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Evaluation of the therapeutic and protective efficacy of doramectin against psoroptic scabies in cattle
veterinary parasitology ELSEVIER
Veterinary Parasitology 72 (1997) 79-89
Evaluation of the therapeutic and protective efficacy of dorarnectin against psoroptic scabies in cattle B.C. Clymer a, T.H. Janes b, M.E. McKenzie c,, a Agricultural Training and Technology, Amarillo, 79114 TX, USA CA VL, Amarillo, 79114 TX, USA ¢ Animal Health Group, Pfizer, 235 East 42nd Street, 3rd Floor, New York, 10017 NY, USA
Abstract Three studies were conducted to evaluate the therapeutic and protective efficacy of doramectin when given by injection at a dose of 200 / x g / k g against induced P s o r o p t e s ovis infestations of cattle. The first study investigated therapeutic efficacy. Mite infestations were established on 15 test animals held in stanchions by transfer of material from infested donor calves. Test animals were then allotted on the basis of mite counts to a treatment group (10 animals) which received doramectin and a control group (5 animals) which received saline. Skin scrapings were collected for mite counts on the day before treatment and on days 7, 14, 21 and 28 after treatment. Efficacy assessed on the basis of the proportion of animals cured by day 28 was 100%. The second study was designed to determine the duration of protective efficacy. Forty-eight scabies-free heifers were allotted to a treated group of 32 which received doramectin, and a control group of 16 which remained untreated. These treatment groups were each divided into eight subgroups. Commencing on treatment day and continuing at weekly intervals for 7 weeks, a subgroup of animals from each treatment was placed in stanchions and challenged by transfer of material from infested donor calves. Skin scrapings for mite counts were collected 7 and 14 days later. Infestations were successfully established on all untreated control calves. Doramectin prevented the establishment of infestation for three weeks and significantly ( P < 0.05) reduced infestation levels for an additional two weeks. The third study established the duration of residual protection conferred by doramectin and ivermectin under contact transmission. Ninety-six scabies-free heifers were divided into two equal treatment groups. Animals in one group received doramectin and animals in the other group received ivermectin at its recommended dose of 200 / x g / k g by subcutaneous injection. Each treatment group was then divided into eight subgroups of six animals. Commencing on treatment
* Corresponding author. Tel.: + 1-212-573-5438; fax: + 1-212-808-8968. 0304-4017/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. Pll S0304-40 17(97)00080-0
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day and continuing at weekly intervals for 7 weeks, a subgroup of animals from each treatment was exposed to purposely infested seeder animals for one week. Three animals from each treatment subgroup were then placed in individual stanchions in which grooming was prevented and the other three were placed together in a pen where normal grooming behavior was permitted. Skin scrapings for mite counts were collected at weekly intervals for up to 4 weeks. Doramectin provided complete protection against infestation for five weeks compared to four weeks for ivermectin. These periods were not influenced by grooming behavior. © 1997 Elsevier Science B.V. Keywords: Doramectin; Ivermectin; Cattle: Acarina; Psoroptes ovis; Protective efficacy
I. Introduction Psoroptic scabies of cattle is a widely-distributed disease problem in temperate regions of the world and can cause a significant economic loss to the cattle industry in those regions. The disease is particularly prevalent in southern Brazil and Argentina (Nunez and Moltedo, 1985) where it frequently takes a severe form and can be responsible for serious losses from mortality and impaired productivity. In other areas where the disease is recognized, including Europe and North America, it occurs sporadically in a less severe form, but if left untreated can lead to significant economic loss particularly in intensively-managed beef animals held in confinement. In the USA, outbreaks of scabies mites are most commonly associated with feedlots and are, therefore, most frequently encountered in the beef-producing states in the midwest and southwest regions of the country. Control measures for psoroptic scabies usually involve mass treatment with acaricidal agents when cattle are being transported from one location to another, especially if originating in an area where the parasite disease is endemic. In intensive beef production systems such as feedlots, treatment is given at arrival prior to placement in feedlot pens. This control strategy is most effective where treatment has a reliable curative effect and provides some residual protection against reinfestation during the finishing period. The introduction of the avermectin class of parenterally-active, broad spectrum antiparasitics has provided the producer with agents that possess a unique combination of convenience-of-use and effectiveness in the treatment and control of psoroptic scabies. The therapeutic efficacy of ivermectin (Ivomec TM MSD Ag Vet), the first commercially-available avermectin, and doramectin (Dectomax TM Pfizer Inc.), a more recent introduction, have been documented by Benz et al. (1989) and Logan et al. (1993), respectively. Published information on the protective efficacy of these avermectins are sparse. The residual protection of injectable ivermectin in experimental challenge models was investigated by Meleney et al. (1982) but comparable data on doramectin are not available. The series of studies reported in this paper were conducted in part to evaluate the protective efficacy of doramectin but also to profile its overall activity against a single laboratory-adapted strain of Psoroptes ovis in the context of assessing the drug's potential for the treatment and control of psoroptic scabies in US feedlots.
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2. Materials and methods Three studies were conducted. The first was designed to evaluate the therapeutic efficacy of doramectin, the second was designed to evaluate the protective efficacy of doramectin and the third was designed to establish the duration of protection conferred by doramectin and ivermectin under conditions of contact transmission. 2.1. Animals
Scabies-free mixed-breed beef heifer calves were used in these studies. At selection, test animals were between 6 and 18 months of age, and their body weights ranged from 134-287 kg. The animals were held either individually in stanchions or in groups in pens during study periods. 2.2. Treatments
Doramectin (Dectomax TM Pfizer Inc.) was administered as a 1% injectable solution at a dose of 200 p g / k g in each of the three studies. In the first study, control animals were treated with physiological saline solution at a dose of 1 m l / 5 0 kg. In the second study, the control animals received no injections. Ivermectin (Ivomec TM MSD Ag Vet) was administered in the commercially available formulation at its recommended dose of 200 / z g / k g in the third study. All treatments were given by subcutaneous injection into the midline of the neck. 2.3. Challenge infestation procedure
The source of psoroptic mites used for inducing infestations was common to all three studies and was a laboratory-adapted strain that has been maintained at Clymer Research and Consulting (CRC) for many years. The mites are raised year round on bovine hosts. In the first two studies, challenge infestation was by means of direct transfer of material from infested donor animals. A 2.5 cm ( ~ 1 in.) square of hair and underlying skin debris were removed by scraping from an active lesion. The collected material was placed on the withers of the candidate animal and held in place by drawing up approximately 2.5 cm of hair up and around the deposition site and knotting with a rubber band. This procedure was repeated two to five times on each animal. In the third study, seeder animals were experimentally infested in the same way. When the seeder animals had reached an infestation/lesion severity that involved at least 60% of the body surface of each calf and then following confirmation of active infestations, these animals were used to infest the test animals. Contact between the seeder animals and the treated principal animals was ensured since each contact pen had only one feed bunk.
