Anthelmintic resistance in equine parasites--current evidence and knowledge gaps.

G Model VETPAR-7073; No. of Pages 9

ARTICLE IN PRESS Veterinary Parasitology xxx (2014) xxx–xxx

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Anthelmintic resistance in equine parasites—Current evidence and knowledge gaps M.K. Nielsen a,∗ , C.R. Reinemeyer b , J.M. Donecker c , D.M. Leathwick d , A.A. Marchiondo e , R.M. Kaplan f a

M.H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA East Tennessee Clinical Research, Inc. , Rockwood, Tennessee, USA Zoetis, Outcomes Research, 707 Parkway Boulevard, Reidsville, NC, USA d AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand e Zoetis, Global Therapeutics Research, 3333 Portage Road, Kalamazoo, MI, USA f Department of Infectious Diseases, University of Georgia, Athens, Georgia, USA b c

a r t i c l e

i n f o

Keywords: Horse Anthelmintic resistance Diagnosis Cyathostomin Parascaris eqourum

a b s t r a c t Anthelmintic resistance is becoming increasingly prevalent among equine nematode parasites. The first reports documenting resistance were published in the 1960s, just a short time after introduction of the first modern anthelmintics phenothiazine and thiabendazole. Several factors are known to influence development of resistance, but evidence specific to equine parasites is limited. Most current knowledge and applications have been extrapolated from research with trichostrongylid parasites of sheep. The number of cyathostomin species co-infecting horses adds to the complexity of investigating drug resistance but, given their apparent limited biological diversity, viewing these in a unispecific context remains a pragmatic approach. Factors affecting resistance development in cyathostomins include parasite seasonality, life span and fecundity, host immunity, and the existence of encysted stages. Further, parasite refugia have been shown to play a vital role in resistance development in other parasites, and likely is also important in equine parasites. Specific genetic factors for drug resistance and possible modes of inheritance have been identified for trichostrongylid nematodes, but it is widely accepted that several more remain undiscovered. Current evidence with equine and ruminant parasites suggests that fitness is not significantly compromised in drug resistant strains. Attempts to develop in vitro and molecular assays for diagnosing anthelmintic resistance in equine nematodes have had only limited success, standardized guidelines are sorely needed for performing the fecal egg count reduction test in horse populations. Taken together, this review illustrates the complexity of understanding anthelmintic resistance in equine nematodes, and emphasizes the need for further research. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Control of helminth parasite infection in horses is considered an essential aspect of routine management in

∗ Corresponding author. Tel.: +1 859 218 1103; fax: +1 859 257 8542. E-mail address: [email protected] (M.K. Nielsen).

equine establishments. Over the past 50 years, a number of broad spectrum endoparasiticides have been available for control of these parasites. Many (e.g., thiabendazole, dichlorvos) have been withdrawn for commercial reasons, but anthelmintic resistance has been reported for all of the drug classes that are currently marketed (Table 1). Cyathostomin resistance to benzimidazoles and tetrahydropyrimidines is highly prevalent, especially as

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Please cite this article in press as: Nielsen, M.K., et al., Anthelmintic resistance in equine parasites—Current evidence and knowledge gaps. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2013.11.030


ARTICLE IN PRESS M.K. Nielsen et al. / Veterinary Parasitology xxx (2014) xxx–xxx

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Table 1 Equine endoparasiticides in the United States, initial market appearance and first published report of anthelmintic resistance involving equine parasites in the United States. Equine endoparasiticide

Initial market appearance (USA)

Initial resistance report (USA)

Phenothiazine Piperazine Benzimidazoles Tetrahydropyrimidines Macrocyclic lactones

1940 1955 1961a 1976b 1984c

Cyathostomins (Drudge and Elam, 1961) Cyathostomins (Drudge et al., 1988) Cyathostomins (Drudge and Lyons, 1965) Cyathostomins (Chapman et al., 1996) Parascaris equorum (Craig et al., 2007)

a b c

Thiabendazole. Pyrantel pamoate. Ivermectin.

