Veterinary Parasitology 209 (2015) 278–280
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Haemonchus contortus resistance to monepantel in sheep R. Van den Brom ∗ , L. Moll, C. Kappert, P. Vellema Department of Small Ruminant Health, GD Animal Health, Arnsbergstraat 7, 7418 EZ Deventer, The Netherlands
a r t i c l e
i n f o
Article history: Received 31 October 2014 Received in revised form 23 February 2015 Accepted 24 February 2015 Keywords: Monepantel Zolvix® Resistance Haemonchus contortus Sheep The Netherlands
a b s t r a c t In a sheep farm in the Netherlands with a suspected Haemonchus contortus resistance to monepantel (Zolvix® , Novartis Animal Health), a fecal egg count reduction test was carried out in two groups of lambs, according to the method of the World Association for the Advancement of Veterinary Parasitology. Group 1 was the untreated control group, and group 2 was treated with monepantel at the manufacturer’s recommended dose rate. Efficacy of treatment with monepantel was 0%. Larval identification of pre- and post-treatment coprocultures revealed 100% H. contortus larvae. On this farm, after a perceived reduction in efficacy of ivermectin and doramectin, the sheep farmer started using monepantel in July 2012, and since then, monepantel was used as the sole anthelmintic. Breeding sheep were treated twice each year in 2013 and 2014, and lambs two times in 2012, four times in 2013, and three times in 2014, before monepantel resistance was suspected, and confirmed three weeks later. Although the frequency of monepantel treatments on this farm was relatively high with treatments on thirteen separate occasions in two years time, possibly establishing favorable conditions for a competitive advantage for resistant H. contortus, it is remarkable that resistance to monepantel was established in such a very short period. This study confirms, to the best of our knowledge, the first case of H. contortus resistance to monepantel occurring in the field. © 2015 Elsevier B.V. All rights reserved.
Parasitic gastroenteritis is a common problem in small ruminants, interfering with animal health and welfare, and causing substantial economic losses (Sargison, 2012). To cope with this problem, broad-spectrum anthelmintics in three groups with a different mode of action have been used in the field for decades, the benzimidazoles (group 1), imidazothiazoles (group 2), and macrocyclic lactones (group 3). Recently, two new classes of anthelmintics have been introduced. In 2008, monepantel, the first anthelmintic in the group of the amino-acetonitrile derivatives (AAD, group
∗ Corresponding author at: Department of Small Ruminant Health, GD Animal Health, P.O. Box 9, 7400 AA Deventer, The Netherlands. Tel.: +31 570 660556; fax: +31 570 660 119. E-mail address: [email protected] (R. Van den Brom). http://dx.doi.org/10.1016/j.vetpar.2015.02.026 0304-4017/© 2015 Elsevier B.V. All rights reserved.
4) was announced (Kaminsky et al., 2008), and in 2010, a combination of derquantel and abamectin (group 5) was released (Little et al., 2010). The amino-acetonitrile derivatives form a relatively new group of anthelmintics, and monepantel as its only representative was launched in 2009 in New Zealand (Scott et al., 2013). Several studies showed that monepantel was fully effective against multiple resistant nematodes (Kaminsky et al., 2008, 2011; George et al., 2012; Sager et al., 2012). Therefore, monepantel was presented as a solution in case of multiple resistance to anthelmintics in all three classical groups (Kaminsky et al., 2008). However, within two years after its introduction, resistance to Teladorsagia circumcincta and Trichostrongylus colubriformis was confirmed in goats and sheep in New Zealand (Scott et al., 2013).
