than in the cattle treated with Eprinomectin ERI at Days 56,84 and 120. For the controls, the mean plasma pepsinogen concentration rose from 307 mU/mL tyrosine at turnout to 2827 mU/mL tyrosine at removal from pasture on Day 120. For the Eprinomectin ER1-treated cattle, the mean plasma pepsinogen concentration was 260 mU/mL tyrosine at turnout and did not rise higher than 1604 mU/mL tyrosine at housing.

Table 3

Mean nematode worm counts for pairs of tracers which grazed with the principals for the indicated intervals, were removed from pasture and humanely euthanized after housing for 20/21 days - Study 1 (Hertfordshire, UK).

Interval (study days) pairs of tracers grazed

0-28 28-56 56-84a 84-98b

Cooperia oncophora 3672 22,123 72,349 125,966

Cooperia surnabada 298 387 5151 107,384

Cooperia spp., L4c 30 1200 4370 39,600

Nematodirus helvetianus 800 6140 4980 85,200

Nematodirus spp., L4 140 2510 1520 6800

Ostertagia lyrata 20 1070 1956 5927

Ostertagia ostertagi 1981 98,050 68,164 202,733

Trichostrongylus axei 0 0 730 0

a Necropsied three and seven days after removal of calves from pasture. b Necropsied one day after removal of calves from pasture. c Fourth stage larvae, developing and/or inhibited.

In addition to the fecal egg and larval counts of the control cattle in all studies from Day 28 forward, the parasite counts of the tracer cattle in Studies 1-4 (Tables 3-6) further documented that the pastures remained contaminated with infective nematode larvae and confirmed that challenge exposure was present for the entire grazing period. The mean total tracer cattle nematode recoveries at necropsy indicated a heavier challenge in the two studies conducted in Europe (Tables 3 and 4) compared to the studies conducted in the USA (Tables 5 and 6).

At necropsy 27-30 days after removal from pasture, cattle treated with Eprinomectin ERI and allowed to graze naturally infected pasture for 120 days had significantly (p<0.05) fewer of the following nematodes than the vehicle-treated (control) cattle with overall reduction of nematode counts by >92% (Table 7):

Dictyocaulus viviparus (adults and fourth-stage larvae), Bunostomum phlebotomum, Cooperia curticei, Cooperia oncophora, Cooperia punctata, Cooperia surnabada, Cooperia spp. inhibited fourth-stage larvae, Haemonchus contortus, Haemonchus placei, Haemonchus spp. inhibited fourth-stage larvae, Nematodirus helvetianus, Nematodirus spp. inhibited fourth-stage larvae, Oesophagostomum radia-tum, Oesophagostomum spp. inhibited fourth-stage larvae, Ostertagia leptospicularis, Ostertagia lyrata, Ostertagia ostertagi, Ostertagia spp. inhibited fourth-stage larvae, Trichostrongylus axei, Trichostrongylus colubriformis, Trichostrongylus spp. inhibited fourth-stage larvae, Trichuris discolor, and Trichuris ovis.

Pre-treatment bodyweight was similar for the Eprinomectin ERI-treated and ERI vehicle-treated (control) animals in all study groups. During the 120-day grazing period following treatment, Eprinomectin ERI-treated

Table 4

Mean nematode worm counts for pairs of tracers which grazed with the principals for the indicated intervals, were removed from pasture and humanely euthanized after housing for 21/22 days - Study 2 (Upper Bavaria, Germany).

Interval (study days) pairs of tracers grazed

-35 to -7a 0-28 28-56 56-84 84-112

Dictyocaulus viviparus 0 186 1592 687 434

Bunostomum phlebotomum 0 40 0 50 50

Cooperia curticei 9465 1795 3320 10,230 7745

Cooperia oncophora 12,660 15,239 8787 39,381 13,640

Cooperia punctata 1580 1900 8540 24,125 21,875

Cooperia surnabada 2541 2686 1198 6325 2626

Cooperia spp., L4b 0 0 0 520 195

Haemonchus contortus 10 245 40 95 50

Nematodirus battus 90 40 0 10 0

Nematodirus helvetianus 0 2650 820 530 560

Nematodirus spp., L4 645 15 80 240 280

Oesophagostomum radiatum 0 68 18 65 57

Ostertagia circumcincta 1019 0 0 0 0

Ostertagia leptospicularis 4231 1227 2887 3950 4996

Ostertagia lyrata 0 0 0 11 24

Ostertagia ostertagi 16,227 2639 7938 25,580 23,616

Ostertagia pinnata 172 0 0 0 0

Ostertagia trifurcata 172 0 0 0 0

Ostertagia spp., L4 11,000 200 40 935 24,015

Strongyloides papillosus 0 0 15 390 0

Trichostrongylus axei 516 530 655 1870 3485

Trichostrongylus colubriformis 220 0 0 0 0

Trichuris discolor 0 18 32 7 10

a Necropsied immediately after removal of calves from pasture. b Fourth stage larvae, developing and/or inhibited.

