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Keywords:

  • horse;
  • cyathostomin;
  • anthelmintic resistance;
  • diagnostic tools;
  • sensitivity testing

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References

Cyathostomins represent a potential cause of equine morbidity and have become the main focus of endoparasite control in managed horses. All grazing horses are at risk of infection with cyathostomins; therefore, the application of appropriate management measures is essential. Anthelmintics currently comprise the main method of control for equine nematodes and the ready availability of these products in some countries has resulted in their use becoming dissociated from veterinary involvement. This is concerning given the levels of anthelmintic resistance that have been recorded in cyathostomin populations. It is important that veterinarians re-establish control over the implementation of parasite control programmes, a major objective of which should be the preservation of anthelmintic efficacy. This article details the principles of cyathostomin control in horses with particular reference to anthelmintic resistance, and the use and interpretation of diagnostic tests for detecting cyathostomins and identifying anthelmintic resistance.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References

Virtually all grazing horses are exposed to cyathostomins. Although over 50 species have been described, most horses are infected with 5–10 common species (Ogbourne 1976; Reinemeyer et al. 1984). Infection with cyathostomins may result in wide ranging clinical signs including vague malaise, colic, weight loss and anorexia. Furthermore, ‘larval cyathostominosis’, characterised by rapid weight loss, oedema and diarrhoea, may result in death in up to 50% of the cases that present with this syndrome (Uhlinger 1990; Hillyer and Mair 1997; Love et al. 1999; Lyons et al. 2000). It should be emphasised, however, that severe clinical disease resulting from cyathostomins is uncommon in the horse population at large and that the perceived incidence of larval cyathostominosis does appear to vary geographically. As most cyathostomin burdens in horses are low and rarely result in clinical disease, recommendations for targeted anthelmintic dosing should be instituted to reduce infection load.

Cyathostomins have a direct, nonmigratory life cycle. Third stage larvae (L3) ingested from pasture penetrate the wall of the large intestine, where they undergo development before re-emerging and maturing to adults within the lumen. Female adult worms release eggs that are shed in faeces. These hatch to release first stage larvae, which undergo 2 moults to become infective L3. In some instances, L3 enter a state of inhibited development as early L3 (EL3) in the host for months to years (Murphy and Love 1997). It is not known why larvae become inhibited, although cold conditioning of L3 prior to ingestion, gradual accumulation of L3 infection, host immunity and population density of the parasites within the intestinal wall and lumen have been proposed as contributory factors (Love et al. 1999; Matthews 2008). Anthelmintic treatment that primarily targets luminal stages may serve as a stimulus for mucosal emergence by reducing the luminal nematode population. During autumn and winter, EL3 and other mucosal stages constitute the major cyathostomin burden. Control of cyathostomins is complicated because horses acquire variable, incomplete immunity, requiring some therapeutic intervention throughout their lives (Matthews 2008). Furthermore, faecal egg counts (FECs) underestimate the true parasite burden when larval populations predominate and luminal adult burdens are low (Dowdall et al. 2002). Finally, evidence obtained thus far indicates that EL3 stages are not particularly susceptible to most anthelmintics (Love and McKeand 1997).