R. Van den Brom et al. / Veterinary Parasitology 209 (2015) 278–280
Introduction of monepantel in the Netherlands took place in 2011, and in 2014, lambs on a Dutch sheep farm became unthrifty, lethargic, had anemia, and seven out of 131 lambs died within 10 days after treatment with monepantel. A post-treatment egg count in a pooled fecal sample, collected 14 days after treatment, resulted in high strongyle-type egg counts. Monepantel resistance was suspected and a study was conducted aiming (1) at investigating whether or not resistance was existing, and (2) at identifying the genus and species of nematodes involved by larval culture, before and after treatment. On the farm with sixty Swifter and Swifter crossbred ewes, the farmer started using monepantel (Zolvix® , 2.5% Oral Solution for Sheep, 2.5 mg/kg bodyweight, Novartis Animal Health) in July 2012, after a perceived reduction in efficacy of ivermectin and doramectin. Since then, monepantel was used as the sole anthelmintic. Breeding ewes were treated twice each year in 2013 and 2014, and lambs two times in 2012, four times in 2013, and three times until July 2014. Following the last treatment, lambs did not respond well to treatment and seven died within 10 days after treatment. Subsequently, two groups of 10 lambs were randomly selected out of a flock of approximately eighty lambs. Lambs were weighed, marked and individually identified. Lambs in group 1 remained as untreated controls. Lambs in group 2 were treated with monepantel, at the manufacturer’s recommended dose rate. Before (day 0) and after (day 10) treatment individual fecal samples were collected. A fecal egg count reduction test (FECRT) was carried out according to the method of the World Association for the Advancement of Veterinary Parasitology (WAAVP) (Coles et al., 1992). Fecal egg counts (FEC) were performed using a modified McMaster method with a sensitivity of 17 eggs per gram (EPG), as also was performed in an earlier Dutch study on anthelmintic resistance (Van den Brom et al., 2013). The percentage reduction was calculated according to the following formula: Efficacy = 100 × (1 − arithmetic mean EPG of the treated group/arithmetic mean EPG of the control group). A composite larval culture was made of each group, and coprocultures were incubated for one week under controlled circumstances at 28 ◦ C, and thereafter processed according to the method described by Roberts and O’Sullivan (1950). For each group, a maximum of one hundred larvae were identified according to Eckert (1960). Fecal egg count reductions (FECR) were calculated using the RESO FECRT analysis program version 4 (http://sydney. edu.au/vetscience/sheepwormcontrol/) for Excel. Interpretation of the FECRT results were as described by the WAAVP (Coles et al., 1992). Fecal egg count results are shown in Table 1. At day 0 and day 10, all fecal samples of the lambs, both in the control and the monepantel group, were positive for strongyle-type eggs with a mean post-treatment EPG of 9185 and 13,352, respectively. The calculated efficacy of monepantel treatment was 0%. Larval identification of pre- and post-treatment coprocultures revealed 100% H. contortus larvae in the treated group, and 100 and 99% H. contortus larvae in the control group, respectively. The remaining larvae were identified as Trichostrongylus species.
279
This study confirms, to the best of our knowledge, the first case of H. contortus resistance to monepantel under field conditions. This resistance occurred on a sheep farm in the Netherlands, within two years after its first use. On this farm, the sheep farmer started using monepantel in July 2012, and since then, monepantel was used as the sole anthelmintic on thirteen different occasions, four occasions in ewes and nine in lambs. Although monepantel resistance had already been found under experimental conditions in Caenorhabditis elegans and in H. contortus before its introduction in 2009 (Kaminsky et al., 2008; Rufener et al., 2009, 2010), Kaminsky et al. (2013) did not find a change in the genetic make-up of H. contortus after its exposure to subcurative levels of monepantel for 16 generations in sheep, suggesting that resistance to monepantel in H. contortus was not likely to develop within a few generation cycles. Nevertheless, in a study in New Zealand, resistance to T. circumcincta and T. colubriformis was confirmed in goats and sheep, after only using this anthelmintic