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Determination of ivermectin efficacy against cyathostomins and Parascaris equorum on horse farms using selective therapy
The Veterinary Journal 188 (2011) 44–47
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Determination of ivermectin efficacy against cyathostomins and Parascaris equorum on horse farms using selective therapy Mette L. Larsen a, Christian Ritz b, Stig L. Petersen c, Martin K. Nielsen a,* a
Department of Large Animal Sciences, Faculty of Life Sciences, University of Copenhagen, DK-2630 Taastrup, Denmark Department of Basic Sciences and Environment, Faculty of Life Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark c EquiLab Laboratory, DK-3550 Slangerup, Denmark b
a r t i c l e
i n f o
Article history: Accepted 5 March 2010
Keywords: Ivermectin Egg reappearance Efficacy Parascaris equorum Strongyles
a b s t r a c t Ivermectin resistance has recently been described in Parascaris equorum and there have been reports from several countries of a shortened egg reappearance period (ERP) following ivermectin treatment for cyathostomins. This study was aimed at determining the efficacy of ivermectin in treating cyathostomins and P. equorum in Danish horses. A total of 196 animals were selected from 52 farms, all of which were using a selective anthelmintic treatment strategy. ERP was investigated with weekly samples from 96 horses from nine farms. Horses were treated with ivermectin oral paste by their owners at an estimated dose rate of 0.2 mg/kg. Overall, faecal egg counts were reduced 10–14 days after treatment by 96.9% and 100% for P. equorum and cyathostomins, respectively. Mean faecal egg count reductions at 4 and 6 weeks post treatment were 99.5% and 96.9%, respectively. No signs of developing ivermectin resistance were found in either cyathostomins or P. equorum in the studied horses. Ó 2010 Elsevier Ltd. All rights reserved.
Introduction Cyathostomins are ubiquitous parasites of grazing horses, and are considered the most important parasites in horses (Love et al., 1999) although large strongyles are still a potential threat when present. In foals and young horses, the roundworm Parascaris equorum also constitutes a major threat to equine health (Clayton, 1978). Over recent decades, increasing resistance of cyathostomins to several groups of anthelmintics has been reported (Kaplan, 2002, 2004). Furthermore, recent reports suggest that in some instances the cyathostomin egg reappearance period (ERP) following treatment with ivermectin has been reduced to 4–5 weeks (Lyons et al., 2008; Molento et al., 2008; von Samson-Himmelstjerna et al., 2007) from the 9 weeks initially reported (Borgsteede et al., 1993). Lyons et al. (2009) showed that this reduction was due to apparent ivermectin resistance in the luminal L4 stage. Reports from several countries of P. equorum isolates showing apparent ivermectin resistance have also been published (Boersema et al., 2002; Hearn and Peregrine, 2003; Schougaard and Nielsen, 2007; Veronesi et al., 2009). These developments constitute a major concern for the equine industry as ivermectin is the most widely used equine anthelmintic (Nielsen et al., 2006), and it is unclear when, or even if, a new anthelmintic class will be launched for equine usage. To delay further development of anthelmintic resistance, recommendations have been made to reduce treatment intensity, and to * Corresponding author. Tel.: +45 35332842; fax: +45 35332880. E-mail address: [email protected] (M.K. Nielsen). 1090-0233/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2010.03.009
base treatments on parasite surveillance (Kaplan, 2002; Nielsen et al., 2007). One widely recommended strategy is selective therapy, where faecal samples are taken from all horses and treatment is given only to individuals exceeding a predetermined cut-off egg count (Duncan and Love, 1991; Krecek et al., 1994; Little et al., 2003; Matthee and McGeoch, 2004). The use of selective therapy has been supported by changes in legislation and several European countries, including Denmark, Sweden, The Netherlands, Finland and Italy, now disallow prophylactic treatment and require a diagnosis before anthelmintics can be administered. In Denmark, where such legislation has been in place since 1999, the majority of horse establishments base their parasite control programs on selective therapy with faecal samples taken from all horses twice yearly (Nielsen et al., 2006). However, it remains unclear whether these restrictions have affected the development of anthelmintic resistance. The aims of this study were to assess the current efficacy of ivermectin against P. equorum and cyathostomins on Danish farms practicing selective therapy, and to determine cyathostomin ERP after ivermectin treatment in these horses. Materials and methods The study was carried out between September 1st and November 1st 2008.
