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Efficacy of closantel, albendazole and levamisole on an ivermectin resistant strain of Haemonchus contortus in sheep
veterinary parasitology ELSEVIER
Veterinary Parasitology 73 (1997) 65-71
Efficacy of closantel, albendazole and levamisole on an ivermectin resistant strain of Haemonchus contortus in sheep R.M. Waruiru
*
Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine (Kabete Campus). University of Nairobi, P.O. Box 29053, Nairobi, Kenya Received 19 November 1996; accepted 4 April 1997
Abstract Following evidence of reduced efficacy of oral ivermectin in a field population of Haemonchus contortus on a sheep farm in Kenya, this strain of the parasite was submitted to a controlled anthelmintic test. One hundred and twenty worm-free lambs were randomly split into two groups of 60. Each lamb in the first group was infected with 5000 third stage larvae (L 3) of the suspected resistant strain the remaining 60 lambs were each infected with 5000 (L 3) of a H. contortus strain of known susceptibility to the major groups of anthelmintic compounds used in sheep. On day 28 post-infection, each group was subdivided according to egg counts and at random into six sub-groups of ten lambs, each of which was treated with closantel at 5.0 mg k g - l, albendazole at 5.0 mg kg 1, levamisole at 7.5 mg k g - i , oral ivermectin at 0.2 mg k g - I or injectable (Inj.) ivermectin at 0.2 mg k g - ~, or was left as an untreated control. At slaughter, 10 days later, all the anthelmintics had resulted in reduced worm burdens in animals infected with the susceptible strain (albendazole 99.0%, levamisole 99.5%, closantel, Inj. and oral ivermectin 100%). By contrast, in the lambs infected with the suspected resistant strain, closantel was 100% effective, but inj. ivermectin, oral ivermectin, albendazole and levamisole reduced worm counts by 47.6%, 24.2%, 38.5% and 41.4%, respectively. Anthelmintic resistance to the other chemical groups had been confirmed previously on this farm and although benzimidazoles and levamisole had not been used for the last 4 years, this strain was still highly resistant to albendazole and levamisole; closantel remained as the only drug evaluated which was still effective against this population of H. contortus. © 1997 Elsevier Science B.V. Keywords: Haemonchus eontortus; Drug resistance; Sheep nematoda; Control methods-nematoda; Benzimidazole; Albendazole, Ivermectin; Levamisole, Closantel
* Corresponding author. Tel.: 254-2-631340; fax: 254-2-631487. 0304-4017/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 0 1 7 ( 9 7 ) 0 0 0 6 5 - 4
66
R.M. Waruiru / Veterinary Parasitology 73 (1997) 65-71
1. Introduction
Over the last 10-20 years, anthelmintic resistance of sheep nematodes to the widely used benzimidazole and levamisole groups of drugs has become an increasingly important problem in the major sheep-rearing areas of the world (Waller, 1987). The discovery of a chemically distinct group of anthelmintics, the avermectins (Burg et al., 1979), provided a welcome alternative. However, subsequent to the introduction of the avermectins in the early 1980s resistant sheep nematodes have now been identified. Ivermectin resistance has been selected for experimentally in both Haemonchus contortus (Egerton et al., 1988), where a high level of resistance was detected after seven generations of exposure to one-tenth of the normal recommmended dose, and in Trichostrongylus colubriformis (Giordano et al., 1988), after four generations had been subjected to various ivermectin dose rates. Under field conditions ivermectin resistance has appeared in H. contortus in sheep and goats in South Africa (Carmichael et al., 1987), in Brazil (Echevarria and Trindade, 1989) in the USA (Craig and Miller, 1990) and Kenya (Mwamachi et al., 1995). Ivermectin resistance also appears to be developing in Australia in 1-1. contortus and T. columbriformis (Le Jambre, 1993). Because of the widespread occurrence of resistance to the broad spectrum anthelmintics in H. contortus, closantel is being used widely to control this parasite in sheep and goats. Unfortunately, there are reports of resistance to closantel also, in South Africa (Van Wyk and Malan, 1988), Australia (Rolfe et al., 1990) and recently in Kenya (Mwamachi et al., 1995). The objective of this experiment was to evaluate the anthelmintic sensitivity of the suspected avermectin resistant strain to closantel, albendazole and levamisole treatments under controlled test conditions; a strain known to be susceptible was included for comparison. 2. Materials and methods 2.1. Experimental design