for a period of two years and on seventeen separate occasions (Scott et al., 2013). In the present study, H. contortus resistance was demonstrated after monepantel had been used on at most thirteen separate occasions within two years. Results from both studies suggest that resistance to monepantel is able to develop in a limited number of generation cycles of nematodes. On the farm in our study, the frequency of monepantel treatments was relatively high. Generally, under Dutch circumstances, ewes are advised to be treated only once a year after lambing and lambs are advised to be treated based on possibility of using clean pastures in combination with fecal egg counts. Nevertheless, it is unknown why resistance was established in such a very short period. In the New Zealand study (Scott et al., 2013), the presence of goats was suggested to be a significant factor. Firstly, goats have a lesser ability to establish an effective immunity to gastrointestinal nematodes as adults, requiring treatment of all age classes more often, resulting in a lesser potential role for refugia. Secondly, because goats have a different metabolism, medicines are possibly cleared more quickly, and parasites may be exposed to lower concentrations of anthelmintics for shorter periods especially if a sheep dosage is used. On the farm in our study, no goats were present, and therefore, goats did not play a role in resistance development. Both the New Zealand farm and the farm in our study have in common that monepantel was used as the sole anthelmintic, while Kaminsky et al. (2013) strongly recommended not to use monepantel without rotation with other anthelmintics. Interestingly, in both the New Zealand study (Scott et al., 2013) and our study, efficacy levels of monepantel were unexpectedly low, with 0%, 0% and 9% efficacy for H. contortus, T. circumcincta and T. colubriformis, respectively. In contrast, resistance to other anthelmintincs seems to develop more gradually, resulting in higher efficacy levels when resistance is first confirmed. Whether or not this is influenced by the different mode of action of monepantel is unknown. Although there are still unknown issues, we can conclude from our finding that this rapid and strong resistance development to one of the newest anthelmintic drugs reinforces the urgent need to restrict the use of
280
R. Van den Brom et al. / Veterinary Parasitology 209 (2015) 278–280
Table 1 Results of fecal egg counts (number of positive samples, mean eggs per gram, range (minimum–maximum)) and results of fecal egg count reduction test (reduction percentage and 95% CL). Strongyle type
Prevalence Mean Range Reduction Upper 95% CL Lower 95% CL
Control
Monepantel
Day 0
Day 10
Day 0
Day 10
10/10 10,507 3667–16550
10/10 9185 5867–14483 NA
10/10 12,690 5817–20600
10/10 13,352 4433–26233 0.0 3.7 0.0
CL, confidence level; NA, not applicable.
anthelmintics, and to reconsider gastrointestinal nematode control in small ruminants. Contributors All authors were involved in the study design. LM and CK sampled and treated the animals. LM did the data analysis. RVDB wrote the initial version of the manuscript, and all other authors gave their input and approved the final manuscript. Conflict of interest All authors declare to have no conflicts of interest regarding the information provided in this manuscript. Acknowledgements Monitoring of Small Ruminant Health is financially supported by the Dutch Ministry of Economic Affairs (EZ) and the Product Board for Livestock and Meat (PVV). We would like to thank our colleagues Coen Hegeman and Edwin Tuller, for performing the fecal egg counts and larval cultures and identification. Additionally, we would like to thank the farmer and his wife for their cooperation. References Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World Association for the Advancement of Veterinary Parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 44, 35–44. Eckert, J., 1960. Die Diagnose des Magen-darmstrongylidenbefalles des Schafes durch Differenzierung der freilebenden dritten Larven. Zbl. Vet. Med. 7, 612–630 [article in German].
