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Prevalence of Parascaris equorum infection in foals on French stud farms and first report of ivermectin-resistant P. equorum populations in France
Veterinary Parasitology 188 (2012) 185–189
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Prevalence of Parascaris equorum infection in foals on French stud farms and first report of ivermectin-resistant P. equorum populations in France Claire Laugier a,∗ , Corinne Sevin a , Sébastien Ménard b , Karine Maillard b a b
Anses, Dozulé Laboratory for Equine Diseases, 14430 Goustranville, France Frank Duncombe Laboratory, IFR 146 ICORE – University of Caen Basse-Normandie, 14053 Caen cedex 4, France
a r t i c l e
i n f o
Article history: Received 19 September 2011 Received in revised form 17 February 2012 Accepted 28 February 2012 Keywords: Parascaris equorum Prevalence Foals France Resistance
1. Introduction Parascaris equorum is a nematode belonging to the family Ascarididae and primarily affects foals. In adult horses, infection is rare and less intense because protective immunity against P. equorum starts to develop by the age of 6 months. The prepatency period is approximately 10–15 weeks (Clayton, 1986). Horses ingest infective eggs that have been shed in the surrounding environment by the previous years’ infected foals. Infection can lead to respiratory symptoms, poor growth, ill thrift accompanied by a rough hair coat and bouts of diarrhoea or colic. When worm burdens are numerous, adult ascarids occasionally cause intestinal obstruction, intussusception or even rupture. The prevalence of P. equorum in foals has been assessed in various countries (e.g., USA, Germany, Poland, Sweden).
∗ Corresponding author at: French Agency for Food, Environmental and Occupational Health Safety, Laboratory for Equine Diseases, RD 675, 14430 Goustranville, France. Tel.: +33 231797946; fax: +33 231392137. E-mail address: [email protected] (C. Laugier). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2012.02.022
Prevalence is generally high and varies from 22.4% to 80% (Rieder et al., 1995; Gawor, 1996; Lyons and Tolliver, 2004; Osterman Lind and Christensson, 2009). However, to date, little is known about the prevalence of P. equorum in foals in France. In most horse breeding regions, young horses are regularly treated against P. equorum until 12 months of age. The main anthelmintics used in horses belong to three different drug classes: benzimidazole compounds, tetrahydropyrimidines (pyrantel) and macrocyclic lactones (MLs: ivermectin and moxidectin). Macrocyclic lactones are particularly important in the control of equine intestinal parasites because they have a broad spectrum activity (i.e., they are nematocidal and insecticidal) and they also target migrating larval stages of large strongyles and P. equorum. These drugs are frequently used in foals and young horses. Since the first description of an ML treatment failure in the Netherlands in 2002 (Boersema et al., 2002), several studies have reported ML-resistant P. equorum in various European countries (von Samson-Himmelstjerna et al., 2007; Schougaard and Nielsen, 2007; Lindgren et al., 2008; Veronesi et al., 2009;) and also in North America and Brazil
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C. Laugier et al. / Veterinary Parasitology 188 (2012) 185–189
Table 1 Prevalence of P. equorum eggs in fecal samples of 455 foals from 15 breeding farms in France. Farm
Breed
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
TBa TB TB TB TB TB TB TB TB TB TB FTb FT FT FT
Number of foals sampled
Overall a b
Mean age (month)
Prevalence (%)
38 62 79 45 48 28 12 12 11 19 10 29 21 18 23
Number of infected foals 6 20 25 14 7 14 1 6 5 2 4 2 16 4 13
4 6 5.5 5 6 6 9 6 3.5 4 6 8 3.5 3.5 8
15.8 32.3 31.6 31.1 14.6 50.0 8.3 50.0 45.5 10.5 40.0 6.9 76.2 22.2 56.5
455
139
5.5
30.5
TB Thoroughbred. FT French Trotter.
(Slocombe et al., 2007; Lyons et al., 2008; Molento et al., 2008). In France, several equine veterinary practitioners have reported apparent ivermectin treatment failures in nursing foals and weanlings. The purpose of this study was first to estimate the prevalence of P. equorum in foals from 15 large stud farms in France and then to investigate the efficacy of ivermectin using the faecal egg count reduction (FECR) test in three farms with suspected resistant ascarid populations. This study was conducted in Normandy, France, which is the leading horse breeding region in France with roughly 10,500 foal births per year.
