Efficacy of abamectin against ivermectin-resistant

Veterinary Parasitology 121 (2004) 277–283

Efficacy of abamectin against ivermectin-resistant strain of Trichostrongylus colubriformis in sheep Alka, R.M. Gopal, K.S. Sandhu∗ , P.K. Sidhu Department of Epidemiology and Preventive Veterinary Medicine, Punjab Agricultural University, Ludhiana 141004, Punjab, India Received 4 November 2003; received in revised form 19 February 2004; accepted 8 March 2004

Abstract The efficacy of two formulations of abamectin, i.e. oral and injectable was determined against ivermectin-resistant strain of T. colubriformis in sheep. Twenty-four lambs were infected with 10,000 third stage larvae of ivermectin-resistant strain of T. colubriformis. Twenty-four days postinfection, the lambs were divided randomly into four groups of six animals each according to egg counts. The first group was left untreated and kept as a control. The second group was treated with ivermectin (oral) at 0.2 mg kg−1 body weight. The third group was treated with oral formulation of abamectin at 0.2 mg kg−1 body weight. The fourth group was treated with injectable formulation of abamectin at 0.2 mg kg−1 body weight. Fecal egg count and controlled slaughter tests were employed to determine the efficacy of abamectin (oral and injection) against ivermectin-resistant strain of T. colubriformis in sheep. Reduction in arithmetic mean fecal egg counts achieved by ivermectin (oral), abamectin (oral) and abamectin (injection) was 66, 98 and 76%, respectively 10 days after treatment. Ivermectin (oral), abamectin (oral) and abamectin (injection) reduced arithmetic mean worm burden by 63, 97 and 74%, respectively. The findings demonstrated that abamectin oral formulation was more effective than abamectin injection against ivermectin-resistant strain of T. colubriformis in sheep. © 2004 Elsevier B.V. All rights reserved. Keywords: Trichostrongylus colubriformis; Ivermectin; Abamectin; Drug resistance; Efficacy; Sheep-nematoda

1. Introduction Gastrointestinal nematodosis is a major health problem in sheep. It severely affects animal production and inflicts serious economic losses. The major gastrointestinal nematodes ∗ Corresponding author. Tel.: +91-161-2406241; fax: +91-161-2400955. E-mail address: [email protected] (K.S. Sandhu).

0304-4017/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2004.03.007

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Alka et al. / Veterinary Parasitology 121 (2004) 277–283

include Trichostrongylus, Haemonchus and Teladorsagia spp. Under intensive management system, anthelmintic drugs are used routinely to control nematode infections due to their relative ease of application and high efficacy. However, the frequent use of anthelmintics may result in rapid development of anthelmintic-resistant nematodes (Martin et al., 1984). Development of resistance in parasites of sheep is throwing a major challenge to worm control practices. Resistance to benzimidazole anthelmintics was first reported in Australia (Smeal et al., 1968). The introduction of ivermectin in the early 1980s brought a revolution in the control of animal parasites and was accepted as a potent anthelmintic (Campbell and Benz, 1984). Unfortunately, resistance has developed to ivermectin and first indication of ivermectin resistance was reported from South Africa (Carmichael et al., 1987) against Haemonchus contortus isolated from sheep. Subsequently, there has been worldwide incidence of ivermectin resistance in various species of nematodes (Gopal et al., 1999). This burgeoning problem of ivermectin resistance in sheep parasites has drawn the attention of veterinarians and farmers to opt for alternative drugs and control strategies. Trials evaluating the efficacy of new compounds against ivermectin-resistant nematodes may assist in delaying the onset of ivermectin resistance. Abamectin (avermectin B1 ) and ivermectin are members of macrocyclic lactone anthelmintics derived from the bacterium Streptomyces avermitilis. Abamectin is a precursor to ivermectin. It differs from ivermectin in having a double-bond at the C22–23 position. Both drugs seem to share common mode of action, but they differ in their activity against nematode infections, abamectin being more active against nematodes than ivermectin (Shoop et al., 1995). Abamectin is highly effective against gastrointestinal nematodes of cattle (Heinz-Mutz et al., 1993; Kaplan et al., 1994) and ivermectin-resistant strain of Teladorsagia circumcincta in sheep (Leathwick et al., 2000). No previous report studying the efficacy of abamectin against ivermectin-resistant strain of Trichostrongylus colubriformis could be traced in literature. Therefore, the present study was planned to evaluate the efficacy of both oral and injectable formulations of abamectin against ivermectin-resistant strain of T. colubriformis in sheep.

2. Materials and methods The experiment was carried out in the Department of Epidemiology and Preventive Veterinary Medicine, Punjab Agricultural University, Ludhiana, India. Twenty-four mixed breed lambs of 8–10-week-old were purchased from Government Sheep Breeding Farm, Mattewara, Punjab (India). The lambs were effectively drenched with albendazole (Alzol Vet, Vets Farma Ltd., India) at 10 mg kg−1 body weight 7 days prior to administration of infection. The lambs were housed in an isolation area in semi-enclosed, sheltered and concrete-based units under strict quarantine conditions and fed a ration of chopped straw and concentrates with free access to water throughout the experiment. The lambs were acclimatized to diet and environment for 7 days before administration of infection. The ivermectin-resistant strain of T. colubriformis was obtained from Massey University, New Zealand. The infection with 10,000 infective larvae of the ivermectin-resistant strain of T. colubriformis was introduced to all lambs 7 days after drenching with albendazole.


