Tutorial Article an Update on Cyathostomins: Anthelmintic Resistance and Worm Control J

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Tutorial Article An update on cyathostomins: Anthelmintic resistance and worm control J. B. MATTHEWS Department of Veterinary Clinical Science, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian EH25 9RG; and Division of Parasitology, Moredun Research Institute, Pentlands Science Park, Midlothian EH26 0PZ, UK. Keywords: horse; cyathostomins; drug resistance

Summary Background

Intestinal nematodes are an important cause of equine Parasitic nematodes that infect the large intestine of horses disease. Of these parasites, the Cyathostominae are the can have a substantial impact on their health and welfare. Of most important group, both in terms of their prevalence these parasites, the Cyathostominae (also known as and their pathogenicity. Cyathostomin infections are cyathostomins or small strongyles) are now the most complex and control is further complicated by ever- significant group (Love et al. 1999). Virtually all grazing horses increasing levels of resistance to some of the commonly are exposed to cyathostomins at some stage in their lives and used anthelmintics. There are no new equine many animals encounter infection all of their lives. The anthelmintics under development, so it is imperative Cyathostominae consist of 52 different species, of which more that the efficacy of any currently-effective drug classes than 40 species have been described in horses (Lichtenfels et be maintained for as long as possible. al. 2002). In individual animals, 10–12 common species usually It is believed that the proportion of refugia (i.e. the comprise most of the parasite burden (i.e. >90%) with the percentage of parasites not exposed to a drug at each remainder composed of rarer species (Ogbourne 1976). treatment) is one of the most crucial factors in Similar patterns of species proportions have been observed in determining the rate at which anthelmintic resistance diverse geographic regions, with the common species being develops. It is important, therefore, that levels of ubiquitous: for example, 3 species, Cylicostephanus refugia be taken into account when designing longibursatus, Cyathostomum catinatum and Cylicocyclus nematode control programmes for horses. This can be nassatus, are particularly common and these often account for assisted by knowledge of the local epidemiology of the 70–80% of the total cyathostomin burden (Kaplan 2002). infection, supplemented by faecal egg count analysis to Cyathostomins have a direct, nonmigratory life cycle: third identify those animals that are making the major stage larvae (L3) are ingested from pasture and these stages contribution to pasture contamination. This type of penetrate the wall of the large intestine (Fig 1). In the rational nematode control requires equine veterinary intestinal wall, the larvae undergo a number of stages of surgeons to get involved in designing and implementing deworming programmes. The advice given must be Adults caecum and colon based on a combination of knowledge of cyathostomin L5 Eggs in faeces biology and epidemiology as well as an awareness of moult the parasite population’s current drug sensitivity and a hatch emerge sound history of husbandry at the establishment. DL4 L1 in faeces Minimum As anthelmintic resistance will be the major 2 weeks constraint on the future control of cyathostomins, moult moult researchers are now actively investigating this area. LL3 L2 in faeces Studies are underway to develop tests that will enable Inhibited larval moult earlier detection of anthelmintic resistance and an assay development ingested EL3 Penetrate wall L3 that will help identify those horses that require of caecum translates anthelmintic treatments targeted at intestinal wall and colon onto pasture larvae. Fig 1: Cyathostomin life cycle. EVE 08-029 Matthews 17/9/08 09:41 Page 3

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development: first, as L3, then as fourth stage larvae (L4). The means that they may need to receive periodic deworming L4 subsequently re-emerge from the intestinal mucosa to treatments throughout their lives. For this reason, complete development to L5, which then mature to adults, in anthelmintics have become the cornerstone of equine the intestinal lumen. The female adult worms release eggs nematode control programmes for the past 40 years. that are shed in the faeces and then hatch to release first stage However, it is likely that overuse of these anthelmintics, larvae (L1). In faeces, the L1 undergo 2 moults and the combined with under-dosing, has resulted in widespread resulting infective L3 migrate onto pasture to await ingestion. resistance to some classes of drug. Because no new equine In some animals, the L3 are thought to enter a state of