2. Materials and methods The prevalence of P. equorum infection was estimated on a total of 455 foals aged from 3 to 9 months from 11 Thoroughbred horse farms and four French Trotter horse farms (Table 1). Most foals included in the study (80%) were Thoroughbreds and their mean age was 5.5 months. The number of foals sampled in each farm varied from 10 to 79. Samples were taken from July to October 2010. During this period, the dams and their foals spent most of their time outdoors in paddocks or pastures. In the 15 stud farms, deworming protocols in foals were roughly similar: the first treatment was administered at 1 month of age and subsequent treatments were given monthly or bimonthly until 18 months. During the first 6–8 months, the use of ivermectin was exclusive or very frequent; then, alternation with the two other drug classes (benzimidazoles or pyrantel) was implemented.
Finally, foals generally received three administrations of ivermectin between birth and weaning. In three of the 15 farms (nos. 6, 13 and 15), equine veterinary practitioners had reported apparent ivermectin treatment failure upon observing massive ascarid infection during autopsy (farm no. 15) or abundant egg excretion (farms nos. 6 and 13) despite regular and/or recent ivermectin treatments. From these three farms, 36 foals naturally infected with P. equorum and shedding at least 100 eggs per gram faeces (epg) were selected to test the effectiveness of ivermectin against this roundworm. Individual faecal samples were collected, depending on the foal’s age, rectally or from the top of newly deposited faeces using veterinary gloves. All faecal samples were stored under refrigeration and examined within 48 h of collection. Faecal egg counts were determined by a modified McMaster technique (Raynaud, 1970) with a saturated solution of sodium chloride (specific gravity 1.20) as the flotation solution and a detection limit of 50 epg. The influence of breed and age on P. equorum prevalence was analysed using a Chi-square test. To analyse the influence of age, foals were grouped into three age classes: 3–4 months, 5–7 months and 8–9 months (Table 2). Differences in faecal egg output between the three age groups were tested using an analysis of variance on log-transformed data (Statview software package 1998 SAS Institute Inc.). For all statistical analyses, we applied a 5% significance level (p < 0.05). In the three farms suspected of harbouring resistant ascarids, the effectiveness of ivermectin was assessed using the FECR test based on individual animals between
Table 2 Age distribution of P. equorum infection and mean fecal egg counts (FEC) of 455 foals from 15 breeding farms in France. Age (months)
Number of foals sampled
Number of infected foals
Prevalence (%)
Mean FEC (S.D.)
3–4 5–7 8–9
134 249 72
51 77 11
38.1 30.9 15.3
596.6 (1052.3) 333.9 (777.2) 186.5 (281.9)
Overall
455
139
30.5
388 (832.7)
C. Laugier et al. / Veterinary Parasitology 188 (2012) 185–189
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Table 3 Results of the Faecal egg count reduction (FECR) Test performed on three stud farms: mean pre- and post- (14 days) IVM treatment FEC, standard deviations (SD), maximum pre- and post-treatment FEC, FECR percentages and lower confidence limits (LCL). Farm ID (no. of foals)
Mean FEC (SD) pre-treatment
Max FEC pre-treatment
Mean FEC (SD) post-treatment
Max FEC post-treatment
FECRa (LCL)
FECRb
FECRc (LCL)
6 (12) 13 (14) 15 (10)
441.67 (362.96) 457.14 (608.28) 576 (755.96)
1400 2300 2600
295.83 (426.13) 203.57 (606.52) 184 (327.35)
1400 2300 1100
39.58 (14.93) 58.24 (29.20) 68.94 (54.80)
32.94 55.34 68.02
30 (−59) 53 (−95) 64 (−16)
a b c
Arithmetic means of transformed individual FECRs (Pook et al., 2002) (method 2). Arithmetic means of individual FECRs (Kochapakdee et al., 1995). Arithmetic means of individual FECRs (Kochapakdee et al., 1995) and LCL calculated on 2000 bootstrap resamplings (BootStreat 2.0; Cabaret, 2011).
pre-treatment (day 0) and post-treatment (day 14) faecal egg counts (FEC). Foals were treated orally with ivermectin paste at 0.2 mg/kg body weight. To adjust dosage, body weights were estimated individually using a girth measuring tape or adapted scales. For each foal, the FECR was calculated using the following formula: FECR = (pre-treatment FEC − post-treatment FEC)/pre-treatment FEC. FECR and the lower 95% confidence limits (LCL) were calculated for each stud farm as described by Pook et al. (2002) (i.e., method 2, based √ on transformed individual FECR values: arcsin FECR) and Kochapakdee et al. (1995). For this latter method, the anthelmintic efficacy and confidence interval were established on two thousand bootstrap resamples using a specific program (BootStreat 2.0; Cabaret, 2011). As in other studies (i.e., Kaplan et al., 2004; Veronesi et al., 2010), P. equorum populations were considered susceptible when FECR was ≥90% and 95% LCL > 90%; were suspected of being resistant when FECR ranged from 80% to 90% and the 95% LCL was <90% and were considered resistant when FECR was ≤80% and the 95% LCL was <90%.