George, S.D., George, A.J., Stein, P.A., Rolfe, P.F., Hosking, B.C., Seewald, W., 2012. The comparative efficacy of abamectin, monepantel and an abamectin/derquantel combination against fourth-stage larvae of a macrocyclic lactone resistant Teladorsagia spp. isolate infecting sheep. Vet. Parasitol. 188, 190–193. Kaminsky, R., Ducray, P., Jung, M., Clover, R., Rufener, L., Bouvier, J., Schorderet Weber, S., Wenger, A., Wieland-Berghausen, S., Goebel, T., Gauvry, N., Pautrat, F., Skripsky, T., Froelich, O., Komoin-Oka, C., Westlund, B., Sluder, A., Maser, P., 2008. A new class of anthelmintics effective against drug-resistant nematodes. Nature 452, 176–180. Kaminsky, R., Bapst, B., Stein, P.A., Strehlau, G.A., Allan, B.A., Hosking, B.C., Rolfe, P.F., Sager, H., 2011. Differences in efficacy of monepantel, derquantel and abamectin against multiresistent nematodes of sheep. Parasitol. Res. 109, 19–23. Kaminsky, R., Pradervand, E., Beech, R., Nilson, D., Rufener, L., 2013. Drug sensitivity monitoring of anthelmintics including the recently available monepantel. In: Proceedings 8th International Sheep Veterinary Congress, Rotorua, New Zealand, 18–23 February 2013. Little, P.R., Hodge, A., Watson, T.G., Seed, J.A., Maeder, S.J., 2010. Field efficacy and safety of an oral formulation of the novel combination anthelmintic, derquantel–abamectin, in sheep in New Zealand. N. Z. Vet. J. 58, 121–129. Roberts, F.H.S., O’Sullivan, P.J., 1950. Methods for egg counting and larval cultures for strongyles infesting the gastrointestinal tract of cattle. Aust. J. Agr. Res. 1, 99–102. Rufener, L., Maser, P., Roditi, I., Kaminsky, R., 2009. Haemonchus contortus acetylcholine receptors of the DEG/3 subfamily and their role in sensitivity to monepantel. PLoS Pathog. 5, e1000380. Rufener, L., Keiser, J.L., Kaminsky, R., Maser, P., Nilsson, D., 2010. Phylogenomics of ligand-gated ion channels predicts monepantel effect. PLoS Pathog. 6, e1001091. Sager, H., Bapst, B., Strehlau, G.A., Kaminsky, R., 2012. Efficacy of monepantel, derquantel and abamectin against adult stages of a multiresistent Haemonchus contortus isolate. Parasitol. Res. 111, 2205–2207. Sargison, N.D., 2012. Pharmaceutical treatments of gastrointestinal nematode infections of sheep – future of anthelmintic drugs. Vet. Parasitol. 189, 79–84. Scott, I., Pomroy, W.E., Kenyon, P.R., Smith, G., Adlington, B., Moss, A., 2013. Lack of efficacy of monepantel against Teladorsagia circumcincta and Trichostrongylus colubriformis. Vet. Parasitol. 198, 166–171. Van den Brom, R., Moll, L., Borgsteede, F.H.M., van Doorn, D.C.K., Lievaart Peterson, K., Dercksen, D.P., Vellema, P., 2013. Multiple anthelmintic resistance of Haemonchus contortus, including a case of moxidectin resistance, in a Dutch sheep flock. Vet. Rec. 173, 552.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Haemonchus contortus resistance to monepantel in sheep R. Van den Brom ∗ , L. Moll, C. Kappert, P. Vellema Department of Small Ruminant Health, GD Animal Health, Arnsbergstraat 7, 7418 EZ Deventer, The Netherlands
a r t i c l e
i n f o
Article history: Received 31 October 2014 Received in revised form 23 February 2015 Accepted 24 February 2015 Keywords: Monepantel Zolvix® Resistance Haemonchus contortus Sheep The Netherlands
a b s t r a c t In a sheep farm in the Netherlands with a suspected Haemonchus contortus resistance to monepantel (Zolvix® , Novartis Animal Health), a fecal egg count reduction test was carried out in two groups of lambs, according to the method of the World Association for the Advancement of Veterinary Parasitology. Group 1 was the untreated control group, and group 2 was treated with monepantel at the manufacturer’s recommended dose rate. Efficacy of treatment with monepantel was 0%. Larval identification of pre- and post-treatment coprocultures revealed 100% H. contortus larvae. On this farm, after a perceived reduction in efficacy of ivermectin and doramectin, the sheep farmer started using monepantel in July 2012, and since then, monepantel was used as the sole anthelmintic. Breeding sheep were treated twice each year in 2013 and 2014, and lambs two times in 2012, four times in 2013, and three times in 2014, before monepantel resistance was suspected, and confirmed three weeks later. Although the frequency of monepantel treatments on this farm was relatively high with treatments on thirteen separate occasions in two years time, possibly establishing favorable conditions for a competitive advantage for resistant H. contortus, it is remarkable that resistance to monepantel was established in such a very short period. This study confirms, to the best of our knowledge, the first case of H. contortus resistance to monepantel occurring in the field. © 2015 Elsevier B.V. All rights reserved.
