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Comparative efficacy of moxidectin and ivermectin against hypobiotic and encysted cyathostomes and other equine parasites
veterinary parasitology ELSEVIER
Veterinary Parasitology 53 (1994) 83-90
Comparative efficacy of moxidectin and ivermectin against hypobiotic and encysted cyathostomes and other equine parasites L i h u a Xiao*, R.P. Herd, G.A. M a j e w s k i Department of Veterinary Preventive Medicine, The Ohio State University College of Veterinary Medicine, 1900 Coffey Road, Columbus, OH 43210, USA (Accepted 18 August 1993 )
Abstract Efficacies of moxidectin and ivermectin were compared in four groups of eight ponies with natural parasite infections: placebo (Control), oral moxidectin gel at 0.3 mg kg-1 of body weight (Mox 0.3), oral moxidectin gel at 0.4 mg kg-1 of body weight (Mox 0.4), and oral ivermectin paste at 0.2 mg kg- ~of body weight (Ivermectin). Fecal samples were taken 0 and 2 weeks after treatment. Animals were necropsied and worms were collected 2 weeks after treatment. Moxidectin and ivermectin showed similar efficacy (99%) against adult cyathostomes, Strongylus spp., Triodontophorus spp. and Habronema muscae. Both drugs were also more than 98% effective against luminal cyathostome and Oxyuris equi fourth stage larvae (L4). Neither drug was effective (0-10.1%) against hypobiotic early third stage cyathostome larvae (EL3). Moxidectin was moderately effective (62.6-79.1%) in removing encysted eyathostome late third stage larvae (LL3) and L4, whereas ivermectin was ineffective (0%) against these stages. By contrast, ivermectin was 95.4% effective against Gasterophilus spp. third instar stage, whereas moxidectin was only 0-20.4% effective. Key words: Cyathostomidae; Horse; Control methods-Nematoda; Moxidectin; Iverrnectin; Hypobiosis
1. Introduction H y p o b i o t i c early third stage larvae (EL3) a n d encysted c y a t h o s t o m e late third stage (LL3) a n d fourth stage larvae (L4) present a m a j o r obstacle for the control *Corresponding author at: Immunology Branch, Mail Stop F-12, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA. 0304-4017/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved SSDI 0304-4017 ( 93 ) 00589-Q
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of equine parasites. Benzimidazole anthelmintics (fenbendazole and oxfendazole) were reportedly effective against encysted late third stage and fourth stage larvae ofcyathostomes (reviewed by Reinemeyer, 1986). However, the accuracy of the technique used to quantify numbers of tissue larvae was questioned (Reinemeyer and Herd, 1986). Furthermore, increasing benzimidazole resistance is likely to have rendered these anthelmintics ineffective on farms where they have been used intensively. Thus, new strategies to control hypobiotic and encysted cyathostome larvae are needed. One anthelmintic of potential efficacy against encysted cyathostome larvae is moxidectin. It is a milbemycin that possesses a 16-membered macrocyclic lactone ring, but lacks the disaccharide on Position 13, a characteristic of abamectin, ivermectin and doramectin (Carter et al., 1987). Although ivermectin has no efficacy against hypobiotic and encysted cyathostome larvae at regular or elevated doses (Eysker et al., 1992; Klei et al., 1993 ), limited studies with moxidectin indicate that it may be effective. Efficacies of 71.7-85.1% and 58.9-95.2% against hypobiotic EL3 and encysted LLa/L4, respectively, were obtained with moxidectin at 0.3 mg kg -1, 0.4 nag kg -1 and 0.5 mg kg -1 of body weight (DiPietro et al., 1992 ). Similar results (84-92% against hypobiotic EL3 and 78-92% against LL3/L4) were achieved by others (Bello et al., 1992; French et al., 1992 ). However, Reinemeyer and Agnilar (1992) and Reinemeyer and Tineo (1993) found that efficacies of moxidectin against encysted LL3 and L4 were variable (069.8%), and there was no efficacy of moxidectin against hypobiotic EL3. This study was designed to compare the efficacy of moxidectin and ivermectin against gastrointestinal parasites of horses, with special attention to the effects on hypobiotic and encysted larvae.
2. Materials and methods
2.1. Animals and experimental design Thirty-two mature ponies with natural infections of internal parasites were purchased from a commercial supplier. After being housed and fed hay for 4-12 weeks, they were assigned to eight replicates according to sex, body weight, pretreatment fecal egg counts (eggs per gram, epg) and strongyle larval identification. Within replicates, ponies were randomly allocated to one of four treatment groups, each with eight animals. Group 1 (Control) was given a placebo of oral gel. Group 2 (Mox 0.3) was treated with moxidectin oral gel (American Cyanamid Company, Princeton, New Jersey) at 0.3 mg kg- 1of body weight. Group 3 (Mox 0.4) was treated with moxidectin at 0.4 mg kg -I. Group 4 (Ivermectin) was treated with ivermectin (Eqvalan, Merck & Co., Inc., Rahway, New Jersey) at 0.2 mg kg- 1. Fecal egg counts (epg) and differential strongylid larval cultures were conducted on each animal before drug treatment (0 days) and 14 days after treatment. All animals were necropsied 14 days after treatment for total and differential worm counts.