Horses All investigated horses were from farms which had been using selective therapy for at least 2 years prior to the study, and which had been using ivermectin as the only anthelmintic for that period. On all farms faecal samples were taken from all
45
M.L. Larsen et al. / The Veterinary Journal 188 (2011) 44–47 horses twice a year, and individuals exceeding a faecal egg count (FEC) of 200 strongyle eggs per gram of faeces (EPG) or having a positive P. equorum egg count were treated. Farms were selected through a referral laboratory analysing faecal samples submitted from various regions in Denmark. A total of 196 horses from 52 farms were used in the study. Egg reappearance period The ERP following ivermectin treatment was determined on nine farms where at least 10 horses were treated. A total of 96 horses were included in the study. On the nine farms, 69 untreated horses with FECs <200 EPG were kept as a control group. There were P5 untreated horses per farm. Faecal egg counts All FECs were determined by the McMaster method with a detection limit of 50 EPG (Roepstorff and Nansen, 1998). Larval cultures were performed on two individual horses sampled at random from each study farm to determine if any large strongyle species were present. Treatment In Denmark, owners are allowed to give medical treatments to their horses only after passing an authorised course in medicine handling. The veterinarian prescribing the drug specifies the dosage, and the dosing is then carried out by the owner. Thus the liveweight of each horse was estimated by its owner who then gave their horse an oral dose of 0.2 mg/kg ivermectin paste (Noromectin vet, Biovet ApS). After ivermectin treatment, the first author then estimated each horse’s weight using a girth tape. Ivermectin efficacy Faecal samples were collected for faecal egg count reduction tests (FECRT) on the day of ivermectin treatment and 10–14 days post treatment. FECR was determined for each individual horse as:
linear models, mixed procedure). FEC post treatment was chosen as the response variable and efficacies were determined post modelling. FEC were analysed as a quantitative discrete variable. Explanatory variables were farm, horse, gender, date, dosage, age, FEC prior to treatment, mean FEC on the farm and proportion of treated horses on the farm. Horse and farm were chosen as random variables. Results were interpreted at the 0.05 level, and variables that were not significant were excluded to refine the model.
Results Only cyathostomins were identified in the larval cultures. The large majority of the horses were dosed correctly, or marginally overdosed, according to the weight estimated by weight girth tape measurements. Eight horses were marginally under dosed. The weight estimations are presented in Fig 1. Ivermectin efficacy One hundred and seventeen horses (59%) were excreting strongyle eggs only. These horses were from 10 different farms and had a mean age of 8.7 years (95% confidence interval (CI): 7.6–9.4) and mean FEC of 491 EPG (95% CI: 421–562). Seventy-nine (40%) of the treated horses were excreting P. equorum eggs prior to treatment. These horses were from 42 farms and had a mean age of 2.1 years (95% CI: 1.6–2.5). A majority of these horses (84%) were co-infected with strongyles; mean pre-treatment strongyle FEC was 323 EPG (95% CI: 262–385). The overall mean FECR 10–14 days following ivermectin treatment were 96.9% and 100% for P. equorum and strongyles, respectively.
FECR ¼ ðFECpretreatment —FECposttreatment Þ FECpretreatment 100% Arithmetic means for each group and each farm were determined from individual FECR. Mean FECRT cut-off values selected for establishing the presence of resistance were 90% for P. equorum and 95% for strongyles (von Samson-Himmelstjerna et al., 2007). Strongyle egg reappearance Weekly FECs were performed from weeks 2 to 6 post treatment and FECR% were calculated. Cut-off values selected for declaring a shortened ERP was a farm mean FECR of <90% 6 weeks or fewer post treatment (von Samson-Himmelstjerna et al., 2007). Statistical analyses Data were analysed using SAS 9.1.3 software (SAS Institute). The distribution of data was examined by the univariate procedure, which showed that FEC post treatment and FECR were not normally distributed (P = 0.0016 and P < 0.0001, respectively). Hence, data were analysed using a variance component model (mixed
Strongyle egg reappearance Horses in this part of the study had a mean age of 9.0 (95% CI: 7.9–10.1) years. The results are presented in Table 1. One horse on one farm was shedding 200 EPG by week 4. By week 5, three farms and by week 6, five farms had horses shedding eggs. However, FECR remained high throughout the study period; the mean farm FECR at 6 weeks post treatment was 96.9%. Of the five farms where horses were shedding eggs, only one had a <90% reduction in FEC, and two other farms had an FEC that was higher but not significantly different from 90%. In the untreated group the mean FEC increased from 88 to 333 during the study.
Table 1 Arithmetic mean reductions in faecal egg counts (FECR) after treatment with ivermectin (0.2 mg/kg). Numbers in parentheses are 95% confidence limits for mean FECR. FECR% post treatment
Fig. 1. Results of girth tape measurements as performed by one of the investigators compared to the bodyweight estimations of the horse’s owner. Numbers on axes are in kg. The dotted lines on each side of the trend line represent deviations of ±25 kg (- - -) and 50 kg (), respectively.
