A survey of macrocyclic lactone efficacy in Australian cyathostomin populations

Veterinary Parasitology: Regional Studies and Reports 8 (2017) 127–132

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A survey of macrocyclic lactone efficacy in Australian cyathostomin populations

MARK

A.M. Beasleya,⁎, A.C. Kotzeb, K. Allena, G.T. Colemana a b

School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia CSIRO Agriculture and Food, Queensland Bioscience Precinct, St Lucia, QLD, Australia

A R T I C L E I N F O

A B S T R A C T

Keywords: Ivermectin Macrocyclic lactones Cyathostomins Strongyles Resistance Egg reappearance period

The macrocyclic lactone (ML) drugs are central to the control of equine strongyles but recent international reports raise concerns about reduced efficacy of these drugs against cyathostomins. The objectives of the present study were firstly, to evaluate the efficacy of ML drugs against cyathostomins on a cross-section of Australian horse farms, and secondly, to determine the egg reappearance period (ERP) following treatment of horses with MLs. A total of 419 horses on 43 properties were treated orally with ivermectin, abamectin or moxidectin, at recommended dose rates and drug efficacy was determined using the faecal egg count reduction test. Efficacy of 100% at 14 days post-treatment was reported on all of the 43 farms. ERP following ivermectin treatment was 6 weeks on two properties and ERP following moxidectin treatment was 12 weeks on a third property. These ERPs are shorter than those reported at the time of commercial release of these drugs which likely reflects changing drug susceptibility of the cyathostomin populations tested. Ongoing surveillance of drug efficacy and ERPs should be part of an integrated management approach to equine worm control that prioritises the preservation of anthelmintic efficacy.

1. Introduction Due to favourable climatic conditions in many regions (particularly coastal regions) of Australia, cyathostomin worms present an ongoing threat to horse health. Although generally less pathogenic than large strongyles, cyathostomins can cause significant disease. In addition to the larval cyathostominosis syndrome associated with mass emergence of encysted larvae, characterised by diarrhoea and weight loss, cyathostomins have also been associated with anorexia and colic (Love et al., 1999). Given the impact of these parasites, there is a need to manage the use of effective anthelmintics to delay the emergence and spread of drug resistance (Matthews, 2014). Drugs available to treat cyathostomins in Australia include benzimidazoles (BZs), macrocyclic lactones (MLs) and tetrahydropyrimidines (THPs). Suppressive treatment programs throughout the 1960′s, 70′s and 80′s used BZs to target Strongylus vulgaris and other large strongyles but led to widespread BZ-resistance among cyathostomin populations (Kaplan and Vidyashankar, 2012). BZ-based products, sometimes in combination with other drug groups such as THPs, remain widely available in Australia. ML drugs, including ivermectin (IVM), abamectin (ABM) and moxidectin (MOX), currently constitute > 50% of the equine anthelmintic products registered in Australia (Public Chemical

⁎

Registration Information System Search, Australian Pesticides and Veterinary Medicines Authority). MOX has a prolonged anthelmintic effect compared to IVM and ABM, and greater efficacy against mucosal stages of cyathostomins (Bairden et al., 2006). The third drug class for worm control in equines, the THPs, includes pyrantel and morantel. Both are included in a range of combination anthelmintic products with either MLs or BZs, however, neither are currently available as singleactive products. This very limited selection of effective chemical treatments for cyathostomins, and the apparent absence of new drug classes under development for the equine market, underlines the importance of sustaining the efficacy of the ML drugs. An essential part of managing ML resistance is proactively monitoring drug efficacy, a practice advised but seldom undertaken on Australian properties. The emergence of ML resistance among cyathostomins was predicted almost two decades ago by Sangster (1999), but reports of resistance, as measured by the faecal egg count reduction test (FECRT), are still scarce in the literature. Since the first suspected case was reported from the UK following treatment of donkeys with MOX (Trawford et al., 2005), reports from Brazil (Molento et al., 2008; Canever et al., 2013), the UK (Relf et al., 2014), Italy (Traversa et al., 2009), Germany (Von Samson-Himmelstjerna et al., 2005) and New Zealand (Bishop et al., 2014) have demonstrated a reduced efficacy of

