- Email: [email protected]
The efficacy of eprinomectin against induced infections of immature (fourth larval stage) and adult nematode parasites in cattle
veterinary parasitology ELSEVIER
Veterinary Parasitology 73 (1997) 119-128
The efficacy of eprinomectin against induced infections of immature (fourth larval stage) and adult nematode parasites in cattle S.R. Pitt a,*, W.K. Langholff b, J.S. Eagleson b, S. Rehbein c " MSDRL Veterinary Laboratory, Highfield Farm, Mangrove Lane, Hertford, Hertfordshire SGI3 8QJ, UK b Merck and Co., PO Box 2000, Rahway, NJ 07065, USA c MSDRL Veterinary Laboratory, Kathrinenhof Walchensteer. 8-12, 8311 Rohrdorf-Lauterbach, Germany
Received 6 August 1996; accepted 19 February 1997
Abstract Eprinomectin is a new endectocide of the avermectin chemical group developed for use in cattle. To establish its effectiveness against Cooperia spp., Dictyocaulus viviparus, Haemonchus contortus, Nematodirus helvetianus, Oesophagostomum radiatum, Ostertagia ostertagi, Trichostrongylus axei and Trichostrongylus colubriformis (immature infections only), six trials were conducted in Europe at two trial sites. In each trial, animals were artificially infected with a number of nematode species and treated with eprinomectin. In three trials, the nematodes were expected to be at the fourth larval stage of development at the time of treatment, and at the adult stage at the time of treatment in the other three trials. When compared with the untreated control groups, efficacy against the adult and immature nematode infections was > 99% for all the nematode species examined. There was no difference in efficacy between groups of animals with clipped hair at the application site, and those with long hair. Subsequent examination of the application sites, both visual and by palpation, showed no gross reactions to the applied treatments. © 1997 Elsevier Science B.V. Keywords." Avermectin; Milbemycin; Eprinomectin; Efficacy; Nematode: Ostertagia; Cooperia; Dictyocaulus
1. Introduction The a v e r m e c t i n / m i l b e m y c i n group of c o m p o u n d s has proved to be remarkably effective against the e c o n o m i c a l l y significant endo- and ectoparasites of several animal
Corresponding author. 0304-4017/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0304-4017(97)00035-6
120
S.R. Pitt et al. / Veterinary Parasitology 73 (1997) 119-128
species, including cattle. Currently, there are f o u r m e m b e r s o f the avermectin/milbemycin family (ivermectin, abamectin, doramectin and moxidectin) used for treating cattle, and they are available in a range of formulations (Shoop et al., 1995). The efficacy of ivermectin, abamectin, doramectin and moxidectin has been widely reported (Benz et al., 1989; Soil et al., 1988; Baggott et al., 1994; Eagleson et al., 1995; Benz and Cox, 1989; Heinze-Mutz et al., 1993; Kaplan et al., 1994; Goudie et al., 1993; Jones et al., 1993; Yazwinski et al., 1994; Ranjan et al., 1992;Ranjan et al., 1994; Couvillion and Guerino, 1992; Williams et al., 1992). Any new avermectin/milbemycin compound for use in cattle must have a high efficacy against endoparasites that are commonly encountered when grazing infected pasture, including the lungworm Dictyocaulus viviparus. Eprinomectin is a new endectocide of the avermectin/milbemycin family (Mrozik et al., 1995), developed for use in cattle, and formulated as a pour-on for use at a dose rate of 500 m c g / k g bodyweight (Shoop et al., 1996a,b). It is not excreted in significant amounts in milk, and is therefore potentially of use in treating all classes of cattle including lactating dairy cows (Shoop et al., 1996a,b). To establish its effectiveness against the nematode parasites commonly encountered by grazing cattle, six trials with induced infections were conducted. The trials were conducted at two sites in Europe: Hertfordshire in the UK and Bavaria in Germany. Brief descriptions of the trials are given in Table 1. This paper focuses on the endectocide efficacy of eprinomectin at the recommended dose rate of 500 m c g / k g .
Table 1 Description of trial and animals used to evaluate the efficacy of eprinomectin against artificial nematode infections in calves Trial Breed Sex Age at Description Weight range number start of of trial at allocation (kg) trial (months) 1
Friesian
M
4
2
Hereford cross Friesian
F
8
3
Schwarzbunte
M
6
4
Friesian
M
7
5
Welsh Black
6M 18 F
5
6
Schwarzbunte
M
6
Efficacy against immature endoparasites, Hertfordshire Efficacy against immature endoparasites, Hertfordshire Efficacy against immature endoparasites, Bavaria Efficacy against adult endoparasites, Hertfordshire Efficacy against adult endoparasites and effect of hair length, Hertfordshire Efficacy against adult endoparasites, Bavaria
103-140 138-179 144-205 186-248 108-153
184-259
S.R. Pitt et al. / Veterina~. Parasitology 73 (1997) 119-128
121
Three trials were undertaken to establish efficacy of eprinomectin against immature nematode infections, and three against established adult infections. An additional purpose of trial 5 was to determine what effect hair length at the application site had on the efficacy against endoparasites of this pour-on formulation.