One hundred and twenty indigenous sheep of mixed breeds, sexes and aged between 8 to 9 months were housed in pens with concrete floors at the Kabete Campus, University of Nairobi. Lucerne hay, a commercial sheep concentrate, mineral salts and water were available ad libitum. On arrival, faecal samples were collected and examined by a modified McMaster technique (Ministry of Agriculture, Fisheries and Food, 1986), for the presence of helminth ova. The lambs had a mean faecal egg count of 675 (0-4700) eggs per gram-1 (epg) faeces and were immediately drenched with fenbendazole (Panacur®-Hoechst, Munich, Germany, 5 mg kg -l body weight), this treatment was repeated after 7 days. A second faecal examination 14 days later showed all animals to be negative for nematode eggs. The lambs were then split at random into two groups of 60 (Groups R and S). The Group R animals were infected with 5000 third stage larvae (L 3) of the suspected resistant strain of H. contortus (R.M. Waruiru, unpublished data, 1995). Following isolation, sheep infected with this field strain had been subjected to treatment with oral ivermectin at 0.2 mg kg ~ and the survivors of this treatment produced the
67
R.M. Waruiru/ Veterinary Parasitology 73 (1997) 65-71
Table 1 Allocation of animals to treatment groups Group Treatment
Dose (mg kg- ~)
Animal per treatment
Weightof heaviest animal (kg)
R infected with suspected avermectin resistant strain
R1 Closantel R2 Ivermectin(injectable)a R3 Ivermectin(oral) R4 Albendazole R5 Levamisole R6 Control
5.0 0.2 0.2 5.0 7.5 -
10 10 10 10 10 10
18.3 17.3 20.1 17.1 18.7 20.1
S infected with susceptible strain
S1 Closantel $2 Ivermectin(injectable) $3 Ivermectin(oral) $4 Albendazole $5 Levamisole $6 Control
0.2 0.2 5.0 7.5 5.0 -
10 10 10 10 10 10
19.2 18.4 17.5 19.0 17.7 16.8
aDrug administered subcutaneously; all other drugs given orally. suspected resistant larvae used in the present experiment. No benzimidazoles or levamisole treatments had been applied for at least 4 years, ivermectin being the only drug used on the farm where the suspected resistant strain was isolated. Previously, however, both groups of anthelmintics had been used fairly intensively to control H. contortus and resistance to BZs and LEV had been confirmed (Waruiru et al., 1991). The animals in Group S were infected with 5000 L 3 of a susceptible strain of H. contortus. At day 28 post-infection, animals in both groups were allocated, according to their faecal egg counts and at random, to one of the six treatments as shown in Table 1. Animals were treated with closantel (Flukiver®-Janssen Pharmeceutica, Beerse, Belgium, 5 mg kg -1 body weight), Inj. ivermectin (Ivomec®-MSD Agvet, Rahway, NJ, USA, 0.2 mg kg-1 body weight), oral ivermectin (Oramec®-MSD Agvet, 0.2 mg k g - i body weight), albendazole (Valbazen®-Smithkline, Beecham, U.K, 5 mg kg -1 bodyweight) and levamisole (Nilverm®-Wellcome Kenya Ltd., Nairobi, 7.5 mg kg-1 body weight), respectively, according to the manufacturer's recommendations. All animals in a given group received a standard dose based on the heaviest animal in the group. Ten animals in each group were the untreated controls (Table 1). The animals were slaughtered on day 10 after treatment (day 39 post-infection) and total worm burdens estimated as recommended by W o o d et al. (1995). 2.2. Statistics
Faecal egg and worm counts were tranformed according to the expression y = log10 ( c o u n t + 1) and analysis of variance performed for testing the main effects and interactions between strain of parasite and treatment. Taking into consideration the significant strain versus treatment interaction, multiple comparisons were conducted using Fisher's least significant difference test.