Farm number
Week 2 (%)
Week 3 (%)
Week 4 (%)
Week 5 (%)
Week 6 (%)
1 2
100 100
100 100
100 100
3 4
100 100
100 100
5 6 7
100 100 100
100 100 100
100 95.8 (87.7–104.0) 100 100 100
8
100
100
100
100 94.9 (84.6–105.0) 100 96.6 (91.4–101.7) 100 100 98.5 (95.8–101.3) 100
9
100
100
100
100
100 89.6 (72.0–107.1) 100 92.6 (83.9–101.2) 100 100 99.2 (97.5–100.8) 93.8 (81.5–106.0) 96.9 (90.8–103.0)
Mean
100
100
99.5 (99.3–99.8)
98.9 (98.5–99.3)
96.9 (96.1–97.7)
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M.L. Larsen et al. / The Veterinary Journal 188 (2011) 44–47
Discussion The present study provides information on the efficacy of the most commonly used anthelmintic on farms using selective therapy under prescription-only restrictions. Ivermectin efficacy was high against both strongyles and P. equorum, and we found no evidence of shortened strongyle ERP. The findings were supported by the high egg counts from untreated control horses on the farms, which rules out any season- or weather-dependent influences on the worms. This contrasts with the increasing body of evidence from other countries which clearly shows ivermectin resistance in P. equorum (Boersema et al., 2002; Hearn and Peregrine, 2003; Schougaard and Nielsen, 2007; Veronesi et al., 2009) and signs of developing resistance in strongyles (Lyons et al., 2008; Molento et al., 2008; von Samson-Himmelstjerna et al., 2007). Anthelmintic resistance is a natural biological consequence of treatment, and it is unrealistic to assume that it can be avoided completely. However, certain strategies, particularly reduced treatment frequency and maintenance of refugia, are predicted to reduce the selective pressures for resistance and delay its occurrence (Nielsen et al., 2007). The primary intention behind the Danish restrictions on anthelmintic usage was to delay the development of resistance for as long as possible. This study provides a contribution to the body of evidence illustrating the potential outcomes of this approach. ERP after ivermectin treatment has attracted considerable attention in recent equine studies. Ten years ago, Sangster (1999) proposed that a shortened ERP could be interpreted as a first sign of anthelmintic resistance. Over the past 5 years, evidence of shortened ivermectin ERP has been gathered from the US (Lyons et al., 2008), Brazil (Molento et al., 2008) and Europe (von SamsonHimmelstjerna et al., 2007). However, interpretation of these findings is complicated by a lack of consensus in the definition of the ERP. Some studies have defined ERP as the week of the first positive egg count post treatment (Dudeney et al., 2008; Little et al., 2003; Lyons et al., 2008; Molento et al., 2008). Others have used a fixed threshold of the mean egg count, such as 100 or 200 EPG (Boersema et al., 1996; Jacobs et al., 1995; Mercier et al., 2001). Finally, a third definition of ERP is to use the FECRT to calculate weekly efficacies and then use 80% (Tarigo-Martinie et al., 2001) or 90% (Boersema et al., 1995; Borgsteede et al., 1993; von SamsonHimmelstjerna et al., 2007) efficacy as the cut-off level. We chose the latter definition for several reasons. Firstly, using a fixed threshold will be highly biased by pre-treatment egg count levels. Horses tend to return to the same egg count level, and low/moderate egg shedders may never reach the threshold. Secondly, waiting for the first horse to shed eggs is also highly biased by pre-treatment egg count levels. Such a horse could be an outlier or an extreme high shedder. Even with full ivermectin efficacy, high egg shedders can be shedding a few eggs post treatment. Finally, using the FECRT makes sense, since (in essence) ERP is a measure of drug efficacy during the weeks after treatment. Using this method, results will not be biased by pre-treatment egg count levels. The best cut-off value to use depends on the anthelmintic used. Since ivermectin can be expected to have an efficacy of >99%, a 90% cut-off appears to be a fairly conservative choice. Given the increased focus on this area we strongly urge the scientific community to reach agreement on the definition of ERP. The very first surveys of ivermectin ERP were done 15 years ago by Dutch investigators, who found an ERP of 6–9 weeks and average efficacy >90% 63 days post treatment (Borgsteede et al., 1993). Using another definition of ERP, positive FEC were found 6 weeks post treatment and a mean of 100 EPG 9 weeks post treatment (Boersema et al., 1996). For practical reasons, the present study
was performed for a 6 week period. Although a few more weeks would have been useful to determine the ivermectin ERP in our study population, there was no evidence of a shortened ERP on any of the farms tested. A recent Danish study reported P. equorum resistant to ivermectin on one stud farm (Schougaard and Nielsen, 2007). This contrasts with the present study where an overall high efficacy was observed on all farms. The age range of horses with ascarid infection studied here was 6 months to 4 years with a mean of 2.1 years, while the studies reporting avermectin/milbemycin resistance all involved foals. Observations suggest that the efficacy of anthelmintics against P. equorum increase with animal age, however, it remains uncertain whether age-related changes in host immune response, age-related differences in drug metabolism, or the relative age of the parasite burden within individual horses might be responsible for this observation. Our findings suggest that ivermectin remains a reliable choice for treating ascarid infections, but FECRT should be performed continuously on a yearly basis to ensure that treatment failure is not occurring. Administering paste anthelmintics is a task that with very few exceptions lies in the hand of horse owners or stable managers. This carries a potential risk of under dosing horses, which could accelerate development of anthelmintic resistance. However, our results suggest that with adequate instruction (as defined by the requirement for training in medicine handling) clients are capable of dosing correctly. Only a few horses were marginally under dosed, and the majority of overdosed horses were overdosed intentionally by the owners. Conclusions The present study provided no evidence of developing ivermectin resistance in the strongyle and ascarid populations we examined. Whether this can be explained by the strict Danish regulations on anthelmintic use remains uncertain, but it is likely that many years of selective therapy have substantially reduced selective forces for resistance as work in other European countries has suggested that ivermectin resistance is increasing. Clearly, more studies are needed to evaluate the long-term consequences of the selective therapy approach so that further refinement of this parasite control strategy can be achieved. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgement The authors are highly grateful to Dr. Ray M. Kaplan, University of Georgia for critically reviewing the manuscript. References Boersema, J.H., Borgsteede, F.H.M., Eysker, M., Saedt, I., 1995. The reappearance of strongyle eggs in feces of horses treated with pyrantel embonate. Veterinary Quarterly 17, 18–20. Boersema, J.H., Eysker, M., Maas, J., vanderAar, W.M., 1996. Comparison of the reappearance of strongyle eggs in foals, yearlings, and adult horses after treatment with ivermectin or pyrantel. Veterinary Quarterly 18, 7–9. Boersema, J.H., Eysker, M., Nas, J.W.M., 2002. Apparent resistance of Parascaris equorum to macrocylic lactones. Veterinary Record 150, 279–281. Borgsteede, F.H.M., Boersma, J.H., Gaasenbeek, C.P.H., Vanderburg, W.P.J., 1993. The reappearance of eggs in feces of horses after treatment with ivermectin. Veterinary Quarterly 15, 24–26. Clayton, H.M., 1978. Ascariasis in foals. Veterinary Record 102, 553–556.
