Determination of Anthelmintic Efficacy Against Equine Cyathostomins and Parascaris Equorum in France T

304 EQUINE VETERINARY EDUCATION / AE / JUNE 2013

Original Article Determination of anthelmintic efficacy against equine cyathostomins and Parascaris equorum in France T. Geurden*, J.-M. Betsch†, K. Maillard‡, B. Vanimisetti§, M. D’Espois¶ and B. Besognet¶ Pfizer Animal Health, Zaventem, Belgium; †Clinique Vétérinaire Equine de Méheudin, Ecouché, France; ‡Laboratoire Départemental Frank Duncombe, St Contest, France; §Pfizer Animal Health, Kalamazoo, Michigan, USA; and ¶Pfizer Animal Health, Paris, France. *Corresponding author email: [email protected]

Keywords: horse; small strongyles; Parascaris; faecal egg count reduction test; anthelmintic resistance

Summary expected cyathostomin egg reappearance period of IVM This article reports the results of a faecal egg count reduction and to a lesser extent of MOX has been reported (von test on 4 farms in France, as an integrated part of the routine Samson-Himmelstjerna et al. 2007; Molento et al. 2008; deworming strategy against horse cyathostomins and Traversa et al. 2009; Lyons et al. 2011a,b). Recently, a large Parascaris equorum. Treatment with fenbendazole (FBZ) or survey conducted in Europe showed the occurrence of ivermectin (IVM) was evaluated in yearlings on Farms 1 and 2 multiple drug resistance in equine cyathostomins (Traversa and treatment with pyrantel embonate (PYR) was tested on et al. 2009, 2012), with indications of potential resistance to Farms 3 and 4. Calculation of the arithmetic mean faecal egg MLs. Cyathostomin resistance to BZs in France has been count reduction and the 95% confidence intervals (95% CI) reported since the early 1990s (Collobert et al. 1996; around the mean was performed using bootstrap analysis. For Collobert-Laugier et al. 2002; Traversa et al. 2012). equine cyathostomins, resistance to FBZ was found with an The current study aimed to evaluate the efficacy arithmetic mean reduction of 48.8% (95% CI: 1.9–69.3%). On of the anthelmintics used in the routine treatment programmes Farms 1 and 2, horses with reduced efficacy were identified. PYR against both cyathostomins and P. equorum on 4 farms in was found to be effective against cyathostomins, with an France, using a faecal egg count reduction test (FECRT). arithmetic mean reduction of 95.3% (95% CI: 84.6–99.8%), as well as IVM (100%). For P. equorum, both FBZ and PYR were Materials and methods effective (100% reduction). The efficacy of IVM, however, was Four farms in the Basse Normandy region of western France low (45.5%; 95% CI: 0–96.3%). These results confirm that FBZ (Orne department) were visited in order to evaluate the resistance in equine cyathostomins is present in France and that efficacy of the anthelmintic compounds used in the anthelmintic resistance to IVM is present in P. equorum. This deworming programmes on these specific farms by FECRT study underlines the necessity to evaluate the efficacy of horse (Coles et al. 2006; von Samson-Himmelstjerna 2012). One of deworming strategies on a regular basis under field conditions. the farms (Farm 2) had been investigated before for a potential decreased efficacy of IVM against P. equorum (Laugier et al. 2011); the other 3 were neighbouring farms. Introduction Yearling horses (age 8–15 months) were examined for the Routine anthelmintic treatment in horses, especially in faecal excretion of parasite eggs, and all positive horses (>200 combination with low refugia, is considered to contribute to the eggs/g) were treated by the local veterinary practitioner with selection of drug-resistant nematodes (Nielsen et al. 2010). Since either fenbendazole (FBZ at 7.5 mg/kg bwt per os, Panacur the first report by Boersema et al. (2002), selected populations of oral paste)1 or ivermectin (IVM at 0.2 mg/kg bwt per os, Parascaris equorum have been reported to survive treatment Eqvalan paste)2 on Farms 1 and 2, or pyrantel embonate (PYR with macrocyclic lactones (MLs; Hearn and Peregrine 2003; Coles at 6.6 mg/kg bwt per os, Strongid oral paste for horses)3 on et al. 2006; von Samson-Himmelstjerna et al. 2007; Schougaard Farms 3 and 4. These drugs were being used in the routine and Nielsen 2007; Slocombe et al. 2007, 2008; Lindgren et al. treatment on these farms. The weight of the animals was 2008; Lyons et al. 2008; Molento et al. 2008; Lind and Christensson recorded using a girth tape. The horses with positive faecal 2009; Näreaho et al. 2011). There are few data on the efficacy of egg counts (FEC) for P. equorum after IVM treatment (Farms 1 anthelmintics against P. equorum in France, although Laugier and 2) were to receive PYR. et al. (2011) recently reported decreased efficacy of ivermectin Faecal samples were collected from each horse on the (IVM) against P. equorum on 2 farms. Anthelmintic resistance (AR) day of treatment (before dosing) and 14 days after dosing to of P. equorum is mainly found on farms with frequent deworming determine individual pretreatment and post treatment FECs. of foals (van Doorn et al. 2007). Control of ML resistant P. equorum Each faecal sample was placed into a labelled plastic bag isolates can be accomplished by treatment with benzimidazoles and examined within 24 h using a quantitative modified (BZD) or pyrantel (PYR), although a few ML-resistant isolates also McMaster technique (sensitivity = 50 eggs/g of faeces; Sloss exhibited AR to PYR (Reinemeyer 2012). et al. 1994). In many regions, AR of cyathostomins to BZD and to a lesser The efficacy of treatment was evaluated based on extent to pyrantel has been reported (von Samson- the reduction between pre- and post treatment FEC for each Himmelstjerna 2012). MLs such as IVM and moxidectin (MOX) horse separately, and also on the arithmetic mean reduction are the most effective molecules, although a shorter than between pre- and post treatment FEC per treatment for the