documented in the southeastern USA (Kaplan et al., 2004). Benzimidazole resistance has been reported world-wide in cyathostomins (reviewed by Kaplan, 2004), and recent European studies have documented signs of pyrantel resistance as well (Traversa et al., 2009; Nielsen et al., 2013b). Of further concern are recent reports of an apparent reduction of cyathostomin egg reappearance periods (ERP) following treatment with ivermectin or moxidectin (Lyons et al., 2008a, 2011). Sangster (1999) has suggested that a shortening of the ERP is the first indication of developing anthelmintic resistance. This hypothesis is alarming in light of two recent studies in which shortened ERPs following ivermectin or moxidectin treatment were associated with survival of luminal, fourth-stage cyathostomin larvae in juvenile horses (Lyons et al., 2009, 2010). In addition to cyathostomins, populations of Parascaris equorum that are resistant to ivermectin and moxidectin have been reported from multiple locations around the world (Boersema et al., 2002; Hearn and Peregrine, 2003; von Samson-Himmelstjerna et al., 2007; Schougaard and Nielsen, 2007; Lind and Christensson, 2009; Veronesi et al., 2010; Nareaho et al., 2011; Laugier et al., 2012). A study in central Kentucky also reported a lack of efficacy by pyrantel pamoate against P. equorum (Lyons et al., 2008b), illustrating the potential for ascarids to develop resistance to other drug classes as well. Over the past decade, the pharmaceutical industry has developed and marketed three new drug classes for controlling nematode parasites in other domestic animals. These are emodepside (cyclo-octadepsipeptide class), monepantel (amino-acetonitrile derivatives), and derquantel (spiroindoles). However, it remains unknown whether these classes are safe and effective in horses, which will ultimately determine their suitability for the equine market. Historically, the macrocyclic lactones remain the drug class most recently introduced for equine use, but ivermectin was first marketed about 30 years ago (Table 1). Because no new equine anthelmintics are imminent, it is critical that further development of resistance be delayed as much as possible. The increasing prevalence of anthelmintic resistance has fostered recommendations for surveillance-based approaches to parasite control and reduced frequency of anthelmintic treatment (Herd et al., 1985; Kaplan and Nielsen, 2010). Over the past 20 years, the practice of selective therapy has been promoted, i.e., treating only those animals with egg counts that exceed a predetermined threshold value (Gomez and Georgi, 1991;

Kaplan and Nielsen, 2010). Contemporaneously, the introduction of prescription-only restrictions on anthelmintics in Denmark appears to have stimulated veterinary involvement in equine parasite control and increased the routine use of fecal egg counts (Nielsen et al., 2006). Since 2006, a European Union directive has led to implementation of similar, restrictive legislation in other European countries. A survey performed in the United States in 1998 determined that equine parasite control depended predominantly on frequent anthelmintic treatment, performed year-round, with little or no use of fecal surveillance (Anonymous, 1998). However, this trend appears to be changing recently as many U.S. veterinary practices have adopted surveillance-based control programs. The American Association of Equine Practitioners (AAEP) has published updated guidelines for parasite control (Nielsen et al., 2013a) which espouse surveillance and decreased use of anthelmintics. These attempts to establish a more sustainable approach to parasite control have highlighted the need for applied research to generate evidence supporting various control recommendations (reviewed by Nielsen, 2012). The objective of this article is to review current information about anthelmintic resistance in equine parasites. Special emphasis will be given to the unique biology of cyathostomins, factors affecting the development of resistance, and methods for detection of resistance. Within each topic, we will identify gaps in the scientific knowledge required to make substantial advances in sustainable parasite control.