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2.2. Worm count procedures After euthanasia, the stomach, small intestine and large intestine were separated, opened longitudinally, and the contents of each collected in separate containers. The luminal surface of each organ was scrubbed to free any attached parasites, and the mucosa was inspected for lesions and for remaining parasites which were removed, fixed and identified. The contents of the stomach and small intestine were washed over a No. 100 sieve (aperture 150/tm) and fixed in 5% formalin. The sieved stomach and small intestine contents were then inspected under 3×magnification for Gasterophilus third instar stage, Habronema spp., Draschia sp. and Parascaris equorum. Ten percent of the stomach contents were also examined under a dissection scope at 10 X magnification for Trichostrongy-
lus axei. Ten percent of the large intestinal contents were retained and fixed in 5% formalin. They were sieved through a No. 100 sieve and examined under 10×magnification for strongyle adults and late fourth stage larvae. A separate 0.01% aliquot of the large intestinal contents was sieved through stacked No. 60 (aperture 240/zm) and No. 325 (aperture 45/zm) sieves. Contents retained by the No. 325 sieve were then examined under 20 × magnification for cyathostome E L 3 and adult Probstmayria vivipara. The cecum and the first half of the ventral colon mucosa were separated from the remaining large intestine. They were weighed separately. Five percent of the mucosa (full thickness) was taken from the haustra (gut wall between taenial bands) of each organ. After being cut to sections of 5 cm by 5 cm in size, the harvested mucosa was transilluminated (Reinemeyer and Herd, 1986), and L L 3 and early L 4 w e r e counted. The mucosa was then scraped from all sections, and digested for up to 4 h at 40°C in pepsin/HC1 solution (Eysker et al., 1984). The whole digest was sieved through a No. 325 sieve, and a 5-20% aliquot of the digest examined under 20 x magnification for E L 3 (including Triodontophorus spp. ). All parasites recovered from the stomach, small intestine and large intestine were enumerated and identified to genus, species and developmental stages by the descriptions of Lichtenfels ( 1975 ) and Lichtenfels and Klei ( 1988 ).
2.3. Data analyses Fecal egg counts were statistically evaluated after logarithmic transformation (log (counts + 1 ) ). Sources of error for analysis of covariance for the fecal egg count analyses were pretreatment (covariate), treatment, and replicate. Because the covariate was not significant at the 0.10 level, analysis of variance was used. Worm counts were also evaluated after a similar logarithmic transformation. A two-way analysis of variance was performed accounting for both treatment and replicate. Group means were compared by an unprotected Least Significant Difference procedure. Percent efficacy was calculated according to the following formula:
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% Efficacy = 1O0 X (A - B ) / A , where A is the geometric mean of the number of eggs or worms in the control group, and B is the geometric mean of the number of eggs or worms in the treated group. 3. Results Mean fecal strongylid egg counts (epg) before and after treatment are shown in Table 1. The average pretreatment fecal strongyle egg counts were not significantly different among groups ( P > 0.05 ). Treatment with moxidectin at 0.3 mg kg -1 or 0.4 mg kg -1, or ivermectin at 0.2 mg kg -1 reduced strongylid epg by 99.8% or more 14 days after treatment, compared with the means ofpretreatment or the untreated Control group ( P < 0.05). One pony in the Mox 0.3 group and one in the Ivermectin group had ascarid eggs at 0 days but was negative at 14 days. Larvae identified in fecal cultures prior to treatment were mostly cyathostomes, Poteriostomum spp.; Triodontophorus spp., Strongylus vulgaris, StrongyTable 1 G e o m e t r i c m e a n s ( + SD ) of fecal strongyle egg c o u n t s of p o n i e s treated with 0.3 mg k g - 1 m o x i d e c t i n ( M o x 0.3), 0.4 m g kg -~ m o x i d e c t i n ( M o x 0.4), 0.2 m g kg - I i v e r m e c t i n ( I v e r m e c t i n ) , or pl a c e bo (Control) Group
Eggs per g r a m Day 0
Placeb o M o x 0.3 M o x 0.4 Ivermectin
437.9 361.7 462.3 456.8
Efficacy D a y 14
+ + + +
11.2 a 10.2 a 2.4 a 8a
698.4 + 13.6 a 0b 0.5 + 2.1 b 0.7 + 1.8 b
100% 99.9% 99.9%
N u m b e r s w i t h different superscripts in the s a m e c o l u m n are significantly different ( P < 0.05 ). Table 2 Geometric means ( + SD) of non-cyathostome parasites recovered from ponies treated with 0.3 mg kg- ~moxidectin (Mox 0.3 ), 0.4 mg kg- 1 moxidectin (Mox 0.4 ). 0.2 mg kg- 1 ivermectin (Ivermectin), or placebo (Control) Parasite
Control
Mox 0.3
Mox 0.4
Ivermectin
Gasterophilusintestinalis Habronemarnuscae Oxyurisequiadults OxyurisequiL42 Strongylusvulgaris Strongylusedentatus Triodontophorusspp.