Table 5

Mean nematode worm counts for pairs of tracers which grazed with the principals for the indicated intervals, were removed from pasture and humanely euthanized after housing for 21/22 days - Study 3 (Arkansas, USA).

Interval (study days) pairs of tracers grazed

-35 to -7a 0-28 28-56 56-84 84-112

Dictyocaulus viviparus 4 0 0 0 5

Bunostomum phlebotomum, L4b 17 0 0 0 0

Cooperia oncophora 4709 8035 3914 3196 0

Cooperia punctata 1616 2931 1847 18,535 12,176

Cooperia surnabada 1352 978 473 261 0

Cooperia spp., L4 1897 63 2 331 145

Haemonchus placei 0 380 143 283 513

Haemonchus spp., L4 0 10 11 0 31

Nematodirus helvetianus 100 627 552 1611 0

Nematodirus spp., L4 227 49 101 650 20

Oesophagostomum radiatum 0 101 40 331 0

Oesophagostomum radiatum, L4 <1 11 <1 40 22

Ostertagia lyrata 8 22 40 3 2

Ostertagia ostertagi 268 2517 1689 3752 501

Ostertagia spp., L4 2160 718 25 173 2

Trichostrongylus axei 5 37 39 14 16

Trichostrongylus colubriformis 0 34 0 0 0

Trichuris spp. 0 2 81 0 0

a Necropsied immediately after removal of calves from pasture. b Fourth stage larvae, developing and/or inhibited.

Table 6

Mean nematode worm counts for pairs of tracers which grazed with the principals for the indicated intervals, were removed from pasture and humanely euthanized after housing for 21/22 days - Study 4 (Idaho, USA).

Interval (study days) pairs oftracers grazed

-35 to -5a 0-28 28-56 56-84 84-112

Cooperia oncophora 50 0 0 120 20

Nematodirus helvetianus 10 30 60 510 0

Nematodirus spp., L4b 0 0 40 0 0

Ostertagia ostertagi 70 60 80 470 1320

Ostertagia spp., L4 250 30 0 0 10

Trichuris spp. 0 0 13 5 8

a Necropsied immediately after removal of calves from pasture. b Fourth stage larvae, developing and/or inhibited.

cattle gained on average between 4.8 and 41.0 kg more than the respective controls. Day 120 bodyweight and weight gain until Day 120 was significantly (p <0.05) greater for animals treated with Eprinomectin ERI than for the control animals in three studies (Table 8).

4. Discussion

The design of the long-term natural nematode challenge studies with the use of one pasture for all animals and pairs of tracer cattle grazing the same pasture excluded the factor of "pasture" (Bransby, 1993) and as a result also provided a precise assessment of the level of pasture infectivity with the species that were most abundant throughout the course of the study.

In all studies, Eprinomectin ERI-treated animals were subjected to continuous nematode larval and/or egg challenge throughout the 120-day grazing period, as shown by control egg and larval counts and/or by the numbers of nematodes recovered from the tracer cattle. Nematode recoveries from tracer cattle in Studies 1-4 indicated that nematode challenge was severe in the two studies conducted in Europe, while challenge was lower in Studies 3 and 4. The tracer animal data also confirmed that the

identified spectrum of nematodes, including Cooperia spp. and O. ostertagi as main components of the total gastrointestinal parasite burdens, was representative of those species known to cause clinical disease or subclinical parasitism and poor productivity in grazing cattle in temperate regions (Sutherland and Scott, 2010).

Based on repeated fecal examinations throughout all studies and nematode recoveries from the cattle removed from pasture 120 days after treatment administration, this series of studies conducted at different geographical locations in the USA and in Europe consistently demonstrated a high level of continuous efficacy of Eprinomectin ERI against a wide range of gastrointestinal and pulmonary nematode parasites of grazing cattle. A weight gain advantage of the Eprinomectin ERI treatment was evident in comparison to vehicle-treated controls. The results of the necropsy of the study animals after removal from pasture confirmed the results of the fecal examinations. Administration of Eprinomectin ERI not only provides efficacious treatment of existing adult, normally developing and inhibited immature nematode stages (Hunter et al., 2013; Rehbein et al., 2013) as has been documented for the eprinomectin pour-on formulation (Shoop et al., 1996; Gogolewski et al., 1997a,b; Pitt et al., 1997; Williams et al.,

Table 7

Burden of gastrointestinal and pulmonary nematodes of cattle after grazing for 120 days following treatment.