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2.4. Experimental design 2.4.1. Study 1 Fifteen calves were selected for test, ranked according to adult mite counts and randomly assigned to two groups; one of ten animals and the other of five animals. On the following day (day 0), each animal in the larger group received its dose of doramectin and each animal in the other group received saline. Calves were kept in individual steel stanchions on concrete floors for the duration of the study. Two skin scrapings of approximately 1 square inch each for mite counts were taken from each animal on the day prior to treatment and 7, 14, 21 and 28 days after treatment. Scrapings were made from suspect areas. If no suspect areas were available, random scrapings were taken from the withers area. 2.4.2. Stud), 2 Forty-eight scab-free animals were selected and allocated at random on the basis of body weight to either a treated group of 32 animals or a control group of 16 animals. Each animal in the treated group received doramectin on study day 0 while animals in the control group remained untreated. Animals in each of these groups were then allotted according to body weight into subgroups of four animals and two animals for the treated group and control group, respectively. Commencing on treatment day, one subgroup of animals from each treatment group was placed in stanchions and challenged with P. ovis from the donor calves according to the procedure already described. Skin scrapings were collected for mite counts 7 days and 14 days later. The whole procedure was repeated with new subgroups at weekly intervals until the last subgroup was challenged seven weeks after the doramectin treatment. 2.4.3. Study 3 A total of 156 animals were used in this study; 104 of these were designated as test animals for assignment to treatment groups and 52 were designated as seeder animals to provide challenge infestation. The seeder animals were experimentally infested in subgroups of six animals each week as described earlier. The 104 test animals were randomly allocated on the basis of body weight to one of two equal groups. Animals in one group were treated with doramectin and animals in the other group received ivermectin. Each treatment group was then divided into eight subgroups of six animals each with the remaining four animals forming a replacement pool for any test animal that dropped out of the study prior to its challenge infestation. Commencing on treatment day (day 0) and repeating at weekly intervals for seven weeks, a new subgroup of animals of each treatment group and a new subgroup of infested seeder animals were divided between three replicate contact exposure pens such that each pen contained two seeder animals, two doramectin-treated animals and two ivermectin-treated animals. After an exposure period of seven days, one of each pair of treated animals from each exposure pen was placed in individual stanchions and the other placed by treatment group in an observation pen (three animals per pen). This was done to determine the influence grooming might have on the establishment of mite infestations in the respective treatment groups. Skin scrapings for mite counts were
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collected 7 and 14 days later in the case of stanchioned animals and 7, 14, 21 and 28 days later in the case of penned animals. 2.5. Mite sampling and counting procedures
Duplicate epidermal scrapings of one square inch area were collected from active lesions. In the absence of active lesions, random scrapings of the withers area were taken. Mites were recovered from each sample using a vacuum assisted separation technique and counted by life cycle stage using a dissecting microscope. Live mites were differentiated as larvae or adults, nymphal stages being included in the adult count. 2.6. Statistical analysis
A log transformation (ln(x + 1)) was applied to mite count data prior to any analysis because of the non-normality of the distribution of mite burdens. Analysis of variance (general linear model; GLM) was conducted on log mite counts. Geometric mean counts were estimated for treatment groups from the log counts. In each analysis, a protected least significant difference (LSD) test was done. If the model was not significant then no pair-wise comparisons were done. All analyses were completed using the SAS ® (SAS Institute, Cary, NC, USA). The 5% level of significance ( P < 0.05) was used in each study. In the first (therapy) study, counts of each stage for each duplicate sample were summed to produce a total live mite count for each sampling day. The transformed data were then analysed to determine if the mean log counts differed significantly between treatments. Treatment differences were then compared using a split plot repeated measures test. The cure rate (percentage of animals with no detectable mites on day 28) was also calculated for each treatment group and analysed using Fisher Exact Test (2-tail). In the second study, the number of mites of each stage was summed for the duplicate samples and analysis of variance carried out separately on each stage. The model partitioned the total variance into that due to regimen (the combination of treatment and challenge subgroup), animal within regimen, week after infestation that mites were counted and the interaction of regimen and week counted. The analysis was done in order to account for simple effects of regimen X week counted and the non-homogeneity of variance that might be observed (Kirk, 1982). If the interaction of the regimen and week counted was significant then a separate analysis was performed for each week counted. In the third study, counts of each mite stage for each duplicate sample for each sampling day were summed for each animal within each subgroup. For analysis, the model contained only a term for regimen, which was the treatment and the exposure subgroup combined. Treatment subgroups housed in stanchions and treatment subgroups housed in pens were analysed separately. Significance was determined if estimates of least square means were different from zero.
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3. Results
3.1. Study 1 Mite count data are presented in Table 1. The pretreatment (day - 1 ) mite counts confirmed the presence of active infestation in all animals prior to treatment. Mites were found in variable numbers in every subsequent sample taken from saline-treated controls showing that infestation persisted throughout the study period. In contrast, no mites were found beyond post-treatment day 7 in samples from doramectin-treated animals indicating complete elimination of infestation and a 100% cure rate.