15.2+7.0" 1.1+1.3" 18.6+26.4" 128.9+65.1" 2.8+4.0 a 2.7+2.9" 5.5_+7.9"
18.0_+2.7a (0) 0b (100%) 2.2+-7.6 "'b (88.2%) 0 b (100%) 0b (100%) 0b (100%) 0 b (100%)
12.1_+3.3a (20.4%) 0b (100%) 0b (100%) 0b (100%) 0b (100%) 0b (100%) 0b (100%)
0.7+0.6 b (95.4%) 0b (100%) 0b (100%) 0b (100%) 0b (100%) 0 b (100%) 0b (100%)
Numbers with different superscripts in the same row are significantlydifferent (P < 0.05 ). "Percent effieacies of anthelminthic treatments are in parentheses. bFourth stage larvae.
0b (100%) 0b (100o/o) 0b (100%) 0b (100%) 0b (100%) 0b ( 100% ) 0b (100%)
108.1 _+48.6 ~ 1133.1 _+16.8 ~ 4.2 + 20.6 ~ 1780.3 +_24.0 ~ 120.5 +__59.2 a 3.1 -+0 * 3.9 + 18.0 ~
Numbers with different superscripts in the same row are significantly different (P
0b (100%) 0b (100%)
13566.5_+7.9 (0) 128.7+28.9 (79.1%) 0.0+5.5 b (99.7%) 0b (100%) 0b (100%) 0b (100%)
Mox 0.3
5031.9 _+34.8 a 289.7 _+36.4 ~
3347.9+5.9 615.5+4.2 312.3+42.1 a 13351.7 -+ 48.1 ~ 799.4+ 65.4 ~ 40.1 _+59.2 a
EL3 Mucosal LIa2/L4 Luminal L43 Adults
Cylicocyclus nassatus Cylicocyclus leptostomus Cylicostephanus longibursatus Cylicostephanus goldi Cylicostephanus calicatus Cylicostephanus minutus Cyathostomum labiatum Cyathostomum catinatum Cyathostomum pateratum Cyathostomum labratum Cyathostomum coronatum
Control
Parasite
0.8 +4.1 b (99.3%) 0b (100%) 0b (100%) 0b (100%) 0~' (100%) 0b ( 100% ) 0b (100%)
1.1 -+ 7.3 b (100%) 0 b (100%)
14183.0+5.0 (0) 230.0+ 13.7 (62.6%) 0.1 +0.3 b (100%) 1.2 -+ 8.6 b (100%) 0.8 -+4.1 b (99.9%) 0 b ( 100% )
Mox 0.4
0 b (100%) 0 b (100%) 0b (100%)
0 b (100%) 0b (100%) 0b (100%)
0b (100%) 0b (100%)
3010.6_+7.1 (10,1%) 617.1 _+23.8 (0) 3.5-+ 15.0 b (98.8%) 0b (100%) 0b (100%) 0 b ( 100% )
Ivermectin
Table 3 Geometric means ( _ SD) of cyathostome adults and larvae recovered from ponies treated with 0.3 mg kg-~ moxidectin (Mox 0.3), 0,4 mg kgmoxidectin (Mox 0.4), 0.2 mg kg- t ivermectin (Ivermectin) or placebo (Control)
O~ "-4
I
k,n
5
88
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lus edentatus and Strongylus equinus were also found. No animals in the Mox 0.3 group, one animal in the Mox 0.4 group, and two animals in the Ivermectin group had a few cyathostome larvae in their fecal cultures 14 days after treatment. Moxidectin and ivermectin had similar efficacy against luminal nematodes (Table 2 ). Both were 100% efficient against Habronema muscae, Triodontophorus spp. (Triodontophorus tenuicollis, Triodontophorus brevicauda and Triodontophorus serratus), S. vulgaris, S. edentatus adults and Oxyuris equi L 4 ( P < 0.05 ). The efficacy of moxidectin at 0.3 mg kg-1 against O. equi adults was moderate (88.2%), but moxidectin at a higher dose and ivermectin were 100% effective. Efficacy of these two drugs in removing Gasterophilus intestinalis third instar stage was 95.4% for ivermectin and ineffective for moxidectin. Infection rates of Parascaris equorum, Probstmayria vivipara and Anoplocephalaperfoliata were too low to permit determination of efficacy. Both anthelmintics were highly effective (99.3% or above) against Cylicocyclus leptostomus, Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus calicatus, Cylicostephanus goldi, Cylicostephanus minutus, Cyathostomum labiatum, Cyathostomum catinatum, Cyathostomum pateratum, Cyathostomum labratum and Cyathostomum coronatum (Table 3, P<0.05). Some Cylicocyclus insigne, Cylicocyclus ultrajectinus and Poteriostomum imparidentatum adults were also present in control animals, but their numbers and infection rates were not high enough to allow the detection of statistical differences. Efficacies of moxidectin and ivermectin against larval stages of cyathostomes were stage and drug dependent (Table 3). Both anthelmintics were highly effective (98%) in removing luminal cyathostome t4, but ineffective in killing hypobiotic EL3 (in luminal contents and digests). Ivermectin had no efficacy against encysted LL 3 and L4 as detected by transillumination. By contrast, animals in the two moxidectin treatment groups had a 62.6-79.1% reduction in encysted LL 3 and L4. These reductions were not significantly different from control values (P>0.05).