anthelmintics appear to be near commercial development, it is inhibited development (also known as hypobiosis) in the imperative that the efficacy of any currently-effective drug intestinal wall. Parasites that enter this state are described as classes be maintained for as long as possible. early L3 (EL3) and these are thought to have important roles in the pathogenesis and epidemiology of cyathostominosis. The Cyathostomins and anthelmintic resistance specific cues involved in triggering the inhibited state are unknown, but could involve host immunity, cold conditioning Three classes of anthelmintic are available for controlling of L3 prior to ingestion and/or the population density of cyathostomin infections: the benzimidazoles (BZs: i.e. parasites present within the intestinal wall and in the lumen. fenbendazole, oxfendazole, oxibendazole), the EL3 can persist in the intestinal wall from months to years tetrahydropyrimidines (THPs: i.e. pyrantel salts) and the (Murphy and Love 1997) and, during autumn/winter season in macrocyclic lactones (MLs: ivermectin and moxidectin). When northern temperate climates, these larvae and the other each anthelmintic class was initially introduced, it was shown mucosal stages (late L3 [LL3] and developing L4 [DL4]), can to have an excellent efficacy against adult cyathostomins, as constitute the major proportion of the cyathostomin burden well as large strongyles. However, over the last decade or so, (Duncan et al. 1998; Dowdall et al. 2002). A consequence of it has become clear that cyathostomin resistance to the BZ the high proportions of mucosal stage larvae is that horses can drugs, in particular, is geographically widespread (Kaplan have burdens to the order of several million nematode larvae, 2002). In contrast, the large strongyles do not seem to have yet show negative or low (i.e. <100 eggs per gram, EPG) acquired drug resistance. The reason for this difference in faecal egg counts (FECs, Dowdall et al. 2002). long-standing drug sensitivity between these 2 groups of Horses with high levels of infection can develop clinical nematodes is unknown but may be associated with symptoms of disease. The clinical signs range from chronic fundamental differences in their life cycles. Drug resistance in weight loss to colic, diarrhoea and death (reviewed by Love cyathostomins does not seem to be associated with particular et al. 1999). If mucosal larvae suddenly re-emerge in large species: morphological studies, which have investigated those numbers (i.e. several million parasites), severe pathological species that survive drug treatment, have indicated that 8 and consequences ensue and a colitis syndrome known as larval 10 of the commonest 12 species were resistant to pyrantel cyathostominosis occurs (Giles et al. 1985). This disease and BZs, respectively (Kaplan 2002). Also, when eggs from presents as sudden onset weight loss, oedema and/or BZ-resistant populations taken before and after fenbendazole diarrheoa and can be fatal in up to 50% cases (Love et al. (FBZ) treatment were identified using molecular techniques, 1999). The exact mechanisms underlying this larval no particular species bias was observed in the surviving reactivation process are unknown, but are likely to involve host population (Hodgkinson et al. 2005). immunity and nematode population density (which, of course, Anthelmintic resistance is defined as ‘when a greater can be affected by anthelmintic treatments) at the site of frequency of individuals in a parasite population, usually infection. The larval cyathostominosis syndrome does not affected by a dose or concentration of a compound, is no seem to be associated with the presence of particular longer affected, or a greater concentration of drug is required cyathostomin species (Hodgkinson et al. 2003). to reach a certain level of efficacy’ (Prichard et al. 1980). Horses vary markedly in their susceptibility to cyathostomin infection; most animals regulate infection relatively well and Drug susceptible consequently have low levels of infection, which is reflected in Drug resistant their FECs. Fewer animals have larger nematode burdens with Several drug moderate to high FECs (i.e. >500 EPG). These varying levels of Drug treatment treatments infection intensity generally persist throughout an animal’s life (Nielsen et al. 2006). As a result, cyathostomin populations are over-dispersed and so the majority of the parasites are often present in the minority of the host population. Consequently, it is only a relatively small number of individual horses that Most worms contribute to the bulk of pasture contamination and this is an Worms with resistance- left after Progeny of resistant conferring mutation(s) treatment worms mate resulting in important concept to consider when instituting nematode represent low proportion population consisting control programmes that minimise selection pressure for drug of total population mostly of resistant worms resistance (see later). The fact that horses do not acquire Fig 2: Schematic representation of selection for drug resistance 100% immunity to cyathostomins (Chapman et al. 2002) in parasitic nematodes. EVE 08-029 Matthews 17/9/08 09:41 Page 4