3. Results The prevalence of P. equorum in the 15 stud farms varied from 6.9% to 76.2% (Table 1). The overall prevalence was 30.5%. Variations in prevalence according to age are given in Table 2. Prevalence was higher in foals aged 3–4 months and lower in those aged 8–9 months. However, there were no significant differences between the two breeds. Egg excretion was generally low and 66.9% of infected foals showed FEC values of ≤200 epg (Fig. 1). For 14 of the 139 infected foals, FECs were ≥1000 epg and reached a maximum of 7800 epg. Faecal egg outputs were not significantly different among the three age groups. The results of the FECR tests in the three suspect stud farms are presented in Table 3. FECRs varied from 30–39.6% (farm no. 6) to 64–68.9% (farm no. 15). In the three farms, the overall efficacy of ivermectin against P. equorum was 52–54.29%. For six foals, the number of eggs excreted post-treatment was equal or greater than the pre-treatment egg count, indicating that ivermectin was completely ineffective. Before ivermectin treatment, 14 foals (38.9%) had positive strongyles egg counts that ranged from 50 to 3300 (mean 574). After treatment, none of them excreted strongyle eggs.
Fig. 1. Frequency distribution of ascarid faecal egg counts from 139 infected foals.
4. Discussion In other studies, the prevalence of P. equorum based on egg counts has been reported to range from 22.4% to 80% in foals (Rieder et al., 1995; Gawor, 1996; Lyons and Tolliver, 2004; Osterman Lind and Christensson, 2009). This study shows a prevalence of 30.5% in 455 foals, most of which were Thoroughbreds and aged from 3 to 9 months. This value is similar to those given by Lyons and Tolliver (2004) and Lyons et al. (2006), who found 22.4% and 39%, respectively, in Thoroughbred foals of the same age class. The FECR test is the standard method for detecting anthelmintic resistance in cyathostomins. This test has not been validated for P. equorum. However, it is the only test currently available for quantifying the removal of reproductive adult female worms (Reinemeyer, 2009). Therefore, the FECR test has been used in many studies investigating resistance in P. equorum (Slocombe et al., 2007; von Samson-Himmelstjerna et al., 2007; Veronesi et al., 2010). There are several methods for calculating the reduction in the number of eggs and confidence intervals. These methods are either based on geometric means or arithmetic means, with or without controls (i.e., non-treated animals) (Presidente, 1985; Coles et al., 1992; Pook et al., 2002). Our study was conducted in the field on valuable horses; it was not possible to have a sufficient number of infected, non-treated foals in each of the three farms. The calculations were therefore carried out using methods that
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do not require controls (Kochapakdee et al., 1995; Pook et al., 2002). The FECR test results strongly suggest that P. equorum populations on the three tested farms were ivermectinresistant. The demonstration of resistance in young racehorses in France is not surprising because resistance has already been reported in North America and in several European countries where parasite control practices for juvenile horses are similar to those used in France. In many large French stud farms, foals are treated every 4–8 weeks and treatment often includes ivermectin. This practice is likely to accelerate the onset of resistance in P. equorum. If the intervals between treatments are shorter than the prepatency period (75–80 days), ivermectinsusceptible worms cannot reproduce and are eliminated and the number of refugia in paddocks and pastures is reduced. Furthermore, according to Reinemeyer (2009), the MLs would have a larvicidal effect and so, all the life stages harboured in the host would undergo selective pressure. International travel and trade of horses may also promote the spread of resistant P. equorum populations from one country to another. Various studies conducted in Europe (i.e., Germany, Italy, Sweden), Canada and the United States have assessed the effectiveness of several anthelmintics on P. equorum in foals. The results show that for certain ascarid populations, the effectiveness of ivermectin may be low (from 0 to 33.5%), whereas fenbendazole and pyrantel are always effective (90–100%) (Slocombe et al., 2007; von SamsonHimmelstjerna et al., 2007; Lindgren et al., 2008). These latter two active compounds belong to the benzimidazole and tetrahydropyrimidine drug classes, respectively, and are therefore potential alternatives for the treatment of ascarid infection in horses. Resistance to pyrantel pamoate has been reported, but only in North America (Kentucky, USA) (Lyons et al., 2008) and no resistance to benzimidazoles has ever been described. One or two faecal examinations should be carried out during the foals’ first 12 months to check the effectiveness of parasite control programmes and the active compounds used. Drug resistance is generally irreversible. Thus, if MLresistant ascarid populations have been detected in a horse population, this drug class should not be the only treatment used for controlling ascarids. Drug classes with different modes of action should then