2. Materials and methods 2.1. Animals and general husbandry
Within each trial, the animals used were of similar breed, age, size and obtained from the same local sources. All animals used had either been reared and maintained under conditions designed to prevent accidental infection with nematode parasites; thus, were parasite-naive at the start of the trims (established by pretrial faecal strongylid egg counts), or they were treated with fenbendazole at twice the manufacturer's recommended dose rate at least 42 days before the day the animals were artificially infected. Each animal was uniquely identified with a numbered ear tag. Table 1 gives details of the animals used in each trial. For the duration of each triM, the animals were housed together, but individually tethered in stalls with solid partitions to prevent both selfgrooming and grooming by other animals. The environmental conditions for all stalls within each trial were identical. The animals were fed according to local practice, the same for each animal within a trial. Water was available at all times from automatic drinkers or buckets. 2.2. Nematode infections and allocation
For the four trims conducted in Hertfordshire, the larvae administered were all local field isolates that had been cultured for at least 2 yrs (except for D. viuiparus which was obtained directly from Intervet UK Ltd.). All the species were passaged at least annually. In the Bavarian trials the larvae used had been locally collected and propagated, with the exception of D. viviparus (obtained from Leipzig University) and H. contortus (from Hoechst, Frankfurt/Main). The animals were infected with third-stage larvae according to the schedule outlined in Table 2 in accordance with W A A V P guidelines. The doses of larvae were administered orally over the back of the tongue using a plastic test tube. The tube was then rinsed with distilled water, and the rinsate administered in the same manner. The allocation details for each trial are shown in Table 3. On the day of allocation, the animals were weighed and ranked (within sex groups in the case of trial 5) byl descending bodyweight. Within rank, sequential replicates of animals were formed. Within each replicate, animals were assigned to a treatment group by lottery. The stallsl were divided into blocks the same size as the number of animals in a replicate. Eachl animal in a replicate was then assigned to a stall within a stall block by lottery. For trial 5, one treated group (Group 2) of animals had the hair over the application site clippedl to a length of approximately 1 cm before treatment on Day 0. This area extended from the withers to rump and down each side to a line approximately three quarters of thei
20000(-6) 10000(-6) 15000(-6) 10000 ( - 28) 10000(-28)
15000(-28)
1 2 3 4 5
6
1000(-28)
1000(-6) 1000(-6) 1000(-6) 1000 ( - 28) 1000(-28)
D.v.
7000(-28)
5000(-6) 5000(-6) 5000(-6) 5000 ( - 28) -
H.c. 1000(-17) 1000(-16) 2000(-17) 1500(-38)
3333(-28)
O.r.
10000(-6) 3000(-6) 663(-6) 5000 ( - 28)
N.h.
10000(-28) 11383(-28)
20000(-6) 10000(-6) 15000(-6) 10000 ( - 28)
0.o.
10000(-28) 15000(-28)
20000(-6) 10000(-6) 15000(-6) 10000 ( - 28)
T.a.
-
10000(-6) 15000(-6) -
T.c.
colubriformis.
C. spp., Mixed Cooperia species; D.v.. D. viviparus; H.c., H. contortus; N.h., N. helvetianus; O.r., O. radiatum; 0.o., O. ostertagi; T.a., T. axei; T.c., T.
Day of infection relative to treatment day in parentheses.
C. spp.
Trial number
Table 2 Number of larvae administered to the animals in each trial
J t,4 oo
123
S.R. Pitt et al. / Veterina~ Parasitology 73 (1997) 119-128
Table 3 Allocation details Trial Day of Animals Number of Treatment number allocation relative on trial replicates group to treatment day (Day 0) 1
1
12
6
2
- 1
12
6
3
0
12
6
4
- I
12
6
5
- 8
24
8
6
0
12
6
Treatmentgroup (all eprinomectin dose rates were 500 mcg/kg)
2×6 animals 1: untreated control 2: eprinomectin 2 × 6 animals 1: control vehicle 2: eprinomectin 2 × 6 animals 1: control vehicle 2: eprinomectin 2 X 6 animals 1: untreated control 2: eprinomectin 3 × 8 animals 1: control vehicle 2: eprinomectin (clipped) 3: eprinomectin (long hair) 2 × 6 animals 1: control vehicle 2: eprinomectin
Day of necropsy relative to day of treatment (Day 0) 14 21 21 14 14 (reps 1-4) 15 (reps 5-8) 14
way down the flank. The average hair length along the application site for Groups 1 (untreated control) and 3 (treated unclipped) was 4.9 cm and 4.7 cm, respectively (group arithmetic means). 2.3. Treatments
Animals within a replicate were either treated with eprinomectin at 500 m c g / k g bodyweight in a topical formulation, or with the vehicle solution (except for trials 1 and 4, where no vehicle solution was administered to the control animals). Details of the treatment groups and dose rates for each group are shown in Table 3. At the time of treatment on Day 0, the animals in trials 4, 5 and 6 were expected to be harbouring adult stages of the nematode species with which they had previously been infected (this expectation being based on the time elapsed between infection with the parasite larvae and the day of treatment; see Table 2). Those in trials 1, 2 and 3 were expected to be harbouring immature stages (fourth larval stage larvae) of the nematodes at the time of treatment. All dose volumes were calculated using bodyweights measured on Day-1 or on the day of treatment. Each solution (vehicle or eprinomectin pour-on) was applied from a height of 5 - 1 0 cm in a line from the tailhead to the withers. A n assessment w a s made o f any loss of formulation due to dripping. Before treatment, the application s i t e was examined visually and by palpation for any skin or haircoat abnormality. All t h e animals were examined for any adverse reaction to the applied treatments intermittently for 4 h after treatment, and then daily until the end of the trials. The application sites were examined visually and by palpation for any significant changes to the hair a n d / o r skin at 7-day intervals. 2.4. Necropsy procedures
The day of necropsy relative to the day of treatment (Day 0) for each trial is shown in Table 3. The animals were euthanized by captive-bolt stunning followed by exsanguina-
124
S.R. Pitt et al. / VeterinaryParasitology 73 (1997) 119-128
tion. For each animal, observations were made of the application site before and after reflection of the skin both visually and by palpation to assess any gross changes present. The abomasum (all trials), small intestine (all trials), large intestine (trials 1, 2, 3 and 6) and lungs with trachea attached (all trials) were removed separately from each animal. The contents of the abomasum and small intestine were individually washed out, collected and resuspended to a fixed volume, and 5 × 1% aliquots were retained for counting. The contents of the large intestine were washed out over a 300-/zm screen and a 100% aliquot (or two 10% aliquots for trials 6 and 3) retained for counting. In addition, the washed abomasa were separately digested in 1% hydrochloric acid and pepsin at room temperature overnight (or tap water at 37°C for 20 h in trials 6 and 3), and any additional nematodes were recovered and counted from a retained 100% aliquot (or two 10% aliquots in trials 6 and 3). Adult lungworms were washed out of the lungs and a 100% aliquot was counted using a modified version of the Inderbitzen lung perfusion technique (Oakley, 1980). All aliquots were counted entirely. The numbers and species of nematode parasitic worms counted in each organ were recorded. 2.5. Statistical methods In all trials, the parasite counts for each animal were calculated by multiplying the number of nematodes actually counted in each location by the aliquot factor. The counts were then transformed to the natural logarithm of (count + 1) for analysis and calculation of geometric means. Treatment comparisons were made for each parasite using the modified Friedman procedure, a nonparametric test for randomized block designs, which leads to an exact probability of finding a statistic as extreme as, or more extreme than, the one observed. The trial was used as the blocking factor; parasite counts were ranked within each trial. The probability value for the test statistic was calculated based on a X 2 distribution with one degree of freedom. For trial 5, independent treatment comparisons were made comparing treated to control, and treated clipped vs. treated nonclipped.