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Anthelmintic resistance in parasitic nematodes is an inherited 2000; Lind et al. 2007). In some areas, the prevalence of characteristic that is passed from one generation of worms to resistance approaches 100% (Kaplan 2004). The the next via the genome. Parasitic nematodes, such as the aforementioned studies report efficacy recorded after BZ cyathostomins, have extremely high levels of genetic diversity administration at a single (i.e. adulticidal) dose rate. FBZ, (Gilleard and Beech 2007). This feature, combined with their administered at the standard dose rate for 5 consecutive exceptionally large population sizes (often millions of parasites days, is also licensed for treatment against mucosal stage per group of animals), means that within populations, larvae with efficacies against the various stages quoted as resistance-conferring mutations may be present at the time of EL3, >91% and LL3/DL4, >99% (Duncan et al. 1998). There first exposure to a drug or can develop relatively quickly have been no controlled efficacy or critical trials published thereafter. This is a particular issue for drugs for which the that detail the larvicidal efficacy of the 5 day FBZ regime steps from a susceptible to a resistant genotype are relatively against BZ-resistant cyathostomins. However, continuous egg simple; for example, where an alteration in nucleotide shedding has been detected in BZ-resistant populations sequence at a single site, known as a single nucleotide following a 5 day course of FBZ treatment (Chandler et al. polymorphism, results in the drug becoming ineffective. In the 2000). In a further study (Hodgkinson et al. 2005), stabled presence of such straightforward resistance-conferring horses, which had been shown previously to harbour BZ- alterations, each time a horse is treated with the associated resistant adult parasites, were treated with pyrantel to kill the drug, the majority of worms that survive are resistant. adult worm population (the average FECRT after pyrantel Following repeated treatments with the same compound, the treatment was 98%) and 7 days after pyrantel treatment, resistant genotype will eventually constitute a larger were administered with a 5 day course of FBZ. Egg shedding proportion of the population and the drug ceases to be was observed within 28 days. As these horses did not have effective (Fig 2). Compounding this, host movements the opportunity to acquire new infections, these results amongst different populations of animals (for example, provide evidence, albeit indirect, that the anthelmintics between studs) will encourage high rates of gene flow administered did not kill many of the mucosal stages. Thus, it ensuring that, in the absence of appropriate treatment and was likely that the eggs excreted represented progeny of quarantine procedures, the inherited resistance traits are mucosal larvae that had survived both pyrantel and 5 days of spread geographically. FBZ. The observations made in these 2 studies suggest that Multi-drug resistance (MDR) to all 3 major anthelmintic use of the 5 day FBZ regime is inadvisable in cyathostomin classes is now a major concern in small ruminant farming populations shown previously to be BZ-resistant by FECRT (Kaplan 2004) and was recently identified in a cyathostomin following a single treatment. Indeed, the levels of resistance population in Brazil (Molento et al. 2008). In some regions, the reported for BZ drugs are such that it is probably best extent of anthelmintic resistance in sheep nematodes is such recommended that, should BZs be selected for use in horses, that small ruminant farming is becoming untenable (Sargison then the efficacy of treatment must be regularly monitored by et al. 2007). Worryingly, reversion to drug susceptibility does FECRT. This is particularly relevant on premises where BZ not seem to occur in parasitic nematodes, even when a anthelmintics have been used with regularity in the past. population has not been exposed to a particular drug class for a number of years (Jackson and Coop 2000). For these Pyrantel resistance reasons, anthelmintic resistance, particularly MDR, must be avoided, or at least delayed, in equine nematode populations. Pyrantel salts were developed for use in horses in the 1970s. Thus far, the incidence of drug resistance in cyathostomins is Compared to the BZ class of anthelmintics, pyrantel salts have not as severe as that observed for the nematodes of sheep and a lower efficacy