be used as alternatives (benzimidazoles, pyrantel). However, MLs should not be entirely banned from parasite control programmes because they have a broad spectrum activity. In stud farms where cyathostomins resistant to benzimidazoles and pyrantel coexist with ML-resistant P. equorum (farm no. 6, data not shown), the use of piperazine to control parasite infections in foals and young horses should be considered. The development of resistance is a natural biological consequence of drug treatments; it is practically inevitable. However, certain practices can help hinder resistance and/or its spread. These control practices include a decrease in the frequency of treatments and the maintenance of refugia, i.e., parasite populations that are not exposed to the selective pressure of treatments and thereby represent a pool of susceptible populations. Thus,
elaboration of parasite control programmes becomes more complex as they have now two objectives: to ensure good health and proper growth in foals and at the same time forestall the development of drug resistance. Treatment protocols must therefore seek to achieve a compromise between these two goals. The reduction of deworming frequency necessarily requires increased faecal monitoring in horses and the implementation of control practices to break the parasite’s life cycle and limit the risk of re-infection. In this context, proper sanitation in horse stalls and barns (frequent removal of manure matter along with appropriate cleaning and disinfection) and removing faeces from pastures and paddocks at least once a week are essential good hygiene measures because they help eliminate the sources of contamination. 5. Conclusion This study conducted in 15 large stud farms located in Normandy, provided the first estimate of the prevalence of P. equorum in foals in France. The FECR test results from three farms demonstrate that ivermectin-resistant P. equorum populations are present in France. To determine the extent of this resistance in France, additional studies are needed to investigate more stud farms, particularly those that have been using MLs for several years to treat foals. This study highlights the importance for horse breeders and veterinary practitioners to regularly monitor the effectiveness of anthelmintics using the FECR test and also to promote sustainable non-chemotherapeutic methods to control parasite infections in stud farms. Conflict of interest The author has no financial or personal relationship with other people or organisations that could inappropriately influence or bias this work. Acknowledgements We wish to thank Pauline Crescent for her participation in data analysis, Lydia Baudet for her help in preparing the manuscript and the tables, Aymeric Hans for his review of the manuscript and the Basse-Normandie Regional Council for its financial support. References Boersema, J.H., Eysker, M., Nas, J.W., 2002. Apparent resistance of Parascaris equorum to macrocyclic lactones. Vet. Rec. 150, 279–281. Cabaret, J. 2011. http://wcentre.tours.inra.fr/sfpar/stat.htm. Clayton, H.M., 1986. Ascarids. Recent advances. Vet. Clin. North Am. 2, 313–327. 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. Gawor, J.J., 1996. Occurrence of Parascaris equorum in foals and adult horses under different breeding conditions. Wiad. Parazytol. 42, 213–219.
C. Laugier et al. / Veterinary Parasitology 188 (2012) 185–189 Kaplan, R.M., Klei, T.R., Lyons, E.T., Lester, G., Courtney, C.H., French, D.D., Tolliver, S.C., Vidyashankar, A.N., Zao, Y., 2004. Prevalence of anthelmintic resistant cyathostomes on horse farms. J. Am. Vet. Med. Assoc. 225, 903–910. Kochapakdee, S., Pandey, V.S., Pralomkarn, W., Choldumrongkul, S., Ngampongsai, W., Lawpetchara, A., 1995. Anthelmintic resistance in goats in southern Thailand. Vet. Rec. 137, 124–125. Lindgren, K., Ljungvall, O., Nilsson, O., Ljungström, B.L., Höglund, J., 2008. Parascaris equorum in foals and in their environment on a Swedish farm, with notes on treatment failure of ivermectin. Vet. Parasitol. 151 (2–4), 337–343. Lyons, E.T., Tolliver, S.C., 2004. Prevalence of parasite eggs (Strongyloides westeri, Parascaris equorum and strongyles) and oocyts (Eimeria leuckarti) in the feces of Thoroughbred foals on 14 farms in central Kentucky in 2003. Parasitol. Res. 92, 400–404. Lyons, E.T., Tolliver, S.C., Collins, S.S., 2006. Field studies on endoparasites of Thoroughbred foals on seven farms in central Kentucky in 2004. Parasitol. Res 98, 496–500. Lyons, E.T., Tolliver, S.C., Ionita, M., Collins, S.S., 2008. Evaluation of parasiticidal activity of fenbendazole, ivermectin, oxibendazole, and pyrantel pamoate in horse foals with emphasis on ascarids (Parascaris equorum) in field studies on five farms in Central Kentucky in 2007. Parasitol. Res. 103 (2), 287–291. Molento, M., Antunes, J., Bentes, R.N., 2008. Anthelmintic resistance in Brazilian horses. Vet. Rec. 162, 384–385. Osterman Lind, E., Christensson, D., 2009. Anthelmintic efficacy on Parascaris equorum in foals on Swedish studs. Acta Vet. Scand. 51, 45–48. Pook, J.F., Power, M.L., Sangster, N.C., Hodgson, J.L., Hodgson, D.R., 2002. Evaluation of tests for anthelmintic resistance in cyathostomes. Vet. Parasitol. 106, 331–343.