3.2. Study 2 Mite count data are summarized in Table 2. There was no significant interaction between treatment subgroups and week counted for larval and adult mites, therefore, the geometric mean values for larval and adult mites are the mean of the 7- and 14-day collections combined. There was a significant interaction between treatment subgroups and week counted for mite eggs, and therefore, separate GLM analyses were performed for the 7-day and the 14-day scrapings. For simplicity, only one set of egg data has been
Table 1 Study 1: Counts of P. ovis and percentage of calves with no mites detected on day 28 Treatment
Animal number
Total mites counted in duplicate samples Day-1
Saline
472 115 483 17 485 37 486 10 487 241 Geometric mean 45
Doramectin 469 73 473 4 475 6 476 15 477 132 478 32 480 5 492 451 493 3 496 8 Geometric mean 20 p a value 0.1126
Day7 40 12 11 9 18 16 0 0 0 0 0 2 0 0 0 0 < 1 0.0001
Day 14
Day 21
Day 28
86 32 23 35 25 36
161 28 4 6 22 20
162 b 5 2 6 10
0 0 0 0 0 0 0 0 0 0 0 0.0001
0 0 0 0 0 0 0 0 0 0 0 0.0001
0 0 0 0 0 0 0 0 0 0 0 0.0001
Percentage of animals with no mites detected on day 28 0
100
0.001
a Significance level of testing the null hypothesis (H 0) (control group = treated group) on log (mite count + 1) or percentage of calves with no detectable mites on day 28. b Calf died on day 26.
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Table 2 Study 2: Geometric mean counts of P. ovis eggs, larvae and nymphs+adults on samples collected 7 and 14 days post-challenge Treatment
None Doramectin None Doramectin None Doramectin None Doramectin None Doramectin None Doramectin None Doramectin None Doramectin
Day of Number challenge of animals infestation 0 2 4 7 2 4 13 2 4 21 2 4 28 2 4 35 2 4 42 2 4 49 2 4
Geometric mean counts a Eggs day 14 Larvae day 7+day 14 Nymphs+adults day 7+day 14 19.4 0b 10.6 0b 12.9 0b 7.1 0b 38.5 1.4 b 17.7 7.6 113.7 34.4 119.9 33.9
28.8 0b 14.2 0b 12.3 < 0.1 b 15.7 0b 28.5 2.2 b 10.9 3.4 b 26.2 21.6 32.9 20.2
48.7 < 0.1 b 26.7 < 0.1 b 21.7 0b 23.9 0.5 b 29.1 2.5 b 16.5 4.7 b 40.7 35.8 43.2 39.1
a For egg data, there was a significant interaction between day of scraping and regimen, day 14 results are presented since these were, overall, the highest counts. For larvae and nymphs +adults, there was no significant difference between the day 7 and day 14 counts so these values were combined. b Means within each pair significantly different from each other (P < 0.05).
presented in T a b l e 2, and the 14-day data was c h o s e n since, in general, e g g s w e r e present in greater n u m b e r s at the later t i m e point than the earlier t i m e point. All d e v e l o p m e n t a l stages o f the m i t e life c y c l e w e r e r e c o v e r e d in substantial n u m b e r s f r o m all subgroups o f n o n - m e d i c a t e d calves indicating successful e s t a b l i s h m e n t o f a p r o g r e s s i v e infestation at e a c h c h a l l e n g e point. Significant infestations did not b e g i n to establish in d o r a m e c t i n - t r e a t e d animals until 28 days after treatment and m i t e burdens did not reach statistical e q u i v a l e n c e ( P > 0.05) to controls until 42 days after treatment.
3.3. Study 3
G e o m e t r i c m e a n m i t e counts, c o m b i n e d f r o m each post-infestation day, for treatment subgroups are s h o w n for stanchioned animals in T a b l e 3 and for p e n n e d animals in T a b l e 4. M i t e s did not establish on i v e r m e c t i n - t r e a t e d animals that w e r e initially e x p o s e d to infestation on days 0, 7, 14, 21 or 28 days post-treatment. Significant ( P < 0.05) n u m b e r s o f adult mites (and eggs) were, h o w e v e r , r e c o v e r e d f r o m the ivermectin-treated s u b g r o u p o f animals e x p o s e d 35 days after treatment w h e t h e r these animals w e r e subsequently h e l d in stanchions or pens. A c t i v e mites w e r e not found on doramectintreated animals until 42 days after treatment w h e n significant ( P < 0.05) numbers o f adult mites (and e g g s ) w e r e r e c o v e r e d f r o m the subgroup o f animals w h i c h w e r e held in
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Table 3 Study 3: Geometric mean counts of P. orris eggs, larvae and n y m p h s + a d u l t s on samples collected from stanchioned animals 7 and 14 days after removal from exposure to challenge infestation Treatment
Doramectin Ivermectin Doramectin lvermectin Doramectin Ivermectin Doramectin Ivermectin Doramectin Ivermectin Doramectin lvermectin Doramectin Ivermectin Doramectin Ivermectin
Day of first exposure to challenge infestation 0 7 14 21 28 35 ~ 42 49
Number of of animals
Geometric mean counts (combined day 7 + 14) Eggs
Larvae
Nymphs + adults
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
(/ 0 0 0 0 0 0 0 0 (I 0 2.1 ~ 0.3 2. I a 7.1 '~ 1.0
0 0 0 0 0 0 0 0 0 0 0 0.4 0.3 2.3 " 3.3 ¢' 1.0
0 (/ 0 0 0 0 (/ 0 0 0 0 1.5 1.5 1.2 3.9 0.3
~ ~ a ~
Value is significantly greater than zero where P < 0.05. h Actual challenge infestation day was day 37 due to a blizzard.
Table 4 Study 3: Geometric mean counts of P. ol,is eggs, larvae and n y m p h s + a d u l t s on samples collected from penned animals 7, 14, 21 and 28 days after removal from exposure to challenge infestation Treatment
Doramectin Ivermectin Doramectin lvermectin Doramectin lvermectin Doramectin Ivermectin Doramectin Ivermectin Doramectin lvermectin Doramectin lvermectin Doramectin lvermectin
Day of challenge infestation 0 7 14 21 28 35 ~ 42 49
Number of animals
Geometric mean counts (combined days 7, 14, 21, 28) Eggs
Larvae
Nymphs + adults
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
0 0 0 0 0 0 0 0 0,3 0 0 17.2 a 0.3 3.5 ~ 0.4 1.5
0 0 0 0 0 0 0 0 0 0 0 5.8 a 0 2.9 " 0.3 1.1
0 0 0 0 0 0 0 0 0 0 0 8.4 a 0.4 2.0 0.3 0.6
a Value is significantly greater than zero where P < 0.05. b Actual challenge infestation day was day 37 due to a blizzard.