against cyathostomins (Lyons et al. 1974). goats; however, with continued over-use of the current drugs, This ‘lesser’ efficacy is thought to have been a contributing the state of affairs in sheep may be one that will have to be factor in the slower development of resistance to this drug faced in future in equine practice. class in equine nematode populations. This is because a relatively larger proportion of parasites are left unaffected by Benzimidazole resistance the drug at each treatment (Kaplan 2002). Recently, however, reports of pyrantel-resistant cyathostomins have become The BZs, introduced in the 1960s, were the first class of broad relatively commonplace, particularly in the USA, where spectrum anthelmintic marketed for use in horses. pyrantel tartrate has been licensed as an in-feed, low dose Unfortunately, resistance to this class of anthelmintic is now daily treatment for horses for over a decade (Valdez et al. widespread and has been recorded in over 21 countries 1995). This continual treatment regime is probably an (Lyons et al. 1999). On the basis of faecal egg count reduction important factor in the relatively high levels of pyrantel tests (FECRTs), varying levels of efficacy have been reported resistance (i.e. >40%) now observed in the USA (Kaplan et al. for BZ drugs (Kaplan 2002), including complete failure to 2004). Reduced sensitivity to pyrantel salts has now been reduce egg counts (Drogemuller et al. 2004). Studies that reported in Europe, where the daily treatment regime is not have examined the prevalence of BZ resistance indicate that it licensed (Comer et al. 2006; Lind et al. 2007; Traversa et al. is present on more than 70% of the premises examined (for 2007). These observations suggest that selection pressure for example, Fisher et al. 1992; Craven et al. 1998; Várady et al. pyrantel resistance occurs in cyathostomins when this drug is EVE 08-029 Matthews 17/9/08 09:41 Page 5

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administered at the standard single dose. Ominously, moxidectin (MOX) 3 weeks later. The MOX treatment reduced resistance to both BZ and pyrantel anthelmintics has been the animal’s FEC to zero; however, the FEC was observed to recorded in single populations of cyathostomins (Little et al. increase within 3 weeks of a second MOX treatment 2003; Drogemuller et al. 2004; Traversa et al. 2007). administered 8 weeks later. This description provides a good Therefore, as for the BZ class, the efficacy of pyrantel salts example of how not to manage anthelmintic resistance (i.e. should be investigated on a regular basis by FECRT, especially administering consecutive treatments of drugs of the same where there is a history of regular use of the drug. class which do not appear to be working) and should be interpreted with caution as the observations were made in a Macrocyclic lactone resistance single horse. Nevertheless, these 2 indications of potential IVM resistance are especially relevant as the newer generation ML, Ivermectin (IVM), the first commercially-available ML, was MOX, has a wider spectrum of activity against mucosal discovered via a screening programme at Merck in the mid- cyathostomins than IVM (Bairden et al. 2001) and will leave 1970s (Geary 2005). This drug quickly dominated the equine fewer refugia at each drug treatment. anthelmintics market, mostly on the basis of its excellent It is also of relevance to mention here that there have been efficacy against migrating large strongyle larvae. It is perceived a number of reports of treatment failure of IVM against that widespread use of IVM has contributed to the dramatic Parascaris equorum in foals (Schougaard and Nielsen 2007; reduction in the incidence of large strongyle infections (Boxell Slocombe et al. 2007). However, a summary of these findings et al. 2004). In contrast, the incidence of cyathostomins has and the impact this will have on future control of this not decreased in horses. This is possibly because mucosal nematode in young animals is outside the scope of this review stages, especially EL3, are not particularly susceptible to IVM and should form the basis of a separate article. (Eysker et al. 1992; Klei et al. 1993; Xiao et al. 1994). As MOX was the second ML marketed for use in horses and detailed above, EL3 are important in the pathogenesis of larval was introduced in the late 1990s. As IVM and MOX are in cyathostominosis, so controlling these stages is imperative in same drug class, they have many similarities. However, one the prevention of cyathostomin-associated disease. major difference is that MOX is licensed for high efficacy (for Although IVM has been the most commonly used example, 90.8%) against EL3 at a single dose (Bairden et al. anthelmintic in horses for the previous 2 decades, until very 2001). It should be noted, however, that these data