2. Important factors associated with parasite and host biology For theoretical considerations of the development and management of anthelmintic resistance, it would be far simpler to consider the cyathostomins as a single species. Computer models of resistance selection have been developed for single nematode species in other hosts (Leathwick et al., 1992; Barnes et al., 1995), but complications grew exponentially when the number of parasite targets was increased, and as potential interactions approached infinity (Dobson et al., 2011). Regrettably, our current knowledge of cyathostomin biology is insufficient to determine whether a simplistic, “unispecific” approach would be valid. Various aspects of cyathostomin biology are discussed herein from the perspective of their impact on resistance.




2.1. Prevalence The subfamily Cyathostominae infecting all equids comprises approximately 50 species distributed among 14 genera (see Lichtenfels et al., 2008). Despite such potential variety, ∼80% of the small strongyles in a typical infection belong to only five species (Ogbourne, 1976; Reinemeyer et al., 1984; Mfitilodze and Hutchinson, 1990; Lichtenfels et al., 2001). Furthermore, 10 species usually constitute ∼98% of the cyathostomin populations in stereotypic infections (Ogbourne, 1976; Reinemeyer et al., 1984; Mfitilodze and Hutchinson, 1990; Lichtenfels et al., 2001). As early as 1982, six of the 10 most prevalent species were already known to be resistant to benzimidazole anthelmintics (Wescott et al., 1982). However, the relative rankings of cyathostomin species have remained fairly consistent over the past four decades, so resistance has apparently had little impact on their prevalence. These observations support the contention that the various species differ little in their ability to develop resistance, or in the effects thereof on their standing within a mixed population. 2.2. Life cycles Despite their taxonomic breadth, the cyathostomins do not exhibit much biological diversity. All species in this subfamily reside as adults in the lumen of the equine large intestine, and presumably utilize similar nutrient sources. Subtle niche differences must exist, however, because adults of many species have distinct site preferences, with their greatest numbers occurring in the cecum, ventral colon, or dorsal colon. Eggs of various cyathostomin species are virtually indistinguishable, and with few exceptions, their third larval stages exhibit eight intestinal cells. The small strongyles all have similar life cycles, with third and fourth larval stages encysting for varying periods within the mucosa or submucosa of the cecum or ventral colon. In zebras, cyathostomin larvae have also been found to encyst in the small intestinal mucosal walls (Scialdo-Krecek et al., 1983; Krecek et al., 1987a,b, 1994). It is not known whether the mucosa and submucosa represent equal options for an individual larva, or if species are rigidly programmed to invade only one or the other. As a practical consequence, it seems logical that species which encyst in the submucosa would be more pathogenic, because excystment should elicit more tissue damage if initiated in a deeper layer of the gut lining. This hypothesis was supported in one study in which pronounced inflammatory reaction of the large intestine following larvicidal treatment with fenbendazole was only associated with larvae encysted in the submucosa (Steinbach et al., 2006). A putative link between encystment site and pathogenicity may serve as a useful basis for prioritizing control efforts among the various species. A similar argument could be made for greater pathogenicity by the larger species (e.g., Cylicocyclus insigne) compared to those that are significantly smaller (e.g., Cylicostephanus minutus and C. longibursatus). Some parasitologists consider Cylicocyclus insigne to be uniquely pathogenic, but perhaps only because it is the sole cyathostomin species with a grossly distinctive, blood-red, L4 stage.