M.L. Larsen et al. / The Veterinary Journal 188 (2011) 44–47 Dudeney, A., Campbell, C., Coles, G., 2008. Macrocyclic lactone resistance in cyathostomins. Veterinary Record 163, 163–164. Duncan, J.L., Love, S., 1991. Preliminary observations on an alternative strategy for the control of horse strongyles. Equine Veterinary Journal 23, 226–228. Hearn, F.P.D., Peregrine, A.S., 2003. Identification of foals infected with Parascaris equorum apparently resistant to ivermectin. Journal of the American Veterinary Medical Association 223, 482–485. Jacobs, D.E., Hutchinson, M.J., Parker, L., Gibbons, L.M., 1995. Equine cyathostome infection – suppression of faecal egg output with moxidectin. Veterinary Record 137, 545. Kaplan, R.M., 2002. Anthelmintic resistance in nematodes of horses. Veterinary Research 33, 491–507. Kaplan, R.M., 2004. Drug resistance in nematodes of veterinary importance: a status report. Trends in Parasitology 20, 477–481. Krecek, R.C., Guthrie, A.J., van Nieuwenhuizen, L.C., Booth, L.M., 1994. A comparison between the effects of conventional and selective antiparasitic treatments on nematode parasites of horses from two management schemes. Journal of South Africa Veterinary Association 64, 97–100. Little, D., Flowers, J.R., Hammerberg, B.H., Gardner, S.Y., 2003. Management of drugresistant cyathostominosis on a breeding farm in central North Carolina. Equine Veterinary Journal 35, 246–251. Love, S., Murphy, D., Mellor, D., 1999. Pathogenicity of cyathostome infection. Veterinary Parasitology 85, 113–121. Lyons, E.T., Tolliver, S.C., Collins, S.S., 2009. Probable reason why small strongyle EPG counts are returning ‘‘early” after ivermectin treatment of horses on a farm in Central Kentucky. Veterinary Parasitology 104, 569–574. Lyons, E.T., Tolliver, S.C., Ionita, M., Lewellen, A., Collins, S.S., 2008. Field studies indicating reduced activity of ivermectin on small strongyles in horses on a farm in Central Kentucky. Parasitology Research 103, 209–215. Matthee, S., McGeoch, M.A., 2004. Helminths in horses: use of selective treatment for the control of strongyles. Journal of the South African Veterinary Association – Tydskrif Van Die Suid-Afrikaanse Veterinere Vereniging 75, 129–136.
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Mercier, P., Chick, B., Alves-Branco, F., White, C.R., 2001. Comparative efficacy, persistent effect, and treatment intervals of anthelmintic pastes in naturally infected horses. Veterinary Parasitology 99, 29–39. Molento, M.B., Antunes, J., Bentes, R.N., Coles, G.C., 2008. Anthelmintic resistant nematodes in Brazilian horses. Veterinary Record 162, 384–385. Nielsen, M.K., Kaplan, R.M., Thamsborg, S.M., Monrad, J., Olsen, S.N., 2007. Climatic influences on development and survival of free-living stages of equine strongyles: implications for worm control strategies and managing anthelmintic resistance. The Veterinary Journal 174, 23–32. Nielsen, M.K., Monrad, J., Olsen, S.N., 2006. Prescription-only anthelmintics – a questionnaire survey of strategies for surveillance and control of equine strongyles in Denmark. Veterinary Parasitology 135, 47–55. Roepstorff, A., Nansen, P., 1998. Epidemiology, Diagnosis and Control of Helminth Parasites of Swine. Food and Agriculture Organization of the United Nations, Rome. Sangster, N.C., 1999. Pharmacology of anthelmintic resistance in cyathostomes: will it occur with the avermectin/milbemycins? Veterinary Parasitology 85, 189– 201. Schougaard, H., Nielsen, M.K., 2007. Apparent ivermectin resistance if Parascaris equorum in foals in Denmark. Veterinary Record 160, 439–440. Tarigo-Martinie, J.L., Wyatt, A.R., Kaplan, R.M., 2001. Prevalence and clinical implications of anthelmintic resistance in cyathostomes of horses. Journal of the American Veterinary Medical Association 218, 1957–1960. Veronesi, F., Moretta, I., Moretti, A., Fioretti, D.P., Genchi, C., 2009. Field effectiveness of pyrantel and failure of Parascaris equorum egg count reduction following ivermectin treatment in Italian horse farms. Veterinary Parasitology 161, 138– 141. von Samson-Himmelstjerna, G., Fritzen, B., Demeler, J., Schuermann, S., Rohn, K., Schnieder, T., Epe, C., 2007. Cases of reduced cyathostomin egg-reappearance period and failure of Parascaris equorum egg count reduction following ivermectin treatment as well as survey on pyrantel efficacy on German horse farms. Veterinary Parasitology 144, 74–80.