TABLE 1: The arithmetic mean (mean) and range of faecal egg counts for both cyathostomins and Parascaris equorum, along with the number (n) of positive animals before treatment with fenbendazole (FBZ), ivermectin (IVM) or pyrantel (PYR), and the efficacy (eff) per farm

Cyathostomins Parascaris equorum

Treatment n Mean Range eff n Mean Range eff

Farm 1 IVM 5 3680 1400–6600 100% 3 2833 100–5800 51.8% FBZ 6 1983 300–4600 39.5% 3 600 400–800 100% Farm 2 IVM 9 790 300–2600 100% 5 900 100–2300 42.2% FBZ 9 1455 200–3300 57.2% 9 255 100–500 100% Farm 3 PYR 18 728 100–2100 92.3% 4 200 100–400 100% Farm 4 PYR 6 1367 300–3900 100% 1 100 NA 100%

entire population in this study. A bootstrap analysis approach TABLE 2: The overall arithmetic mean (mean) pre- and post based on Hosking et al. (2010) was used to estimate the treatment faecal egg counts (FEC) for both cyathostomins and arithmetic mean FEC reduction per treatment and the 95% Parascaris equorum, along with the number (n) of positive animals confidence intervals (95% CI), as follows: 1) Using the original before and after treatment with fenbendazole (FBZ), ivermectin (IVM) or pyrantel (PYR), and the efficacy (eff) and 95% confidence data set for each drug consisting of n1 pretreatment and a interval (95% CI) corresponding set of post treatment count values, n1 new values were sampled with replacement for each of the pre- Pretreatment Post treatment Efficacy and post treatment counts; 2) efficacy was then calculated as 100*(arithmetic mean pretreatment count - arithmetic mean Mean FEC n Mean FEC n Eff 95% CI post treatment count)/(arithmetic mean pretreatment count); Cyathostomins 3) Steps 1 and 2 were repeated 10,000 times; 4) the boundaries FBZ 1666.7 15 853 12 48.8 1.9–69.3 of the 95% CI for efficacy were given as the 2.5 and 97.5 PYR 887.5 24 41.6 3 95.3 84.6–99.8 percentiles of the distribution of the calculated efficacies from IVM 1878.6 14 1.8 0 100 NA the 10,000 sample data sets. The algorithm was implemented in Parascaris equorum SAS v9.24, and the percentiles were calculated using the Proc FBZ 341.7 12 0 0 100 NA Univariate procedure in SAS. PYR 913.9 5 0 0 100 NA The mean FECRs were interpreted for all the compounds as IVM 1757 8 957 6 45.5 0–96.3 follows: 1) resistance present if FECR <95%; 2) resistance suspected if FECR is Ն95% and the lower confidence limit <90%; and 3) no resistance if FECR Ն95% and lower confidence limit retrieved from pasture and were not treated. Farm 2 was the >90%, in accordance with methods recommended by the farm on which AR P. equorum was previously detected World Association for the Advancement of Veterinary (Laugier et al. 2011). An overview of the results of the FECRT per Parasitology for the detection of anthelmintic resistance in treatment and per parasite is presented in Table 2. The horses. For BZD and PYR, a threshold of 90% was put forward in individual faecal egg counts and reduction after treatment for horses (Coles et al. 2006). those horses with a reduction <100% is provided in Table 3.