3. Results The results for the three trials conducted to determine efficacy against immature nematode infections are shown in Table 4 and the results for the three trials conducted to determine efficacy against adult nematode infections in Table 5. When compared with the untreated control groups, efficacy against the adult and immature nematode infections was > 99% for all nematode species investigated (Cooperia spp., D. viviparus, H. contortus, Nematodirus helvetianus, Oesophagostomum radiatum, Ostertagia ostertagi, Trichostrongylus axei and Trichostrongylus colubriformis [immature infections only]). These reductions were significant ( P < 0.001) for all species in all the trials. There was no significant difference ( P > 0.1) in efficacy between groups for animals with clipped hair (1 cm length) at the application site, and those with long hair (4.7 cm length) in trial 5. There was no loss of formulation due to dripping in any of the trials. Subsequent observations by visual examination and palpation of the application sites during the trial
100 < 0.001
Pooled statistics % efficacy P control v eprinomectinb 100 < 0.001
0.0
17.6 (3-34)
0.0
37.0 (17-99)
0.0
85.5 (6-237)
D.v.
> 99 < 0.001
0.0
17,9 (0-295)
0.0
100.0 (0-1560)
1.8 (0-20)
2142.8 (1420-3160)
H.c.
100 < 0.001
0.0
15.5 (0-530)
0.0
237.0 (0-1260)
0.0
3033,1 (2000-4700)
N.h.
All induced infections were expected to be fourth larval stage at the time of treatment. Ranges in parentheses. aGeometric means based on transformation to In (count + l). bProbability values from modified Friedman test. c This was a combination infection of C, oncophora, C. punctata and C. surnabada.
0.0
8741.6 c (5880-11240)
0.0
2854.0 (20-10760)
0.0
9979.8 (7020-15200)
C.o.
500 m c g / k g eprinomectin~
3 Control ~
500 m c g / k g eprinomectin a
2 Control a
500 m c g / k g eprinomectina
1 Control a
100 < 0.001
0.0
944.0 (510-1540)
0.0
140.0 (84-182)
0.0
610.1 (315-841)
O.r.
> 99 < 0.001
1.2 (0-20)
6116.8 (3585-8715)
< 1.0 (0-60)
7945.0 (6580-10120)
55.4 (0-300)
12640.8 (6480-22540)
0.o.
> 99 < 0.001
0.0
4362.6 (2920-6585)
0.0
2297.0 (1580-3580)
3.6 (0-20)
5016.9 (2720-8380)
T.a.
Table 4 Group nematode worm burdens at necropsy for animals expected to be harbouring immature (fourth larval stage) larvae at the time of treatment
100 < 0.001
0.0
3176.3 (850-5900)
0.0
491.0 (40-1640)
-
-
T.c.
/
.~
2"
> 99 < 0.001
0.9 (0-50)
6259.3 d (5330-7160)
13.9/21.9 ¢ (0-760/0-540) ~
100 < 0.001
0.0
4.9 (0-104)
0.0/0.0 ~
65.6 (5-218)
0.0
41.9 (16-119)
D.v.
100 < 0.001
0.0
22.1 (0-430)
0.0
917.7 (540-1840)
H.c.
100 < 0.001
0.0
42.1 (0-1610)
0
1130.6 (120-2260)
N.h.
100 < 0.001
0.0
421.9 (360-490)
O.r.
> 99 < 0.001
0.0
3753.8 (745-6600)
(o-o/o-2o) c
O.O/O.Y
4411.0 (2840-6180)
0.0
4412.4 (3520-5500)
O.o.
> 99 < 0.001
0.0
4998.6 (2780-6840)
0.0/0.0 c
3489.2 (2620-4880)
0.7 (0-20)
2424.0 (1600-3260)
T.a.
~Clipped hair at application site/normal hair length. There was no significant difference in adult worm counts at necropsy between the two groups ( P > 0.1). dThis was a combination infection of C. oncophora, C. punctata and C. surnabada.
All induced infections were expected to be adult at the time of treatment. Ranges in parentheses. aGeometric means based on transformation to In (count + 1). bprobability values from modified Friedman test.
Pooled statistics % efficacy P control v eprinomectin b
500 m c g / k g eprinomectin ~
6 ControP
500 m c g / k g eprinomectin ~
4869.0 (2720-6200)
12.0 (0-260)
500 m c g / k g eprinomectin~
5 Control a
4910.6 (3860-6380)
C.o.
ControP
4
Table 5 Group nematode worm burdens at necropsy for animals expected to be harbouring adults at the time of treatment
t.o
I
e~
S.R. Pitt et al. / Veterinary Parasitology 73 (1997) 119-128
127
and at necropsy showed that the formulation was well tolerated by all the animals with no adverse reactions to treatment being seen either ante- or post-mortem.