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Among parasites of domestic animals, cyathostomins also exhibit the longest periods of arrested development of any nematode that does not migrate beyond the gut. Ponies that were dewormed frequently and held under conditions which obviated reinfection continued to develop patent infections for up to 2.5 years after initial confinement (Smith, 1976a,b). But, it is unknown whether the various species differ in the duration of their arrestment, or if only a few can persist for such an extended period. Further elucidation of life cycle details and pathogenic potential will determine whether individual species can serve as valid markers for the behavior of a population in toto. 2.3. Seasonality Cyathostomins undergo arrestment at the early third larval stage during seasons when climatic conditions are unfavorable for development and/or persistence of environmental stages. As a general pattern, larvae arrest through winter in northern temperate climates, and during summer in southern temperate climates. Work with equids in tropical regions has generated somewhat conflicting evidence on larval arrestment. One study demonstrated an apparent absence of arrestment (Eysker and Pandey, 1987), while others generated evidence of winter arrestment (Krecek et al., 1987a,b, 1989). These observations may reflect different seasonal patterns in parasite transmission. In contrast to the perennial, interval deworming approaches that have dominated strongyle control efforts for the past 50 years, chemical intervention is more logical if applied during seasons when environmental conditions support larval development. These issues are related to the concept of refugia, which is subsequently discussed in greater detail herein. 2.4. Life span and fecundity Surprisingly little is known about the longevity of individual cyathostomins. Studies in sheep have determined that life spans vary widely among trichostrongylid species (Leathwick et al., 1997). Seasonal egg count patterns and other population data suggest that small strongyles typically produce two generations per year in temperate regions (Reinemeyer et al., 1986). Longer life spans would result in greater contamination of the environment with eggs carrying resistant gene alleles, and the establishment of newly ingested larvae will determine the rate at which parasite generations change over time. Explicit evidence on longevity will be required to accurately model cyathostomin population dynamics, resistance selection, and clinical management. It appears that cyathostomin species differ substantially in relative fecundity. In a recent study, groups of 25 female worms representing 15 different strongylid species were dissected, and all eggs within the uteri were counted. The results illustrated a positive correlation between worm size and numbers of eggs, and it was concluded that larger worms were likely to be more fecund (Kuzmina et al., 2012).




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2.5. Host immunity Experimental evidence indicates that successful establishment of newly ingested larvae is largely influenced by host immunity. The rate of establishment of incoming larvae is difficult to assess in cyathostomins because variable portions may undergo arrested development at the early third larval (EL3 ) stage, and all larvae eventually undergo encystment before they can mature into reproductive adults. One study compared cyathostomin populations in a cohort of naturally-infected ponies to a group of parasite naïve ponies kept in stall confinement. Subsequent experimental infection of these animals resulted in a significantly higher burden of hypobiotic EL3 s in the previously exposed group (Chapman et al., 2002). Another study by the same group revealed that EL3 s occurred in higher numbers in older ponies compared to yearlings, but no differences were observed between age groups in the numbers of developing encysted larvae (late L3 and L4 stages) (Chapman et al., 2003). Taken together, these studies strongly suggest that host immunity plays an important role in regulating the process of arrested development of cyathostomins. Therefore, one may conclude that host immunity also plays a significant role in modulating the selective forces of anthelmintic treatment on cyathostomin populations. The complex dynamics affecting the development of anthelmintic resistance in horses leave us with many questions that need further study. In the absence of detailed information, it appears that the cyathostomins are more similar to each other, in terms of pathogenicity, epidemiology, and population dynamics, than other nematodes which also share a subfamily and host organ, but in an alternate host, e.g., Haemonchus contortus and Teladorsagia circumcincta in sheep. Studying the cyathostomins from a unispecific context may lead to immediate practical advances, whereas treating their perceived complexity as an insurmountable obstacle will only reinforce the status quo. 3. Other factors affecting development of anthelmintic resistance The development of anthelmintic resistance is a highly complex process that is influenced by host, parasite, and environmental factors. Parasite variables are the most complex, and in addition to the biological factors and population dynamics discussed previously, these include differences in genetic mechanisms and mutation rates among species, numbers of genes involved, concurrent effects on fitness of resistant worms, drug pharmacokinetics, patterns and frequency of anthelmintic treatments, and the actual genetic and epigenetic mechanisms of resistance, including the mode of inheritance. Host factors include immunity and anthelmintic pharmacokinetics, whereas frequency of deworming and climatic variables must be listed among the environmental influences. This collection of theoretical factors is largely extrapolated from studies with trichostrongylid nematodes of sheep; very little original information has been generated for equine parasites. Rather than offering an exhaustive review, the objective of this section is to elaborate on selected factors