Parasitic gastroenteritis is a common problem in small ruminants, interfering with animal health and welfare, and causing substantial economic losses (Sargison, 2012). To cope with this problem, broad-spectrum anthelmintics in three groups with a different mode of action have been used in the field for decades, the benzimidazoles (group 1), imidazothiazoles (group 2), and macrocyclic lactones (group 3). Recently, two new classes of anthelmintics have been introduced. In 2008, monepantel, the first anthelmintic in the group of the amino-acetonitrile derivatives (AAD, group
∗ Corresponding author at: Department of Small Ruminant Health, GD Animal Health, P.O. Box 9, 7400 AA Deventer, The Netherlands. Tel.: +31 570 660556; fax: +31 570 660 119. E-mail address: [email protected] (R. Van den Brom). http://dx.doi.org/10.1016/j.vetpar.2015.02.026 0304-4017/© 2015 Elsevier B.V. All rights reserved.
4) was announced (Kaminsky et al., 2008), and in 2010, a combination of derquantel and abamectin (group 5) was released (Little et al., 2010). The amino-acetonitrile derivatives form a relatively new group of anthelmintics, and monepantel as its only representative was launched in 2009 in New Zealand (Scott et al., 2013). Several studies showed that monepantel was fully effective against multiple resistant nematodes (Kaminsky et al., 2008, 2011; George et al., 2012; Sager et al., 2012). Therefore, monepantel was presented as a solution in case of multiple resistance to anthelmintics in all three classical groups (Kaminsky et al., 2008). However, within two years after its introduction, resistance to Teladorsagia circumcincta and Trichostrongylus colubriformis was confirmed in goats and sheep in New Zealand (Scott et al., 2013).
R. Van den Brom et al. / Veterinary Parasitology 209 (2015) 278–280
Introduction of monepantel in the Netherlands took place in 2011, and in 2014, lambs on a Dutch sheep farm became unthrifty, lethargic, had anemia, and seven out of 131 lambs died within 10 days after treatment with monepantel. A post-treatment egg count in a pooled fecal sample, collected 14 days after treatment, resulted in high strongyle-type egg counts. Monepantel resistance was suspected and a study was conducted aiming (1) at investigating whether or not resistance was existing, and (2) at identifying the genus and species of nematodes involved by larval culture, before and after treatment. On the farm with sixty Swifter and Swifter crossbred ewes, the farmer started using monepantel (Zolvix® , 2.5% Oral Solution for Sheep, 2.5 mg/kg bodyweight, Novartis Animal Health) in July 2012, after a perceived reduction in efficacy of ivermectin and doramectin. Since then, monepantel was used as the sole anthelmintic. Breeding ewes were treated twice each year in 2013 and 2014, and lambs two times in 2012, four times in 2013, and three times until July 2014. Following the last treatment, lambs did not respond well to treatment and seven died within 10 days after treatment. Subsequently, two groups of 10 lambs were randomly selected out of a flock of approximately eighty lambs. Lambs were weighed, marked and individually identified. Lambs in group 1 remained as untreated controls. Lambs in group 2 were treated with monepantel, at the manufacturer’s recommended dose rate. Before (day 0) and after (day 10) treatment individual fecal samples were collected. A fecal egg count reduction test (FECRT) was carried out according to the method of the World Association for the Advancement of Veterinary Parasitology (WAAVP) (Coles et al., 1992). Fecal egg counts (FEC) were performed using a modified McMaster method with a sensitivity of 17 eggs per gram (EPG), as also was performed in an earlier Dutch study on anthelmintic resistance (Van den Brom et al., 2013). The percentage reduction was calculated according to the following formula: Efficacy = 100 × (1 − arithmetic mean EPG of the treated group/arithmetic mean EPG of the control group). A composite larval culture was made of each group, and coprocultures were incubated for one week under controlled circumstances at 28 ◦ C, and thereafter processed according to the method described by Roberts and O’Sullivan (1950). For each group, a maximum of one hundred larvae were identified according to Eckert (1960). Fecal egg count reductions (FECR) were calculated using the RESO FECRT analysis program version 4 (http://sydney. edu.au/vetscience/sheepwormcontrol/) for Excel. Interpretation of the FECRT results were as described by the WAAVP (Coles et al., 1992). Fecal egg count results are shown in Table 1. At day 0 and day 10, all fecal samples of the lambs, both in the control and the monepantel group, were positive for strongyle-type eggs with a mean post-treatment EPG of 9185 and 13,352, respectively. The calculated efficacy of monepantel treatment was 0%. Larval identification of pre- and post-treatment coprocultures revealed 100% H. contortus larvae in the treated group, and 100 and 99% H. contortus larvae in the control group, respectively. The remaining larvae were identified as Trichostrongylus species.