Anthelmintics

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References

Three classes of anthelmintic are licensed for the control and treatment of nematodes in horses; benzimidazoles (BZs), tetrahydropyrimidines (THPs) and macrocyclic lactones (avermectins and milbemycins, MLs). With no new anthelmintic classes expected to be available for use in horses in the short to medium term, current anthelmintics must be used appropriately to preserve their efficacy. Original controlled efficacy trials revealed noteworthy differences in the activities of the various anthelmintic classes against different cyathostomin life cycle stages in drug-sensitive populations. For example, a single dose of fenbendazole (FBZ) (5 mg/kg bwt) was shown to exhibit 100% efficacy againstadult cyathostomins (Colglazier et al. 1977), whilst a dose of 7.5 mg/kg bwt administered daily for 5 consecutive days provided >95% efficacy vs. total mucosal larvae, including >91% efficacy against inhibited EL3 (Duncan et al. 1998). The THP pyrantel was shown to eliminate 89–96% of adult cyathostomins in a single dose administered orally (Lyons et al. 1974). Ivermectin (IVM) at a dose rate of 0.2 mg/kg bwt was demonstrated to have excellent activity against adult cyathostomins (>99% efficacy) and luminal larvae (98% efficacy), but appeared to have minimal effect on EL3, even at a high (up to 1 mg/kg bwt) dose rate (Klei et al. 1993; Xiao et al. 1994). On the other hand, some studies demonstrated that a single dose of moxidectin (MOX) (0.4 mg/kg bwt) had a ‘persistent’ effect and was shown to be highly effective against all life cycle stages including 90.8% efficacy against EL3 (Reinemeyer et al. 2003; Bairden et al. 2006). It should be noted, however, that variable efficacy of MOX against EL3 has been reported in the literature; for example Eysker et al. (1997) and Xiao et al. (1994) demonstrated less effect on EL3 stage cyathostomins. The discrepancies in MOX efficacy reported possibly reflect differences in experimental design amongst studies; for example, the chosen interval between anthelmintic dosing and necropsy (reviewed by Matthews 2008).

The egg reappearance period (ERP) is the period post anthelmintic dosing, during which egg shedding remains negligible, or below a certain threshold (Duncan 1985). This period varies with anthelmintic class. ERPs in anthelmintic sensitive populations can vary but were previously generally specified as 6–8 weeks for BZs, 6 weeks for pyrantel, 8–10 weeks for IVM and >13 weeks for MOX (Herd and Gabel 1990; Borgsteede et al. 1993; Mercier et al. 2001). However, ERPs appear to have shortened over time and, in some cases, considerably in the last decade (von Samson-Himmelstjerna et al. 2007; Lyons et al. 2008; Molento et al. 2008). Despite this many owners and veterinary surgeons are still instituting interval anthelmintic treatments on the basis of the ERPs described many years ago. Knowledge regarding these changes in ERP needs to be transferred to the appropriate end-users and, importantly, linked to the fact that observed reductions in ERP post anthelmintic dosing can be a first indication of the development of drug resistance within nematode populations.

Anthelmintic resistance

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References

Anthelmintic resistance is now a serious problem in cyathostomins. ‘Resistance is present when there is a greater frequency of individuals within a population that can tolerate doses of compound than in normal populations of the same species and is heritable’ (Prichard et al. 1980). Parasitic nematodes are predisposed to development of anthelmintic resistance due to their considerable population sizes, high levels of genetic diversity and relatively rapid generation rates (Gilleard and Beech 2007). Resistance traits can then be disseminated fairly rapidly through populations via animal movements and high rates of gene flow. Resistance is currently considered a permanent trait, with little supporting evidence for reversion to susceptibility (Jackson and Coop 2000).

Resistance in adult stage cyathostomins to the BZ thiabendazole was first reported in the USA in 1965 (reviewed by Lyons et al. 1999). Resistance to a single (adulticidal) dose of other members of the BZ class has subsequently been described at other geographical locations (Drudge et al. 1979; Craven et al. 1998; Lyons et al. 1999). The relatively high level of BZ resistance is unsurprising, given that these products represent the first class of broad spectrum anthelmintic approved for equine use in the 1960s. Some studies have found the presence of BZ resistance to be almost ubiquitous in cyathostomins; for example, BZ-resistant populations were identified on 97.7% of USA farms tested (Kaplan et al. 2004). In other studies, the prevalence rate of BZ resistance in equine nematode populations has not been as high as that reported in the USA study: 72% of farms tested in Sweden were shown to harbour BZ-resistant parasites (Kaplan et al. 2004; Osterman Lind et al. 2007). High levels of egg shedding have also been recorded following administration of a 5 day FBZ course, suggesting that such treatment is inadvisable where lack of efficacy of a single BZ dose has already been detected (Chandler et al. 2000; Chandler and Love 2002; Rossano et al. 2010).