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stanchions (Table 3). However, burdens in the animals held in pens were not statistically greater than zero in the 42-day subgroup or any subsequent subgroup (Table 4).
4. Discussion Doramectin given by injection at its recommended dose of 200 ~ g / k g was completely effective in eliminating infestations of this laboratory-adapted strain of P. ovis (Table 1) mimicking results obtained with parasites from other sources reported by Logan et al. (1993). Avermectins exert their antiparasitic effect by producing a rapid non-spastic paralysis in target parasites which impairs their ability to survive on the host resulting in elimination and death (Hart, 1986; Harrow et al., 1991). In the case of P. ovis, it is postulated that foraging mites acquire their disabling dose of drug primarily by ingestion while feeding on tissue fluids from damaged skin. Therefore, in order to be effective at eliminating all stages of the mite life cycle including unhatched eggs and achieve this curative effect, the drug must persist at effective concentrations in tissue fluids for long enough to allow the larval stage to emerge from the most recently laid eggs and commence to feed. The life cycle of P. ovis is usually completed on cattle within 10-12 days although in the author's experience this can take as long as 17 days. The absence of live mites on treated animals from 14-28 days post-treatment indicates that doramectin is available to the mites at effective concentrations for the necessary duration, a conclusion supported by pharmacokinetic data showing that doramectin persists in plasma for 35 days (Toutain et al., 1997). As might be expected, this persistency of doramectin also prevents the establishment of reinfestation for a period of time following a single treatment. This property was demonstrated in the second study in which a single treatment of 200 /xg/kg provided complete protection against infestation for three weeks and partial protection for an additional two weeks (Table 2). The challenge infestation procedure used in this study which involved the direct transfer of mite-infested material to animals prevented from self grooming represents a severe test of the protective capacity of the drug. In the face of a more natural challenge, by exposure to infested animals, as adopted in the third study, the period of complete protection conferred by doramectin was extended to five weeks (Tables 3 and 4). This was compared with a protection period of four weeks conferred by ivermectin in this study. Meleney et al. (1982) estimated the duration of residual protection of ivermectin to be 21 days. However, in their study, they used direct challenge of stanchioned animals every 3 days as the infestation method. It is interesting to observe that both ivermectin and doramectin demonstrate a longer period of residual protection with natural challenge than with artificial challenge suggesting that the former is the better technique to use for predicting the protective efficacy of avermectins under field use. The third study was designed also to investigate the effects of grooming behavior on the observed efficacy of the test drugs. It is not unusual for grooming activities that occur within groups of animals penned together to elicit a self cure of scabies mite infestations. Comparison of the results from stanchioned animals (Table 3) where grooming was prevented and penned animals (Table 4) where normal grooming activi-
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ties were permitted, showed no difference in the period of complete protection conferred by either doramectin (5 weeks) or ivermectin (4 weeks). However, there was a difference between the two agents in respect of how soon, thereafter, significant mite infestations established. In the case of ivermectin, mite counts significantly greater than zero (complete protection) were observed one week later (at 5 weeks), whereas, doramectin prevented establishment of infestations significantly greater than zero through to the last observation point (at 7 weeks). The longer duration of residual protection provided by doramectin compared with ivermectin can be explained by differences in the plasma pharmacokinetic profiles of the two drugs. In a comparative study using commercial formulations, Toutain et al. (1997) found that for all the key parameters (area under the curve, elimination half life, absorption half life, and mean residence time), values for doramectin were all significantly ( P < 0.02) greater than those for ivermectin. Because of their ease-of-use and broad spectrum of activity that encompasses both arthropod and nematode parasites, avermectins have already gained wide acceptance for use in feedlot arrival programs for the control of parasitic diseases. Although reliable curative efficacy is critical, protective efficacy also plays an important role in the successful application of avermectin agents in the control of psoroptic scabies mites under normal field conditions. Usually in feedlots, cattle arrive from several sources at different times, and under these circumstances, treated animals may have contact with untreated animals. In these situations, the longer period of protection offered by doramectin provides the feedlot operator with additional insurance against disease outbreaks occurring later in the finishing period. It is concluded from this series of studies that doramectin at its recommended dose of 200 / z g / k g is highly effective in the treatment and control of psoroptic scabies mite infestations of cattle and its prolonged residual protection renders the drug particularly valuable in feedlot applications.
Acknowledgements These investigations were sponsored by Pfizer Inc., New York, NY, USA. The authors wish to thank Dr. G.M. Steele and Mr. D.J. Walstrom for their help in coordinating these studies and Mr. R.M. Jones for his help with the preparation of the manuscript.
References 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. Harrow, I.D., Gration, K.A.F., Evans, N.A., 1991. Neurobiologyof arthropod parasites. Parasitology 102, $59-$69. Hart, R.J., 1986. Mode of action of agents used against arthropod parasites. In: Campbell, W.C., Rew, R.S. (Eds.), Chemotherapyof Parasitic Diseases. Plenum, New York, pp. 585-601.
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Kirk, R.E., 1982. Experimental Design: Procedures for the Behavioral Sciences. Brooks/Cole Publishing, Belmont, 94002 CA, pp. 365-378. Logan, N.B., Weatherley, A.J., Phillips, F.E., Wilkins, C.P., Shanks, D.J., 1993. Spectrum of activity of doramectin against cattle mites and lice. Vet. Parasitol. 49, 67-73. Meleney, W.P., Wright, F.C., Guillot, F.S., 1982. Residual protection against cattle scabies offered by ivermectin. Am. J. Vet. Res. 43, 1767-1769. Nunez, J.L., Moltedo, H.L., 1985. Sarna Psoroptica en Ovinos y Bovinos. Editorial Hemisferio Sur S.A., Buenos Aires, Argentina. Toutain, P.L., Upson, D., Terhune, T., McKenzie, M.E., 1997. Comparative pharmacokinetics of doramectin and ivermectin in cattle. Vet. Parasitol.