which illustrate the complexity of resistance development, and to emphasize the dearth of specific evidence regarding equine parasites. 3.1. Refugia The term parasite refugia has become widely accepted in large animal parasitology. The term refugia denotes any subpopulation of parasites that is not exposed to an anthelmintic at the time of treatment. This includes pre-parasitic stages in the environment, parasites of herd members that are left untreated when a selective treatment is applied, and parasitic stages within the animal that are unaffected by treatment due to physical, physiologic, or pharmacokinetic factors. The subpopulation left in refugia represents a reservoir of unselected genes which includes those alleles imparting anthelmintic susceptibility. Refugia ensure that susceptible worms are available to mate with resistant worms that have survived treatment. When the numbers of parasites in refugia are increased, the rate of resistance development in the parasite population will be reduced correspondingly. This contention is supported by field studies with parasites of sheep (Martin et al., 1981; Waghorn et al., 2008; Leathwick et al., 2012), as well as by computer simulations which mimic sheep parasite dynamics (Barnes et al., 1995; Leathwick, 2012). Although parasite refugia has been promoted as beneficial to equine parasite control (Nielsen et al., 2007; Matthews, 2008; Kaplan and Nielsen, 2010), not a single published field or computer modeling study has demonstrated the utility of this concept in equine parasites. Regardless, it can be argued that the parasite biology and host-parasite interactions of equine nematodes have more similarities than differences with those in ruminant models. In the absence of evidence to the contrary, it remains a useful working hypothesis to consider refugia as a beneficial factor in delaying equine anthelmintic resistance. Despite the similarities, cyathostomin biology differs from that of ruminant trichostrongylids in some key areas. Most important is the subpopulation of encysted cyathostomin larvae, which has no direct equivalent in ruminant trichostrongyles. Cyathostomin third and fourth stage larvae can remain encysted for years (Gibson, 1953), and only selected anthelmintic classes or dosages are effective against these stages. These mucosal stages comprise a portion of the refugia whenever non-larvicidal treatments are applied (Kaplan, 2002; Matthews, 2008). It has even been suggested that this “mucosal refugia” may partially explain why ivermectin resistance has taken more than 20 years to develop in cyathostomins (Kaplan, 2002; Matthews, 2008). This remains a valid hypothesis, but no experimental evidence currently exists to support this assertion. Resistance to macrocyclic lactone anthelmintics is now widespread among P. equorum populations around the world (Boersema et al., 2002; Hearn and Peregrine, 2003; Schougaard and Nielsen, 2007; Veronesi et al., 2010; Nareaho et al., 2011; Laugier et al., 2012). Arguably, the refugia of Parascaris equorum should be large because females are very fecund, and ascarid eggs can survive for years in the environment (Fairbairn, 1957). Refugia within the host, however, is non-existent after macrocyclic