4. Discussion Formulations of earlier generation avermectins and milbemycins had less than complete efficacy against certain nematode species at this use level with Nematodirus acting as a dose-limiting parasite for abamectin and ivermectin (Kaplan et al., 1994 and Benz and Cox, 1989) and Cooperia being a dose-limiting parasite for moxidectin and doramectin (Ranjan et al., 1992; Jones et al., 1993; Taylor et al., 1993; and Yazwinski et al., 1994). Demonstration of consistent efficacy of > 99% against adult and immature parasites of these species, as well as against the other important nematode parasites of the gastrointestinal tract and lungs of cattle, confirms that eprinomectin applied in the topical formulation at 500 m c g / k g does not have a similar limiting parasite among the economically important cattle parasites at this dose level. Further, the excretion, metabolism and residue profile of this product is such that it is not only safe for use in all classes and ages of cattle, but the product may even be used on lactating dairy cattle without any requirement for milk withholding (Shoop et al., 1996a,b). These factors combined with the virtually complete efficacy of eprinomectin against all species of adult and immature nematodes of cattle tested in these studies reinforces the contention that this compound is the most efficacious in the class (Shoop et al., 1996a,b).
References Baggott, D.G., Ross, D.B., Preston, J.M., Gross, S.J., 1994. Nematode burdens and productivity of grazing cattle treated with a prototype sustained-releasebolus containingivermectin.Vet. Rec. 135, 503-506. Benz, G.W., Cox, J.L., 1989. Use of abamectinin cattle. In: Campbell,W.C. (Ed.), Ivermectinand Abamectin. Springer, New York, pp. 230-233. Benz, G.W., Roncalli, R.A., Gross, S.J., 1989. Use of ivermectin in cattle, sheep, goats and swine. In: Campbell, W,C. (Ed.), Ivermectinand Abamectin.Springer, New York pp. 215-229. Couvillion,C.E. and Guerino, F., 1992. Dose Titration of Moxidectin Against Naturally Acquired Nematode Infections in Cattle. Proc. 37th Annu. Meeting Am. Assoc. Vet. Parasitol., WA, 32. Eagleson, J.S., Cramer, L.G. and Farrington,D.O., 1995. Efficacy of the IvermectinSustainedRelease Bolus in AnimalsWeighingGreater Than 300 kg at the Time of Treatment. 15th Int. Conf. of the World Assoc. for the Advancementof Vet. Parasitol. Goudie, A.C., Evans, N.A., Gration, K.A.F., Bishop, B.F., Gibson, S.P., Holdom, K.S., Kaye, B., Wicks, S.R.. Lewis, D., Weatherley, A.J., 1993. Doramectin--a potent novel endectocide. Vet. Parasitol. 49, 5-15. Heinze-Mutz, E.M., Pitt, S.R., Bairden, K., Baggott, D.G., Armour, J., Barth, D., Cramer, L.G., 1993. Efficacy of abamectin against nematodes in cattle. Vet. Rec. 132, 35-37. Jones, R.M., Logan, N.B., Weatherley,A.J., Little, A.S., Smothers, C.D., 1993, Activityof doramectinagainst nematode parasites of cattle. Vet. Parasitol. 49, 27-37. Kaplan, R.M., Courtney, C.H., Kunkle, W.E., Zeng, Q.Y., Jernigan, A.D., Eagleson, J.S., 1994. Efficacy of injectableabamectinagainst gastrointestinaltract nematodes and lungwormsof cattle. Am. J. Vet. Res. 55, 353-357. Mrozik, H., Eskola, P., Byron, H., Arison, B.O.. Linn, A.L., Matzuk, A., Shih, T.L., Tischler, M., Waksmunski, F.S., Wyvratt, M.J., Blizzard, G.M.M., Fisher, M.H., Shoop, W.L., Egerton, J.R., 1995.
128
S.R. Pitt et al. / Veterinary Parasitology 73 (1997) 119-128
4'-deoxy-4'-aminoavermectins with potent broad spectrum antiparasitic activities. Bioorg. Med. Chem. Lett. 5 (20), 2435-2440. Oakley, G.A., 1980. The recovery of Dic~ocaulus viviparus from bovine lungs by lung perfusion: a modification of Inderbitzens method. Res. Vet. Sci. 29, 395. Ranjan, S., Trudeau, R.K., Prichard, R.K., Von Kutzleben, R., Carrier, D., 1992. Efficacy of moxidectin against naturally acquired nematode infections in cattle. Vet. Parasitol. 41,227-231. Ranjan, S., Trudeau, R.K., Prichard, R.K., Von Kutzleben, R., 1994. Efficacy of Moxidectin pour-on against natural nematode infections in cattle. Proc. 39th Annu. Meeting Am. Assoc. Vet. Parasitol., CA A7 20-20. Shoop, W.L., Mrozik, H., Fisher, M.H., 1995. Structure and activity of avermectins and milbemycins in animal health. Vet. Parasitol. 59, 139-156. Shoop, W.L., Egerton, R., Eary, C.H., Haines, H.W., Michael, B.F., Mrozik, H., Eskola, P., Fisher, M.H., Slayton, L., Ostlind, D.A., Skelly, B.J., Fulton, R.K., Barth, D., Costa, S., Gregory, L.M., Campbell, W.C., Seward, R.L., Turner, M.J., 1996a. Eprinomectin: a novel avermectin for use as a topical endectocide for cattle. Int. J. Parasitol. (in press). Shoop, W.L., DeMontigny, P., Fink, D.W., Williams, J.B., Egerton, J.R., Mrozik, H., Fisher, M.H., Skelly, B.J., Turner, M.J., 1996b. Efficacy in sheep and pharmacokinetics in cattle that led to the selection of eprinomectin as a topical endectocide for cattle. Int. J. Parasitol. (in press). Soil, M.D., Carmichael, J.H., Harvey, R.G., 1988. Prophylactic efficacy of sustained-release ivermectin against induced nematode infestations in cattle. J. S. Afr. Vet. Assoc. 59, 9-11. Taylor, S.M., Kenny, J., Edgar, H., 1993. Comparison of the efficacy of injectable and topical moxidectin for the reduction of faecal egg counts of cattle. Vet. Rec, 133, 216-217. Williams, J.C., Barras, S.A., Wang, G.T., 1992. Efficacy of moxidectin against gastrointestinal nematodes of cattle. Vet. Rec. 131,345-347. Yazwinski, T.A., Featherstone, H., Tucker, C., 1994. Effectiveness of doramectin for treatment of experimentally induced gastrointestinal tract larval nematode infections in calves. Am. J. Vet. Res. 55, 820-821.