4. Discussion
The efficacy of moxidectin at 0.3 mg kg-1 and 0.4 mg kg-1 against most gastrointestinal parasites of ponies was similar to that of ivermectin at 0.2 mg kg- 1. Both drugs were highly efficacious against nematode adults and luminal cyathostome and O. equi L4, but ineffective against cyathostome EL3. Moxidectin and ivermectin, however, differed in their efficacies against encysted cyathostome LL 3 and L4 and Gasterophilus sl0p. third instar stage. Moxidectin was moderately effective in removing encysted cyathostome LL 3 and L4 and ineffective against Gasterophilus spp. third instar stage in this trial. By contrast, ivermectin was highly efficacious against Gasterophilus slap. and ineffective against encysted cyathostome LL3 and L4. Results of this study confirmed previous observations that ivermectin had no efficacy against hypobiotic and encysted cyathostome larvae (Eysker et al., 1992;
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Klei et al., 1993). They also agreed with the results of Reinemeyer and Aguilar (1992) and Reinemeyer and Tineo (1993) that moxidectin had no efficacy against hypobiotic E L 3 and variable efficicies against encysted LL3 and L4. They, however, differed from the moderate to high efficacies against hypobiotic E L 3 (71.7-92%) and encysted L L 3 and L4 (58.9-95.2%) reported by some other researchers (BeUo et al., 1992; DiPietro et al., 1992; French et al., 1992 ). The poor efficacy of moxidectin against Gasterophilus spp. in this trial was in contrast to results reported in other studies. Although moxidectin had poor efficacy against G. intestinalis third instar stage at 0.3 mg kg-1 in some prior studies (Reinemeyer and Aguilar, 1992; DiPietro et al., 1992; French et al., 1992 ), it had high efficacy (87.7-100%) at 0.4 and 0.5 mg kg-' (Reinemeyer and Aguilar, 1992; Bello et al., 1992; DiPietro et al., 1992; French et al., 1992; Lyons et al., 1992; Reinemeyer and Tineo, 1993). Moxidectin was ineffective in the present study against G. intestinalis third instar stage at both 0.3 and 0.4 mg kg- 1. Lyons et al. (1992) reported that one of six batches of moxidectin oral gel (0.4 mg kg- 1) had low efficacy (53%) against G. intestinalis in one animal. It is unknown whether the poor results obtained in the present study were due to batch differences. Reasons for the different efficacies of moxidectin and ivermectin against encysted cyathostome L L 3 and L4 and Gasterophilus spp. are not clear. Although moxidectin and ivermectin are chemically related, minor structural changes may have changed the mode of action, because moxidectin is highly effective in removing ivermectin-resistant Haemonchus contortus in sheep (Craig et al., 1992; Pankavich et al., 1992 ) and Teladorsagia circumcincta/trifurcata in goats (Pomroy et al., 1992).
Acknowledgements The authors thank the American Cyanamid Company for partial financial support and Dr. Craig R. Reinemeyer for technical advice.
References Bello, T.R., Laningham, J.E.T. and Aguilar, R., 1992. Efficacy of moxidectin against equine parasites. Proc. 37th Annual Meeting Am. Assoc. Vet. Parasitol., 2-4 August 1992, Boston, MA. American Association of Veterinary Parasitologists, p. 50. Carter, G.T., Nietsche, J.A. and Borders, D.B., 1987. Structure determination of LL-F28249-alpha, beta, gamma and lambda, potent antiparasitic macrolides from Streptomyces cyaneogriseus ssp. noncyanogenus. J. Chem. Soc. Chem. Commun., 6: 402-404. Craig, T.M., Hatfield, T.A., Pankavich, J.A. and Wang, G.T., 1992. Efficacy of moxidectin against an ivermectin-resistantstrain ofHaemonchus contortus in sheep. Vet. Parasitol., 41: 329-333. DiPietro, J.A., Paul, A.J., Ewert, K.M., Todd Jr., K.S., Lock, T.F. and Aguilar, R., 1992. Moxidectin gel: a new equine endectocide. Proc. 38th Annual Conf. Am. Assoc. Equine Pract., 30 November2 December 1992, Orlando, FL. American Association of Equine Practitioners, Lexington, KY, pp. 311-316. Eysker, M., Jansen, J. and Mirck, M.H., 1984. Inhibited development of Cyathostominae in the horse in the early third stage. Res. Vet. Sci., 37: 355-356.