R,M, Waruiru / Veterinary Parasitology 73 (1997) 65-71
68
3. Results Results of total worm counts and faecal egg counts are shown in Tables 2 and 3, respectively. These results show clear differences in the efficacy of ivermectin, albendazole and levamisole against the two strains. Table 2 Mean worm burdens a (range) of sheep infected with two strains of H. contortus and their percentage reduction after treatment with closantel, ivermectin, albendazole and levamisole compared with untreated controls Strain
Treatment
Mean worm count
Reduction (%)
S
Closantel Ivermectin (injectable) Ivermectin (oral) Albendazole Levamisole Control
0b 0b 0b 24 b 14 b 2702 d
(0-0) (0-0) (0-0) (0-210) (0-1 I0) ( 1701-3480)
100 100 100 99.0 99.5 -
R
Closantel Ivermectin (injectable) Ivermectin (oral) Albendazole Levamisole Control
0b 649 c 1033 e 838 c 799 ~ 1363 c
(0-0) (24-1970) (61-2314) (310-2960) (281 - 1772) (484- 2280)
100 47.6 34.2 38.5 41.4 -
S, ivermectin susceptible; R, ivermectin resistant. aValues shown are arithimetic mean worm counts, but comparisons were made on log-transformed data. b'C'dResults followed by different superscript letters are significantly different ( P < 0.05).
Table 3 Effect of closantel, ivermectin, albendazole and levamisole on faecal egg output a in sheep infected with two strains of H. contortus Strain
Treatment
Mean egg count (range)
Closantel Ivermectin (injectable) Ivermectin (oral) Albendazole Levamisole Control
2843 b 2576 b 2684 b 2357 b 3408 b 3675 b
(1100-4200) (900-3700) (1300-3400) (700-2900) (1400-4400) (1000-4500)
0b 0b 0b 47 b 8b 3727 d
(0-0) (0-0) (0-0) (0-400) (0-100) (1100-4300)
100 100 100 98.7 99.8 + 1.4
Closantel Ivermectin (injectable) Ivermectin (oral) Albendazole Levamisole Control
1489 c 1541 c 1391 c 1441 ~ 1610 c 1827 c
(300-2700) (600-3100) (400-2100) (600-3000) (700-3200) (400-3100)
0b 3300 1489 e 275 a 3150 2118 e
(0-0) (0-800) (300-2900) (0-700) (100-1100) (600-3000)
100 84.4 29.7 87.0 85.1 + 15.9
Day 0
Reduction (%) Day 10
S, ivermecfin susceptible; R, ivermectin resistant. aValues shown are arithimetic mean worm egg counts, but comparisons were made on log-transformed data. b'C'dResults followed by different superscript letters in the same column are significantly different (P < 0.05).
R.M. Waruiru / Veterinary_ Parasitology 73 (1997) 65-71
69
From the worm burdens of the controls, there were obvious differences in establishment rates of the two strains: 54% of the susceptible against 27.3% for the suspected resistant strain. This was reflected in the epg counts on the day of treatment (Day 0) which were also significantly lower in animals infected with the suspected resistant strain ( P < 0.05). 10 days later there was no statistical difference in epg ( P > 0.05) between the two control groups, although the suspected resistant strain still showed a much lower output than the susceptible strain (Table 3). The susceptible strain was equally susceptible to closantel, ivermectin, albendazole and levamisole, while the suspected resistant strain showed a high level of resistance to ivermectin, albendazole and levamisole. With the suspected resistant strain, there was no significant difference ( P > 0.05) in worm burden reduction between groups treated with Inj. ivermectin, oral ivermectin, albendazole and levamisole, these drugs showed an efficacy of 47.6%, 24.2% 38.5% and 41.4%, respectively. Closantel reduced worm counts of this resistant strain by 100% (Table 2). Faecal egg count (epg) was reduced by almost 100% by all five drugs in the animals infected with the susceptible strain. In the suspected resistant strain, closantel was also 100% effective, while there was insignificant ( P > 0.05) reduction in epg in the animals treated with oral ivermectin (Table 3). Inj. ivermectin, albendazole and levamisole significantly ( P < 0.05) reduced epg counts by 84.4%, 87.0% and 85.1%, respectively. However, these reductions were not reflected in the worm counts (Table 3).