Veterinary Parasitology 73 (1997) 119-128
The efficacy of eprinomectin against induced infections of immature (fourth larval stage) and adult nematode parasites in cattle S.R. Pitt a,*, W.K. Langholff b, J.S. Eagleson b, S. Rehbein c " MSDRL Veterinary Laboratory, Highfield Farm, Mangrove Lane, Hertford, Hertfordshire SGI3 8QJ, UK b Merck and Co., PO Box 2000, Rahway, NJ 07065, USA c MSDRL Veterinary Laboratory, Kathrinenhof Walchensteer. 8-12, 8311 Rohrdorf-Lauterbach, Germany
Received 6 August 1996; accepted 19 February 1997
Abstract Eprinomectin is a new endectocide of the avermectin chemical group developed for use in cattle. To establish its effectiveness against Cooperia spp., Dictyocaulus viviparus, Haemonchus contortus, Nematodirus helvetianus, Oesophagostomum radiatum, Ostertagia ostertagi, Trichostrongylus axei and Trichostrongylus colubriformis (immature infections only), six trials were conducted in Europe at two trial sites. In each trial, animals were artificially infected with a number of nematode species and treated with eprinomectin. In three trials, the nematodes were expected to be at the fourth larval stage of development at the time of treatment, and at the adult stage at the time of treatment in the other three trials. When compared with the untreated control groups, efficacy against the adult and immature nematode infections was > 99% for all the nematode species examined. There was no difference in efficacy between groups of animals with clipped hair at the application site, and those with long hair. Subsequent examination of the application sites, both visual and by palpation, showed no gross reactions to the applied treatments. © 1997 Elsevier Science B.V. Keywords." Avermectin; Milbemycin; Eprinomectin; Efficacy; Nematode: Ostertagia; Cooperia; Dictyocaulus
1. Introduction The a v e r m e c t i n / m i l b e m y c i n group of c o m p o u n d s has proved to be remarkably effective against the e c o n o m i c a l l y significant endo- and ectoparasites of several animal
Corresponding author. 0304-4017/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0304-4017(97)00035-6
120
S.R. Pitt et al. / Veterinary Parasitology 73 (1997) 119-128
species, including cattle. Currently, there are f o u r m e m b e r s o f the avermectin/milbemycin family (ivermectin, abamectin, doramectin and moxidectin) used for treating cattle, and they are available in a range of formulations (Shoop et al., 1995). The efficacy of ivermectin, abamectin, doramectin and moxidectin has been widely reported (Benz et al., 1989; Soil et al., 1988; Baggott et al., 1994; Eagleson et al., 1995; Benz and Cox, 1989; Heinze-Mutz et al., 1993; Kaplan et al., 1994; Goudie et al., 1993; Jones et al., 1993; Yazwinski et al., 1994; Ranjan et al., 1992;Ranjan et al., 1994; Couvillion and Guerino, 1992; Williams et al., 1992). Any new avermectin/milbemycin compound for use in cattle must have a high efficacy against endoparasites that are commonly encountered when grazing infected pasture, including the lungworm Dictyocaulus viviparus. Eprinomectin is a new endectocide of the avermectin/milbemycin family (Mrozik et al., 1995), developed for use in cattle, and formulated as a pour-on for use at a dose rate of 500 m c g / k g bodyweight (Shoop et al., 1996a,b). It is not excreted in significant amounts in milk, and is therefore potentially of use in treating all classes of cattle including lactating dairy cows (Shoop et al., 1996a,b). To establish its effectiveness against the nematode parasites commonly encountered by grazing cattle, six trials with induced infections were conducted. The trials were conducted at two sites in Europe: Hertfordshire in the UK and Bavaria in Germany. Brief descriptions of the trials are given in Table 1. This paper focuses on the endectocide efficacy of eprinomectin at the recommended dose rate of 500 m c g / k g .
Table 1 Description of trial and animals used to evaluate the efficacy of eprinomectin against artificial nematode infections in calves Trial Breed Sex Age at Description Weight range number start of of trial at allocation (kg) trial (months) 1
Friesian
M
4
2
Hereford cross Friesian
F
8
3
Schwarzbunte
M
6
4
Friesian
M
7
5
Welsh Black
6M 18 F
5
6
Schwarzbunte
M
6
Efficacy against immature endoparasites, Hertfordshire Efficacy against immature endoparasites, Hertfordshire Efficacy against immature endoparasites, Bavaria Efficacy against adult endoparasites, Hertfordshire Efficacy against adult endoparasites and effect of hair length, Hertfordshire Efficacy against adult endoparasites, Bavaria
103-140 138-179 144-205 186-248 108-153
184-259
S.R. Pitt et al. / Veterina~. Parasitology 73 (1997) 119-128
121
Three trials were undertaken to establish efficacy of eprinomectin against immature nematode infections, and three against established adult infections. An additional purpose of trial 5 was to determine what effect hair length at the application site had on the efficacy against endoparasites of this pour-on formulation.