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Presidente, P.J.A., 1985. Methods for detection of resistance to anthelmintics. In: Anderson, N., Waller, P.J. (Eds.), Resistance in Nematodes to Anthelmintic Drugs. CSIRO Division of Animal Health, Glebe, NSW, Australia, pp. 13–28. Raynaud, J.P., 1970. Etude de l’efficacité d’une technique de coproscopie quantitative pour le diagnostic et le contrôle des infestations parasitaires des bovins, ovins, équins et porcins. Ann. Parasitol. Hum. Comp. 85, 321–342. Reinemeyer, C.R., 2009. Diagnosis and control of anthelmintic-resistant Parascaris equorum. Parasite Vectors 2 (Suppl. 2), S8. Rieder, N., Beelitz, P., Gothe, R., 1995. Incidence of Parascaris equorum in foals and their mares after strategic use of wide-spectrum anthelmintics for several years. Tierarztl. Prax. 23, 53–58. Schougaard, H., Nielsen, M.K., 2007. Apparent ivermectin resistance of Parascaris equorum in foals in Denmark. Vet. Rec. 160, 439–440. Slocombe, J.O.D., De Gannes, R.V.G., Lake, M.C., 2007. Macrocyclic lactoneresistant Parascaris equorum on stud farms in Canada and effectiveness of fenbendazole and pyrantel pamoate. Vet. Parasitol. 145 (3–4), 371–376. Veronesi, F., Moretta, I., Moretti, A., Pergili Fioretti, D., Genchi, C., 2009. Field effectiveness of pyrantel and failure of Parascaris equorum egg count reduction following ivermectin treatment in Italian horse farms. Vet. Parasitol. 161, 138–141. Veronesi, F., Pergili Fioretti, D., Genchi, C., 2010. Are macrocyclic lactones useful drugs for the treatment of Parascaris equorum infections in foals? Vet. Parasitol. 172, 164–167. von Samson-Himmelstjerna, G., Fritzen, B., Demeler, J., Schürmann, 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. Vet. Parasitol. 144 (3–4), 74–80.
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Prevalence of Parascaris equorum infection in foals on French stud farms and first report of ivermectin-resistant P. equorum populations in France Claire Laugier a,∗ , Corinne Sevin a , Sébastien Ménard b , Karine Maillard b a b
Anses, Dozulé Laboratory for Equine Diseases, 14430 Goustranville, France Frank Duncombe Laboratory, IFR 146 ICORE – University of Caen Basse-Normandie, 14053 Caen cedex 4, France
a r t i c l e
i n f o
Article history: Received 19 September 2011 Received in revised form 17 February 2012 Accepted 28 February 2012 Keywords: Parascaris equorum Prevalence Foals France Resistance
1. Introduction Parascaris equorum is a nematode belonging to the family Ascarididae and primarily affects foals. In adult horses, infection is rare and less intense because protective immunity against P. equorum starts to develop by the age of 6 months. The prepatency period is approximately 10–15 weeks (Clayton, 1986). Horses ingest infective eggs that have been shed in the surrounding environment by the previous years’ infected foals. Infection can lead to respiratory symptoms, poor growth, ill thrift accompanied by a rough hair coat and bouts of diarrhoea or colic. When worm burdens are numerous, adult ascarids occasionally cause intestinal obstruction, intussusception or even rupture. The prevalence of P. equorum in foals has been assessed in various countries (e.g., USA, Germany, Poland, Sweden).