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Twenty-four days post-infection, the lambs were ranked on the basis of their fecal egg counts and randomly divided into four groups of six animals each, so that each group had almost similar mean fecal egg counts. The first untreated group was kept as control. The second group was treated with oral formulation of ivermectin (Ivomec liquid for sheep and goats 0.08% (w/v); MSDAgvet, New Zealand) at 0.2 mg kg−1 body weight. The third group was treated with oral formulation of abamectin (Genesis oral drench 0.1% (w/v); Ancare, New Zealand) at 0.2 mg kg−1 body weight. The fourth group received injectable formulation of abamectin (Genesis injection 1% (w/v); Ancare, New Zealand) at 0.2 mg kg−1 body weight by subcutaneous route. Fecal samples were collected directly from the rectum of lambs and fecal egg counts estimated at 0, 7 and 14 and days post-infection and 0, 3, 5, 7 and 10 days post-treatment by a modified McMaster technique (Whitlock, 1948); 2 g of feces were homogenized in 28 ml of saturated NaCl solution, sieved and the two samples of filtrate pipetted into both chambers of McMaster counting slide. The slide was examined under light microscope at 100×. The total number of eggs were multiplied by 50 to calculate eggs per gram of feces. The lambs were slaughtered 10 days after treatment and the small intestine removed from each animal. The intestine was cut along its length and washed under tap water into a bucket. The washed intestine was then immersed in a solution containing pepsin (20 g), concentrated hydrochloric acid (10 ml) and water (600 ml) and incubated at 37 ◦ C for 2 h. Five percent aliquots of small intestine contents and pepsin digests were collected for worm counting. The aliquots were preserved in 5% formalin for worm counting. Total worm counts were done using stereo zoom microscope (RSM-8; Radical Instruments, India). Efficacy of each treatment was determined by calculating percent reduction in arithmetic mean fecal egg count (Coles et al., 1992) and percent reduction in arithmetic mean worm count (McKenna, 1998) of treated group in comparison to untreated group using formulae: T2 C1 Percentage reduction in faecal egg count = 1 − × 100 × T1 C2 T Percentage reduction in worm count = 1 − × 100 C T and C indicate the mean egg count/worm count for treatment and control group, respectively. The subscripts 1 and 2 designate mean egg count before and after treatment, respectively. Multiple comparisons of fecal egg counts or worm counts at slaughter were made on log (count + 1) data using Tukey’s test. 3. Results The mean establishment rate of infection in sheep infected with ivermectin-resistant strain of T. colubriformis was 46% following administration of 10,000 infective larvae of ivermectin-resistant strain of T. colubriformis to each lamb. Fecal egg count and controlled efficacy tests were employed to determine the efficacy of abamectin against ivermectinresistant strain of T. colubriformis in sheep.

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Treatment

Eggs per gram of feces 0 DPTb

3 DPT

5 DPT

7 DPT

10 DPT

Untreated Ivermectin % Reduction

4448.8 (1250–5977) 4630 (1600–10500)

4367 (2335–6100) 1440 (750–2200) 68.31

3881.8 (2409–4900) 1190.4 (450–1650) 70.53

4539.2 (2300–7196) 1330 (1100–1900) 71.85

4250 (2050–6050) 1490 (1400–1700) 66.31

Abamectin(oral) % Reduction

4099 (1950–10100)

930∗∗ (50–3900) 76.89

100∗∗ (0–200) 97.20

60∗∗ (0–150) 98.56

80∗∗ (0–150) 97.96

Abamectin (injection) % Reduction

4522.2 (2100–7850)

1062.8 (350–2950) 76.06

840 (100–1500) 78.71

890 (0–2600) 80.71

1050 (0–1950) 75.69

Values in parentheses represent the range of mean fecal egg counts. a n, number of animals in each group. b Days post-treatment. ∗∗ Values are significantly different from control group (P > 0.05) on the respective treatments.


Table 1 Mean and percentage reduction in fecal egg counts of lambs infected with ivermectin-resistant strain of T. colubriformis following administration of ivermectin, abamectin (oral) or abamectin (injection) (n = 6)a


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Table 2 Mean and percentage reduction in nematode worm counts of lambs infected with ivermectin-resistant strain of T. colubriformis following administration of ivermectin, abamectin (oral) and abamectin (injection) (n = 6) Treatment group

Nematode worm count

% Reduction

Untreated Ivermectin Abamectin (oral) Abamectin (injection)

4592 (4100–5140) 1712 (960–2480) 144∗∗ (0–400) 1204 (200–3560)

62.72 96.86 73.78

Values in parentheses represent the range of mean worm counts. n, number of animals in each group. ∗∗ Values are significantly different from control group (P > 0.05).