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Determination of ivermectin efficacy against cyathostomins and Parascaris equorum on horse farms using selective therapy Mette L. Larsen a, Christian Ritz b, Stig L. Petersen c, Martin K. Nielsen a,* a
Department of Large Animal Sciences, Faculty of Life Sciences, University of Copenhagen, DK-2630 Taastrup, Denmark Department of Basic Sciences and Environment, Faculty of Life Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark c EquiLab Laboratory, DK-3550 Slangerup, Denmark b
a r t i c l e
i n f o
Article history: Accepted 5 March 2010
Keywords: Ivermectin Egg reappearance Efficacy Parascaris equorum Strongyles
a b s t r a c t Ivermectin resistance has recently been described in Parascaris equorum and there have been reports from several countries of a shortened egg reappearance period (ERP) following ivermectin treatment for cyathostomins. This study was aimed at determining the efficacy of ivermectin in treating cyathostomins and P. equorum in Danish horses. A total of 196 animals were selected from 52 farms, all of which were using a selective anthelmintic treatment strategy. ERP was investigated with weekly samples from 96 horses from nine farms. Horses were treated with ivermectin oral paste by their owners at an estimated dose rate of 0.2 mg/kg. Overall, faecal egg counts were reduced 10–14 days after treatment by 96.9% and 100% for P. equorum and cyathostomins, respectively. Mean faecal egg count reductions at 4 and 6 weeks post treatment were 99.5% and 96.9%, respectively. No signs of developing ivermectin resistance were found in either cyathostomins or P. equorum in the studied horses. Ó 2010 Elsevier Ltd. All rights reserved.
Introduction Cyathostomins are ubiquitous parasites of grazing horses, and are considered the most important parasites in horses (Love et al., 1999) although large strongyles are still a potential threat when present. In foals and young horses, the roundworm Parascaris equorum also constitutes a major threat to equine health (Clayton, 1978). Over recent decades, increasing resistance of cyathostomins to several groups of anthelmintics has been reported (Kaplan, 2002, 2004). Furthermore, recent reports suggest that in some instances the cyathostomin egg reappearance period (ERP) following treatment with ivermectin has been reduced to 4–5 weeks (Lyons et al., 2008; Molento et al., 2008; von Samson-Himmelstjerna et al., 2007) from the 9 weeks initially reported (Borgsteede et al., 1993). Lyons et al. (2009) showed that this reduction was due to apparent ivermectin resistance in the luminal L4 stage. Reports from several countries of P. equorum isolates showing apparent ivermectin resistance have also been published (Boersema et al., 2002; Hearn and Peregrine, 2003; Schougaard and Nielsen, 2007; Veronesi et al., 2009). These developments constitute a major concern for the equine industry as ivermectin is the most widely used equine anthelmintic (Nielsen et al., 2006), and it is unclear when, or even if, a new anthelmintic class will be launched for equine usage. To delay further development of anthelmintic resistance, recommendations have been made to reduce treatment intensity, and to * Corresponding author. Tel.: +45 35332842; fax: +45 35332880. E-mail address: [email protected] (M.K. Nielsen). 1090-0233/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2010.03.009
base treatments on parasite surveillance (Kaplan, 2002; Nielsen et al., 2007). One widely recommended strategy is selective therapy, where faecal samples are taken from all horses and treatment is given only to individuals exceeding a predetermined cut-off egg count (Duncan and Love, 1991; Krecek et al., 1994; Little et al., 2003; Matthee and McGeoch, 2004). The use of selective therapy has been supported by changes in legislation and several European countries, including Denmark, Sweden, The Netherlands, Finland and Italy, now disallow prophylactic treatment and require a diagnosis before anthelmintics can be administered. In Denmark, where such legislation has been in place since 1999, the majority of horse establishments base their parasite control programs on selective therapy with faecal samples taken from all horses twice yearly (Nielsen et al., 2006). However, it remains unclear whether these restrictions have affected the development of anthelmintic resistance. The aims of this study were to assess the current efficacy of ivermectin against P. equorum and cyathostomins on Danish farms practicing selective therapy, and to determine cyathostomin ERP after ivermectin treatment in these horses. Materials and methods The study was carried out between September 1st and November 1st 2008.
Horses All investigated horses were from farms which had been using selective therapy for at least 2 years prior to the study, and which had been using ivermectin as the only anthelmintic for that period. On all farms faecal samples were taken from all
45
M.L. Larsen et al. / The Veterinary Journal 188 (2011) 44–47 horses twice a year, and individuals exceeding a faecal egg count (FEC) of 200 strongyle eggs per gram of faeces (EPG) or having a positive P. equorum egg count were treated. Farms were selected through a referral laboratory analysing faecal samples submitted from various regions in Denmark. A total of 196 horses from 52 farms were used in the study. Egg reappearance period The ERP following ivermectin treatment was determined on nine farms where at least 10 horses were treated. A total of 96 horses were included in the study. On the nine farms, 69 untreated horses with FECs <200 EPG were kept as a control group. There were P5 untreated horses per farm. Faecal egg counts All FECs were determined by the McMaster method with a detection limit of 50 EPG (Roepstorff and Nansen, 1998). Larval cultures were performed on two individual horses sampled at random from each study farm to determine if any large strongyle species were present. Treatment In Denmark, owners are allowed to give medical treatments to their horses only after passing an authorised course in medicine handling. The veterinarian prescribing the drug specifies the dosage, and the dosing is then carried out by the owner. Thus the liveweight of each horse was estimated by its owner who then gave their horse an oral dose of 0.2 mg/kg ivermectin paste (Noromectin vet, Biovet ApS). After ivermectin treatment, the first author then estimated each horse’s weight using a girth tape. Ivermectin efficacy Faecal samples were collected for faecal egg count reduction tests (FECRT) on the day of ivermectin treatment and 10–14 days post treatment. FECR was determined for each individual horse as:
linear models, mixed procedure). FEC post treatment was chosen as the response variable and efficacies were determined post modelling. FEC were analysed as a quantitative discrete variable. Explanatory variables were farm, horse, gender, date, dosage, age, FEC prior to treatment, mean FEC on the farm and proportion of treated horses on the farm. Horse and farm were chosen as random variables. Results were interpreted at the 0.05 level, and variables that were not significant were excluded to refine the model.