Resistance of cyathostomins to THPs appears to have been slower to develop. This has been proposed to reflect the comparatively lower larvicidal efficacy of THPs resulting in proportions of parasites not exposed to the effects of selection pressure (Dargatz et al. 2000). Alternatively, the prevalence of THP resistance may be underestimated considering the relative lack of studies investigating the field efficacy of this class of anthelmintic in horses. Widespread resistance has been described in the USA where the availability of daily, in-feed pyrantel treatments, licensed since the 1990s (Dipietro 1992), may have accelerated the development of resistance (Tarigo-Martinie et al. 2001; Slocombe and de Gannes 2006). Pyrantel resistance has now also been reported in Europe (Comer et al. 2006; Osterman Lind et al. 2007) and single populations of cyathostomins have been identified that display both BZ and pyrantel resistance (Kaplan et al. 2004; Traversa et al. 2007).

Despite IVM dominating the equine anthelmintics market over the last 2 decades, resistance to IVM in cyathostomins was reported only relatively recently. This may reflect its lack of efficacy vs. mucosal larvae or the involvement of more complex genetic mechanisms for resistance development (Sangster 1999). Reductions in the ERP post treatment, initially to 6 weeks (Tarigo-Martinie et al. 2001) and then to 4–5 weeks, have been reported in the USA, Brazil and Europe (von Samson-Himmelstjerna et al. 2007; Lyons et al. 2008; Molento et al. 2008). The ML anthelmintic MOX is the only remaining product effective against EL3 for which widespread drug resistance has not yet been reported; therefore, preservation of its efficacy is important. However, MOX-resistant cyathostomins have been detected in 2 populations of equids based at the UK Donkey Sanctuary (Trawford et al. 2005) and a shortened strongyle ERP has been reported after administration of MOX to horses in Kentucky (Rossano et al. 2010). Resistance to MOX is considered inevitable and the possible reasons for this have been reviewed previously (Sangster 1999; Matthews 2008). Most worryingly, concurrent resistance to all 3 classes of anthelmintic in single cyathostomin populations has been indicated in Brazil, where both IVM and MOX failed to reduce cyathostomin faecal egg counts (FECs) adequately 28 days after dosing (Molento et al. 2008). The molecular mechanisms underlying ML resistance in nematode species are currently not understood.

Approaches to parasite control to counter anthelmintic resistance

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References

Despite recommendations being described for selective targeted therapy nearly 2 decades ago (Duncan and Love 1991; Gomez and Georgi 1991), there has been a significant lag period in their implementation. Studies in the last decade have shown that veterinarians have become increasingly dissociated from equine parasite control, providing advice on only 29–34% of yards questioned (Earle et al. 2002; Comer et al. 2006). It is essential that veterinarians re-establish involvement in parasite control programmes by designing evidence-based sustainable, targeted and prophylactic protocols. The principle objective of such programmes should be to decrease selection pressure for anthelmintic resistance, whilst still maintaining equine health and performance by reducing nematode infection intensity. Differences in environmental influences, pasture management principles and herd dynamics imply that no single protocol can be applied uniformly; however, an enhanced understanding of the principle aims of control, specific target parasites, appropriate diagnostic tests (and their limitations), along with a knowledge of the parasite populations' anthelmintic sensitivity provide the necessary concepts on which to base a best-practice control programme.