279
This study confirms, to the best of our knowledge, the first case of H. contortus resistance to monepantel under field conditions. This resistance occurred on a sheep farm in the Netherlands, within two years after its first use. On this farm, the sheep farmer started using monepantel in July 2012, and since then, monepantel was used as the sole anthelmintic on thirteen different occasions, four occasions in ewes and nine in lambs. Although monepantel resistance had already been found under experimental conditions in Caenorhabditis elegans and in H. contortus before its introduction in 2009 (Kaminsky et al., 2008; Rufener et al., 2009, 2010), Kaminsky et al. (2013) did not find a change in the genetic make-up of H. contortus after its exposure to subcurative levels of monepantel for 16 generations in sheep, suggesting that resistance to monepantel in H. contortus was not likely to develop within a few generation cycles. Nevertheless, in a study in New Zealand, resistance to T. circumcincta and T. colubriformis was confirmed in goats and sheep, after only using this anthelmintic for a period of two years and on seventeen separate occasions (Scott et al., 2013). In the present study, H. contortus resistance was demonstrated after monepantel had been used on at most thirteen separate occasions within two years. Results from both studies suggest that resistance to monepantel is able to develop in a limited number of generation cycles of nematodes. On the farm in our study, the frequency of monepantel treatments was relatively high. Generally, under Dutch circumstances, ewes are advised to be treated only once a year after lambing and lambs are advised to be treated based on possibility of using clean pastures in combination with fecal egg counts. Nevertheless, it is unknown why resistance was established in such a very short period. In the New Zealand study (Scott et al., 2013), the presence of goats was suggested to be a significant factor. Firstly, goats have a lesser ability to establish an effective immunity to gastrointestinal nematodes as adults, requiring treatment of all age classes more often, resulting in a lesser potential role for refugia. Secondly, because goats have a different metabolism, medicines are possibly cleared more quickly, and parasites may be exposed to lower concentrations of anthelmintics for shorter periods especially if a sheep dosage is used. On the farm in our study, no goats were present, and therefore, goats did not play a role in resistance development. Both the New Zealand farm and the farm in our study have in common that monepantel was used as the sole anthelmintic, while Kaminsky et al. (2013) strongly recommended not to use monepantel without rotation with other anthelmintics. Interestingly, in both the New Zealand study (Scott et al., 2013) and our study, efficacy levels of monepantel were unexpectedly low, with 0%, 0% and 9% efficacy for H. contortus, T. circumcincta and T. colubriformis, respectively. In contrast, resistance to other anthelmintincs seems to develop more gradually, resulting in higher efficacy levels when resistance is first confirmed. Whether or not this is influenced by the different mode of action of monepantel is unknown. Although there are still unknown issues, we can conclude from our finding that this rapid and strong resistance development to one of the newest anthelmintic drugs reinforces the urgent need to restrict the use of
280
R. Van den Brom et al. / Veterinary Parasitology 209 (2015) 278–280
Table 1 Results of fecal egg counts (number of positive samples, mean eggs per gram, range (minimum–maximum)) and results of fecal egg count reduction test (reduction percentage and 95% CL). Strongyle type
Prevalence Mean Range Reduction Upper 95% CL Lower 95% CL
Control
Monepantel
Day 0
Day 10
Day 0
Day 10
10/10 10,507 3667–16550
10/10 9185 5867–14483 NA
10/10 12,690 5817–20600
10/10 13,352 4433–26233 0.0 3.7 0.0
CL, confidence level; NA, not applicable.