∗ Corresponding author at: French Agency for Food, Environmental and Occupational Health Safety, Laboratory for Equine Diseases, RD 675, 14430 Goustranville, France. Tel.: +33 231797946; fax: +33 231392137. E-mail address: [email protected] (C. Laugier). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2012.02.022
Prevalence is generally high and varies from 22.4% to 80% (Rieder et al., 1995; Gawor, 1996; Lyons and Tolliver, 2004; Osterman Lind and Christensson, 2009). However, to date, little is known about the prevalence of P. equorum in foals in France. In most horse breeding regions, young horses are regularly treated against P. equorum until 12 months of age. The main anthelmintics used in horses belong to three different drug classes: benzimidazole compounds, tetrahydropyrimidines (pyrantel) and macrocyclic lactones (MLs: ivermectin and moxidectin). Macrocyclic lactones are particularly important in the control of equine intestinal parasites because they have a broad spectrum activity (i.e., they are nematocidal and insecticidal) and they also target migrating larval stages of large strongyles and P. equorum. These drugs are frequently used in foals and young horses. Since the first description of an ML treatment failure in the Netherlands in 2002 (Boersema et al., 2002), several studies have reported ML-resistant P. equorum in various European countries (von Samson-Himmelstjerna et al., 2007; Schougaard and Nielsen, 2007; Lindgren et al., 2008; Veronesi et al., 2009;) and also in North America and Brazil
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C. Laugier et al. / Veterinary Parasitology 188 (2012) 185–189
Table 1 Prevalence of P. equorum eggs in fecal samples of 455 foals from 15 breeding farms in France. Farm
Breed
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
TBa TB TB TB TB TB TB TB TB TB TB FTb FT FT FT
Number of foals sampled
Overall a b
Mean age (month)
Prevalence (%)
38 62 79 45 48 28 12 12 11 19 10 29 21 18 23
Number of infected foals 6 20 25 14 7 14 1 6 5 2 4 2 16 4 13
4 6 5.5 5 6 6 9 6 3.5 4 6 8 3.5 3.5 8
15.8 32.3 31.6 31.1 14.6 50.0 8.3 50.0 45.5 10.5 40.0 6.9 76.2 22.2 56.5
455
139
5.5
30.5
TB Thoroughbred. FT French Trotter.
(Slocombe et al., 2007; Lyons et al., 2008; Molento et al., 2008). In France, several equine veterinary practitioners have reported apparent ivermectin treatment failures in nursing foals and weanlings. The purpose of this study was first to estimate the prevalence of P. equorum in foals from 15 large stud farms in France and then to investigate the efficacy of ivermectin using the faecal egg count reduction (FECR) test in three farms with suspected resistant ascarid populations. This study was conducted in Normandy, France, which is the leading horse breeding region in France with roughly 10,500 foal births per year.
2. Materials and methods The prevalence of P. equorum infection was estimated on a total of 455 foals aged from 3 to 9 months from 11 Thoroughbred horse farms and four French Trotter horse farms (Table 1). Most foals included in the study (80%) were Thoroughbreds and their mean age was 5.5 months. The number of foals sampled in each farm varied from 10 to 79. Samples were taken from July to October 2010. During this period, the dams and their foals spent most of their time outdoors in paddocks or pastures. In the 15 stud farms, deworming protocols in foals were roughly similar: the first treatment was administered at 1 month of age and subsequent treatments were given monthly or bimonthly until 18 months. During the first 6–8 months, the use of ivermectin was exclusive or very frequent; then, alternation with the two other drug classes (benzimidazoles or pyrantel) was implemented.
Finally, foals generally received three administrations of ivermectin between birth and weaning. In three of the 15 farms (nos. 6, 13 and 15), equine veterinary practitioners had reported apparent ivermectin treatment failure upon observing massive ascarid infection during autopsy (farm no. 15) or abundant egg excretion (farms nos. 6 and 13) despite regular and/or recent ivermectin treatments. From these three farms, 36 foals naturally infected with P. equorum and shedding at least 100 eggs per gram faeces (epg) were selected to test the effectiveness of ivermectin against this roundworm. Individual faecal samples were collected, depending on the foal’s age, rectally or from the top of newly deposited faeces using veterinary gloves. All faecal samples were stored under refrigeration and examined within 48 h of collection. Faecal egg counts were determined by a modified McMaster technique (Raynaud, 1970) with a saturated solution of sodium chloride (specific gravity 1.20) as the flotation solution and a detection limit of 50 epg. The influence of breed and age on P. equorum prevalence was analysed using a Chi-square test. To analyse the influence of age, foals were grouped into three age classes: 3–4 months, 5–7 months and 8–9 months (Table 2). Differences in faecal egg output between the three age groups were tested using an analysis of variance on log-transformed data (Statview software package 1998 SAS Institute Inc.). For all statistical analyses, we applied a 5% significance level (p < 0.05). In the three farms suspected of harbouring resistant ascarids, the effectiveness of ivermectin was assessed using the FECR test based on individual animals between
Table 2 Age distribution of P. equorum infection and mean fecal egg counts (FEC) of 455 foals from 15 breeding farms in France. Age (months)
Number of foals sampled
Number of infected foals
Prevalence (%)
Mean FEC (S.D.)