2. Materials and methods 2.1. Animals and general husbandry
Within each trial, the animals used were of similar breed, age, size and obtained from the same local sources. All animals used had either been reared and maintained under conditions designed to prevent accidental infection with nematode parasites; thus, were parasite-naive at the start of the trims (established by pretrial faecal strongylid egg counts), or they were treated with fenbendazole at twice the manufacturer's recommended dose rate at least 42 days before the day the animals were artificially infected. Each animal was uniquely identified with a numbered ear tag. Table 1 gives details of the animals used in each trial. For the duration of each triM, the animals were housed together, but individually tethered in stalls with solid partitions to prevent both selfgrooming and grooming by other animals. The environmental conditions for all stalls within each trial were identical. The animals were fed according to local practice, the same for each animal within a trial. Water was available at all times from automatic drinkers or buckets. 2.2. Nematode infections and allocation
For the four trims conducted in Hertfordshire, the larvae administered were all local field isolates that had been cultured for at least 2 yrs (except for D. viuiparus which was obtained directly from Intervet UK Ltd.). All the species were passaged at least annually. In the Bavarian trials the larvae used had been locally collected and propagated, with the exception of D. viviparus (obtained from Leipzig University) and H. contortus (from Hoechst, Frankfurt/Main). The animals were infected with third-stage larvae according to the schedule outlined in Table 2 in accordance with W A A V P guidelines. The doses of larvae were administered orally over the back of the tongue using a plastic test tube. The tube was then rinsed with distilled water, and the rinsate administered in the same manner. The allocation details for each trial are shown in Table 3. On the day of allocation, the animals were weighed and ranked (within sex groups in the case of trial 5) byl descending bodyweight. Within rank, sequential replicates of animals were formed. Within each replicate, animals were assigned to a treatment group by lottery. The stallsl were divided into blocks the same size as the number of animals in a replicate. Eachl animal in a replicate was then assigned to a stall within a stall block by lottery. For trial 5, one treated group (Group 2) of animals had the hair over the application site clippedl to a length of approximately 1 cm before treatment on Day 0. This area extended from the withers to rump and down each side to a line approximately three quarters of thei
20000(-6) 10000(-6) 15000(-6) 10000 ( - 28) 10000(-28)
15000(-28)
1 2 3 4 5
6
1000(-28)
1000(-6) 1000(-6) 1000(-6) 1000 ( - 28) 1000(-28)
D.v.
7000(-28)
5000(-6) 5000(-6) 5000(-6) 5000 ( - 28) -
H.c. 1000(-17) 1000(-16) 2000(-17) 1500(-38)
3333(-28)
O.r.
10000(-6) 3000(-6) 663(-6) 5000 ( - 28)
N.h.
10000(-28) 11383(-28)
20000(-6) 10000(-6) 15000(-6) 10000 ( - 28)
0.o.
10000(-28) 15000(-28)
20000(-6) 10000(-6) 15000(-6) 10000 ( - 28)
T.a.
-
10000(-6) 15000(-6) -
T.c.
colubriformis.
C. spp., Mixed Cooperia species; D.v.. D. viviparus; H.c., H. contortus; N.h., N. helvetianus; O.r., O. radiatum; 0.o., O. ostertagi; T.a., T. axei; T.c., T.
Day of infection relative to treatment day in parentheses.
C. spp.
Trial number
Table 2 Number of larvae administered to the animals in each trial
J t,4 oo
123
S.R. Pitt et al. / Veterina~ Parasitology 73 (1997) 119-128
Table 3 Allocation details Trial Day of Animals Number of Treatment number allocation relative on trial replicates group to treatment day (Day 0) 1
1
12
6
2
- 1
12
6
3
0
12
6
4
- I
12
6
5
- 8
24
8
6
0
12
6
Treatmentgroup (all eprinomectin dose rates were 500 mcg/kg)
2×6 animals 1: untreated control 2: eprinomectin 2 × 6 animals 1: control vehicle 2: eprinomectin 2 × 6 animals 1: control vehicle 2: eprinomectin 2 X 6 animals 1: untreated control 2: eprinomectin 3 × 8 animals 1: control vehicle 2: eprinomectin (clipped) 3: eprinomectin (long hair) 2 × 6 animals 1: control vehicle 2: eprinomectin
Day of necropsy relative to day of treatment (Day 0) 14 21 21 14 14 (reps 1-4) 15 (reps 5-8) 14
way down the flank. The average hair length along the application site for Groups 1 (untreated control) and 3 (treated unclipped) was 4.9 cm and 4.7 cm, respectively (group arithmetic means). 2.3. Treatments
Animals within a replicate were either treated with eprinomectin at 500 m c g / k g bodyweight in a topical formulation, or with the vehicle solution (except for trials 1 and 4, where no vehicle solution was administered to the control animals). Details of the treatment groups and dose rates for each group are shown in Table 3. At the time of treatment on Day 0, the animals in trials 4, 5 and 6 were expected to be harbouring adult stages of the nematode species with which they had previously been infected (this expectation being based on the time elapsed between infection with the parasite larvae and the day of treatment; see Table 2). Those in trials 1, 2 and 3 were expected to be harbouring immature stages (fourth larval stage larvae) of the nematodes at the time of treatment. All dose volumes were calculated using bodyweights measured on Day-1 or on the day of treatment. Each solution (vehicle or eprinomectin pour-on) was applied from a height of 5 - 1 0 cm in a line from the tailhead to the withers. A n assessment w a s made o f any loss of formulation due to dripping. Before treatment, the application s i t e was examined visually and by palpation for any skin or haircoat abnormality. All t h e animals were examined for any adverse reaction to the applied treatments intermittently for 4 h after treatment, and then daily until the end of the trials. The application sites were examined visually and by palpation for any significant changes to the hair a n d / o r skin at 7-day intervals. 2.4. Necropsy procedures
The day of necropsy relative to the day of treatment (Day 0) for each trial is shown in Table 3. The animals were euthanized by captive-bolt stunning followed by exsanguina-
124
S.R. Pitt et al. / VeterinaryParasitology 73 (1997) 119-128
tion. For each animal, observations were made of the application site before and after reflection of the skin both visually and by palpation to assess any gross changes present. The abomasum (all trials), small intestine (all trials), large intestine (trials 1, 2, 3 and 6) and lungs with trachea attached (all trials) were removed separately from each animal. The contents of the abomasum and small intestine were individually washed out, collected and resuspended to a fixed volume, and 5 × 1% aliquots were retained for counting. The contents of the large intestine were washed out over a 300-/zm screen and a 100% aliquot (or two 10% aliquots for trials 6 and 3) retained for counting. In addition, the washed abomasa were separately digested in 1% hydrochloric acid and pepsin at room temperature overnight (or tap water at 37°C for 20 h in trials 6 and 3), and any additional nematodes were recovered and counted from a retained 100% aliquot (or two 10% aliquots in trials 6 and 3). Adult lungworms were washed out of the lungs and a 100% aliquot was counted using a modified version of the Inderbitzen lung perfusion technique (Oakley, 1980). All aliquots were counted entirely. The numbers and species of nematode parasitic worms counted in each organ were recorded. 2.5. Statistical methods In all trials, the parasite counts for each animal were calculated by multiplying the number of nematodes actually counted in each location by the aliquot factor. The counts were then transformed to the natural logarithm of (count + 1) for analysis and calculation of geometric means. Treatment comparisons were made for each parasite using the modified Friedman procedure, a nonparametric test for randomized block designs, which leads to an exact probability of finding a statistic as extreme as, or more extreme than, the one observed. The trial was used as the blocking factor; parasite counts were ranked within each trial. The probability value for the test statistic was calculated based on a X 2 distribution with one degree of freedom. For trial 5, independent treatment comparisons were made comparing treated to control, and treated clipped vs. treated nonclipped.