lactone treatment because ivermectin and moxidectin affect all parasitic life cycle stages within the host, including migrating larvae (Reinemeyer, 2012). In addition, frequent treatment at intervals less than the egg reappearance period of Parascaris simultaneously reduces the environmental refugia because only resistant worms are able to reproduce. Although the level of refugia is clearly important in the development of anthelmintic resistance, its ultimate impact can be modified by numerous factors. Climatic conditions regulate the development and survival of preparasitic, free-living stages, and thus have the greatest impact on the magnitude of available refugia. Generic data on strongylid larval bionomics are available for horses (reviewed by Nielsen et al., 2007), but a majority of these studies are over 20 years old, and some were published 50 years ago. Very few environmental studies have been conducted, and little or no information exists on possible species differences. 3.2. Genetic factors Modes of inheritance are obviously important for understanding the development of anthelmintic resistance in parasite populations. However, these remain unknown for any parasites of horses. Studies in sheep demonstrate that Herculean efforts are required to achieve this level of understanding. Research has shown that resistance to ivermectin in Teladorsagia circumcincta behaves as a fully dominant character, whereas resistance to moxidectin is largely recessive (Sutherland et al., 2002). However, during the drug’s persistent efficacy phase, resistance to moxidectin is fully dominant (Sutherland et al., 2003), so interpreting the relative selectivity of different active compounds is even further complicated (Leathwick and Sutherland, 2002). Moreover, there is general agreement that resistance is multigenic, and except for beta-tubulin mutations associated with benzimidazole resistance, numerous genetic and epi-genetic mechanisms remain undiscovered (Beech et al., 2011). Cyathostomin infections typically involve multiple species, but it remains unknown whether the same molecular mechanisms and modes of inheritance can be assumed across all cyathostomin species. Furthermore, some observations suggest that different stages of the same parasite species may express differing levels of resistance to a given drug. The shortened cyathostomin egg reappearance periods following treatment with ivermectin or moxidectin have been associated with selective survival of luminal fourth stage larvae, suggesting resistance in this specific stage (Lyons et al., 2009, 2010). Possible explanations include differences in parasite metabolism, expression of enzyme pathways, or receptor molecules, but these remain purely hypothetical at this time. 3.3. Parasite fitness A question of universal interest is whether anthelmintic resistance is associated with changes in the relative fitness of parasite populations. Resistance would effectively provide its own negative selection pressure if it

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concurrently compromised worm fitness. If resistant worms were indeed less fit, resistant populations would likely revert to susceptibility if left unexposed to the anthelmintic for a sufficiently long period. However, there is little evidence to suggest that resistance imparts a significant fitness deficit. In trichostrongylids of sheep, no clear evidence has been found that benzimidazole (BZD) resistance is associated with fitness deficits (Maingi et al., 1990; Elard et al., 1998). Furthermore, no reversion back to susceptibility was seen following non-use of benzimidazole drugs for six years in a benzimidazoleresistant population of Haemonchus contortus (Borgsteede and Duyn, 1989), or after passage of benzimidazoleresistant populations of H. contortus and Trichostrongylus colubriformis for 12 generations without drug-selection (Hall et al., 1982). Similarly, limited observational data for cyathostomins do not support compromised fitness because BZD-resistant cyathostomins remained resistant after being left untreated for approximately 20 years (Lyons et al., 2007). In another study, benzimidazole-resistant cyathostomins were exposed to bimonthly treatments with pyrantel pamoate for eight years, which resulted in pyrantel resistance but did not change the benzimidazole resistance status of the worm population (Lyons et al., 2001). Altogether, there is no evidence of a clear fitness loss associated with benzimidazole resistance in nematode parasites of livestock. Likewise, it does not appear that reversion occurs in parasites with resistance to the macrocyclic lactone class, however similar experiments have not been conducted for this drug class. In contrast, there is limited evidence that partial reversion may occur in levamisole-resistant populations (Waller et al., 1985). Ultimately, whether or not resistance confers a fitness deficit will depend on the genetic changes required to confer the resistant phenotype, and this will differ for every drug class. It has previously been hypothesized that resistance alleles are already present in parasite populations in very low numbers prior to introduction of a new and fully efficacious anthelmintic (Beech et al., 1994). The fact that these alleles are rare could be explained by an associated fitness deficit that has prevented these alleles from occurring in higher numbers. The difficulties with finding any clear signs of such fitness loss could then be explained by co-selection for fitness characteristics in the parasite population, as selection for anthelmintic resistance proceeds over time (Prichard, 1990). However, more recent data convincingly shows that most resistance mutations are not pre-existing, but more often occur as random independent spontaneous mutations that are occurring constantly in nematode populations (Silvestre et al., 2009; Gilleard et al., 2012). Thus, resistance alleles are mostly due to contemporaneous mutations that then are rapidly selected for with high drug selection pressure. Overall, it cannot be ruled out that the genetic changes that confer resistance also confer a fitness deficit to the resistant worm, but even if this is true it seems to be unimportant at the clinical level if the worms can compensate and gain other fitness characteristics by cross-breeding with susceptible worms in the course of the evolution of resistance. Thus, limited reversion to susceptibility remains a possibility for some anthelmintic classes,