4. Discussion The results obtained in this study clearly confirm iverrnectin resistance of a field strain of H. contortus (R.M. Waruiru, unpublished data, 1995). Further, the results indicate that this strain has retained benzimidazole and levamisole resistance despite the fact that benzimidazoles and levamisole had not been used for at least 4 years on the farm where it was isolated. Albendazole was highly efficient against the susceptible strain (99.0%), but for the resistant strain, although treatment depressed egg output by 87.0%, it only reduced worm burdens by 38.5%. These results confirm previous observations on the effect of benzimidazoles (BZs) on BZ-resistant strains, i.e. a temporary suppression of worm egg counts without a corresponding worm loss (Hotson et al., 1970). Ivermectin was highly effective against the susceptible strain, but in the resistant strain its efficacy ranged from 24.2% (oral ivermectin) to 47.6% (Inj. ivermectin). The difference in efficacy of the two formulations of ivermectin is consistent with the findings of McKellar and Marriner (1987) that treatment with injectable ivermectin resulted in a higher and more sustained level of the drug than oral ivermectin. Similar results were obtained by Craig and Miller (1990) in Angora goats. They found no difference in faecal egg counts between a group treated with oral ivermectin and controls, but a significant difference with injectable ivermectin. In the present study, of the four chemically distinct anthelmintics used, closantel was the only drug which proved highly effective against both the susceptible as well as the suspected resistant strain. Thus, closantel may be useful in preventing production losses
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R.M. Waruiru / Veterinary Parasitology 73 (1997) 65-71
on sheep farms where H. contortus has developed resistance to benzimidazole, levamisole and ivermectin classes of anthelmintics. One of the most interesting features of closantel is its persistent anthelmintic effect, which, however is variable, depending on the dose rate and the route of administration and also on the host species (Guerrero, 1984). Under field conditions in a tropical environment closantel, at an oral dose of 7.5 mg kg- 1 gave virtually complete protection against H. contortus for at least 4 weeks in sheep (Owen, 1988). However, there is some uncertainty about the development of resistance against closantel since selection of resistant worms may be enhanced if the sustained activity is associated with a prolonged period of decreasing drug concentration (Dash, 1986). In order to preserve the full potential of closantel it is recommended that in Kenya the drug be used for strategic treatment in alternation with broad-spectrum anthelmintics.
Acknowledgements This study was supported by a grant from the Agriculture Research Fund (Grant No. A F R / L S K P / R C - U S A I D / l 1 3 2 / 1 ) under the auspices of the Kenya Agriculture Research Institute (KARI) and the author is most grateful. Thanks are also extended to Mr. J.W. Ngotho for his excellent technical assistance.
References Burg, R.W., Miller, B.M., Baker, E.E., Birbaum, J., Curfie, S.A., Hartman, R., Kong, Y.-L., Monaghan, R.L., Olson, G., Putter, I., Tunac, J.B., Wallick, H., Stapley, E.O., Oiwa, R., Omura, S., 1979. Avermectins, new family of potent anthelmintic agents: producing organism and fermentation. Antimicrob. Agents Chemother. 15, 361-367. Carmichael, I., Visser, R., Schneider, D., Stoll, M., 1987. Haemonchus contortus resistance to ivermectin. J. S. Aft. Vet. Assoc. 58, 93. Craig, T.M., Miller, D.K., 1990. Resistance by Haemonchus contortus to ivermectin in Angora goats. Vet. Rec. 126, 580. Dash, K.M., 1986. Control of helmithosis in lambs by strategic treatment with closantel and broad-spectrum anthelmintics. Aust. Vet. J. 65, 4-8. Echevarria, F.A.M., Trindade, G.N.P., 1989. Anthelmintic resistance by Haemonchus contortus to ivermectin in Brazil: a preliminary report. Vet. Rec. 124, 147-148. Egerton, J.R., Suhayda, D., Eary, C.H., 1988. Laboratory selection of Haemonchus contortus for resistant to ivermectin. J. Parasitol. 74, 614-617. Giordano, D.J., Tritschler, J.P., Coles, G.C., 1988. Selection of ivermectin resistant Trichostrongylus colubriformis in lambs. Vet. Parasitol. 30, 139-148. Guerrero, J., 1984. Closantel: a review of its antiparasitic activity. Prev. Vet. Med. 2, 317-327. Hotson, I.K., Campbell, N.J., Smeal, M.G., 1970. Anthelmintic resistance in Trichostrongylus colubriformis. Aust. Vet. J. 46, 356-360. I2 Jambre, L.F., 1993. Genetics of parasitic nematodes. J. Cell. Biochem. (Suppl. 17c) 103. McKellar, Q.A., Marriner, S.E., 1987. Comparison of the athelmintic efficacy of oxfendazole or ivermectin administered orally and ivermectin administered subcutaneously to sheep during the periparturient period. Vet. Rec. 120, 383-386. Ministry of Agriculture, Fisheries and Food, 1986. Manual of Veterinary Parasitological Laboratory Techniques. HMSO, London, pp. 231.