were in recently, there were no reports of resistance to this drug. It is contrast to earlier results that suggested a lower efficacy of possible that, because IVM may not gain access to mucosal MOX against these stages (Xiao et al. 1994; Eysker et al. larvae which live in cysts in the intestinal wall, these stages are 1997). The differences between these studies have never been not directly exposed to the drug and hence are not subjected entirely resolved, but may be due to variation in time scale that to selection pressure (Sangster 1999). Such unexposed the horses were necropsied after MOX treatment. In the 2001 parasites are described as being ‘in refugia’ (van Wyk 2001). study, where high efficacy was shown, horses were necropsied Another reason for the continuing sensitivity of cyathostomins 8 weeks after MOX therapy (Bairden et al. 2001) compared to IVM is that the underlying mechanisms of resistance to the with 5 weeks (Eysker et al. 1997) and 2 weeks (Xiao et al. ML class may be complex. For example, several DNA 1994). The variation in necropsy timings could impact the mutations may be required before the nematode acquires the study results in 2 ways: full resistant genotype and/or the worm may need to undergo (i) At the earlier time points, although EL3 were affected by additional genetic alterations such as changes in gene MOX, their presence could still be accounted for in expression levels that affect proteins that remove the drug parasite enumerations; i.e. insufficient time had elapsed from the parasite. Although an increased complexity of for dead/damaged parasites to be resorbed or expelled; genetic alterations may delay resistance acquisition, (ii) MOX may only be effective against later developmental parasitologists believe that ML resistance is inevitable stages (i.e. LL3, DL4). By the time the nematode burdens (Sangster 1999). were assessed 8 weeks post treatment, EL3, not affected Indeed, evidence of reduced efficacy is starting to emerge by the drug had developed into MOX-sensitive LL3 or DL4 for IVM: in a study where an IVM-FECRT was performed at a which were then damaged/killed by active MOX still number of equine premises in Germany, the egg reappearance present due to its long persistence in the tissues. period (ERP) was reduced from 8 to 5 weeks on 2 out of 6 farms (von Samson-Himmelstjerna et al. 2007b). As There is also the possibility, albeit less likely, that the anthelmintic resistance is often first observed as a reduced ERP, differences observed between the studies were due to a this may be a first indication of resistance in these populations. variation in mucosal digestion techniques required for EL3 More recently, lack of IVM efficacy against adult nematodes enumeration (Eysker and Klei 1999). Regardless of these was described in a single horse in Australia (Edward and differences, one parameter that has been highly repeatable over Hoffman 2008). Here, treatment with an IVM-praziquantel a wide range of studies is the prolonged ERP (at least 13 weeks) combination had no effect on the animal’s FEC and a follow- following MOX treatment (Boersema et al. 1998; Mercier et al. up treatment with abamectin reduced FEC by only 75%. The 2001). This feature indicates that MOX has a substantial impact horse was subsequently treated with oxibendazole, after on mucosal larvae irrespective of the actual stage that it has a which the egg counts increased, and finally was administered nematocidal effect against. Because of its excellent effect EVE 08-029 Matthews 17/9/08 09:41 Page 6

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against mucosal larvae, the efficacy of MOX should be Low faecal preserved for as long as possible because, should resistance to Drug susceptible egg count Drug resistant DO NOT treat this drug develop and spread, there will be no good options left High faecal for control of cyathostomin intestinal wall stages. egg count That cyathostomins have the genetic capability to develop drug treat resistance to MOX has been highlighted recently in the UK where reduced efficacy of MOX was observed in donkeys (Trawford et al. 2005). A reduced FECR (<90% mean Progeny from Horse A mix reduction) was observed in 2 groups of donkeys after they had with mostly susceptible pool 1 of L3 already on pasture and been treated orally with MOX (Equest) administered at the L3 derived from nontreated correct dose rate. This FECRT followed a period during which horses an injectable MOX formulation, designed for use in cattle, had been administered per os (Coles 2006). These circumstances highlight the fact that the recommended formulation should be used for all deworming treatments and that the drug Fig 3: Maintaining refugia by targeted selective treatments. should be administered at the correct dose rate for the appropriate species. Aligned with the observation in donkeys, are still administered in a nonrational manner. For example, in are the laboratory-based studies which demonstrated that the a racing yard survey in the UK, 84% horses were still administration of MOX to horses over time decreased the level administered with anthelmintic even when their FECs were of genetic diversity in the common cyathostomin species, <50 EPG (Comer et al. 2006)! In a separate study (Earle et al. C. nassatus, derived from that population (Tandon et al. 2005). 