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Evaluation of the therapeutic and protective efficacy of dorarnectin against psoroptic scabies in cattle B.C. Clymer a, T.H. Janes b, M.E. McKenzie c,, a Agricultural Training and Technology, Amarillo, 79114 TX, USA CA VL, Amarillo, 79114 TX, USA ¢ Animal Health Group, Pfizer, 235 East 42nd Street, 3rd Floor, New York, 10017 NY, USA
Abstract Three studies were conducted to evaluate the therapeutic and protective efficacy of doramectin when given by injection at a dose of 200 / x g / k g against induced P s o r o p t e s ovis infestations of cattle. The first study investigated therapeutic efficacy. Mite infestations were established on 15 test animals held in stanchions by transfer of material from infested donor calves. Test animals were then allotted on the basis of mite counts to a treatment group (10 animals) which received doramectin and a control group (5 animals) which received saline. Skin scrapings were collected for mite counts on the day before treatment and on days 7, 14, 21 and 28 after treatment. Efficacy assessed on the basis of the proportion of animals cured by day 28 was 100%. The second study was designed to determine the duration of protective efficacy. Forty-eight scabies-free heifers were allotted to a treated group of 32 which received doramectin, and a control group of 16 which remained untreated. These treatment groups were each divided into eight subgroups. Commencing on treatment day and continuing at weekly intervals for 7 weeks, a subgroup of animals from each treatment was placed in stanchions and challenged by transfer of material from infested donor calves. Skin scrapings for mite counts were collected 7 and 14 days later. Infestations were successfully established on all untreated control calves. Doramectin prevented the establishment of infestation for three weeks and significantly ( P < 0.05) reduced infestation levels for an additional two weeks. The third study established the duration of residual protection conferred by doramectin and ivermectin under contact transmission. Ninety-six scabies-free heifers were divided into two equal treatment groups. Animals in one group received doramectin and animals in the other group received ivermectin at its recommended dose of 200 / x g / k g by subcutaneous injection. Each treatment group was then divided into eight subgroups of six animals. Commencing on treatment
* Corresponding author. Tel.: + 1-212-573-5438; fax: + 1-212-808-8968. 0304-4017/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. Pll S0304-40 17(97)00080-0
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day and continuing at weekly intervals for 7 weeks, a subgroup of animals from each treatment was exposed to purposely infested seeder animals for one week. Three animals from each treatment subgroup were then placed in individual stanchions in which grooming was prevented and the other three were placed together in a pen where normal grooming behavior was permitted. Skin scrapings for mite counts were collected at weekly intervals for up to 4 weeks. Doramectin provided complete protection against infestation for five weeks compared to four weeks for ivermectin. These periods were not influenced by grooming behavior. © 1997 Elsevier Science B.V. Keywords: Doramectin; Ivermectin; Cattle: Acarina; Psoroptes ovis; Protective efficacy
I. Introduction Psoroptic scabies of cattle is a widely-distributed disease problem in temperate regions of the world and can cause a significant economic loss to the cattle industry in those regions. The disease is particularly prevalent in southern Brazil and Argentina (Nunez and Moltedo, 1985) where it frequently takes a severe form and can be responsible for serious losses from mortality and impaired productivity. In other areas where the disease is recognized, including Europe and North America, it occurs sporadically in a less severe form, but if left untreated can lead to significant economic loss particularly in intensively-managed beef animals held in confinement. In the USA, outbreaks of scabies mites are most commonly associated with feedlots and are, therefore, most frequently encountered in the beef-producing states in the midwest and southwest regions of the country. Control measures for psoroptic scabies usually involve mass treatment with acaricidal agents when cattle are being transported from one location to another, especially if originating in an area where the parasite disease is endemic. In intensive beef production systems such as feedlots, treatment is given at arrival prior to placement in feedlot pens. This control strategy is most effective where treatment has a reliable curative effect and provides some residual protection against reinfestation during the finishing period. The introduction of the avermectin class of parenterally-active, broad spectrum antiparasitics has provided the producer with agents that possess a unique combination of convenience-of-use and effectiveness in the treatment and control of psoroptic scabies. The therapeutic efficacy of ivermectin (Ivomec TM MSD Ag Vet), the first commercially-available avermectin, and doramectin (Dectomax TM Pfizer Inc.), a more recent introduction, have been documented by Benz et al. (1989) and Logan et al. (1993), respectively. Published information on the protective efficacy of these avermectins are sparse. The residual protection of injectable ivermectin in experimental challenge models was investigated by Meleney et al. (1982) but comparable data on doramectin are not available. The series of studies reported in this paper were conducted in part to evaluate the protective efficacy of doramectin but also to profile its overall activity against a single laboratory-adapted strain of Psoroptes ovis in the context of assessing the drug's potential for the treatment and control of psoroptic scabies in US feedlots.
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2. Materials and methods Three studies were conducted. The first was designed to evaluate the therapeutic efficacy of doramectin, the second was designed to evaluate the protective efficacy of doramectin and the third was designed to establish the duration of protection conferred by doramectin and ivermectin under conditions of contact transmission. 2.1. Animals
Scabies-free mixed-breed beef heifer calves were used in these studies. At selection, test animals were between 6 and 18 months of age, and their body weights ranged from 134-287 kg. The animals were held either individually in stanchions or in groups in pens during study periods. 2.2. Treatments
Doramectin (Dectomax TM Pfizer Inc.) was administered as a 1% injectable solution at a dose of 200 p g / k g in each of the three studies. In the first study, control animals were treated with physiological saline solution at a dose of 1 m l / 5 0 kg. In the second study, the control animals received no injections. Ivermectin (Ivomec TM MSD Ag Vet) was administered in the commercially available formulation at its recommended dose of 200 / z g / k g in the third study. All treatments were given by subcutaneous injection into the midline of the neck. 2.3. Challenge infestation procedure
The source of psoroptic mites used for inducing infestations was common to all three studies and was a laboratory-adapted strain that has been maintained at Clymer Research and Consulting (CRC) for many years. The mites are raised year round on bovine hosts. In the first two studies, challenge infestation was by means of direct transfer of material from infested donor animals. A 2.5 cm ( ~ 1 in.) square of hair and underlying skin debris were removed by scraping from an active lesion. The collected material was placed on the withers of the candidate animal and held in place by drawing up approximately 2.5 cm of hair up and around the deposition site and knotting with a rubber band. This procedure was repeated two to five times on each animal. In the third study, seeder animals were experimentally infested in the same way. When the seeder animals had reached an infestation/lesion severity that involved at least 60% of the body surface of each calf and then following confirmation of active infestations, these animals were used to infest the test animals. Contact between the seeder animals and the treated principal animals was ensured since each contact pen had only one feed bunk.