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Mwamachi, D.M., Audho, J.O., Thorpe, W., Baker, R.C., 1995. Evidence for multiple anthelmintic resistance in sheep and goats reared under the same management in coastal Kenya. Vet. Parasitol. 60, 303-313. Owen, I.L., 1988. Field trials with closantel and Haemonchus contortus in sheep in Papua New Guinea. Aust. Vet. J. 65,267-270. Rolfe, P.F., Boray, J.C., Fitzgibbon, C., Parsons, G., Kemsley, P., Sangster, N., 1990. Closantel resistance in Haemonchus contortus from sheep. Aust. Vet. J. 67, 29-31. Van Wyk, J.A., Malan, F.S., 1988. Resistance in field strains of Haemonchus contortus to ivermectin, closantel, rafoxanide and the bezimidazoles in South Africa. Vet. Rec. 123, 226-228. Waller, P,J., 1987. Anthelmintic resistance and the future for roundworm control. Vet. Parasitol. 25, 177-191. Waruiru, R.M., Maingi, N., Gichanga, E.J., 1991. The prevalence of anthelmintic resistance in sheep in three districts of Kenya. Bull. Anim. Hlth. Pro. Afr. 39, 423-428. Wood, I.B., Amaral, N.K., Bairden, K., Dancan, J.L., Kassai, T., Malone, J.B. Jr., Pankavich, J.A., Reinecke, R.K., Slocombe, O., Taylor, S.M., Vercruysse, J., 1995. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) second edition of guidelines for evaluating the efficacy of anthelmintics in ruminants (bovine, ovine, caprine). Vet. Parasitol. 58, 181-213.
Veterinary Parasitology 73 (1997) 65-71
Efficacy of closantel, albendazole and levamisole on an ivermectin resistant strain of Haemonchus contortus in sheep R.M. Waruiru
*
Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine (Kabete Campus). University of Nairobi, P.O. Box 29053, Nairobi, Kenya Received 19 November 1996; accepted 4 April 1997
Abstract Following evidence of reduced efficacy of oral ivermectin in a field population of Haemonchus contortus on a sheep farm in Kenya, this strain of the parasite was submitted to a controlled anthelmintic test. One hundred and twenty worm-free lambs were randomly split into two groups of 60. Each lamb in the first group was infected with 5000 third stage larvae (L 3) of the suspected resistant strain the remaining 60 lambs were each infected with 5000 (L 3) of a H. contortus strain of known susceptibility to the major groups of anthelmintic compounds used in sheep. On day 28 post-infection, each group was subdivided according to egg counts and at random into six sub-groups of ten lambs, each of which was treated with closantel at 5.0 mg k g - l, albendazole at 5.0 mg kg 1, levamisole at 7.5 mg k g - i , oral ivermectin at 0.2 mg k g - I or injectable (Inj.) ivermectin at 0.2 mg k g - ~, or was left as an untreated control. At slaughter, 10 days later, all the anthelmintics had resulted in reduced worm burdens in animals infected with the susceptible strain (albendazole 99.0%, levamisole 99.5%, closantel, Inj. and oral ivermectin 100%). By contrast, in the lambs infected with the suspected resistant strain, closantel was 100% effective, but inj. ivermectin, oral ivermectin, albendazole and levamisole reduced worm counts by 47.6%, 24.2%, 38.5% and 41.4%, respectively. Anthelmintic resistance to the other chemical groups had been confirmed previously on this farm and although benzimidazoles and levamisole had not been used for the last 4 years, this strain was still highly resistant to albendazole and levamisole; closantel remained as the only drug evaluated which was still effective against this population of H. contortus. © 1997 Elsevier Science B.V. Keywords: Haemonchus eontortus; Drug resistance; Sheep nematoda; Control methods-nematoda; Benzimidazole; Albendazole, Ivermectin; Levamisole, Closantel
* Corresponding author. Tel.: 254-2-631340; fax: 254-2-631487. 0304-4017/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 0 1 7 ( 9 7 ) 0 0 0 6 5 - 4
66
R.M. Waruiru / Veterinary Parasitology 73 (1997) 65-71
1. Introduction