The rationale for targeted anthelmintic therapy

Individual horses differ markedly in their susceptibility to strongyle infections (Morgan et al. 2005). The resultant over-dispersed nature of nematode infections provides an ideal opportunity for sustainable control via selective, targeted therapy (Sangster 2003). On typical equine premises, 20% of horses shed 80% of nematode eggs thereby providing the major contribution to pasture contamination. These apparently more susceptible ‘high shedders’ generally maintain higher lifelong FECs than their herd mates, despite similar exposure levels (Nielsen et al. 2006a; Becher et al. 2010). Once identified by FEC, they can be appropriately targeted with adulticidal anthelmintics to reduce pasture contamination. Conversely, ‘low shedders’ apparently have an innate or acquired ability to control strongyle nematode infections, hence requiring less frequent anthelmintic treatments. Alternatively, horses may be transient low shedders following recent, frequent anthelmintic use, presumably a result of lower pasture infection intensities (Lloyd 2009). When animals that are low shedders are left untreated with anthelmintics, the eggs in their faeces provide an important population of parasites known as refugia. These parasites serve to dilute eggs that have been laid by adult worms that have survived anthelmintic therapy in treated horses. As refugia is considered to be the most influential factor in the development of anthelmintic resistance (van Wyk et al. 1997), blanket treatment of all horses, irrespective of their faecal egg output, is not recommended. Young horses are generally more susceptible to gastrointestinal nematode infections and usually have a higher infection intensity than mature horses (von Samson-Himmelstjerna et al. 2009). Shortened ERPs have been identified after anthelmintic therapy in young horses (Herd and Gabel 1990). Consequently, equine populations with high proportions of younger animals present a challenge for targeted anthelmintic programmes.

Determining cyathostomin burdens for targeted therapy

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References

Faecal egg counts (FECs)

The FEC is an inexpensive test that can be readily performed ‘in-house’ at a practice and requires little in the way of specialised equipment or, once appropriate training has been delivered, advanced skills. Additionally, a number of specialised laboratories offer an inexpensive FEC service. FECs are essential for the delivery of selective, targeted therapy, allowing the identification of horses that require adulticidal treatments. Selective, targeted programmes have been shown to reduce anthelmintic use by up to 75% (Kaplan 2002). FECs are currently underused in equine practice and are often reserved for investigative purposes in clinical cases (Earle et al. 2002).

Repeated analysis of FECs from the same individual may show variability due to the uneven distribution of eggs in faeces and to inherent day-to-day variation in FECs (Uhlinger 1993). However, the level of variability is unlikely to be of sufficient magnitude to influence the classification of individual horses as low or high shedders over time. Thus, most methodologies currently in use should be of sufficient sensitivity to detect FECs to be able to inform on appropriate anthelmintic use. The McMaster (or modified McMaster) test, based on the flotation dilution principle, is the most commonly used technique for detecting nematode eggs. FECPAK1 has recently been developed to enable on-site monitoring and has improved sensitivity due to the examination of a greater volume of faeces (Presland et al. 2005). A centrifugal-flotation technique is the most sensitive, particularly at low egg numbers (Bello and Allen 2009). Higher levels of sensitivity (i.e. detection limits at <50 eggs/g) will aid more robust determination of anthelmintic sensitivity via the faecal egg count reduction test (FECRT), especially where the accurate determination of low FECs after anthelmintic dosing is required. Most accurate results are obtained from samples collected within 12 h of passage. Samples may be refrigerated in airtight containers for up to 120 h without significant alteration in egg numbers (Nielsen et al. 2010).

It should be noted that FECs do not provide an accurate gauge of total nematode burdens within individual animals, especially during autumn/winter when mucosal larval stages are undetectable (Duncan 1974). Despite their limitations, FEC analyses do provide essential information. They are best performed during the grazing season to identify the relative contribution that individual horses make to pasture contamination. All co-grazers should be tested simultaneously after the ERP of the previously administered anthelmintic has elapsed. Individuals with a FEC above a set threshold should be treated synchronously with a licensed anthelmintic, shown previously to be effective, at a dose rate for 100–110% bodyweight estimated by weight tape or scale. Follow-up FECs may then be performed at 2–3 monthly intervals depending on the product used. The frequency of FEC analysis may be reduced subsequent to elucidating the egg excretion dynamics of a given equine population. The most frequently cited FEC cut-off for determining a requirement for treatment is 200–500 eggs/g (Matthews 2008). Since FECs do not detect larval cyathostomins, larvicidal treatment must be administered when appropriate independent of FEC data.