3.1. Fecal egg count reduction test The results of fecal egg counts of sheep infected with ivermectin-resistant strain of T. colubriformis at the time of treatment and 3, 5, 7 and 10 days post-treatment for four treatments of control, ivermectin oral, abamectin oral and abamectin injectable are presented in Table 1. Following albendazole treatment on day −7 the lambs all had negative fecal egg counts during the prepatent period (days 0, 7, 14 post-challenge). It demonstrated that double dose of albendazole was quite effective in rendering the animals worm-free before the start of experiment. Abamectin oral formulation achieved 77, 97, 98.6 and 98% reduction in arithmetic mean fecal egg counts at 3, 5, 7 and 10 days, respectively, after administration. Abamectin S/C injection reduced mean fecal egg counts by 76, 79, 81 and 76% at 3, 5, 7 and 10 days, respectively, after administration. Ivermectin oral formulation showed 68, 70.5, 72 and 66% reduction in arithmetic mean fecal egg counts at 3, 5, 7 and 10 days, respectively, after treatment. Statistical analysis revealed that mean fecal egg counts of control group were not significantly (P > 0.05) different from that of ivermectin oral and abamectin injectable group at all intervals but significantly (P < 0.05) higher than abamectin oral group at 3, 5, 7 and 10 days after treatment. 3.2. Controlled efficacy test Total mean worm burdens for the control, ivermectin oral, abamectin oral and abamectin injectable groups are shown in Table 2 together with the calculated efficacies for each of the treated groups. Ivermectin, abamectin (oral) and abamectin (injection) reduced mean worm burdens by 63, 97 and 74%, respectively. The mean worm count of abamectin oral group was significantly (P < 0.05) less than control, ivermectin oral and abamectin injectable groups whereas mean worm counts of control, ivermectin oral and abamectin injectable group were not significantly (P > 0.05) different from each other.

4. Discussion Anthelmintic resistance is a heritable change in the ability of individual parasite to survive the recommended therapeutic dose of an anthelmintic drug (Taylor and Hunt, 1989).

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The development and spread of anthelmintic resistance is associated with numerous factors viz., frequency of anthelmintic treatments, dosage of anthelmintics and management practices. Selection pressure for resistance increases with the increase in the number of worms surviving the anthelmintic treatment. Strategies to minimize the selection for resistance include sound management practices and effective use of anthelmintics. It is suggested that most effective compound should be used in treatment programs. According to World Association for the Advancement of Veterinary Parasitology (WAAVP) recommendations a highly effective anthelmintic should have efficacy of more than 98% (Wood et al., 1995). Ivermectin and abamectin are broad spectrum anthelmintics, which differ in their efficacy against nematode infections (Shoop et al., 1995). Resistance to ivermectin against T. colubriformis has been reported against experimental and naturally acquired infections in sheep (Shoop et al., 1990; Gopal et al., 1999). The movement of animals harboring ivermectin-resistant parasites from one country to other country has been reported (Varady et al., 1993). Therefore, it is essential to quarantine animals arriving on new land and treat them with an effective drug or with a highly effective combination of drugs (Dobson et al., 2001). To find out alternative control measure, in the present study efficacy of abamectin (oral and injection) was evaluated against ivermectin-resistant strain of T. colubriformis in sheep. The reduced efficacy of ivermectin against ivermectinresistant strain of T. colubriformis confirmed the ivermectin-resistant status of the strain. The results of both fecal egg count and worm count reduction tests demonstrated that abamectin oral formulation was markedly effective against ivermectin-resistant strain of T. colubriformis. Similar results with abamectin oral formulation were achieved against ivermectin-resistant strain of T. circumcincta (Leathwick et al., 2000). The higher efficacy of oral formulation of abamectin against ivermectin-resistant strain of T. colubriformis than ivermectin oral formulation might be due to differences in the structure and pharmacokinetics of two compounds. However, abamectin injection formulation did not significantly reduce fecal egg and worm counts of ivermectin-resistant strain of T. colubriformis. Similar differences in the efficacy of oral and injectable formulations of moxidectin were obtained against ivermectin-resistant strain of T. colubriformis (Gopal et al., 2001). The difference in the efficacy of two formulations of abamectin may be due to differences in their pharmacokinetics. No literature could be traced on pharmacokinetics of abamectin in sheep. The pharmacokinetic studies of abamectin using oral and injectable formulations need to be performed in sheep to explain the difference in the efficacy of two formulations. Side-resistance “a state in which drug-selected population has a gene(s) coding for a mechanism that defeats the toxicity of drugs within a mode of action family” (Shoop, 1993) limits the prolonged use of two or more drugs having similar mode of action. It has been reported that side-resistance occurs between the macrocyclic compounds (Conder et al., 1993; Watson et al., 1996). Although in this study orally administered abamectin had a high efficacy against an ivermectin-resistant strain of T. colubriformis, its prolonged use against resistant nematodes would almost certainly lead to the development of side-resistance against this compound too.


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