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2.4. Experimental design 2.4.1. Study 1 Fifteen calves were selected for test, ranked according to adult mite counts and randomly assigned to two groups; one of ten animals and the other of five animals. On the following day (day 0), each animal in the larger group received its dose of doramectin and each animal in the other group received saline. Calves were kept in individual steel stanchions on concrete floors for the duration of the study. Two skin scrapings of approximately 1 square inch each for mite counts were taken from each animal on the day prior to treatment and 7, 14, 21 and 28 days after treatment. Scrapings were made from suspect areas. If no suspect areas were available, random scrapings were taken from the withers area. 2.4.2. Stud), 2 Forty-eight scab-free animals were selected and allocated at random on the basis of body weight to either a treated group of 32 animals or a control group of 16 animals. Each animal in the treated group received doramectin on study day 0 while animals in the control group remained untreated. Animals in each of these groups were then allotted according to body weight into subgroups of four animals and two animals for the treated group and control group, respectively. Commencing on treatment day, one subgroup of animals from each treatment group was placed in stanchions and challenged with P. ovis from the donor calves according to the procedure already described. Skin scrapings were collected for mite counts 7 days and 14 days later. The whole procedure was repeated with new subgroups at weekly intervals until the last subgroup was challenged seven weeks after the doramectin treatment. 2.4.3. Study 3 A total of 156 animals were used in this study; 104 of these were designated as test animals for assignment to treatment groups and 52 were designated as seeder animals to provide challenge infestation. The seeder animals were experimentally infested in subgroups of six animals each week as described earlier. The 104 test animals were randomly allocated on the basis of body weight to one of two equal groups. Animals in one group were treated with doramectin and animals in the other group received ivermectin. Each treatment group was then divided into eight subgroups of six animals each with the remaining four animals forming a replacement pool for any test animal that dropped out of the study prior to its challenge infestation. Commencing on treatment day (day 0) and repeating at weekly intervals for seven weeks, a new subgroup of animals of each treatment group and a new subgroup of infested seeder animals were divided between three replicate contact exposure pens such that each pen contained two seeder animals, two doramectin-treated animals and two ivermectin-treated animals. After an exposure period of seven days, one of each pair of treated animals from each exposure pen was placed in individual stanchions and the other placed by treatment group in an observation pen (three animals per pen). This was done to determine the influence grooming might have on the establishment of mite infestations in the respective treatment groups. Skin scrapings for mite counts were
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collected 7 and 14 days later in the case of stanchioned animals and 7, 14, 21 and 28 days later in the case of penned animals. 2.5. Mite sampling and counting procedures
Duplicate epidermal scrapings of one square inch area were collected from active lesions. In the absence of active lesions, random scrapings of the withers area were taken. Mites were recovered from each sample using a vacuum assisted separation technique and counted by life cycle stage using a dissecting microscope. Live mites were differentiated as larvae or adults, nymphal stages being included in the adult count. 2.6. Statistical analysis
A log transformation (ln(x + 1)) was applied to mite count data prior to any analysis because of the non-normality of the distribution of mite burdens. Analysis of variance (general linear model; GLM) was conducted on log mite counts. Geometric mean counts were estimated for treatment groups from the log counts. In each analysis, a protected least significant difference (LSD) test was done. If the model was not significant then no pair-wise comparisons were done. All analyses were completed using the SAS ® (SAS Institute, Cary, NC, USA). The 5% level of significance ( P < 0.05) was used in each study. In the first (therapy) study, counts of each stage for each duplicate sample were summed to produce a total live mite count for each sampling day. The transformed data were then analysed to determine if the mean log counts differed significantly between treatments. Treatment differences were then compared using a split plot repeated measures test. The cure rate (percentage of animals with no detectable mites on day 28) was also calculated for each treatment group and analysed using Fisher Exact Test (2-tail). In the second study, the number of mites of each stage was summed for the duplicate samples and analysis of variance carried out separately on each stage. The model partitioned the total variance into that due to regimen (the combination of treatment and challenge subgroup), animal within regimen, week after infestation that mites were counted and the interaction of regimen and week counted. The analysis was done in order to account for simple effects of regimen X week counted and the non-homogeneity of variance that might be observed (Kirk, 1982). If the interaction of the regimen and week counted was significant then a separate analysis was performed for each week counted. In the third study, counts of each mite stage for each duplicate sample for each sampling day were summed for each animal within each subgroup. For analysis, the model contained only a term for regimen, which was the treatment and the exposure subgroup combined. Treatment subgroups housed in stanchions and treatment subgroups housed in pens were analysed separately. Significance was determined if estimates of least square means were different from zero.
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3. Results
3.1. Study 1 Mite count data are presented in Table 1. The pretreatment (day - 1 ) mite counts confirmed the presence of active infestation in all animals prior to treatment. Mites were found in variable numbers in every subsequent sample taken from saline-treated controls showing that infestation persisted throughout the study period. In contrast, no mites were found beyond post-treatment day 7 in samples from doramectin-treated animals indicating complete elimination of infestation and a 100% cure rate.