3. Results The results for the three trials conducted to determine efficacy against immature nematode infections are shown in Table 4 and the results for the three trials conducted to determine efficacy against adult nematode infections in Table 5. When compared with the untreated control groups, efficacy against the adult and immature nematode infections was > 99% for all nematode species investigated (Cooperia spp., D. viviparus, H. contortus, Nematodirus helvetianus, Oesophagostomum radiatum, Ostertagia ostertagi, Trichostrongylus axei and Trichostrongylus colubriformis [immature infections only]). These reductions were significant ( P < 0.001) for all species in all the trials. There was no significant difference ( P > 0.1) in efficacy between groups for animals with clipped hair (1 cm length) at the application site, and those with long hair (4.7 cm length) in trial 5. There was no loss of formulation due to dripping in any of the trials. Subsequent observations by visual examination and palpation of the application sites during the trial
100 < 0.001
Pooled statistics % efficacy P control v eprinomectinb 100 < 0.001
0.0
17.6 (3-34)
0.0
37.0 (17-99)
0.0
85.5 (6-237)
D.v.
> 99 < 0.001
0.0
17,9 (0-295)
0.0
100.0 (0-1560)
1.8 (0-20)
2142.8 (1420-3160)
H.c.
100 < 0.001
0.0
15.5 (0-530)
0.0
237.0 (0-1260)
0.0
3033,1 (2000-4700)
N.h.
All induced infections were expected to be fourth larval stage at the time of treatment. Ranges in parentheses. aGeometric means based on transformation to In (count + l). bProbability values from modified Friedman test. c This was a combination infection of C, oncophora, C. punctata and C. surnabada.
0.0
8741.6 c (5880-11240)
0.0
2854.0 (20-10760)
0.0
9979.8 (7020-15200)
C.o.
500 m c g / k g eprinomectin~
3 Control ~
500 m c g / k g eprinomectin a
2 Control a
500 m c g / k g eprinomectina
1 Control a
100 < 0.001
0.0
944.0 (510-1540)
0.0
140.0 (84-182)
0.0
610.1 (315-841)
O.r.
> 99 < 0.001
1.2 (0-20)
6116.8 (3585-8715)
< 1.0 (0-60)
7945.0 (6580-10120)
55.4 (0-300)
12640.8 (6480-22540)
0.o.
> 99 < 0.001
0.0
4362.6 (2920-6585)
0.0
2297.0 (1580-3580)
3.6 (0-20)
5016.9 (2720-8380)
T.a.
Table 4 Group nematode worm burdens at necropsy for animals expected to be harbouring immature (fourth larval stage) larvae at the time of treatment
100 < 0.001
0.0
3176.3 (850-5900)
0.0
491.0 (40-1640)
-
-
T.c.
/
.~
2"
> 99 < 0.001
0.9 (0-50)
6259.3 d (5330-7160)
13.9/21.9 ¢ (0-760/0-540) ~
100 < 0.001
0.0
4.9 (0-104)
0.0/0.0 ~
65.6 (5-218)
0.0
41.9 (16-119)
D.v.
100 < 0.001
0.0
22.1 (0-430)
0.0
917.7 (540-1840)
H.c.
100 < 0.001
0.0
42.1 (0-1610)
0
1130.6 (120-2260)
N.h.
100 < 0.001
0.0
421.9 (360-490)
O.r.
> 99 < 0.001
0.0
3753.8 (745-6600)
(o-o/o-2o) c
O.O/O.Y
4411.0 (2840-6180)
0.0
4412.4 (3520-5500)
O.o.
> 99 < 0.001
0.0
4998.6 (2780-6840)
0.0/0.0 c
3489.2 (2620-4880)
0.7 (0-20)
2424.0 (1600-3260)
T.a.
~Clipped hair at application site/normal hair length. There was no significant difference in adult worm counts at necropsy between the two groups ( P > 0.1). dThis was a combination infection of C. oncophora, C. punctata and C. surnabada.
All induced infections were expected to be adult at the time of treatment. Ranges in parentheses. aGeometric means based on transformation to In (count + 1). bprobability values from modified Friedman test.
Pooled statistics % efficacy P control v eprinomectin b
500 m c g / k g eprinomectin ~
6 ControP
500 m c g / k g eprinomectin ~
4869.0 (2720-6200)
12.0 (0-260)
500 m c g / k g eprinomectin~
5 Control a
4910.6 (3860-6380)
C.o.
ControP
4
Table 5 Group nematode worm burdens at necropsy for animals expected to be harbouring adults at the time of treatment
t.o
I
e~
S.R. Pitt et al. / Veterinary Parasitology 73 (1997) 119-128
127
and at necropsy showed that the formulation was well tolerated by all the animals with no adverse reactions to treatment being seen either ante- or post-mortem.