3–4 5–7 8–9
134 249 72
51 77 11
38.1 30.9 15.3
596.6 (1052.3) 333.9 (777.2) 186.5 (281.9)
Overall
455
139
30.5
388 (832.7)
C. Laugier et al. / Veterinary Parasitology 188 (2012) 185–189
187
Table 3 Results of the Faecal egg count reduction (FECR) Test performed on three stud farms: mean pre- and post- (14 days) IVM treatment FEC, standard deviations (SD), maximum pre- and post-treatment FEC, FECR percentages and lower confidence limits (LCL). Farm ID (no. of foals)
Mean FEC (SD) pre-treatment
Max FEC pre-treatment
Mean FEC (SD) post-treatment
Max FEC post-treatment
FECRa (LCL)
FECRb
FECRc (LCL)
6 (12) 13 (14) 15 (10)
441.67 (362.96) 457.14 (608.28) 576 (755.96)
1400 2300 2600
295.83 (426.13) 203.57 (606.52) 184 (327.35)
1400 2300 1100
39.58 (14.93) 58.24 (29.20) 68.94 (54.80)
32.94 55.34 68.02
30 (−59) 53 (−95) 64 (−16)
a b c
Arithmetic means of transformed individual FECRs (Pook et al., 2002) (method 2). Arithmetic means of individual FECRs (Kochapakdee et al., 1995). Arithmetic means of individual FECRs (Kochapakdee et al., 1995) and LCL calculated on 2000 bootstrap resamplings (BootStreat 2.0; Cabaret, 2011).
pre-treatment (day 0) and post-treatment (day 14) faecal egg counts (FEC). Foals were treated orally with ivermectin paste at 0.2 mg/kg body weight. To adjust dosage, body weights were estimated individually using a girth measuring tape or adapted scales. For each foal, the FECR was calculated using the following formula: FECR = (pre-treatment FEC − post-treatment FEC)/pre-treatment FEC. FECR and the lower 95% confidence limits (LCL) were calculated for each stud farm as described by Pook et al. (2002) (i.e., method 2, based √ on transformed individual FECR values: arcsin FECR) and Kochapakdee et al. (1995). For this latter method, the anthelmintic efficacy and confidence interval were established on two thousand bootstrap resamples using a specific program (BootStreat 2.0; Cabaret, 2011). As in other studies (i.e., Kaplan et al., 2004; Veronesi et al., 2010), P. equorum populations were considered susceptible when FECR was ≥90% and 95% LCL > 90%; were suspected of being resistant when FECR ranged from 80% to 90% and the 95% LCL was <90% and were considered resistant when FECR was ≤80% and the 95% LCL was <90%.
3. Results The prevalence of P. equorum in the 15 stud farms varied from 6.9% to 76.2% (Table 1). The overall prevalence was 30.5%. Variations in prevalence according to age are given in Table 2. Prevalence was higher in foals aged 3–4 months and lower in those aged 8–9 months. However, there were no significant differences between the two breeds. Egg excretion was generally low and 66.9% of infected foals showed FEC values of ≤200 epg (Fig. 1). For 14 of the 139 infected foals, FECs were ≥1000 epg and reached a maximum of 7800 epg. Faecal egg outputs were not significantly different among the three age groups. The results of the FECR tests in the three suspect stud farms are presented in Table 3. FECRs varied from 30–39.6% (farm no. 6) to 64–68.9% (farm no. 15). In the three farms, the overall efficacy of ivermectin against P. equorum was 52–54.29%. For six foals, the number of eggs excreted post-treatment was equal or greater than the pre-treatment egg count, indicating that ivermectin was completely ineffective. Before ivermectin treatment, 14 foals (38.9%) had positive strongyles egg counts that ranged from 50 to 3300 (mean 574). After treatment, none of them excreted strongyle eggs.
Fig. 1. Frequency distribution of ascarid faecal egg counts from 139 infected foals.