3.2. Study 2 Mite count data are summarized in Table 2. There was no significant interaction between treatment subgroups and week counted for larval and adult mites, therefore, the geometric mean values for larval and adult mites are the mean of the 7- and 14-day collections combined. There was a significant interaction between treatment subgroups and week counted for mite eggs, and therefore, separate GLM analyses were performed for the 7-day and the 14-day scrapings. For simplicity, only one set of egg data has been
Table 1 Study 1: Counts of P. ovis and percentage of calves with no mites detected on day 28 Treatment
Animal number
Total mites counted in duplicate samples Day-1
Saline
472 115 483 17 485 37 486 10 487 241 Geometric mean 45
Doramectin 469 73 473 4 475 6 476 15 477 132 478 32 480 5 492 451 493 3 496 8 Geometric mean 20 p a value 0.1126
Day7 40 12 11 9 18 16 0 0 0 0 0 2 0 0 0 0 < 1 0.0001
Day 14
Day 21
Day 28
86 32 23 35 25 36
161 28 4 6 22 20
162 b 5 2 6 10
0 0 0 0 0 0 0 0 0 0 0 0.0001
0 0 0 0 0 0 0 0 0 0 0 0.0001
0 0 0 0 0 0 0 0 0 0 0 0.0001
Percentage of animals with no mites detected on day 28 0
100
0.001
a Significance level of testing the null hypothesis (H 0) (control group = treated group) on log (mite count + 1) or percentage of calves with no detectable mites on day 28. b Calf died on day 26.
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Table 2 Study 2: Geometric mean counts of P. ovis eggs, larvae and nymphs+adults on samples collected 7 and 14 days post-challenge Treatment
None Doramectin None Doramectin None Doramectin None Doramectin None Doramectin None Doramectin None Doramectin None Doramectin
Day of Number challenge of animals infestation 0 2 4 7 2 4 13 2 4 21 2 4 28 2 4 35 2 4 42 2 4 49 2 4
Geometric mean counts a Eggs day 14 Larvae day 7+day 14 Nymphs+adults day 7+day 14 19.4 0b 10.6 0b 12.9 0b 7.1 0b 38.5 1.4 b 17.7 7.6 113.7 34.4 119.9 33.9
28.8 0b 14.2 0b 12.3 < 0.1 b 15.7 0b 28.5 2.2 b 10.9 3.4 b 26.2 21.6 32.9 20.2
48.7 < 0.1 b 26.7 < 0.1 b 21.7 0b 23.9 0.5 b 29.1 2.5 b 16.5 4.7 b 40.7 35.8 43.2 39.1
a For egg data, there was a significant interaction between day of scraping and regimen, day 14 results are presented since these were, overall, the highest counts. For larvae and nymphs +adults, there was no significant difference between the day 7 and day 14 counts so these values were combined. b Means within each pair significantly different from each other (P < 0.05).
presented in T a b l e 2, and the 14-day data was c h o s e n since, in general, e g g s w e r e present in greater n u m b e r s at the later t i m e point than the earlier t i m e point. All d e v e l o p m e n t a l stages o f the m i t e life c y c l e w e r e r e c o v e r e d in substantial n u m b e r s f r o m all subgroups o f n o n - m e d i c a t e d calves indicating successful e s t a b l i s h m e n t o f a p r o g r e s s i v e infestation at e a c h c h a l l e n g e point. Significant infestations did not b e g i n to establish in d o r a m e c t i n - t r e a t e d animals until 28 days after treatment and m i t e burdens did not reach statistical e q u i v a l e n c e ( P > 0.05) to controls until 42 days after treatment.
3.3. Study 3
G e o m e t r i c m e a n m i t e counts, c o m b i n e d f r o m each post-infestation day, for treatment subgroups are s h o w n for stanchioned animals in T a b l e 3 and for p e n n e d animals in T a b l e 4. M i t e s did not establish on i v e r m e c t i n - t r e a t e d animals that w e r e initially e x p o s e d to infestation on days 0, 7, 14, 21 or 28 days post-treatment. Significant ( P < 0.05) n u m b e r s o f adult mites (and eggs) were, h o w e v e r , r e c o v e r e d f r o m the ivermectin-treated s u b g r o u p o f animals e x p o s e d 35 days after treatment w h e t h e r these animals w e r e subsequently h e l d in stanchions or pens. A c t i v e mites w e r e not found on doramectintreated animals until 42 days after treatment w h e n significant ( P < 0.05) numbers o f adult mites (and e g g s ) w e r e r e c o v e r e d f r o m the subgroup o f animals w h i c h w e r e held in
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Table 3 Study 3: Geometric mean counts of P. orris eggs, larvae and n y m p h s + a d u l t s on samples collected from stanchioned animals 7 and 14 days after removal from exposure to challenge infestation Treatment
Doramectin Ivermectin Doramectin lvermectin Doramectin Ivermectin Doramectin Ivermectin Doramectin Ivermectin Doramectin lvermectin Doramectin Ivermectin Doramectin Ivermectin
Day of first exposure to challenge infestation 0 7 14 21 28 35 ~ 42 49
Number of of animals
Geometric mean counts (combined day 7 + 14) Eggs
Larvae
Nymphs + adults
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
(/ 0 0 0 0 0 0 0 0 (I 0 2.1 ~ 0.3 2. I a 7.1 '~ 1.0
0 0 0 0 0 0 0 0 0 0 0 0.4 0.3 2.3 " 3.3 ¢' 1.0
0 (/ 0 0 0 0 (/ 0 0 0 0 1.5 1.5 1.2 3.9 0.3
~ ~ a ~
Value is significantly greater than zero where P < 0.05. h Actual challenge infestation day was day 37 due to a blizzard.
Table 4 Study 3: Geometric mean counts of P. ol,is eggs, larvae and n y m p h s + a d u l t s on samples collected from penned animals 7, 14, 21 and 28 days after removal from exposure to challenge infestation Treatment
Doramectin Ivermectin Doramectin lvermectin Doramectin lvermectin Doramectin Ivermectin Doramectin Ivermectin Doramectin lvermectin Doramectin lvermectin Doramectin lvermectin
Day of challenge infestation 0 7 14 21 28 35 ~ 42 49
Number of animals
Geometric mean counts (combined days 7, 14, 21, 28) Eggs
Larvae
Nymphs + adults
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
0 0 0 0 0 0 0 0 0,3 0 0 17.2 a 0.3 3.5 ~ 0.4 1.5
0 0 0 0 0 0 0 0 0 0 0 5.8 a 0 2.9 " 0.3 1.1
0 0 0 0 0 0 0 0 0 0 0 8.4 a 0.4 2.0 0.3 0.6
a Value is significantly greater than zero where P < 0.05. b Actual challenge infestation day was day 37 due to a blizzard.