Studya

Control (ERI vehicle)

Eprinomectin ERI

Efficacyd

Probabilitye

Prevalenceb

Prevalence

Dictyocaulus viviparus

2 13/14f

3 13/15

4 15/15

5 4/15

Dictyocaulus viviparus, fourth-stage larvae

4 2/15

5 6/15

Bunostomum phlebotomum

2 9/14f

3 2/15 5 7/15

Capillaria spp. 3 3/15

5 1/15

Cooperia curticei 2 13/14f

Cooperia oncophora

1 14/15

2 13/14f

3 7/15

4 14/15

Cooperia punctata

2 14/14f

3 15/15

Cooperia surnabada 1 2

12/15 11/14f 4/15 8/15

10 5 5 <1

10 <1 <1

1288 2109 21 459

34,183 1785

107 350 5 13

244 199 48 1

616 <1 4

Cooperia spp., inhibited fourth-stage larvae

1 11/15

2 13/14f

3 13/15

4 3/15

Haemonchus contortus

2 9/14f

Haemonchus placei

3 15/15

4 2/15

5 6/15

Haemonchus spp., inhibited fourth stage larvae 3 9/15 5

5 10/15 22

Nematodirus helvetianus

1 7/15 24

2 9/14f 14

3 2/15 1

4 6/15 9

Nematodirus spp., inhibited fourth-stage larvae

1 8/15 24

2 5/14f 5

3 3/15 <1

4 1/15 <1

Oesophagostomum radiatum

1 2/15 <1

2 14/14f 66

3 12/15 70

4 4/15 <1

5 12/15 21

0/15 1/15

2/15 0/15

0/15 0/15 0/15

0/15 0/15

2/15 2/15 7/15 1/15

6/15 15/15

0/15 2/15 4/15 1/15

0/15 1/15

5/15 0/15

7/15 0/15 0/15

0/15 1/15

2/15 2/15 1/15 0/15

0/15 1/15 0/15 0/15

0/15 0/15 3/15 0/15 0/15

0 <1 <1 0

<1 <1 3 <1

0 <1 <1 <1

<1 <1 <1 0

0 <1 0 0

0 0 1 0 0

100% 100%

>99% >99%

84% >99%

>99% 84%

>99% 95%

99% 100%

97% 97%

<0.05 <0.05 <0.05 nah

<0.05 na

<0.05 <0.05 ns'

<0.05 ns

<0.05 <0.05 na

<0.05 <0.05 <0.05 na

<0.05 na

<0.05 <0.05

<0.05 <0.05 na

<0.05 na na na

<0.05 <0.05 na

Table 7 (Continued)

Studya Control (ERI vehicle) Prevalence' GMc Eprinomectin ERI Prevalence GM Efficacyd Probabilitye

Oesophagostomum spp., inhibited fourth-stage larvae

3 14/15 21 4/15 1 95% <0.05

5 3/15 <1 0/15 0 - na

Ostertagia leptospicularis

2 13/14f 698 2/15 <1 >99% <0.05

Ostertagia lyrata

1 14/15 121 0/15 0 100% <0.05

3 15/15 139 4/15 <1 >99% <0.05

5 4/15 2 1/15 <1 - na

Ostertagia ostertagi

1 15/15 12,537 1/15 <1 >99% <0.05

2 14/14f 4961 2/15 <1 >99% <0.05

3 15/15 3381 10/15 8 >99% <0.05

4 15/15 1774 1/15 <1 >99% <0.05

5 15/15 823 2/15 <1 >99% <0.05

Ostertagia spp., inhibited fourth-stage larvae

1 15/15 6261 0/15 0 100% <0.05

2 14/14f 14,703 4/15 4 >99% <0.05

3 9/15 2 0/15 0 100% <0.05

4 1/15 <1 0/15 0 - na

5 15/15 389 1/15 <1 >99% <0.05

Ostertagia spp., developing (late) fourth-stage larvae

3 14/15 9 2/15 <1 98% <0.05

4 4/15 1 0/15 0 - na

Strongyloides papillosus

3 2/15 1 0/15 0 - na

Trichostrongylus axei

2 14/14f 3125 6/15 5 >99% <0.05

3 15/15 108 3/15 <1 >99% <0.05

Trichostrongylus colubriformis

3 6/15 2 0/15 0 100% <0.05

5 1/15 <1 1/15 <1 - na

Trichostrongylus spp.