2002), veterinary surgeons were involved in the design of worm control programmes in only 29% racing yards, whereas Rational nematode control that promotes 42% of yards selected anthelmintics on the basis of veterinary continued drug efficacy advice. This indicated that the practitioners were more likely to give ‘drug-based’ rather than ‘management’ advice on worm A number of surveys and studies performed in the last control. The message from these studies is that veterinary 10 years have shown that many horses are dewormed too surgeons should get more involved in worm control frequently and that clients are not working with their programmes and that they should base their advice on a veterinary surgeons in designing rational worm control combination of the correct knowledge of drug sensitivity of programmes. For example, in a national survey in the USA strongyles (and other parasites) and a sound history of their (Anon 1998: National Animal Health Monitoring System (US), client’s establishment. Equine ‘98), 96.8% of all equine establishments administered Parasite control methods should aim to maintain drug anthelmintics to the majority of their horses in the preceding efficacy for as long as possible. One way of achieving this is to 12 months and 49.2% of these animals received at least reduce selection pressure on the nematode population by 4 treatments per annum. Interestingly, the percentage of ensuring that the majority is not exposed to the drug(s) at establishments in this survey with moderate to high levels of every treatment, i.e. one must try to maintain an adequate faecal egg shedding (i.e. >300 EPG) were similar, regardless of level of refugia. It is currently believed that the proportion of whether or not the majority of horses were dewormed. These refugia in each population is one of the most crucial factors in data suggest that anthelmintic resistance may have been a determining the rate at which resistance develops (van Wyk problem at some of these sites and that the drugs being used 2001; Pomroy 2006). Nematodes in refugia provide a pool of were ineffective. This survey also showed that there was a lack drug-sensitive genes so, with this in mind, treatments should of interaction between horse owners/keepers and their be avoided when pasture refugia levels are low (Nielsen et al. veterinary surgeons with respect to worm control: only 27% 2007). Leaving some animals untreated is another prudent of the study population obtained anthelmintics directly from method of ensuring that there are refugia left in a given their veterinary surgeons with the majority being purchased population (Fig 3). For these reasons, it is important to from feed/veterinary supply stores. In a separate questionnaire understand the life cycle of the parasite as well as its basic study involving 55 yards in Ireland, none of the respondents epidemiology in each geographical region and to consider indicated that they left any animals untreated with those seasons and circumstances whereby refugia are anthelmintics suggesting a lack of knowledge on the diminished (Neislen et al. 2007). importance of refugia (O’Meara and Mulcahy 2002). The interval dosing scheme, which many horse Furthermore, 72% of these participants administered owners/managers still practice, involves administration of anthelmintics at dosing intervals of <8 weeks and only 40% anthelmintics at regular intervals based on the ERP of each admitted to owning an accurate weight measurement device. drug. This regime has been successful in reducing parasite- Confusion with regard to the types of drug class used was also associated disease in horses but is believed to have made a a feature of this study, especially with respect to how to substantial contribution to the development of drug resistance appropriately rotate anthelmintics. Other surveys have shown (Kaplan 2002). Strategic dosing, where anthelmintics are that even when animals are assessed for FEC, anthelmintics administered when parasite levels are thought to be highest, EVE 08-029 Matthews 17/9/08 09:41 Page 7

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Do not treat Moderate to high FEC 0–low FEC

Treat to reduce FEC Total population

Fig 4: Schematic representation of how the over-dispersed nature of cyathostomin populations affects drug treatments designed to reduce pasture contamination.