Over the last 10-20 years, anthelmintic resistance of sheep nematodes to the widely used benzimidazole and levamisole groups of drugs has become an increasingly important problem in the major sheep-rearing areas of the world (Waller, 1987). The discovery of a chemically distinct group of anthelmintics, the avermectins (Burg et al., 1979), provided a welcome alternative. However, subsequent to the introduction of the avermectins in the early 1980s resistant sheep nematodes have now been identified. Ivermectin resistance has been selected for experimentally in both Haemonchus contortus (Egerton et al., 1988), where a high level of resistance was detected after seven generations of exposure to one-tenth of the normal recommmended dose, and in Trichostrongylus colubriformis (Giordano et al., 1988), after four generations had been subjected to various ivermectin dose rates. Under field conditions ivermectin resistance has appeared in H. contortus in sheep and goats in South Africa (Carmichael et al., 1987), in Brazil (Echevarria and Trindade, 1989) in the USA (Craig and Miller, 1990) and Kenya (Mwamachi et al., 1995). Ivermectin resistance also appears to be developing in Australia in 1-1. contortus and T. columbriformis (Le Jambre, 1993). Because of the widespread occurrence of resistance to the broad spectrum anthelmintics in H. contortus, closantel is being used widely to control this parasite in sheep and goats. Unfortunately, there are reports of resistance to closantel also, in South Africa (Van Wyk and Malan, 1988), Australia (Rolfe et al., 1990) and recently in Kenya (Mwamachi et al., 1995). The objective of this experiment was to evaluate the anthelmintic sensitivity of the suspected avermectin resistant strain to closantel, albendazole and levamisole treatments under controlled test conditions; a strain known to be susceptible was included for comparison. 2. Materials and methods 2.1. Experimental design
One hundred and twenty indigenous sheep of mixed breeds, sexes and aged between 8 to 9 months were housed in pens with concrete floors at the Kabete Campus, University of Nairobi. Lucerne hay, a commercial sheep concentrate, mineral salts and water were available ad libitum. On arrival, faecal samples were collected and examined by a modified McMaster technique (Ministry of Agriculture, Fisheries and Food, 1986), for the presence of helminth ova. The lambs had a mean faecal egg count of 675 (0-4700) eggs per gram-1 (epg) faeces and were immediately drenched with fenbendazole (Panacur®-Hoechst, Munich, Germany, 5 mg kg -l body weight), this treatment was repeated after 7 days. A second faecal examination 14 days later showed all animals to be negative for nematode eggs. The lambs were then split at random into two groups of 60 (Groups R and S). The Group R animals were infected with 5000 third stage larvae (L 3) of the suspected resistant strain of H. contortus (R.M. Waruiru, unpublished data, 1995). Following isolation, sheep infected with this field strain had been subjected to treatment with oral ivermectin at 0.2 mg kg ~ and the survivors of this treatment produced the
67
R.M. Waruiru/ Veterinary Parasitology 73 (1997) 65-71
Table 1 Allocation of animals to treatment groups Group Treatment
Dose (mg kg- ~)
Animal per treatment
Weightof heaviest animal (kg)
R infected with suspected avermectin resistant strain
R1 Closantel R2 Ivermectin(injectable)a R3 Ivermectin(oral) R4 Albendazole R5 Levamisole R6 Control
5.0 0.2 0.2 5.0 7.5 -
10 10 10 10 10 10
18.3 17.3 20.1 17.1 18.7 20.1
S infected with susceptible strain
S1 Closantel $2 Ivermectin(injectable) $3 Ivermectin(oral) $4 Albendazole $5 Levamisole $6 Control
0.2 0.2 5.0 7.5 5.0 -
10 10 10 10 10 10
19.2 18.4 17.5 19.0 17.7 16.8