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Eysker, M., Boersema, J.H. and Kooyman, F.N.J., 1992. The effect of ivermectin treatment against inhibited early third stage, late third stage and fourth stage larvae and adult stages of cyathostomes in Shetland ponies and spontaneous expulsion of these helminths. Vet. Parasitol., 42: 295-302. French, D.D., Klei, T.R., Taylor, H.W., Chapman, M.R., Monahan, C. and Aguilar, R., 1992. Efficacy of moxidectin gel against internal parasites of ponies. Proc. 37th Annual Meeting Am. Assoc. Vet. Parasitol., 2-4 August 1992, Boston, MA. American Association of Veterinary Parasitologists, p. 51. Klei, T.R., Chapman, M.R., French, D.D. and Taylor, H.W., 1993. Evaluation of ivermectin at an elevated dose against encysted equine cyathostome larvae. Vet. Parasitol., 47: 99-106. Lichtenfels, J.R., 1975. Helminths of domestic equids. Proc. Helminthol. Soc. Wash., 42: 1-92. Lichtenfels, J.R. and Klei, T.R., 1988. Cylicostephanus torbertae sp. n. (Nematoda: Strongyloidea) from Equus caballus with a discussion of the genera Cylicostephanus, Petrovinema, and Skrjabinodentus. Proc. Helminthol. Soc. Wash., 55: 165-170. Lyons, E.T., Tolliver, S.C., Drudge, J.H., Granstrom, D.E., Collins, S.S. and Stamper, S., 1992. Critical and controlled tests of activity of moxidectin (CL 301,423) against natural infections of internal parasites of equids. Vet. Parasitol., 41: 255-284. Pankavich, J.A., Berger, H. and Simkins, K.L., 1992. Efficacy of moxidectin, nemadectin and ivermectin against an ivermectin-resistant strain of Haemonchus contortus in sheep. Vet. Rec., 130: 241-243. Pomroy, W.E., Whelan, N., Alexander, A.M., West, D.W., Stafford, K., Adlington, B.A. and Calder, S.M., 1992. Multiple resistance in goat-derived Ostertagia and the efficacy of moxidectin and combinations of other anthelmintics. NZ Vet. J., 40:76-78. Reinemeyer, C.R., 1986. Small strongyles: recent advances. Vet. Clin. North Am. Equine Pract., 2: 281-312. Reinemeyer, C.R. and Aguilar, R., 1992. Dose titration study of moxidectin gel as an oral equine anthelmintic. Proc. 37th Annual Meeting Am. Assoc. Vet. Parasitol., 2-4 August 1992, Boston, MA. American Association of Veterinary Parasitologists, p. 72. Re inemeyer, C.R. and Herd, R.P., 1986. A comparison of two techniques for quantitation of encysted cyathostome larvae in the horse. Am J. Vet. Res., 47: 507-509. Reinemeyer, C.R. and Tineo, A.A., 1993. Comparison of the efflcacies of moxidectin gel and ivermectin paste against nematode parasites of horses. Proc. 38th Annual Meeting Am. Assoc. Vet. Parasitol., 17-20 July 1993, Minneapolis, MN. American Association of Veterinary Parasitologists, p. 32.
Veterinary Parasitology 53 (1994) 83-90
Comparative efficacy of moxidectin and ivermectin against hypobiotic and encysted cyathostomes and other equine parasites L i h u a Xiao*, R.P. Herd, G.A. M a j e w s k i Department of Veterinary Preventive Medicine, The Ohio State University College of Veterinary Medicine, 1900 Coffey Road, Columbus, OH 43210, USA (Accepted 18 August 1993 )
Abstract Efficacies of moxidectin and ivermectin were compared in four groups of eight ponies with natural parasite infections: placebo (Control), oral moxidectin gel at 0.3 mg kg-1 of body weight (Mox 0.3), oral moxidectin gel at 0.4 mg kg-1 of body weight (Mox 0.4), and oral ivermectin paste at 0.2 mg kg- ~of body weight (Ivermectin). Fecal samples were taken 0 and 2 weeks after treatment. Animals were necropsied and worms were collected 2 weeks after treatment. Moxidectin and ivermectin showed similar efficacy (99%) against adult cyathostomes, Strongylus spp., Triodontophorus spp. and Habronema muscae. Both drugs were also more than 98% effective against luminal cyathostome and Oxyuris equi fourth stage larvae (L4). Neither drug was effective (0-10.1%) against hypobiotic early third stage cyathostome larvae (EL3). Moxidectin was moderately effective (62.6-79.1%) in removing encysted eyathostome late third stage larvae (LL3) and L4, whereas ivermectin was ineffective (0%) against these stages. By contrast, ivermectin was 95.4% effective against Gasterophilus spp. third instar stage, whereas moxidectin was only 0-20.4% effective. Key words: Cyathostomidae; Horse; Control methods-Nematoda; Moxidectin; Iverrnectin; Hypobiosis
1. Introduction H y p o b i o t i c early third stage larvae (EL3) a n d encysted c y a t h o s t o m e late third stage (LL3) a n d fourth stage larvae (L4) present a m a j o r obstacle for the control *Corresponding author at: Immunology Branch, Mail Stop F-12, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA. 0304-4017/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved SSDI 0304-4017 ( 93 ) 00589-Q
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of equine parasites. Benzimidazole anthelmintics (fenbendazole and oxfendazole) were reportedly effective against encysted late third stage and fourth stage larvae ofcyathostomes (reviewed by Reinemeyer, 1986). However, the accuracy of the technique used to quantify numbers of tissue larvae was questioned (Reinemeyer and Herd, 1986). Furthermore, increasing benzimidazole resistance is likely to have rendered these anthelmintics ineffective on farms where they have been used intensively. Thus, new strategies to control hypobiotic and encysted cyathostome larvae are needed. One anthelmintic of potential efficacy against encysted cyathostome larvae is moxidectin. It is a milbemycin that possesses a 16-membered macrocyclic lactone ring, but lacks the disaccharide on Position 13, a characteristic of abamectin, ivermectin and doramectin (Carter et al., 1987). Although ivermectin has no efficacy against hypobiotic and encysted cyathostome larvae at regular or elevated doses (Eysker et al., 1992; Klei et al., 1993 ), limited studies with moxidectin indicate that it may be effective. Efficacies of 71.7-85.1% and 58.9-95.2% against hypobiotic EL3 and encysted LLa/L4, respectively, were obtained with moxidectin at 0.3 mg kg -1, 0.4 nag kg -1 and 0.5 mg kg -1 of body weight (DiPietro et al., 1992 ). Similar results (84-92% against hypobiotic EL3 and 78-92% against LL3/L4) were achieved by others (Bello et al., 1992; French et al., 1992 ). However, Reinemeyer and Agnilar (1992) and Reinemeyer and Tineo (1993) found that efficacies of moxidectin against encysted LL3 and L4 were variable (069.8%), and there was no efficacy of moxidectin against hypobiotic EL3. This study was designed to compare the efficacy of moxidectin and ivermectin against gastrointestinal parasites of horses, with special attention to the effects on hypobiotic and encysted larvae.