Results Only cyathostomins were identified in the larval cultures. The large majority of the horses were dosed correctly, or marginally overdosed, according to the weight estimated by weight girth tape measurements. Eight horses were marginally under dosed. The weight estimations are presented in Fig 1. Ivermectin efficacy One hundred and seventeen horses (59%) were excreting strongyle eggs only. These horses were from 10 different farms and had a mean age of 8.7 years (95% confidence interval (CI): 7.6–9.4) and mean FEC of 491 EPG (95% CI: 421–562). Seventy-nine (40%) of the treated horses were excreting P. equorum eggs prior to treatment. These horses were from 42 farms and had a mean age of 2.1 years (95% CI: 1.6–2.5). A majority of these horses (84%) were co-infected with strongyles; mean pre-treatment strongyle FEC was 323 EPG (95% CI: 262–385). The overall mean FECR 10–14 days following ivermectin treatment were 96.9% and 100% for P. equorum and strongyles, respectively.
FECR ¼ ðFECpretreatment —FECposttreatment Þ FECpretreatment 100% Arithmetic means for each group and each farm were determined from individual FECR. Mean FECRT cut-off values selected for establishing the presence of resistance were 90% for P. equorum and 95% for strongyles (von Samson-Himmelstjerna et al., 2007). Strongyle egg reappearance Weekly FECs were performed from weeks 2 to 6 post treatment and FECR% were calculated. Cut-off values selected for declaring a shortened ERP was a farm mean FECR of <90% 6 weeks or fewer post treatment (von Samson-Himmelstjerna et al., 2007). Statistical analyses Data were analysed using SAS 9.1.3 software (SAS Institute). The distribution of data was examined by the univariate procedure, which showed that FEC post treatment and FECR were not normally distributed (P = 0.0016 and P < 0.0001, respectively). Hence, data were analysed using a variance component model (mixed
Strongyle egg reappearance Horses in this part of the study had a mean age of 9.0 (95% CI: 7.9–10.1) years. The results are presented in Table 1. One horse on one farm was shedding 200 EPG by week 4. By week 5, three farms and by week 6, five farms had horses shedding eggs. However, FECR remained high throughout the study period; the mean farm FECR at 6 weeks post treatment was 96.9%. Of the five farms where horses were shedding eggs, only one had a <90% reduction in FEC, and two other farms had an FEC that was higher but not significantly different from 90%. In the untreated group the mean FEC increased from 88 to 333 during the study.
Table 1 Arithmetic mean reductions in faecal egg counts (FECR) after treatment with ivermectin (0.2 mg/kg). Numbers in parentheses are 95% confidence limits for mean FECR. FECR% post treatment
Fig. 1. Results of girth tape measurements as performed by one of the investigators compared to the bodyweight estimations of the horse’s owner. Numbers on axes are in kg. The dotted lines on each side of the trend line represent deviations of ±25 kg (- - -) and 50 kg (), respectively.