4. Discussion Formulations of earlier generation avermectins and milbemycins had less than complete efficacy against certain nematode species at this use level with Nematodirus acting as a dose-limiting parasite for abamectin and ivermectin (Kaplan et al., 1994 and Benz and Cox, 1989) and Cooperia being a dose-limiting parasite for moxidectin and doramectin (Ranjan et al., 1992; Jones et al., 1993; Taylor et al., 1993; and Yazwinski et al., 1994). Demonstration of consistent efficacy of > 99% against adult and immature parasites of these species, as well as against the other important nematode parasites of the gastrointestinal tract and lungs of cattle, confirms that eprinomectin applied in the topical formulation at 500 m c g / k g does not have a similar limiting parasite among the economically important cattle parasites at this dose level. Further, the excretion, metabolism and residue profile of this product is such that it is not only safe for use in all classes and ages of cattle, but the product may even be used on lactating dairy cattle without any requirement for milk withholding (Shoop et al., 1996a,b). These factors combined with the virtually complete efficacy of eprinomectin against all species of adult and immature nematodes of cattle tested in these studies reinforces the contention that this compound is the most efficacious in the class (Shoop et al., 1996a,b).
References Baggott, D.G., Ross, D.B., Preston, J.M., Gross, S.J., 1994. Nematode burdens and productivity of grazing cattle treated with a prototype sustained-releasebolus containingivermectin.Vet. Rec. 135, 503-506. Benz, G.W., Cox, J.L., 1989. Use of abamectinin cattle. In: Campbell,W.C. (Ed.), Ivermectinand Abamectin. Springer, New York, pp. 230-233. Benz, G.W., Roncalli, R.A., Gross, S.J., 1989. Use of ivermectin in cattle, sheep, goats and swine. In: Campbell, W,C. (Ed.), Ivermectinand Abamectin.Springer, New York pp. 215-229. Couvillion,C.E. and Guerino, F., 1992. Dose Titration of Moxidectin Against Naturally Acquired Nematode Infections in Cattle. Proc. 37th Annu. Meeting Am. Assoc. Vet. Parasitol., WA, 32. Eagleson, J.S., Cramer, L.G. and Farrington,D.O., 1995. Efficacy of the IvermectinSustainedRelease Bolus in AnimalsWeighingGreater Than 300 kg at the Time of Treatment. 15th Int. Conf. of the World Assoc. for the Advancementof Vet. Parasitol. Goudie, A.C., Evans, N.A., Gration, K.A.F., Bishop, B.F., Gibson, S.P., Holdom, K.S., Kaye, B., Wicks, S.R.. Lewis, D., Weatherley, A.J., 1993. Doramectin--a potent novel endectocide. Vet. Parasitol. 49, 5-15. Heinze-Mutz, E.M., Pitt, S.R., Bairden, K., Baggott, D.G., Armour, J., Barth, D., Cramer, L.G., 1993. Efficacy of abamectin against nematodes in cattle. Vet. Rec. 132, 35-37. Jones, R.M., Logan, N.B., Weatherley,A.J., Little, A.S., Smothers, C.D., 1993, Activityof doramectinagainst nematode parasites of cattle. Vet. Parasitol. 49, 27-37. Kaplan, R.M., Courtney, C.H., Kunkle, W.E., Zeng, Q.Y., Jernigan, A.D., Eagleson, J.S., 1994. Efficacy of injectableabamectinagainst gastrointestinaltract nematodes and lungwormsof cattle. Am. J. Vet. Res. 55, 353-357. Mrozik, H., Eskola, P., Byron, H., Arison, B.O.. Linn, A.L., Matzuk, A., Shih, T.L., Tischler, M., Waksmunski, F.S., Wyvratt, M.J., Blizzard, G.M.M., Fisher, M.H., Shoop, W.L., Egerton, J.R., 1995.
128
S.R. Pitt et al. / Veterinary Parasitology 73 (1997) 119-128
4'-deoxy-4'-aminoavermectins with potent broad spectrum antiparasitic activities. Bioorg. Med. Chem. Lett. 5 (20), 2435-2440. Oakley, G.A., 1980. The recovery of Dic~ocaulus viviparus from bovine lungs by lung perfusion: a modification of Inderbitzens method. Res. Vet. Sci. 29, 395. Ranjan, S., Trudeau, R.K., Prichard, R.K., Von Kutzleben, R., Carrier, D., 1992. Efficacy of moxidectin against naturally acquired nematode infections in cattle. Vet. Parasitol. 41,227-231. Ranjan, S., Trudeau, R.K., Prichard, R.K., Von Kutzleben, R., 1994. Efficacy of Moxidectin pour-on against natural nematode infections in cattle. Proc. 39th Annu. Meeting Am. Assoc. Vet. Parasitol., CA A7 20-20. Shoop, W.L., Mrozik, H., Fisher, M.H., 1995. Structure and activity of avermectins and milbemycins in animal health. Vet. Parasitol. 59, 139-156. Shoop, W.L., Egerton, R., Eary, C.H., Haines, H.W., Michael, B.F., Mrozik, H., Eskola, P., Fisher, M.H., Slayton, L., Ostlind, D.A., Skelly, B.J., Fulton, R.K., Barth, D., Costa, S., Gregory, L.M., Campbell, W.C., Seward, R.L., Turner, M.J., 1996a. Eprinomectin: a novel avermectin for use as a topical endectocide for cattle. Int. J. Parasitol. (in press). Shoop, W.L., DeMontigny, P., Fink, D.W., Williams, J.B., Egerton, J.R., Mrozik, H., Fisher, M.H., Skelly, B.J., Turner, M.J., 1996b. Efficacy in sheep and pharmacokinetics in cattle that led to the selection of eprinomectin as a topical endectocide for cattle. Int. J. Parasitol. (in press). Soil, M.D., Carmichael, J.H., Harvey, R.G., 1988. Prophylactic efficacy of sustained-release ivermectin against induced nematode infestations in cattle. J. S. Afr. Vet. Assoc. 59, 9-11. Taylor, S.M., Kenny, J., Edgar, H., 1993. Comparison of the efficacy of injectable and topical moxidectin for the reduction of faecal egg counts of cattle. Vet. Rec, 133, 216-217. Williams, J.C., Barras, S.A., Wang, G.T., 1992. Efficacy of moxidectin against gastrointestinal nematodes of cattle. Vet. Rec. 131,345-347. Yazwinski, T.A., Featherstone, H., Tucker, C., 1994. Effectiveness of doramectin for treatment of experimentally induced gastrointestinal tract larval nematode infections in calves. Am. J. Vet. Res. 55, 820-821.