2. Materials and methods
2.1. Animals and experimental design Thirty-two mature ponies with natural infections of internal parasites were purchased from a commercial supplier. After being housed and fed hay for 4-12 weeks, they were assigned to eight replicates according to sex, body weight, pretreatment fecal egg counts (eggs per gram, epg) and strongyle larval identification. Within replicates, ponies were randomly allocated to one of four treatment groups, each with eight animals. Group 1 (Control) was given a placebo of oral gel. Group 2 (Mox 0.3) was treated with moxidectin oral gel (American Cyanamid Company, Princeton, New Jersey) at 0.3 mg kg- 1of body weight. Group 3 (Mox 0.4) was treated with moxidectin at 0.4 mg kg -I. Group 4 (Ivermectin) was treated with ivermectin (Eqvalan, Merck & Co., Inc., Rahway, New Jersey) at 0.2 mg kg- 1. Fecal egg counts (epg) and differential strongylid larval cultures were conducted on each animal before drug treatment (0 days) and 14 days after treatment. All animals were necropsied 14 days after treatment for total and differential worm counts.
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2.2. Worm count procedures After euthanasia, the stomach, small intestine and large intestine were separated, opened longitudinally, and the contents of each collected in separate containers. The luminal surface of each organ was scrubbed to free any attached parasites, and the mucosa was inspected for lesions and for remaining parasites which were removed, fixed and identified. The contents of the stomach and small intestine were washed over a No. 100 sieve (aperture 150/tm) and fixed in 5% formalin. The sieved stomach and small intestine contents were then inspected under 3×magnification for Gasterophilus third instar stage, Habronema spp., Draschia sp. and Parascaris equorum. Ten percent of the stomach contents were also examined under a dissection scope at 10 X magnification for Trichostrongy-
lus axei. Ten percent of the large intestinal contents were retained and fixed in 5% formalin. They were sieved through a No. 100 sieve and examined under 10×magnification for strongyle adults and late fourth stage larvae. A separate 0.01% aliquot of the large intestinal contents was sieved through stacked No. 60 (aperture 240/zm) and No. 325 (aperture 45/zm) sieves. Contents retained by the No. 325 sieve were then examined under 20 × magnification for cyathostome E L 3 and adult Probstmayria vivipara. The cecum and the first half of the ventral colon mucosa were separated from the remaining large intestine. They were weighed separately. Five percent of the mucosa (full thickness) was taken from the haustra (gut wall between taenial bands) of each organ. After being cut to sections of 5 cm by 5 cm in size, the harvested mucosa was transilluminated (Reinemeyer and Herd, 1986), and L L 3 and early L 4 w e r e counted. The mucosa was then scraped from all sections, and digested for up to 4 h at 40°C in pepsin/HC1 solution (Eysker et al., 1984). The whole digest was sieved through a No. 325 sieve, and a 5-20% aliquot of the digest examined under 20 x magnification for E L 3 (including Triodontophorus spp. ). All parasites recovered from the stomach, small intestine and large intestine were enumerated and identified to genus, species and developmental stages by the descriptions of Lichtenfels ( 1975 ) and Lichtenfels and Klei ( 1988 ).