aDrug administered subcutaneously; all other drugs given orally. suspected resistant larvae used in the present experiment. No benzimidazoles or levamisole treatments had been applied for at least 4 years, ivermectin being the only drug used on the farm where the suspected resistant strain was isolated. Previously, however, both groups of anthelmintics had been used fairly intensively to control H. contortus and resistance to BZs and LEV had been confirmed (Waruiru et al., 1991). The animals in Group S were infected with 5000 L 3 of a susceptible strain of H. contortus. At day 28 post-infection, animals in both groups were allocated, according to their faecal egg counts and at random, to one of the six treatments as shown in Table 1. Animals were treated with closantel (Flukiver®-Janssen Pharmeceutica, Beerse, Belgium, 5 mg kg -1 body weight), Inj. ivermectin (Ivomec®-MSD Agvet, Rahway, NJ, USA, 0.2 mg kg-1 body weight), oral ivermectin (Oramec®-MSD Agvet, 0.2 mg k g - i body weight), albendazole (Valbazen®-Smithkline, Beecham, U.K, 5 mg kg -1 bodyweight) and levamisole (Nilverm®-Wellcome Kenya Ltd., Nairobi, 7.5 mg kg-1 body weight), respectively, according to the manufacturer's recommendations. All animals in a given group received a standard dose based on the heaviest animal in the group. Ten animals in each group were the untreated controls (Table 1). The animals were slaughtered on day 10 after treatment (day 39 post-infection) and total worm burdens estimated as recommended by W o o d et al. (1995). 2.2. Statistics
Faecal egg and worm counts were tranformed according to the expression y = log10 ( c o u n t + 1) and analysis of variance performed for testing the main effects and interactions between strain of parasite and treatment. Taking into consideration the significant strain versus treatment interaction, multiple comparisons were conducted using Fisher's least significant difference test.
R,M, Waruiru / Veterinary Parasitology 73 (1997) 65-71
68
3. Results Results of total worm counts and faecal egg counts are shown in Tables 2 and 3, respectively. These results show clear differences in the efficacy of ivermectin, albendazole and levamisole against the two strains. Table 2 Mean worm burdens a (range) of sheep infected with two strains of H. contortus and their percentage reduction after treatment with closantel, ivermectin, albendazole and levamisole compared with untreated controls Strain
Treatment
Mean worm count
Reduction (%)
S
Closantel Ivermectin (injectable) Ivermectin (oral) Albendazole Levamisole Control
0b 0b 0b 24 b 14 b 2702 d
(0-0) (0-0) (0-0) (0-210) (0-1 I0) ( 1701-3480)
100 100 100 99.0 99.5 -
R
Closantel Ivermectin (injectable) Ivermectin (oral) Albendazole Levamisole Control
0b 649 c 1033 e 838 c 799 ~ 1363 c
(0-0) (24-1970) (61-2314) (310-2960) (281 - 1772) (484- 2280)
100 47.6 34.2 38.5 41.4 -
S, ivermectin susceptible; R, ivermectin resistant. aValues shown are arithimetic mean worm counts, but comparisons were made on log-transformed data. b'C'dResults followed by different superscript letters are significantly different ( P < 0.05).
Table 3 Effect of closantel, ivermectin, albendazole and levamisole on faecal egg output a in sheep infected with two strains of H. contortus Strain
Treatment
Mean egg count (range)
Closantel Ivermectin (injectable) Ivermectin (oral) Albendazole Levamisole Control
2843 b 2576 b 2684 b 2357 b 3408 b 3675 b
(1100-4200) (900-3700) (1300-3400) (700-2900) (1400-4400) (1000-4500)
0b 0b 0b 47 b 8b 3727 d
(0-0) (0-0) (0-0) (0-400) (0-100) (1100-4300)
100 100 100 98.7 99.8 + 1.4
Closantel Ivermectin (injectable) Ivermectin (oral) Albendazole Levamisole Control
1489 c 1541 c 1391 c 1441 ~ 1610 c 1827 c
(300-2700) (600-3100) (400-2100) (600-3000) (700-3200) (400-3100)
0b 3300 1489 e 275 a 3150 2118 e
(0-0) (0-800) (300-2900) (0-700) (100-1100) (600-3000)
100 84.4 29.7 87.0 85.1 + 15.9
Day 0
Reduction (%) Day 10
S, ivermecfin susceptible; R, ivermectin resistant. aValues shown are arithimetic mean worm egg counts, but comparisons were made on log-transformed data. b'C'dResults followed by different superscript letters in the same column are significantly different (P < 0.05).