Anthelmintics became prescription-only medications in Denmark in 1999. Subsequently, targeted therapy based on routine FECs was implemented and it was observed that the frequency of treatment subsequently decreased over time (Nielsen et al. 2006b). Effective selective, targeted dosing has also been described in Germany and Austria (Nielsen et al. 2006a; Becher et al. 2010). It should be noted that reductions in anthelmintic treatment frequency could potentially lead to increased prevalence of other types of parasite, such as Oxyuris equi, Gasterophilus spp. and, of particular concern, Strongylus spp. However, following a decade of selective, targeted treatment in Denmark, it was noted that the prevalence of Strongylus vulgaris has remained similar to that observed in neighbouring Sweden where selective, targeted treatment regimens have not been instituted at the same level (Nielsen 2009). Little is known about the influence of selective, targeted treatment regimes on the development of anthelmintic resistance, the population dynamics of the parasites or of the risk of parasite-associated clinical disease in horses and further research is required in this area. Better baseline prevalence data are essential to assess the impact of changes in parasite management regimes over time. Underlying this, there is a need for increased reliability of diagnostic techniques (such as FEC analysis) and also for the development of further tools that help in the assessment of parasite burden and anthelmintic sensitivity.

Future diagnostic tests for assessing cyathostomin encysted larval burden

Larval cyathostominosis results from the mass emergence of encysted larval stages from the large intestinal wall. There is currently no routinely available noninvasive method to enumerate cyathostomin encysted larvae, and horses with high burdens may have low or negative FECs (Dowdall et al. 2002). Furthermore, horses with larval cyathostominosis often have no specific discriminating clinical signs. A noninvasive diagnostic test for encysted larvae would assist in the diagnosis of larval cyathostominosis and would aid in the identification of horses requiring larvicidal treatments. Previously, Dowdall et al. (2002, 2004) demonstrated that serum IgG(T) responses to 2 larval native antigen complexes (of 20 and 25 kDa in size) were observed to be significantly higher in clinical cases than in cyathostomin-naïve or -negative animals. Moreover, in experimentally-infected animals, anti-25 kDa complex IgG(T) levels correlated positively with field exposure and, importantly, with burdens of EL3 (r(s) = 0.74, P = 0.015) and total mucosal parasites (r(s) = 0.78, P = 0.010). In naturally infected horses, whose parasite burdens were quantified post mortem, antigen-specific IgG(T) responses were also significantly higher in infected than in uninfected horses (P = 0.0001 and 0.002 for anti-25 and anti-20 kDa responses, respectively). In these horses, anti-25 kDa IgG(T) levels correlated positively with mucosal and luminal burdens (P<0.05). In terms of IgG(T) responses to the 20 kDa antigen complex, levels correlated positively with luminal burdens only (P = 0.0043) (Dowdall et al. 2004). Antigens within the complexes were shown to be specific to the cyathostomin group, with no or limited cross reactivity to other helminth species (Dowdall et al. 2003). As these antigens are time-consuming to prepare and rely on a continuous source of tissue from infected animals, representative recombinant proteins have been produced in Escherichia coli (McWilliam et al. 2010). Recently, genes encoding 2 antigenic proteins, cyathostomin gut associated larval antigen (Cy-GALA) and cyathostomin immuno-diagnostic antigen (Cy-CID), that are produced by encysted larval stages, were isolated and the proteins expressed in E. coli. A number of recombinant Cy-GALA proteins, representative of common cyathostomin species, have now been produced. These proteins exhibit no reactivity to serum from horses specifically infected with other noncyathostomin species, nor does antiserum, raised to one of the proteins, bind to worm extracts from other species. Initial data suggest that recombinant GALA proteins have diagnostic potential and a patent application is under review. Further development of this assay is directed at increasing cyathostomin species coverage for the proteins Cy-GALA and Cy-CID, to ensure that the assay is sensitive to the presence of other, less common cyathostomin species. It is anticipated that a diagnostic assay based on detection of antibodies to a cocktail of these recombinant proteins will be developed for commercial use in the next 3–5 years.