4. Discussion In other studies, the prevalence of P. equorum based on egg counts has been reported to range from 22.4% to 80% in foals (Rieder et al., 1995; Gawor, 1996; Lyons and Tolliver, 2004; Osterman Lind and Christensson, 2009). This study shows a prevalence of 30.5% in 455 foals, most of which were Thoroughbreds and aged from 3 to 9 months. This value is similar to those given by Lyons and Tolliver (2004) and Lyons et al. (2006), who found 22.4% and 39%, respectively, in Thoroughbred foals of the same age class. The FECR test is the standard method for detecting anthelmintic resistance in cyathostomins. This test has not been validated for P. equorum. However, it is the only test currently available for quantifying the removal of reproductive adult female worms (Reinemeyer, 2009). Therefore, the FECR test has been used in many studies investigating resistance in P. equorum (Slocombe et al., 2007; von Samson-Himmelstjerna et al., 2007; Veronesi et al., 2010). There are several methods for calculating the reduction in the number of eggs and confidence intervals. These methods are either based on geometric means or arithmetic means, with or without controls (i.e., non-treated animals) (Presidente, 1985; Coles et al., 1992; Pook et al., 2002). Our study was conducted in the field on valuable horses; it was not possible to have a sufficient number of infected, non-treated foals in each of the three farms. The calculations were therefore carried out using methods that
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do not require controls (Kochapakdee et al., 1995; Pook et al., 2002). The FECR test results strongly suggest that P. equorum populations on the three tested farms were ivermectinresistant. The demonstration of resistance in young racehorses in France is not surprising because resistance has already been reported in North America and in several European countries where parasite control practices for juvenile horses are similar to those used in France. In many large French stud farms, foals are treated every 4–8 weeks and treatment often includes ivermectin. This practice is likely to accelerate the onset of resistance in P. equorum. If the intervals between treatments are shorter than the prepatency period (75–80 days), ivermectinsusceptible worms cannot reproduce and are eliminated and the number of refugia in paddocks and pastures is reduced. Furthermore, according to Reinemeyer (2009), the MLs would have a larvicidal effect and so, all the life stages harboured in the host would undergo selective pressure. International travel and trade of horses may also promote the spread of resistant P. equorum populations from one country to another. Various studies conducted in Europe (i.e., Germany, Italy, Sweden), Canada and the United States have assessed the effectiveness of several anthelmintics on P. equorum in foals. The results show that for certain ascarid populations, the effectiveness of ivermectin may be low (from 0 to 33.5%), whereas fenbendazole and pyrantel are always effective (90–100%) (Slocombe et al., 2007; von SamsonHimmelstjerna et al., 2007; Lindgren et al., 2008). These latter two active compounds belong to the benzimidazole and tetrahydropyrimidine drug classes, respectively, and are therefore potential alternatives for the treatment of ascarid infection in horses. Resistance to pyrantel pamoate has been reported, but only in North America (Kentucky, USA) (Lyons et al., 2008) and no resistance to benzimidazoles has ever been described. One or two faecal examinations should be carried out during the foals’ first 12 months to check the effectiveness of parasite control programmes and the active compounds used. Drug resistance is generally irreversible. Thus, if MLresistant ascarid populations have been detected in a horse population, this drug class should not be the only treatment used for controlling ascarids. Drug classes with different modes of action should then be used as alternatives (benzimidazoles, pyrantel). However, MLs should not be entirely banned from parasite control programmes because they have a broad spectrum activity. In stud farms where cyathostomins resistant to benzimidazoles and pyrantel coexist with ML-resistant P. equorum (farm no. 6, data not shown), the use of piperazine to control parasite infections in foals and young horses should be considered. The development of resistance is a natural biological consequence of drug treatments; it is practically inevitable. However, certain practices can help hinder resistance and/or its spread. These control practices include a decrease in the frequency of treatments and the maintenance of refugia, i.e., parasite populations that are not exposed to the selective pressure of treatments and thereby represent a pool of susceptible populations. Thus,
elaboration of parasite control programmes becomes more complex as they have now two objectives: to ensure good health and proper growth in foals and at the same time forestall the development of drug resistance. Treatment protocols must therefore seek to achieve a compromise between these two goals. The reduction of deworming frequency necessarily requires increased faecal monitoring in horses and the implementation of control practices to break the parasite’s life cycle and limit the risk of re-infection. In this context, proper sanitation in horse stalls and barns (frequent removal of manure matter along with appropriate cleaning and disinfection) and removing faeces from pastures and paddocks at least once a week are essential good hygiene measures because they help eliminate the sources of contamination. 5. Conclusion This study conducted in 15 large stud farms located in Normandy, provided the first estimate of the prevalence of P. equorum in foals in France. The FECR test results from three farms demonstrate that ivermectin-resistant P. equorum populations are present in France. To determine the extent of this resistance in France, additional studies are needed to investigate more stud farms, particularly those that have been using MLs for several years to treat foals. This study highlights the importance for horse breeders and veterinary practitioners to regularly monitor the effectiveness of anthelmintics using the FECR test and also to promote sustainable non-chemotherapeutic methods to control parasite infections in stud farms. Conflict of interest The author has no financial or personal relationship with other people or organisations that could inappropriately influence or bias this work. Acknowledgements We wish to thank Pauline Crescent for her participation in data analysis, Lydia Baudet for her help in preparing the manuscript and the tables, Aymeric Hans for his review of the manuscript and the Basse-Normandie Regional Council for its financial support. References Boersema, J.H., Eysker, M., Nas, J.W., 2002. Apparent resistance of Parascaris equorum to macrocyclic lactones. Vet. Rec. 150, 279–281. Cabaret, J. 2011. http://wcentre.tours.inra.fr/sfpar/stat.htm. Clayton, H.M., 1986. Ascarids. Recent advances. Vet. Clin. North Am. 2, 313–327. 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. Gawor, J.J., 1996. Occurrence of Parascaris equorum in foals and adult horses under different breeding conditions. Wiad. Parazytol. 42, 213–219.
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