Corresponding author. E-mail address: [email protected] (A.M. Beasley).

http://dx.doi.org/10.1016/j.vprsr.2017.03.009 Received 11 September 2016; Received in revised form 16 December 2016; Accepted 20 March 2017 Available online 21 March 2017 2405-9390/ © 2017 Elsevier B.V. All rights reserved.

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guidelines for horses (Coles et al., 1992). FECs were performed using a modified McMaster technique with a lower detection limit of < 10 epg (range 6.2–7.5 epg) and a Whitlock Universal Slide (J A Whitlock & Co., Eastwood, Australia). On day 0, pre-treatment samples were collected from all available horses prior to administration of a commercial equine anthelmintic containing an active ingredient from the ML class (ivermectin, 0.2 mg/ kg, 29 properties; abamectin, 0.2 mg/kg, 10 properties; or moxidectin, 0.4 mg/kg, 4 properties). Samples were sealed in air-tight bags (expelling as much air as possible) and submitted to the laboratory by express post (maximum transit time = 3 days). We acknowledge that without vacuum packaging, truly anaerobic conditions cannot be created and some egg development may have been possible during transit (Nielsen et al., 2010; Sengupta et al., 2016). Post-treatment samples were collected on day 14 only from horses with a pre-treatment FEC of ≥ 150 epg. FECR (%) for each property was calculated according to American Association of Equine Practitioners (AAEP) Parasite Control Guidelines (Nielsen et al., 2016). Using the equation below, the FECR (%) for each horse in the group was calculated individually. Formula used to calculate FECR (%): [(FECpre – FECpost)/FECpre] × 100. The mean reduction for all horses tested was then calculated to determine the percent reduction for the property. While equine-specific criteria are yet to be established to define the presence of ML-resistance, the AAEP Parasite Control Guidelines (Nielsen et al., 2016) recommend using a reduction of mean faecal egg count < 95%. These guidelines are in line with the recommendations of the WAAVP (Coles et al., 1992) for FECRTs in sheep, which include a supporting criterion of a lower confidence limit (LCL) of < 90%. On three farms, further sampling was undertaken during spring to determine the ERP following treatment with either MOX (1 farm) or IVM (2 farms). Faecal samples were collected from individual horses weekly from 2 weeks post-treatment until the ERP was reached. Faecal sampling ceased at 7, 8 and 12 weeks post-treatment for properties A, B and C, respectively. ERP was defined as the period of time from treatment until the FECR (%) was ≤ 90% (von Samson-Himmelstjerna et al., 2007; Larsen et al., 2011; Nielsen et al., 2016; van Doorn et al., 2014). Comparisons among pre-treatment FEC of properties A, B and C were done using one way ANOVA (Kruskal-Wallis Test with Dunn's multiple comparison) (Graphpad Prism v6.05).