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but the evidence available to date suggests that clinically relevant levels of reversion do not occur for most current drug classes once resistance develops.

et al., 2005; Pook et al., 2002; Tandon and Kaplan, 2004). Therefore, it has been concluded that the LDA is not useful for determining anthelmintic resistance in cyathostomins.

4. Diagnosis of anthelmintic resistance

4.4. Larval migration inhibition assay

Following is a brief outline of methods that have been evaluated for diagnosing anthelmintic resistance in horses, and points out the most pressing research needs and gaps in current knowledge. Although a number of in vitro assays have been developed for determining anthelmintic resistance in sheep parasites, none have been validated or proven useful for horses. A molecular assay for detecting mutations associated with benzimidazole drugs has been developed for cyathostomins, but given the ubiquity of BZD resistance in small strongyles, its usefulness is minimal. To date, no diagnostic methods have been developed and validated for detecting anthelmintic resistance in Parascaris equorum. Thus, all the work described herein refers exclusively to cyathostomins.

A third in vitro technology, the larval migration inhibition assay (LMIA), has been developed and refined for sheep parasites (Kotze et al., 2006), but has also been evaluated for cattle (Demeler et al., 2010a,b). In one published report, the LMIA was applied for equine cyathostomins (van Doorn et al., 2010), but the technique was employed to identify larvae suspected of being resistant to ivermectin and did not evaluate the diagnostic properties of the technique. Additional preliminary data have been generated with cyathostomins, but these are unpublished (Matthews et al., 2012). The general impression, however, is that the LMIA is not very useful for cyathostomins. It is important to emphasize that no cyathostomin isolates have been characterized as unequivocally resistant to macrocyclic lactones. Consequently, no positive controls exist for use in molecular or in vitro assays to diagnose ivermectin and moxidectin resistance, and no such assays have been developed to date. In summary, none of the currently available, in vitro assays for diagnosing anthelmintic resistance have proven useful for cyathostomins in horses.

4.1. In vitro assays Several in vitro assays have been developed and validated for diagnosing anthelmintic resistance in trichostrongylid nematodes of small ruminants. Several attempts have been made to modify and validate these methods for use in horses. 4.2. Egg hatch assay The Egg Hatch Assay (EHA) has been widely validated for detecting benzimidazole resistance in ruminant parasites (von Samson-Himmelstjerna et al., 2009). Some studies have illustrated modest utility of EHA in horses (Craven et al., 1999; Wirtherle et al., 2004; von SamsonHimmelstjerna et al., 2002a), but this method is less accurate for cyathostomins than for trichostrongylids, and is restricted to use with drugs of the benzimidazole class. Considering the ubiquitous occurrence of BZD resistance among cyathostomin populations world-wide, screening for resistance to this specific drug class would merely verify a foregone conclusion (Matthews et al., 2012). 4.3. Larval development assay An assay capable of simultaneous detection of resistance to several different drug classes is preferable. A Larval Development Assay (LDA) possesses this quality and is sold commercially as DrenchRite® (Dr. Jennifer Gill, Microbial Screening Technologies, Smithfield, Australia) for use in small ruminants. This technique has been used successfully for small ruminants (Ancheta et al., 2004; Howell et al., 2008; Kaplan et al., 2007), and has recently shown promise for trichostrongylids of cattle as well (Demeler et al., 2012). Several laboratories have evaluated the LDA for cyathostomin parasites, but results were characterized by wide variability, poor reproducibility, a lack of meaningful correlation with the fecal egg count reduction test (FECRT), and narrow resistant-to-susceptible ratios (Lind