4 5/15 2 1/15 <1 - na

Trichostrongylus spp., inhibited fourth stage larvae

3 3/15 <1 0/15 0 - na

5 12/15 27 0/15 0 100% <0.05

Trichuris discolor

2 14/14f 50 7/15 4 92% <0.05

Trichuris ovis

4 12/15 45 6/15 2 95% <0.05

a Study sites: Study 1 - Hertfordshire, UK; Study 2 - Upper Bavaria, Germany; Study 3 - Arkansas, USA; Study 4 - Idaho, USA; Study 5 - Missouri, USA. b Prevalence: Number of cattle infected/Number of cattle in group.

c Geometric mean counts (based on transformation to the natural logarithm of [count + 1]).

d Efficacy = 100 [(geometric mean control - geometric mean Eprinomectin ERl)/geometric mean control].

e Probability from the Wilcoxon rank sum test.

f One animal from the control group died on Study Day 56.

g Not calculated due to a prevalence of <40% in the control group.

h Not analyzed.

1 Not significant at a = 0.05.

1997; Yazwinski et al., 1997), but also prevented the establishment of inhibited larvae of several nematode genera, including the accumulation of the most important arrested early fourth-stage larvae of Ostertagia spp. Before humane euthanasia, all study animals were kept for about four weeks under conditions designed to prevent further nema-tode infections, thus allowing normally developing and inhibited nematode larvae to be distinguished (Williams et al., 1997).

Consistent with the results collected in field studies (Kunkle et al., 2013), the fecal output of nematode eggs and lungworm larvae from Eprinomectin ERI-treated cattle was very low or zero throughout the study period. This effect will be beneficial to reducing pasture contamination. Grazing cattle treated with Eprinomectin ERI are protected from establishment of incoming larvae, and this protection will thus reduce the contamination of the pasture and transmission of nematode parasites.

Table 8

Summary of bodyweight and weight gain data.

Studya Treatment11 Mean Day -3/-1/0 Mean Day 120 Mean weight gain

Bodyweight (kg)c Bodyweight (kg) (kg) to Day 120

Probabilityd Probability Probability

1 Control, n = 15 145.6 161.0 15.4

EpERI, n =15 146.3 176.8 30.5

nse p <0.05 p <0.05

2 Control, n = 15f 139.3 165.8 26.6

EpERI, n =15 139.5 207.1 67.6

ns p <0.05 p <0.05

3 Control, n = 15 165.7 197.2 31.5

EpERI, n =15 165.2 201.5 36.3

ns ns ns

4 Control, n = 15 216.2 246.2 29.9

EpERI, n =15 214.5 250.4 36.0

ns ns ns

5 Control, n = 15 234.3 271.9 37.6

EpERI, n =15 234.9 295.5 60.6

ns p <0.05 p <0.05

a Study sites: Study 1 - Hertfordshire, UK; Study 2 - Upper Bavaria, Germany; Study 3 - Arkansas, USA; Study 4 - Idaho, USA; Study 5 - Missouri, USA. b Control=vehicle-treated; EpERI = Eprinomectin ERI. c Least squares means rounded to one degree of precision post-calculation.

d Significant probability values from Wilcoxon rank sum test comparing Eprinomectin ERI to controls are rounded to 0.05. e Not significant at a = 0.05.

f One animal from the control group died on Study Day 56.

The plasma pepsinogen levels in the treated cattle in Study 2 suggest that the prophylaxis provided by Eprinomectin ERI prevented pathology to the abomasum associated with infection with abomasal parasites, especially O. ostertagi (Vercruysse and Claerebout, 2001).

The high prophylactic anthelmintic efficacy of Eprinomectin ERI demonstrated in the present studies was reflected in an improved rate of weight gain of Eprinomectin ERI-treated versus vehicle-treated cattle. This result confirms the results of other studies using Epri-nomectin ERI under natural challenge conditions (Kunkle et al., 2013) and numerous studies in which an ivermectin intraruminal slow-release bolus has been used to control nematode infection in young stock (e.g., Jacobsen et al., 1995; Pitt et al., 1996; Forbes et al., 2002). This effect is consistent with the growth response following repeated strategic anthelmintic treatments in first season grazing cattle (Shaw et al., 1998). The higher benefit of Eprinomectin ERI treatment on weight gain observed in Studies 1 and 2 compared to Studies 3 and 4 can be explained by the substantially lower nematode challenge in the latter studies as reflected in the total nematode burdens acquired by the tracer cattle and the composition of the nematode population.

In conclusion, the results of the series of controlled studies reported here demonstrate the very high efficacy and acceptability of Eprinomectin ERI when administered sub-cutaneously at 1.0mg/kg bodyweight to cattle against a wide range of gastrointestinal and pulmonary nematode infections under natural nematode challenge conditions for 120 days. With its extended activity, Eprinomectin ERI offers a highly effective product which can be conveniently administered to control the most important parasitic infections of grazing cattle for at least 4 months after treatment.

®All marks are the property of their respective owners.

Conflict of interest statement

The work reported herein was funded by Merial Limited, GA, USA.

All authors were/are current employees (SR, DB, BK, SY, LC, MS) or contractors (EJ, TY) of Merial and assisted with the study design, conduct, data analysis and review of the manuscript.

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