MLs against cyathostomins in FECRTs. More common are reports of shortened egg reappearance periods (ERPs) (von SamsonHimmelstjerna et al., 2007; Dudeney et al., 2008; Molento et al., 2008; Lyons et al., 2008b; Rossano et al., 2010; Lyons et al., 2011; Lyons and Tolliver, 2013), and these are considered evidence of a shift in ML-sensitivity toward resistance. Critical tests have shown a reduced IVM efficacy of only 0–16% against luminal L4 stages (Lyons et al., 2009; Lyons et al., 2010; Lyons and Tolliver, 2013), thereby shortening the time taken for egg shedding to recommence following treatment. There are comparatively few investigative reports into anthelmintic resistance among cyathostomins on Australian horse properties. Pook et al. (2002) reported a 100% FECR following treatment of horses with IVM on 7 NSW properties (n ≥ 4 horses), there has been only a single Australian case report of suspected ML resistance involving an individual horse (Edward and Hoffmann, 2008). In that case, monthly faecal egg counts (FECs) were performed on a 24-year-old horse that was treated 7 times within a 13-month period using various ML worming products. Although FECs were not performed within the recommended time frame of 10–14 days post-treatment, as per FECRT guidelines, the study most notably reported a count of > 2000 epg 3–4 weeks following one IVM treatment and an increase from 200 to 500 epg 3 weeks after a MOX treatment. FEC results of another two horses on the same property were not discussed in detail and the authors reported no evidence of reduced efficacy on 9 other properties investigated. Without further investigations, this case should be considered as anecdotal evidence only. Australian contributions to the global dataset on anthelmintic efficacy against equine helminth populations are scarce. We report here the results of a large survey of ML efficacy across a broad crosssection of Australian equine properties. 2. Materials and methods 2.1. Participating farms A variety of strategies were used to purposively recruit equine properties. Participants were recruited based on their ability to provide a minimum of 10 horses for pre-treatment testing and willingness to perform collections from individual horses on 2 separate occasions. Publicly listed horse studs or stables were contacted directly. Articles were published in equine magazines and newsletters calling for suitable participants, and social media was also utilised to inform horse owners of the study and call for suitable participants. A total of 43 properties were recruited to participate in the study during 2012 and 2013, and were located in regions across four Australian States (Queensland n = 23 properties, New South Wales n = 13, Victoria n = 4, and South Australia n = 3). The number of horses on each farm included in the FECRT ranged from 6 to 15 (median 10) and the total number of horses examined in the study was 419. Age data was provided for 322 of the 419 samples. Age of horses ranged from 3 months up to 37 years of age (mean = 8.8 years, median = 7.0 years). Animal ethics approval for the project was granted by the University of Queensland's animal ethics committee (approval number ANFRA/148/12). Three of the recruited QLD properties (referred to as properties A, B and C) were selected to also undergo an extended period of FEC testing to measure ERP. These properties were all located in the same region of southeast QLD and were selected based on their managers' willingness to comply with the extended weekly sampling protocol. Properties A, B and C differed in their anthelmintic treatment histories and the ages of horses (Table 1).

3. Results The distribution of pre-treatment FECs was overdispersed and strongly skewed to the right (Fig. 1), in line with what is typically observed in parasite count data (Crofton, 1971; Sreter et al., 1994; Galvani, 2003; Scheuerle et al., 2016). The 419 FEC values had a minimum of 150 epg, a median of 524 epg, a maximum of 4269 epg, and a mean ± SE of 723 ± 33 epg. Only 17% of the values were < 200 epg. The mean percent reduction in FEC at 14 days post-treatment on all 42 properties surveyed was 100% following treatment with IVM, ABM or MOX. On the three properties where ERP was measured, pre-treatment FECs ranged from 236 to 1740 epg (mean 667 epg, 95% CI 489–845, n = 6) for property A, 181–2282 epg (mean 1107 epg, 95% CI 704–1510, n = 18) for property B, and 359–918 epg (mean 741 epg, 95% CI 491–991, n = 6) for property C (Fig. 2), and were not significantly different (P = 0.2356). For the IVM treatment properties (A and B), strongylid eggs were first detected in weeks 5 and 4, respectively, and the FECR threshold of 90% was reached between weeks 5 and 6 post-treatment for both properties, indicating an ERP of 6 weeks. For the MOX treatment group (property C), strongylid eggs were first detected at week 9 and the FECR was 90.4% at week 11, and had dropped to 81% by week 12 (Table 2),

2.2. Faecal egg count reduction test and determination of egg reappearance period The FECRT was carried out in accordance with the World Association for the Advancement of Veterinary Parasitology (WAAVP) 128