4.5. Molecular assays An allele-specific PCR assay has been developed for detecting genetic polymorphisms at the beta-tubulin codon 200 of some common cyathostomin species. This codon has been associated with benzimidazole resistance in ruminant trichostrongylid nematodes, but field studies suggested that its role in BZD resistance of cyathostomins may be less significant, and that codon 167 likely plays a more important role (von Samson-Himmelstjerna et al., 2002b; Pape et al., 2003; Lake et al., 2009). For codon 167 polymorphisms, a pyrosequencing assay has been developed that accurately detects mutations at this site (Lake et al., 2009). Thus, there is no single molecular assay for diagnosing benzimidazole resistance in equine parasites. Even if one existed, its clinical usefulness would be minimal, given the ubiquity of resistance to this drug class. No molecular assays exist for any of the other drug classes, despite a substantial clinical need. With the exception of the beta-tubulin mutations associated with BZD resistance, it is generally accepted that specific molecular mechanisms for resistance in other drug classes have not yet been identified (Beech et al., 2011). Furthermore, general molecular associations with resistance, such as the P-glycoprotein (PgP) gene, apparently act independently of drug class. This factor may further complicate the development of molecular assays for specific drug classes. 4.6. Fecal egg count reduction test Currently, the only available method for diagnosing anthelmintic resistance in equine parasites is the fecal egg




count reduction test (FECRT). Although this procedure is widely used, its application is accompanied by a clear lack of consensus and an absence of equine-specific guidelines. Consequently, published studies differ greatly in central elements of study design. Large differences are apparent in designation of group size, fecal egg-counting technique, and cut-off values for classifying resistance to a given drug. Because very few investigators use a common method, it is extremely difficult to compare the results from different studies. A recent review has identified numerous statistical and biological challenges associated with use of FECRT to determine anthelmintic resistance in cyathostomins (Vidyashankar et al., 2012). It is clear that both the study design and the choice of statistical analysis have a huge impact on the accuracy of the method for classifying resistance on farms. In comparison to their ruminant counterparts, equine populations often comprise a variety of breeds, uses, and ages, and are more diverse than animals typically raised to produce meat and fiber. Further, the number of cyathostomin species comprising a typical population likely contributes significantly to the variability observed with the FECRT in horses (Vidyashankar et al., 2012). The performance of any diagnostic test is dependent on the choice of cut-off value for determining a positive test result. However, in the absence of standard guidelines for performing the FECRT in horses, different cut-off values have been used to declare resistance in various studies. For example, Kaplan et al. (2004) employed an 80% mean FECR value, while Traversa et al. (2009, 2012) used 90% plus a combination of the lower 95% confidence limit and the mean FECR. Yet other studies have employed a 95% mean cut-off value (Craven et al., 1999; Larsen et al., 2011), and some authors suggest using different cut-off values for different drug classes (Kaplan and Nielsen, 2010). As mentioned above, the choice of cut-off values for determining resistance is only one important ingredient of the FECRT; a lack of consensus currently exists for many other components as well. This example merely indicates the level of confusion which arises from the lack of standard guidelines in equine parasitology.

5. Concluding remarks In summary, it is clear that the study of anthelmintic resistance in equine nematode parasites offers fruitful opportunities for various scientific specialties. Much of the current understanding has been extrapolated from studies of sheep trichostrongylid parasites, and only limited original evidence is available for equine nematodes. Numerous similarities exist between ruminant and equine parasites, but some major differences have been identified, and the effects of these differences on the development of resistance remain unknown. Attempts to develop in vitro and molecular methods for diagnosing anthelmintic resistance in equine parasites have had only limited success, and more work is needed to standardize the FECRT for equine usage. Similarly, validated methods and guidelines for diagnosing anthelmintic resistance in P. equorum deserve a high priority.

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