Results Discussion The enrolment of the animals was based on a positive FEC prior The present study aimed to evaluate the efficacy of the to treatment, either for P. equorum, cyathostominae or both. standard deworming strategy on 4 farms in France and the On Farms 1 and 2, animals were allocated into treatment results confirm the occurrence of AR in cyathostomins groups based on descending pretreatment FECs. On Farm 1, to treatment with FBZ (Traversa et al. 2012). Similar observations 12 animals were enrolled (6 allocated to IVM and 6 to FBZ); on were recently reported in other European countries (Lind et al. Farm 2, 21 animals were enrolled (10 allocated to IVM and 11 2007; Cernˇanská et al. 2009; Traversa et al. 2009). The efficacy of to FBZ); on Farm 3, 18 animals were enrolled (all allocated to IVM against cyathostomins was confirmed to be high in France PYR); and on Farm 4, 6 animals were enrolled (all allocated to (Traversa et al. 2012). Nevertheless, it is known that the FECRT is PYR). Both for P. equorum and for cyathostominae, all animals not a very sensitive method for the early diagnosis of AR, and included in the FECRT had a pretreatment FEC >200 eggs/g, owners as well as veterinary practitioners need to consider how except for 3 animals with a FEC of 100 eggs/g for P. equorum. to use effective anthelmintics for horses on a case-by-case An overview of the pretreatment faecal egg counts and the basis. The evaluation of treatment efficacy by FECRT should be efficacy per farm is provided in Table 1. incorporated in the routine deworming management on these For P. equorum, the efficacy of FBZ and PYR was very high. farms. In case of suspected AR, based on faecal examination or In the IVM treated animals, however, 6 of the 8 animals with based on a farm anamnesis, more sensitive tools and positive pretreatment FEC were still excreting eggs 15 days approaches to investigate the true resistance status on the farm after treatment, with a mean reduction of 45.5%, indicating should be used (Vidyashankar et al. 2012). AR. Four of these horses were treated with PYR on Day 20 after For all broad-spectrum dewormers in horses except PYR, the IVM treatment and a faecal examination of these 4 horses P. equorum is considered as dose-limiting species and has on Day 35 indicated a 100% efficacy of the PYR treatment hence a lower threshold for the development of AR (data not shown). The 3 remaining horses could not be (Reinemeyer 2012). Pyrantel embonate was indeed found to