provides a more rational approach; however, problems arise Of course an essential part of all nematode control when abnormal patterns of weather lead to early or late peak programmes is to ensure that drug therapies are combined pasture counts or when heavily infected animals are added to with good management: i.e. do not overstock, remove faeces the population (Proudman and Matthews 2000). An regularly from pasture, administer anthelmintic dosages based adaptation to this strategy is to administer anthelmintics at the on the weight of each animal and ensure that newcomers are most appropriate times of the year, based on the life cycle and quarantined and treated with MOX (preferably combined with epidemiology, and to also take into account the levels of praziquantel for tapeworms). These efforts will be effective in infection in individual horses by monitoring individual FECs. reducing reliance on drug treatments and will help decrease Monitoring of FECs will reduce the total number of selection for resistance while minimising infection levels. treatments required in a group of horses, thus allowing a sub- Nowadays, in light of the extent of BZ resistance and population of nematodes to remain in refugia (Fig 4). Regular emerging pyrantel resistance in cyathostomins, it is difficult to FEC analysis will have the advantage that it will identify those rotate drug classes on an annual basis. However, where there horses most susceptible to cyathostomins and the associated is sensitivity to >1 class of anthelmintic, an annual rotation disease syndromes. It will also get the veterinary surgeon programme should be practiced. For example, in the UK, one involved, so that emerging resistance is more likely to be could institute one year of ML treatments, with IVM used for detected. It should be remembered that FECs do not give an adulticidal treatments (based on FEC analysis in late spring accurate estimation of the total nematode burden within each through to autumn) combined with a larvicidal MOX horse (Duncan 1974) especially, for example, in autumn/winter treatment in late autumn/early winter for all horses. As part of in the UK, and are best used to indicate that animal’s potential this programme, an IVM treatment in the spring/summer for contamination of pasture. Hence, FEC results should be could be combined with praziquantel for anti-tapeworm used to identify those animals that require adulticidal therapy. In the second year, the adulticidal IVM treatments treatments. Before committing to undertaking FECs always could be replaced with pyrantel (use once a year at the double keep in mind the ERP for each drug as this will determine how dose for tapeworm control). Annually rotating the class of soon after the last treatment that analysis will be of use drug used for the autumn mucosal cyathostomin treatment (providing there is no resistance to the last drug used!). The may be problematical and, in the absence of a FECRT that ERP for each commonly used drug is as follows: pyrantel and indicated sensitivity to BZ, then MOX is probably best BZs - 4 weeks, IVM - 8 weeks and MOX - 14 weeks. However, administered for this purpose. As many of the adulticidal note that eggs can reappear more quickly in young animals treatments as possible should be administered on the basis of and in those with large nematode burdens. The cut-off EPG FEC analysis and one should consider performing a FECRT value on which to base the decision to treat is still a matter of annually for one of the drugs used. Recent computer debate; between 200 and 500 EPG is often cited and a value modelling studies have indicated that an effective approach between these 2: this is probably a good place to start, for delaying resistance selection may be to treat individuals at depending on the grazing and deworming history of the the same time with 2 chemically distinct drugs (Kaplan 2002) horses sampled. An alternative suggestion is to deworm the however this approach requires additional evaluation before group when the mean FEC is 100–300 EPG (Uhlinger 1993). being recommended routinely, especially as MDR has been However, given the importance of refugia in animals with identified in cyathostomins. lower egg output, it is best to administer anthelmintic based From now on, nematode control programmes should on individual FECs, combined with knowledge of the yard include analysis of drug efficacy by utilising the FECRT. Data history. This is the reason why veterinary surgeons should work derived from FECRTs can be difficult to interpret though, as with their clients in customising worm control programmes on there is often a wide range of efficacy within a single group of a yard-by-yard basis (Proudman and Matthews 2000). horses. For this reason, cut-off values for establishing drug EVE 08-029 Matthews 17/9/08 09:41 Page 8