Farm number
Week 2 (%)
Week 3 (%)
Week 4 (%)
Week 5 (%)
Week 6 (%)
1 2
100 100
100 100
100 100
3 4
100 100
100 100
5 6 7
100 100 100
100 100 100
100 95.8 (87.7–104.0) 100 100 100
8
100
100
100
100 94.9 (84.6–105.0) 100 96.6 (91.4–101.7) 100 100 98.5 (95.8–101.3) 100
9
100
100
100
100
100 89.6 (72.0–107.1) 100 92.6 (83.9–101.2) 100 100 99.2 (97.5–100.8) 93.8 (81.5–106.0) 96.9 (90.8–103.0)
Mean
100
100
99.5 (99.3–99.8)
98.9 (98.5–99.3)
96.9 (96.1–97.7)
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M.L. Larsen et al. / The Veterinary Journal 188 (2011) 44–47
Discussion The present study provides information on the efficacy of the most commonly used anthelmintic on farms using selective therapy under prescription-only restrictions. Ivermectin efficacy was high against both strongyles and P. equorum, and we found no evidence of shortened strongyle ERP. The findings were supported by the high egg counts from untreated control horses on the farms, which rules out any season- or weather-dependent influences on the worms. This contrasts with the increasing body of evidence from other countries which clearly shows ivermectin resistance in P. equorum (Boersema et al., 2002; Hearn and Peregrine, 2003; Schougaard and Nielsen, 2007; Veronesi et al., 2009) and signs of developing resistance in strongyles (Lyons et al., 2008; Molento et al., 2008; von Samson-Himmelstjerna et al., 2007). Anthelmintic resistance is a natural biological consequence of treatment, and it is unrealistic to assume that it can be avoided completely. However, certain strategies, particularly reduced treatment frequency and maintenance of refugia, are predicted to reduce the selective pressures for resistance and delay its occurrence (Nielsen et al., 2007). The primary intention behind the Danish restrictions on anthelmintic usage was to delay the development of resistance for as long as possible. This study provides a contribution to the body of evidence illustrating the potential outcomes of this approach. ERP after ivermectin treatment has attracted considerable attention in recent equine studies. Ten years ago, Sangster (1999) proposed that a shortened ERP could be interpreted as a first sign of anthelmintic resistance. Over the past 5 years, evidence of shortened ivermectin ERP has been gathered from the US (Lyons et al., 2008), Brazil (Molento et al., 2008) and Europe (von SamsonHimmelstjerna et al., 2007). However, interpretation of these findings is complicated by a lack of consensus in the definition of the ERP. Some studies have defined ERP as the week of the first positive egg count post treatment (Dudeney et al., 2008; Little et al., 2003; Lyons et al., 2008; Molento et al., 2008). Others have used a fixed threshold of the mean egg count, such as 100 or 200 EPG (Boersema et al., 1996; Jacobs et al., 1995; Mercier et al., 2001). Finally, a third definition of ERP is to use the FECRT to calculate weekly efficacies and then use 80% (Tarigo-Martinie et al., 2001) or 90% (Boersema et al., 1995; Borgsteede et al., 1993; von SamsonHimmelstjerna et al., 2007) efficacy as the cut-off level. We chose the latter definition for several reasons. Firstly, using a fixed threshold will be highly biased by pre-treatment egg count levels. Horses tend to return to the same egg count level, and low/moderate egg shedders may never reach the threshold. Secondly, waiting for the first horse to shed eggs is also highly biased by pre-treatment egg count levels. Such a horse could be an outlier or an extreme high shedder. Even with full ivermectin efficacy, high egg shedders can be shedding a few eggs post treatment. Finally, using the FECRT makes sense, since (in essence) ERP is a measure of drug efficacy during the weeks after treatment. Using this method, results will not be biased by pre-treatment egg count levels. The best cut-off value to use depends on the anthelmintic used. Since ivermectin can be expected to have an efficacy of >99%, a 90% cut-off appears to be a fairly conservative choice. Given the increased focus on this area we strongly urge the scientific community to reach agreement on the definition of ERP. The very first surveys of ivermectin ERP were done 15 years ago by Dutch investigators, who found an ERP of 6–9 weeks and average efficacy >90% 63 days post treatment (Borgsteede et al., 1993). Using another definition of ERP, positive FEC were found 6 weeks post treatment and a mean of 100 EPG 9 weeks post treatment (Boersema et al., 1996). For practical reasons, the present study
was performed for a 6 week period. Although a few more weeks would have been useful to determine the ivermectin ERP in our study population, there was no evidence of a shortened ERP on any of the farms tested. A recent Danish study reported P. equorum resistant to ivermectin on one stud farm (Schougaard and Nielsen, 2007). This contrasts with the present study where an overall high efficacy was observed on all farms. The age range of horses with ascarid infection studied here was 6 months to 4 years with a mean of 2.1 years, while the studies reporting avermectin/milbemycin resistance all involved foals. Observations suggest that the efficacy of anthelmintics against P. equorum increase with animal age, however, it remains uncertain whether age-related changes in host immune response, age-related differences in drug metabolism, or the relative age of the parasite burden within individual horses might be responsible for this observation. Our findings suggest that ivermectin remains a reliable choice for treating ascarid infections, but FECRT should be performed continuously on a yearly basis to ensure that treatment failure is not occurring. Administering paste anthelmintics is a task that with very few exceptions lies in the hand of horse owners or stable managers. This carries a potential risk of under dosing horses, which could accelerate development of anthelmintic resistance. However, our results suggest that with adequate instruction (as defined by the requirement for training in medicine handling) clients are capable of dosing correctly. Only a few horses were marginally under dosed, and the majority of overdosed horses were overdosed intentionally by the owners. Conclusions The present study provided no evidence of developing ivermectin resistance in the strongyle and ascarid populations we examined. Whether this can be explained by the strict Danish regulations on anthelmintic use remains uncertain, but it is likely that many years of selective therapy have substantially reduced selective forces for resistance as work in other European countries has suggested that ivermectin resistance is increasing. Clearly, more studies are needed to evaluate the long-term consequences of the selective therapy approach so that further refinement of this parasite control strategy can be achieved. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgement The authors are highly grateful to Dr. Ray M. Kaplan, University of Georgia for critically reviewing the manuscript. References Boersema, J.H., Borgsteede, F.H.M., Eysker, M., Saedt, I., 1995. The reappearance of strongyle eggs in feces of horses treated with pyrantel embonate. Veterinary Quarterly 17, 18–20. Boersema, J.H., Eysker, M., Maas, J., vanderAar, W.M., 1996. Comparison of the reappearance of strongyle eggs in foals, yearlings, and adult horses after treatment with ivermectin or pyrantel. Veterinary Quarterly 18, 7–9. Boersema, J.H., Eysker, M., Nas, J.W.M., 2002. Apparent resistance of Parascaris equorum to macrocylic lactones. Veterinary Record 150, 279–281. Borgsteede, F.H.M., Boersma, J.H., Gaasenbeek, C.P.H., Vanderburg, W.P.J., 1993. The reappearance of eggs in feces of horses after treatment with ivermectin. Veterinary Quarterly 15, 24–26. Clayton, H.M., 1978. Ascariasis in foals. Veterinary Record 102, 553–556.