anthelmintics, and to reconsider gastrointestinal nematode control in small ruminants. Contributors All authors were involved in the study design. LM and CK sampled and treated the animals. LM did the data analysis. RVDB wrote the initial version of the manuscript, and all other authors gave their input and approved the final manuscript. Conflict of interest All authors declare to have no conflicts of interest regarding the information provided in this manuscript. Acknowledgements Monitoring of Small Ruminant Health is financially supported by the Dutch Ministry of Economic Affairs (EZ) and the Product Board for Livestock and Meat (PVV). We would like to thank our colleagues Coen Hegeman and Edwin Tuller, for performing the fecal egg counts and larval cultures and identification. Additionally, we would like to thank the farmer and his wife for their cooperation. References Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World Association for the Advancement of Veterinary Parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 44, 35–44. Eckert, J., 1960. Die Diagnose des Magen-darmstrongylidenbefalles des Schafes durch Differenzierung der freilebenden dritten Larven. Zbl. Vet. Med. 7, 612–630 [article in German].
George, S.D., George, A.J., Stein, P.A., Rolfe, P.F., Hosking, B.C., Seewald, W., 2012. The comparative efficacy of abamectin, monepantel and an abamectin/derquantel combination against fourth-stage larvae of a macrocyclic lactone resistant Teladorsagia spp. isolate infecting sheep. Vet. Parasitol. 188, 190–193. Kaminsky, R., Ducray, P., Jung, M., Clover, R., Rufener, L., Bouvier, J., Schorderet Weber, S., Wenger, A., Wieland-Berghausen, S., Goebel, T., Gauvry, N., Pautrat, F., Skripsky, T., Froelich, O., Komoin-Oka, C., Westlund, B., Sluder, A., Maser, P., 2008. A new class of anthelmintics effective against drug-resistant nematodes. Nature 452, 176–180. Kaminsky, R., Bapst, B., Stein, P.A., Strehlau, G.A., Allan, B.A., Hosking, B.C., Rolfe, P.F., Sager, H., 2011. Differences in efficacy of monepantel, derquantel and abamectin against multiresistent nematodes of sheep. Parasitol. Res. 109, 19–23. Kaminsky, R., Pradervand, E., Beech, R., Nilson, D., Rufener, L., 2013. Drug sensitivity monitoring of anthelmintics including the recently available monepantel. In: Proceedings 8th International Sheep Veterinary Congress, Rotorua, New Zealand, 18–23 February 2013. Little, P.R., Hodge, A., Watson, T.G., Seed, J.A., Maeder, S.J., 2010. Field efficacy and safety of an oral formulation of the novel combination anthelmintic, derquantel–abamectin, in sheep in New Zealand. N. Z. Vet. J. 58, 121–129. Roberts, F.H.S., O’Sullivan, P.J., 1950. Methods for egg counting and larval cultures for strongyles infesting the gastrointestinal tract of cattle. Aust. J. Agr. Res. 1, 99–102. Rufener, L., Maser, P., Roditi, I., Kaminsky, R., 2009. Haemonchus contortus acetylcholine receptors of the DEG/3 subfamily and their role in sensitivity to monepantel. PLoS Pathog. 5, e1000380. Rufener, L., Keiser, J.L., Kaminsky, R., Maser, P., Nilsson, D., 2010. Phylogenomics of ligand-gated ion channels predicts monepantel effect. PLoS Pathog. 6, e1001091. Sager, H., Bapst, B., Strehlau, G.A., Kaminsky, R., 2012. Efficacy of monepantel, derquantel and abamectin against adult stages of a multiresistent Haemonchus contortus isolate. Parasitol. Res. 111, 2205–2207. Sargison, N.D., 2012. Pharmaceutical treatments of gastrointestinal nematode infections of sheep – future of anthelmintic drugs. Vet. Parasitol. 189, 79–84. Scott, I., Pomroy, W.E., Kenyon, P.R., Smith, G., Adlington, B., Moss, A., 2013. Lack of efficacy of monepantel against Teladorsagia circumcincta and Trichostrongylus colubriformis. Vet. Parasitol. 198, 166–171. Van den Brom, R., Moll, L., Borgsteede, F.H.M., van Doorn, D.C.K., Lievaart Peterson, K., Dercksen, D.P., Vellema, P., 2013. Multiple anthelmintic resistance of Haemonchus contortus, including a case of moxidectin resistance, in a Dutch sheep flock. Vet. Rec. 173, 552.