2.3. Data analyses Fecal egg counts were statistically evaluated after logarithmic transformation (log (counts + 1 ) ). Sources of error for analysis of covariance for the fecal egg count analyses were pretreatment (covariate), treatment, and replicate. Because the covariate was not significant at the 0.10 level, analysis of variance was used. Worm counts were also evaluated after a similar logarithmic transformation. A two-way analysis of variance was performed accounting for both treatment and replicate. Group means were compared by an unprotected Least Significant Difference procedure. Percent efficacy was calculated according to the following formula:
L. Xiao et al. / Veterinary Parasitology 53 (1994) 83-90
86
% Efficacy = 1O0 X (A - B ) / A , where A is the geometric mean of the number of eggs or worms in the control group, and B is the geometric mean of the number of eggs or worms in the treated group. 3. Results Mean fecal strongylid egg counts (epg) before and after treatment are shown in Table 1. The average pretreatment fecal strongyle egg counts were not significantly different among groups ( P > 0.05 ). Treatment with moxidectin at 0.3 mg kg -1 or 0.4 mg kg -1, or ivermectin at 0.2 mg kg -1 reduced strongylid epg by 99.8% or more 14 days after treatment, compared with the means ofpretreatment or the untreated Control group ( P < 0.05). One pony in the Mox 0.3 group and one in the Ivermectin group had ascarid eggs at 0 days but was negative at 14 days. Larvae identified in fecal cultures prior to treatment were mostly cyathostomes, Poteriostomum spp.; Triodontophorus spp., Strongylus vulgaris, StrongyTable 1 G e o m e t r i c m e a n s ( + SD ) of fecal strongyle egg c o u n t s of p o n i e s treated with 0.3 mg k g - 1 m o x i d e c t i n ( M o x 0.3), 0.4 m g kg -~ m o x i d e c t i n ( M o x 0.4), 0.2 m g kg - I i v e r m e c t i n ( I v e r m e c t i n ) , or pl a c e bo (Control) Group
Eggs per g r a m Day 0
Placeb o M o x 0.3 M o x 0.4 Ivermectin
437.9 361.7 462.3 456.8
Efficacy D a y 14
+ + + +
11.2 a 10.2 a 2.4 a 8a
698.4 + 13.6 a 0b 0.5 + 2.1 b 0.7 + 1.8 b
100% 99.9% 99.9%
N u m b e r s w i t h different superscripts in the s a m e c o l u m n are significantly different ( P < 0.05 ). Table 2 Geometric means ( + SD) of non-cyathostome parasites recovered from ponies treated with 0.3 mg kg- ~moxidectin (Mox 0.3 ), 0.4 mg kg- 1 moxidectin (Mox 0.4 ). 0.2 mg kg- 1 ivermectin (Ivermectin), or placebo (Control) Parasite
Control
Mox 0.3
Mox 0.4
Ivermectin
Gasterophilusintestinalis Habronemarnuscae Oxyurisequiadults OxyurisequiL42 Strongylusvulgaris Strongylusedentatus Triodontophorusspp.
15.2+7.0" 1.1+1.3" 18.6+26.4" 128.9+65.1" 2.8+4.0 a 2.7+2.9" 5.5_+7.9"
18.0_+2.7a (0) 0b (100%) 2.2+-7.6 "'b (88.2%) 0 b (100%) 0b (100%) 0b (100%) 0 b (100%)
12.1_+3.3a (20.4%) 0b (100%) 0b (100%) 0b (100%) 0b (100%) 0b (100%) 0b (100%)
0.7+0.6 b (95.4%) 0b (100%) 0b (100%) 0b (100%) 0b (100%) 0 b (100%) 0b (100%)
Numbers with different superscripts in the same row are significantlydifferent (P < 0.05 ). "Percent effieacies of anthelminthic treatments are in parentheses. bFourth stage larvae.
0b (100%) 0b (100o/o) 0b (100%) 0b (100%) 0b (100%) 0b ( 100% ) 0b (100%)
108.1 _+48.6 ~ 1133.1 _+16.8 ~ 4.2 + 20.6 ~ 1780.3 +_24.0 ~ 120.5 +__59.2 a 3.1 -+0 * 3.9 + 18.0 ~
Numbers with different superscripts in the same row are significantly different (P
0b (100%) 0b (100%)
13566.5_+7.9 (0) 128.7+28.9 (79.1%) 0.0+5.5 b (99.7%) 0b (100%) 0b (100%) 0b (100%)
Mox 0.3
5031.9 _+34.8 a 289.7 _+36.4 ~
3347.9+5.9 615.5+4.2 312.3+42.1 a 13351.7 -+ 48.1 ~ 799.4+ 65.4 ~ 40.1 _+59.2 a
EL3 Mucosal LIa2/L4 Luminal L43 Adults
Cylicocyclus nassatus Cylicocyclus leptostomus Cylicostephanus longibursatus Cylicostephanus goldi Cylicostephanus calicatus Cylicostephanus minutus Cyathostomum labiatum Cyathostomum catinatum Cyathostomum pateratum Cyathostomum labratum Cyathostomum coronatum
Control
Parasite
0.8 +4.1 b (99.3%) 0b (100%) 0b (100%) 0b (100%) 0~' (100%) 0b ( 100% ) 0b (100%)
1.1 -+ 7.3 b (100%) 0 b (100%)
14183.0+5.0 (0) 230.0+ 13.7 (62.6%) 0.1 +0.3 b (100%) 1.2 -+ 8.6 b (100%) 0.8 -+4.1 b (99.9%) 0 b ( 100% )
Mox 0.4
0 b (100%) 0 b (100%) 0b (100%)
0 b (100%) 0b (100%) 0b (100%)
0b (100%) 0b (100%)
3010.6_+7.1 (10,1%) 617.1 _+23.8 (0) 3.5-+ 15.0 b (98.8%) 0b (100%) 0b (100%) 0 b ( 100% )
Ivermectin
Table 3 Geometric means ( _ SD) of cyathostome adults and larvae recovered from ponies treated with 0.3 mg kg-~ moxidectin (Mox 0.3), 0,4 mg kgmoxidectin (Mox 0.4), 0.2 mg kg- t ivermectin (Ivermectin) or placebo (Control)
O~ "-4
I
k,n
5
88
L. Xiao et al. / Veterinary Parasitology 53 (1994) 83-90