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stanchions (Table 3). However, burdens in the animals held in pens were not statistically greater than zero in the 42-day subgroup or any subsequent subgroup (Table 4).
4. Discussion Doramectin given by injection at its recommended dose of 200 ~ g / k g was completely effective in eliminating infestations of this laboratory-adapted strain of P. ovis (Table 1) mimicking results obtained with parasites from other sources reported by Logan et al. (1993). Avermectins exert their antiparasitic effect by producing a rapid non-spastic paralysis in target parasites which impairs their ability to survive on the host resulting in elimination and death (Hart, 1986; Harrow et al., 1991). In the case of P. ovis, it is postulated that foraging mites acquire their disabling dose of drug primarily by ingestion while feeding on tissue fluids from damaged skin. Therefore, in order to be effective at eliminating all stages of the mite life cycle including unhatched eggs and achieve this curative effect, the drug must persist at effective concentrations in tissue fluids for long enough to allow the larval stage to emerge from the most recently laid eggs and commence to feed. The life cycle of P. ovis is usually completed on cattle within 10-12 days although in the author's experience this can take as long as 17 days. The absence of live mites on treated animals from 14-28 days post-treatment indicates that doramectin is available to the mites at effective concentrations for the necessary duration, a conclusion supported by pharmacokinetic data showing that doramectin persists in plasma for 35 days (Toutain et al., 1997). As might be expected, this persistency of doramectin also prevents the establishment of reinfestation for a period of time following a single treatment. This property was demonstrated in the second study in which a single treatment of 200 /xg/kg provided complete protection against infestation for three weeks and partial protection for an additional two weeks (Table 2). The challenge infestation procedure used in this study which involved the direct transfer of mite-infested material to animals prevented from self grooming represents a severe test of the protective capacity of the drug. In the face of a more natural challenge, by exposure to infested animals, as adopted in the third study, the period of complete protection conferred by doramectin was extended to five weeks (Tables 3 and 4). This was compared with a protection period of four weeks conferred by ivermectin in this study. Meleney et al. (1982) estimated the duration of residual protection of ivermectin to be 21 days. However, in their study, they used direct challenge of stanchioned animals every 3 days as the infestation method. It is interesting to observe that both ivermectin and doramectin demonstrate a longer period of residual protection with natural challenge than with artificial challenge suggesting that the former is the better technique to use for predicting the protective efficacy of avermectins under field use. The third study was designed also to investigate the effects of grooming behavior on the observed efficacy of the test drugs. It is not unusual for grooming activities that occur within groups of animals penned together to elicit a self cure of scabies mite infestations. Comparison of the results from stanchioned animals (Table 3) where grooming was prevented and penned animals (Table 4) where normal grooming activi-
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ties were permitted, showed no difference in the period of complete protection conferred by either doramectin (5 weeks) or ivermectin (4 weeks). However, there was a difference between the two agents in respect of how soon, thereafter, significant mite infestations established. In the case of ivermectin, mite counts significantly greater than zero (complete protection) were observed one week later (at 5 weeks), whereas, doramectin prevented establishment of infestations significantly greater than zero through to the last observation point (at 7 weeks). The longer duration of residual protection provided by doramectin compared with ivermectin can be explained by differences in the plasma pharmacokinetic profiles of the two drugs. In a comparative study using commercial formulations, Toutain et al. (1997) found that for all the key parameters (area under the curve, elimination half life, absorption half life, and mean residence time), values for doramectin were all significantly ( P < 0.02) greater than those for ivermectin. Because of their ease-of-use and broad spectrum of activity that encompasses both arthropod and nematode parasites, avermectins have already gained wide acceptance for use in feedlot arrival programs for the control of parasitic diseases. Although reliable curative efficacy is critical, protective efficacy also plays an important role in the successful application of avermectin agents in the control of psoroptic scabies mites under normal field conditions. Usually in feedlots, cattle arrive from several sources at different times, and under these circumstances, treated animals may have contact with untreated animals. In these situations, the longer period of protection offered by doramectin provides the feedlot operator with additional insurance against disease outbreaks occurring later in the finishing period. It is concluded from this series of studies that doramectin at its recommended dose of 200 / z g / k g is highly effective in the treatment and control of psoroptic scabies mite infestations of cattle and its prolonged residual protection renders the drug particularly valuable in feedlot applications.
Acknowledgements These investigations were sponsored by Pfizer Inc., New York, NY, USA. The authors wish to thank Dr. G.M. Steele and Mr. D.J. Walstrom for their help in coordinating these studies and Mr. R.M. Jones for his help with the preparation of the manuscript.
References 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. Harrow, I.D., Gration, K.A.F., Evans, N.A., 1991. Neurobiologyof arthropod parasites. Parasitology 102, $59-$69. Hart, R.J., 1986. Mode of action of agents used against arthropod parasites. In: Campbell, W.C., Rew, R.S. (Eds.), Chemotherapyof Parasitic Diseases. Plenum, New York, pp. 585-601.
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Kirk, R.E., 1982. Experimental Design: Procedures for the Behavioral Sciences. Brooks/Cole Publishing, Belmont, 94002 CA, pp. 365-378. Logan, N.B., Weatherley, A.J., Phillips, F.E., Wilkins, C.P., Shanks, D.J., 1993. Spectrum of activity of doramectin against cattle mites and lice. Vet. Parasitol. 49, 67-73. Meleney, W.P., Wright, F.C., Guillot, F.S., 1982. Residual protection against cattle scabies offered by ivermectin. Am. J. Vet. Res. 43, 1767-1769. Nunez, J.L., Moltedo, H.L., 1985. Sarna Psoroptica en Ovinos y Bovinos. Editorial Hemisferio Sur S.A., Buenos Aires, Argentina. Toutain, P.L., Upson, D., Terhune, T., McKenzie, M.E., 1997. Comparative pharmacokinetics of doramectin and ivermectin in cattle. Vet. Parasitol.