Mast cell counts and their proteinase (chymase/tryptase) expression may also offer predictive value for the determination of total (including mural) cyathostomin burdens. The intestinal immune response to cyathostomin infection is not fully understood, although increased mast cell numbers have been documented in the large intestine of horses naturally infected with cyathostomins, with a linear correlation observed between magnitude of caecal cyathostomin burden and both toluidine blue stained, and chymase- and tryptase-labelled mast cell populations (Pickles et al. 2010). Development of a noninvasive serological or rectal biopsy assay for mast cells or their proteinases is ongoing and could represent a advance in the in vivo diagnosis of active cyathostomin infection in future.

Assessing anthelmintic sensitivity

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References

Anthelmintic sensitivity testing should be the cornerstone of all nematode control programmes. It is important to determine anthelmintic sensitivity in a population and to thoroughly investigate all suspected cases of drug resistance. In the UK, suspected cases of resistance should be reported to the Veterinary Medicines Directorate as a ‘Suspected Adverse Reaction’. Anthelmintic resistance may be indicated by a significantly reduced FECRT, a reduction in the ERP post treatment when compared with that of the original efficacy studies, or by clinical signs of disease in horses where appropriate anthelmintic dosing is thought to have been instituted.

Faecal egg count reduction test

The FECRT compares FECs before and at 10–14 days after anthelmintic treatment. It is simple to perform, can be applied to all anthelmintic classes and is currently the most widely used and suitable screening test for anthelmintic resistance in equine nematodes (Coles et al. 2006; Matthews et al. 2011).

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The FECRT has several limitations. Studies in sheep suggest that it is relatively insensitive for detecting resistance when the proportion of genetically resistant worms is below 25% (Martin et al. 1989). Therefore, low levels of anthelmintic resistance may be missed and, by the time resistance is identifiable by FECRT, resistance genes may be widely disseminated (Taylor and Hunt 1989). The accuracy of the FECRT is also affected by the inherent variability of FEC data (Uhlinger 1993) and, in sheep, it has also been shown to provide poor correlation with actual nematode numbers (Miller et al. 2006). It is assumed that similar limitations will apply to use of the FECRT in horses. It is also difficult to interpret data derived from horses that have low FECs. Pre- and post treatment samples should be handled and processed similarly to decrease unnecessary variability (Nielsen et al. 2010). Performing FECRTs on a large proportion of the population will provide the most accurate representation of anthelmintic sensitivity and it is recommended that a minimum of 6 horses (Coles et al. 2006) or ≥80% of the population should be assessed.

Different FECRT cut-offs are recommended for different anthelmintic classes; 90% for BZs and THPs and 95% for MLs (Kaplan and Nielsen 2010). These levels take into consideration the differing efficacies of the 3 anthelmintic classes in drug-sensitive nematode populations. A mean population FECRT above these cut-offs represents acceptable anthelmintic efficacy, whilst a lower mean FECRT suggests drug resistance (Kaplan and Nielsen 2010). In cases of suspected resistance detected by the FECRT, the test should be repeated. A second observed reduction in efficacy confirms anthelmintic resistance, whereas a FECRT above the accepted thresholds detailed above indicates possible under-dosing or administration error in the initial study. It is also important to consider the number of horses tested, the age distribution of the population and average pretreatment FEC to determine whether the result is likely to be truly representative of the entire population.

In vitro diagnosis of anthelmintic sensitivity

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References

Several in vitro diagnostic tests of anthelmintic sensitivity have been investigated. While these are unlikely to be used in general practice, in future they may be utilised by research laboratories to assay the anthelmintic sensitivity of cyathostomin populations.