TABLE 3: The individual data for horses with less than 100% efficacy contribute in maintaining enough refugia. Similarly in young against Parascaris equorum and cyathostomins after treatment horses, the number of treatments can be reduced (von with ivermectin (IVM), fenbendazole (FBZ) or pyrantel (PYR). The Samson-Himmelstjerna et al. 2009; Fritzen et al. 2010). It is farm number (Farm No.), faecal egg counts before and after common practice to treat foals on a monthly to bimonthly treatment and the percentage reduction (red %) are provided basis, in order to control Strongyloides westeri and/or Farm No. Treatment Before After Red % P. equorum. These frequent treatments enhance the selection for AR, and are probably not warranted. Treating foals at Parascaris equorum bimonthly intervals is the maximum dosing interval for 1 IVM 100 200 0 controlling P. equorum, and larger intervals are preferred (van 1 IVM 2600 3800 0 Doorn et al. 2007; Reinemeyer 2012). It should be clarified that 1 IVM 5800 100 98.3 in the control programmes with larger treatment intervals egg 2 IVM 300 100 66.7 2 IVM 1100 200 81.8 contamination of the environment cannot be prevented. 2 IVM 2300 2300 0 However, the benefits in delaying the onset of AR outweigh the Cyathostomins negative effect of a low infection pressure. Allowing foals to 1 FBZ 300 800 0 be infected can even be considered as beneficial, especially 1 FBZ 500 1700 0 in combination with treatment before these infections 1 FBZ 1800 400 78 with P. equorum become patent, as this pattern of 1 FBZ 2300 1200 48 infection-treatment elicits a natural immune response, and 1 FBZ 2400 2600 0 can as such be considered as a ‘natural vaccination’ 1 FBZ 4600 500 89 (Reinemeyer 2012). As in mature animals, monitoring of the 2 FBZ 800 300 63 excretion level might help in the decision on the need of 2 FBZ 900 1100 0 2 FBZ 1500 1300 13 anthelmintic treatment. 2 FBZ 2100 1200 43 Other management includes the yearly monitoring of the 2 FBZ 3200 900 72 efficacy in resident foals and yearlings by FECRT, as done in this 2 FBZ 3300 800 76 study. If ineffective, the drug to which AR is demonstrated 3 PYR 1500 100 93 should not be used for the management of that particular 3 PYR 600 800 0 parasite species, although it still can be used to control other 3 PYR 400 100 75 parasites on the farm, as illustrated by IVM and BZD in the present study. This implicates a separate and tailored deworming strategy for P. equorum and cyathostomins, and it could be necessary to prioritise ascarid control for foals and be highly efficacious against P. equorum in the present study, yearlings over cyathostomin control (von Samson- and should be considered as a drug-of-choice for this parasite. Himmelstjerna 2012). Furthermore, appropriate measures in Furthermore, the animals positive after IVM treatment that order to prevent the spreading of AR by animal movement for were treated with PYR did not excrete eggs 15 days after the races and mating (Veronesi et al. 2009) should be considered. PYR treatment, confirming the high efficacy of PYR against IVM Treatment of newly introduced animals with effective resistant worms of this parasite (von Samson-Himmelstjerna anthelminthics should be carefully implemented on arrival to a et al. 2007; Slocombe et al. 2007; Lind and Christensson 2009; farm, followed by a quarantine period in order to minimise the Veronesi et al. 2009; Reinemeyer 2012). Nevertheless, AR of spread of potential resistant strains on the pastures. P. equorum against PYR has been reported (Lyons et al. 2008, Furthermore, removing the faeces from pasture weekly or twice 2011c; Näreaho et al. 2011). As the efficacy was 100% on weekly can further reduce P. equorum FECs in horses. both farms, FBZ can also be considered for treatment of In conclusion, this study reports the finding of P. equorum. cyathostomin AR against FBZ and P. equroum AR against IVM, The cyathostomin resistance against FBZ and the 100% during the routine screening of efficacy on 4 farms in France. efficacy of this drug against P. equorum on the same farms The results highlight the benefit of this routine screening seems contradictory, but may be a reflection of the different practice, as AR was identified against anthelmintics that were conditions for development of AR in both parasites, not in the still used in the routine deworming programmes on these least the different age distribution and associated deworming farms. strategies in horses for both parasites. Similar observations have been reported by Lyons et al. (2008), although in the same Authors’ declaration of interests study combined cyathostomin and P. equorum AR was At the time of the study Thomas Geurden, Bindu Vanimisetti, observed on other farms. The AR of P. equorum to IVM and not Melanie D’Espois and Bruno Besognet were all paid employees to FBZ and PYR may be due to the more frequent use of IVM or of Pfizer Animal Health, who funded the study. Employees of to the persistent efficacy of IVM. The so-called tail selection Pfizer Animal Health were involved in the study design, the minimises the refugia within the host, especially for target analysis and interpretation of data, the writing of the species with long prepatent periods, as P. equorum (Sangster manuscript and the decision to submit the manuscript for 1999; Reinemeyer 2012). Yet, it is still uncertain how much the publication. Employees of Pfizer Animal Health were not tail selection contributes to AR development (Sangster 1999), involved in the collection and recording of the data. as IVM is also larvicidal against P. equorum. Anthelmintic treatments in horses should be properly managed in order to prevent the development of AR. Mature Acknowledgements animals may be treated individually according to their egg The authors would like to thank Douglas Rugg for his highly shedding values as opposed to blanket treatments, to appreciated comments on the manuscript.

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