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resistance have not yet been standardised in horses, especially mechanisms are complex and multi-factorial (Prichard 2005; as there are efficacy differences between classes against McCavera et al. 2007), but this work has generated important susceptible cyathostomins. It is recommended that, in future, comparative data on which to base the study of ML resistance different cut-off values be established for each drug type in cyathostomins. As a first step, sequences of potential target (Kaplan 2002). For example, for BZ drugs, a 90% mean genes are being isolated from ML-resistant and -susceptible reduction in FEC (as previously recommended) is probably cyathostomin populations. Such research is complicated by the appropriate for declaring resistance. This level is probably not potential number of species involved and their inherent levels suitable for IVM as treatment consistently gives nearly 100% of variation in DNA sequence (McDonnell et al. 2000), which reduction in FEC at 2 weeks post treatment. This makes the means that development of molecular assays for ML resistance appearance of any eggs in faeces following IVM treatment a will be a long-term process. cause for concern. Hence, the old definition of IVM resistance As indicated above, by using targeted anthelmintic of <95% reduction (with a lower confidence limit of <90%) treatments, selection pressure for ML resistance will be may be rather conservative and a more rigorous definition will reduced. Targeting is facilitated by methods that allow us to be required. Conversely, pyrantel efficacy against detect moderate to highly infected individuals. For egg output, cyathostomins is less than IVM, so this should also be taken this is achievable using FEC analysis, however, for assessment into account. The World Association for the Advancement of of mucosal burdens, no test exists. Recent efforts to develop a Veterinary Parasitology has now set up a working committee serum IgG(T)-based assay to assess mucosal larval burdens to establish new recommendations for the FERCT for each have shown promise (Dowdall et al. 2002, 2003, 2004). anthelmintic used in horses and these will be published in the Antigens identified as being potentially diagnostic are present forthcoming year. The findings will be transmitted back to in extracts of EL3, LL3 and DL4 but it is impossible to generate equine veterinary surgeons in due course. Nevertheless, as sufficient amounts of these for use in a commercial diagnostic there are no alternative practical means to confirm drug assay. A project is currently under way to identify the genes resistance in equine nematodes, the FECRT, despite its that encode these antigens and to express the proteins in limitations, remains the standard practice that should be used. recombinant form in bacteria. In the long-term, these proteins will be produced in large quantities for use in an ELISA-based The future outlook format that can then be developed into a commercial assay.

When anthelmintic treatment failure is detected, either by Concluding remarks observation of clinical disease in recently treated animals or by a low reduction in FEC, it is too late to affect the Increasing levels of anthelmintic resistance will provide a preponderance of resistance genes in the associated nematode challenge for horse owners and their veterinary surgeons in population. As such nematodes do to not revert to drug future. More rational nematode control methods, based on a sensitivity, it is important that we develop methods to detect sound knowledge of the biology and epidemiology of these resistance-associated alleles earlier. Such early detection infections, combined with better diagnostic tests, should requires an understanding of the molecular mechanisms enable the spread of resistance, particularly to the ML class, to behind resistance and on the design of sensitive methods to be decelerated. However, most importantly, this requires the identify these alleles when they are present at low frequency. enthusiastic input of equine veterinary surgeons into Such assays can be used to direct drug use strategically to nematode control programmes on a global basis. maintain high efficacy of, for example, the ML class, for as long as possible. To identify molecular markers, detailed analyses of Acknowledgements the genes that encode the target proteins of the drug or those proteins involved in its removal from the organism are required. The author thanks the Horse Trust, Horserace Betting Levy Currently, we lack the knowledge by which we can measure Board, BBSRC and Donkey Sanctuary for generous funding. the accumulation of the resistance genes in cyathostomins and J.B.M. is funded by the Scottish Executive RERAD and the gaining basic knowledge is crucial (Matthews et al. 2004). Scottish Funding Council. The author also thanks long-term Without this, early ‘genotypic’ diagnosis of resistance will not cyathostomin collaborators Jane Hodgkinson and Ray Kaplan be possible, leaving phenotypic detection (i.e. treatment for their continued enthusiasm. failure) as the only alternative. Some research has been performed to detect alleles associated with BZ resistance in Manufacturer’s address cyathostomins (Clark et al. 2005; von Samson-Himmelstjerna 1 et al. 2007a; Hodgkinson et al. 2008), but it is possibly too late Fort Dodge Animal Health Ltd, Southampton, Hampshire, UK. to develop these for field use given the high levels of BZ resistance already present in cyathostomin populations. The BZ References studies, however, have provided invaluable tools and Anon (1998) National Animal Health Monitoring System (United methodologies with which to start identifying mechanisms of States), USDA: APHIS: VS (1998) Fort Collins. Equine ‘98, Part I: resistance to IVM and MOX. Studies on ML resistance in human Baseline reference of 1998 equine health and management. and ruminant nematodes have indicated that the underlying Information sheet: Internal Parasites and U.S. Horses: Strongyles. EVE 08-029 Matthews 17/9/08 09:41 Page 9

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