The larval migration inhibition test (LMIT)

In the LMIT, nematode L3, cultured from faecal samples, are subjected to migration through a small pore filter in the presence of increasing concentrations of ML, which paralyses drug-sensitive parasites. The LMIT therefore facilitates a comparative, quantifiable analysis amongst populations or within a single population over time. The LMIT has recently been developed as a robust assay for examining ML sensitivity of cyathostomins in the laboratory (Matthews et al. 2011) and is due to undergo inter-laboratory ring-testing to assess its potential as a pre-FECRT screen for ML sensitivity.

The egg hatch test (EHT)

The EHT is designed to detect BZ resistance by determining the concentration of thiabendazole that prevents the hatching of 50% of nematode eggs in a given sample (LeJambre 1976). Correlations between EHT data and other measures of cyathostomin anthelmintic sensitivity have been shown to vary (Craven et al. 1999), possibly because the tests examined the effect of BZs on different life cycle stages (Craven et al. 1999; Konigova et al. 2003). Further detailed validation studies in cyathostomins, including the determination of an appropriate EC50 cut-off for determination of BZ resistance, are required to assess the potential of this test.

A molecular test for BZ resistance?

Benzimidazole resistance in cyathostomins is associated with mutations at codons 167 and 200 of the beta-tubulin gene (Clark et al. 2005; Hodgkinson et al. 2008; Lake et al. 2009). Beta-tubulin is essential for a range of critical processes within nematodes. The resultant reduction in anthelmintic binding allows parasites to survive in lethal BZ concentrations. The structural changes are associated with alterations in the amino acid sequence in the beta-tubulin protein. Mutations at codons 167 and 200 are both relevant to BZ resistance and differential selection at the 2 sites has been observed to occur in FBZ vs. oxibendazole resistant populations (Lake et al. 2009). Codons 167 and 200 are present on different exons and separated by a large intron within the beta-tubulin gene, meaning that 2 distinct molecular assays have had to be designed to analyse DNA sequences at each site. A multi-cyathostomin species, codon 167-based assay has been developed to directly ‘pyrosequence’ individual L3 from field samples of unknown species and BZ sensitivity (Lake et al. 2009). The results indicated substantial increases in homozygous ‘resistant’ genotypes after only a single FBZ treatment.

Environmental control

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References

Increasing prevalence of anthelmintic resistance highlights the importance of appropriate environmental management. Regular removal of faeces (Herd 1986), co-grazing with other species and decreasing stocking density reduce infective L3 loads on pasture. Additionally ‘dose and move’ strategies should be avoided to maintain a population in refugia (Waller 1987). The nematophagous fungus Pochonia chlamydosporia may represent a future biological control method (Braga et al. 2010a,b) but requires much further validation.

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References

The current prevalence and potential pathogenicity of cyathostomins, combined with widespread anthelmintic resistance and an apparent dissociation of veterinarians from parasite control programmes is a cause for concern. Selective, targeted dosing and confirmation of drug efficacy, aided by the use of appropriate diagnostic tests should prevent unnecessary drug administration, aid in the maintenance of refugia and, hopefully, slow the spread of anthelmintic resistance in future.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References

The authors acknowledge the generous financial support of the Horse Trust, Horseracing Betting Levy Board, Thoroughbred Breeders' Association, BBSRC and Donkey Sanctuary for some of the research described here. C.S. is a Horse Trust Clinical Scholar in Equine Medicine.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Anthelmintics
  5. Anthelmintic resistance
  6. Approaches to parasite control to counter anthelmintic resistance
  7. Determining cyathostomin burdens for targeted therapy
  8. Assessing anthelmintic sensitivity
  9. In vitro diagnosis of anthelmintic sensitivity
  10. Environmental control
  11. Conclusion
  12. Conflicts of interest
  13. Acknowledgements
  14. Manufacturer's address
  15. References