lus edentatus and Strongylus equinus were also found. No animals in the Mox 0.3 group, one animal in the Mox 0.4 group, and two animals in the Ivermectin group had a few cyathostome larvae in their fecal cultures 14 days after treatment. Moxidectin and ivermectin had similar efficacy against luminal nematodes (Table 2 ). Both were 100% efficient against Habronema muscae, Triodontophorus spp. (Triodontophorus tenuicollis, Triodontophorus brevicauda and Triodontophorus serratus), S. vulgaris, S. edentatus adults and Oxyuris equi L 4 ( P < 0.05 ). The efficacy of moxidectin at 0.3 mg kg-1 against O. equi adults was moderate (88.2%), but moxidectin at a higher dose and ivermectin were 100% effective. Efficacy of these two drugs in removing Gasterophilus intestinalis third instar stage was 95.4% for ivermectin and ineffective for moxidectin. Infection rates of Parascaris equorum, Probstmayria vivipara and Anoplocephalaperfoliata were too low to permit determination of efficacy. Both anthelmintics were highly effective (99.3% or above) against Cylicocyclus leptostomus, Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus calicatus, Cylicostephanus goldi, Cylicostephanus minutus, Cyathostomum labiatum, Cyathostomum catinatum, Cyathostomum pateratum, Cyathostomum labratum and Cyathostomum coronatum (Table 3, P<0.05). Some Cylicocyclus insigne, Cylicocyclus ultrajectinus and Poteriostomum imparidentatum adults were also present in control animals, but their numbers and infection rates were not high enough to allow the detection of statistical differences. Efficacies of moxidectin and ivermectin against larval stages of cyathostomes were stage and drug dependent (Table 3). Both anthelmintics were highly effective (98%) in removing luminal cyathostome t4, but ineffective in killing hypobiotic EL3 (in luminal contents and digests). Ivermectin had no efficacy against encysted LL 3 and L4 as detected by transillumination. By contrast, animals in the two moxidectin treatment groups had a 62.6-79.1% reduction in encysted LL 3 and L4. These reductions were not significantly different from control values (P>0.05).
4. Discussion
The efficacy of moxidectin at 0.3 mg kg-1 and 0.4 mg kg-1 against most gastrointestinal parasites of ponies was similar to that of ivermectin at 0.2 mg kg- 1. Both drugs were highly efficacious against nematode adults and luminal cyathostome and O. equi L4, but ineffective against cyathostome EL3. Moxidectin and ivermectin, however, differed in their efficacies against encysted cyathostome LL 3 and L4 and Gasterophilus sl0p. third instar stage. Moxidectin was moderately effective in removing encysted cyathostome LL 3 and L4 and ineffective against Gasterophilus spp. third instar stage in this trial. By contrast, ivermectin was highly efficacious against Gasterophilus slap. and ineffective against encysted cyathostome LL3 and L4. Results of this study confirmed previous observations that ivermectin had no efficacy against hypobiotic and encysted cyathostome larvae (Eysker et al., 1992;
L. Xiao et al. / Veterinary Parasitology 53 (1994) 83-90
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Klei et al., 1993). They also agreed with the results of Reinemeyer and Aguilar (1992) and Reinemeyer and Tineo (1993) that moxidectin had no efficacy against hypobiotic E L 3 and variable efficicies against encysted LL3 and L4. They, however, differed from the moderate to high efficacies against hypobiotic E L 3 (71.7-92%) and encysted L L 3 and L4 (58.9-95.2%) reported by some other researchers (BeUo et al., 1992; DiPietro et al., 1992; French et al., 1992 ). The poor efficacy of moxidectin against Gasterophilus spp. in this trial was in contrast to results reported in other studies. Although moxidectin had poor efficacy against G. intestinalis third instar stage at 0.3 mg kg-1 in some prior studies (Reinemeyer and Aguilar, 1992; DiPietro et al., 1992; French et al., 1992 ), it had high efficacy (87.7-100%) at 0.4 and 0.5 mg kg-' (Reinemeyer and Aguilar, 1992; Bello et al., 1992; DiPietro et al., 1992; French et al., 1992; Lyons et al., 1992; Reinemeyer and Tineo, 1993). Moxidectin was ineffective in the present study against G. intestinalis third instar stage at both 0.3 and 0.4 mg kg- 1. Lyons et al. (1992) reported that one of six batches of moxidectin oral gel (0.4 mg kg- 1) had low efficacy (53%) against G. intestinalis in one animal. It is unknown whether the poor results obtained in the present study were due to batch differences. Reasons for the different efficacies of moxidectin and ivermectin against encysted cyathostome L L 3 and L4 and Gasterophilus spp. are not clear. Although moxidectin and ivermectin are chemically related, minor structural changes may have changed the mode of action, because moxidectin is highly effective in removing ivermectin-resistant Haemonchus contortus in sheep (Craig et al., 1992; Pankavich et al., 1992 ) and Teladorsagia circumcincta/trifurcata in goats (Pomroy et al., 1992).
Acknowledgements The authors thank the American Cyanamid Company for partial financial support and Dr. Craig R. Reinemeyer for technical advice.
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