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From the worm burdens of the controls, there were obvious differences in establishment rates of the two strains: 54% of the susceptible against 27.3% for the suspected resistant strain. This was reflected in the epg counts on the day of treatment (Day 0) which were also significantly lower in animals infected with the suspected resistant strain ( P < 0.05). 10 days later there was no statistical difference in epg ( P > 0.05) between the two control groups, although the suspected resistant strain still showed a much lower output than the susceptible strain (Table 3). The susceptible strain was equally susceptible to closantel, ivermectin, albendazole and levamisole, while the suspected resistant strain showed a high level of resistance to ivermectin, albendazole and levamisole. With the suspected resistant strain, there was no significant difference ( P > 0.05) in worm burden reduction between groups treated with Inj. ivermectin, oral ivermectin, albendazole and levamisole, these drugs showed an efficacy of 47.6%, 24.2% 38.5% and 41.4%, respectively. Closantel reduced worm counts of this resistant strain by 100% (Table 2). Faecal egg count (epg) was reduced by almost 100% by all five drugs in the animals infected with the susceptible strain. In the suspected resistant strain, closantel was also 100% effective, while there was insignificant ( P > 0.05) reduction in epg in the animals treated with oral ivermectin (Table 3). Inj. ivermectin, albendazole and levamisole significantly ( P < 0.05) reduced epg counts by 84.4%, 87.0% and 85.1%, respectively. However, these reductions were not reflected in the worm counts (Table 3).
4. Discussion The results obtained in this study clearly confirm iverrnectin resistance of a field strain of H. contortus (R.M. Waruiru, unpublished data, 1995). Further, the results indicate that this strain has retained benzimidazole and levamisole resistance despite the fact that benzimidazoles and levamisole had not been used for at least 4 years on the farm where it was isolated. Albendazole was highly efficient against the susceptible strain (99.0%), but for the resistant strain, although treatment depressed egg output by 87.0%, it only reduced worm burdens by 38.5%. These results confirm previous observations on the effect of benzimidazoles (BZs) on BZ-resistant strains, i.e. a temporary suppression of worm egg counts without a corresponding worm loss (Hotson et al., 1970). Ivermectin was highly effective against the susceptible strain, but in the resistant strain its efficacy ranged from 24.2% (oral ivermectin) to 47.6% (Inj. ivermectin). The difference in efficacy of the two formulations of ivermectin is consistent with the findings of McKellar and Marriner (1987) that treatment with injectable ivermectin resulted in a higher and more sustained level of the drug than oral ivermectin. Similar results were obtained by Craig and Miller (1990) in Angora goats. They found no difference in faecal egg counts between a group treated with oral ivermectin and controls, but a significant difference with injectable ivermectin. In the present study, of the four chemically distinct anthelmintics used, closantel was the only drug which proved highly effective against both the susceptible as well as the suspected resistant strain. Thus, closantel may be useful in preventing production losses
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on sheep farms where H. contortus has developed resistance to benzimidazole, levamisole and ivermectin classes of anthelmintics. One of the most interesting features of closantel is its persistent anthelmintic effect, which, however is variable, depending on the dose rate and the route of administration and also on the host species (Guerrero, 1984). Under field conditions in a tropical environment closantel, at an oral dose of 7.5 mg kg- 1 gave virtually complete protection against H. contortus for at least 4 weeks in sheep (Owen, 1988). However, there is some uncertainty about the development of resistance against closantel since selection of resistant worms may be enhanced if the sustained activity is associated with a prolonged period of decreasing drug concentration (Dash, 1986). In order to preserve the full potential of closantel it is recommended that in Kenya the drug be used for strategic treatment in alternation with broad-spectrum anthelmintics.
Acknowledgements This study was supported by a grant from the Agriculture Research Fund (Grant No. A F R / L S K P / R C - U S A I D / l 1 3 2 / 1 ) under the auspices of the Kenya Agriculture Research Institute (KARI) and the author is most grateful. Thanks are also extended to Mr. J.W. Ngotho for his excellent technical assistance.
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