M.L. Larsen et al. / The Veterinary Journal 188 (2011) 44–47 Dudeney, A., Campbell, C., Coles, G., 2008. Macrocyclic lactone resistance in cyathostomins. Veterinary Record 163, 163–164. Duncan, J.L., Love, S., 1991. Preliminary observations on an alternative strategy for the control of horse strongyles. Equine Veterinary Journal 23, 226–228. Hearn, F.P.D., Peregrine, A.S., 2003. Identification of foals infected with Parascaris equorum apparently resistant to ivermectin. Journal of the American Veterinary Medical Association 223, 482–485. Jacobs, D.E., Hutchinson, M.J., Parker, L., Gibbons, L.M., 1995. Equine cyathostome infection – suppression of faecal egg output with moxidectin. Veterinary Record 137, 545. Kaplan, R.M., 2002. Anthelmintic resistance in nematodes of horses. Veterinary Research 33, 491–507. Kaplan, R.M., 2004. Drug resistance in nematodes of veterinary importance: a status report. Trends in Parasitology 20, 477–481. Krecek, R.C., Guthrie, A.J., van Nieuwenhuizen, L.C., Booth, L.M., 1994. A comparison between the effects of conventional and selective antiparasitic treatments on nematode parasites of horses from two management schemes. Journal of South Africa Veterinary Association 64, 97–100. Little, D., Flowers, J.R., Hammerberg, B.H., Gardner, S.Y., 2003. Management of drugresistant cyathostominosis on a breeding farm in central North Carolina. Equine Veterinary Journal 35, 246–251. Love, S., Murphy, D., Mellor, D., 1999. Pathogenicity of cyathostome infection. Veterinary Parasitology 85, 113–121. Lyons, E.T., Tolliver, S.C., Collins, S.S., 2009. Probable reason why small strongyle EPG counts are returning ‘‘early” after ivermectin treatment of horses on a farm in Central Kentucky. Veterinary Parasitology 104, 569–574. Lyons, E.T., Tolliver, S.C., Ionita, M., Lewellen, A., Collins, S.S., 2008. Field studies indicating reduced activity of ivermectin on small strongyles in horses on a farm in Central Kentucky. Parasitology Research 103, 209–215. Matthee, S., McGeoch, M.A., 2004. Helminths in horses: use of selective treatment for the control of strongyles. Journal of the South African Veterinary Association – Tydskrif Van Die Suid-Afrikaanse Veterinere Vereniging 75, 129–136.
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Mercier, P., Chick, B., Alves-Branco, F., White, C.R., 2001. Comparative efficacy, persistent effect, and treatment intervals of anthelmintic pastes in naturally infected horses. Veterinary Parasitology 99, 29–39. Molento, M.B., Antunes, J., Bentes, R.N., Coles, G.C., 2008. Anthelmintic resistant nematodes in Brazilian horses. Veterinary Record 162, 384–385. Nielsen, M.K., Kaplan, R.M., Thamsborg, S.M., Monrad, J., Olsen, S.N., 2007. Climatic influences on development and survival of free-living stages of equine strongyles: implications for worm control strategies and managing anthelmintic resistance. The Veterinary Journal 174, 23–32. Nielsen, M.K., Monrad, J., Olsen, S.N., 2006. Prescription-only anthelmintics – a questionnaire survey of strategies for surveillance and control of equine strongyles in Denmark. Veterinary Parasitology 135, 47–55. Roepstorff, A., Nansen, P., 1998. Epidemiology, Diagnosis and Control of Helminth Parasites of Swine. Food and Agriculture Organization of the United Nations, Rome. Sangster, N.C., 1999. Pharmacology of anthelmintic resistance in cyathostomes: will it occur with the avermectin/milbemycins? Veterinary Parasitology 85, 189– 201. Schougaard, H., Nielsen, M.K., 2007. Apparent ivermectin resistance if Parascaris equorum in foals in Denmark. Veterinary Record 160, 439–440. Tarigo-Martinie, J.L., Wyatt, A.R., Kaplan, R.M., 2001. Prevalence and clinical implications of anthelmintic resistance in cyathostomes of horses. Journal of the American Veterinary Medical Association 218, 1957–1960. Veronesi, F., Moretta, I., Moretti, A., Fioretti, D.P., Genchi, C., 2009. Field effectiveness of pyrantel and failure of Parascaris equorum egg count reduction following ivermectin treatment in Italian horse farms. Veterinary Parasitology 161, 138– 141. von Samson-Himmelstjerna, G., Fritzen, B., Demeler, J., Schuermann, S., Rohn, K., Schnieder, T., Epe, C., 2007. Cases of reduced cyathostomin egg-reappearance period and failure of Parascaris equorum egg count reduction following ivermectin treatment as well as survey on pyrantel efficacy on German horse farms. Veterinary Parasitology 144, 74–80.