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Table 1 Summary of 3 equine properties (A, B and C) recruited to measure strongylid egg reappearance period. Property

A

B

C

No. of horses Age range

6 Mature/Aged 12–30 yrs Mixed breeds Infrequent (1–2 ML treatments/yr) over previous decade 16 weeks

18 Weanlings 25–35 weeks Australian Stock Horse crosses Frequent (4+) ML treatments per year over previous decade 12 weeks

6 Mostly mature 3–25 yrs Mixed breeds Infrequent (1–2 ML treatments/yr) over previous decade 12 weeks

Set stocked, continuously grazed together on same pasture

Set stocked, continuously grazed together on same pasture

Set stocked, continuously grazed together on same pasture

Breed Anthelmintic treatment history Time since previous anthelmintic treatment⁎ Grazing management

⁎

Previous treatments were IVM or ABM.

1800

A Faecal Egg Count (epg)

Relative frequency (%)

40

30

20

10

0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400

0

Property A (IVM)

1600 1400

Property B (IVM)

1200

Property C (MOX)

1000 800 600 400 200 0

Pre-T 2

3

Faecal egg count (epg)

4

5

6

7

8

9

10 11 12

Fig. 1. Histogram showing the relative frequency of 419 pre-treatment faecal egg counts from 43 properties.

B

indicating an ERP of 12 weeks. 4. Discussion The FECRT results reported here are consistent with the findings of Pook et al. (2002) and show that ML drugs still remain effective against cyathostomins on the 43 properties examined, achieving a 100% reduction in FEC at 14 days post-treatment. ERPs measured on 3 properties, however, were shorter than those reported at the time of commercial release of ML anthelmintics, and are consistent with reports of shortened ERPs in other parts of the world. These findings indicate that ML products remain effective in treating existing cyathostomin populations, but there is cause for concern that ML-resistance may be emerging. Although these results show that ML drugs continue to be effective in treating Australian cyathostomins, evidence of emerging resistance is beginning to accumulate in the literature. A large European survey demonstrated IVM resistance on one Italian farm (46% faecal egg count reduction, or FECR; n = 5 horses), and two UK farms (72% and 89% FECR; n = 5 horses) (Traversa et al., 2009) and, similarly, Relf et al. (2014) reported an 85.7% FECR (n = 6 horses) following IVM treatment on a UK farm. A brief report of < 90% FECR 14 days after IVM treatment has originated from Germany (Von Samson-Himmelstjerna et al., 2005) although precise information on group sizes on individual farms and methods used to calculated drug efficacy was not provided. In New Zealand, Bishop et al. (2014) reported an 84% FECR following treatment of 12 Thoroughbred weanling foals. Of particular concern are Brazilian field studies where a range of anthelmintics representing the three available drug classes all failed to reduce strongylid FECs by ≥ 90% Molento et al. (2008). These authors reported FECRs of 65%, 16%, 84% after treatment of horses (n = 6) with IVM, MOX and ABM respectively. Combined with the additional findings that fenbendazole (FBZ, a BZ) and pyrantel (PYR, a THP) reduced FECs by just 18% and

Faecal Egg Count Reduction (%)

Weeks post-treatment 110 100 90 80 70 60 50 40 30 20 10 0 2

3

4

5

6

7

8

9

10

11

12

Weeks post-treatment Fig. 2. Mean faecal egg counts (A) and faecal egg count reductions (B) showing 95% confidence intervals of three equine groups following treatment with either ivermectin (properties A and B) or moxidectin (property C). The 90% FECR threshold for defining the time when the official egg reappearance period has been reached is marked on (B) by the dashed red line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

7% respectively, these results provide evidence of multi-drug resistant cyathostomins present on a single farm. Similarly, Canever et al. (2013) reported FECRs of − 18%, 59% and 89% for FBZ, PYR and IVM respectively on a single Brazilian farm. In such scenarios, options for worm control would become extremely limited. Furthermore, there is growing evidence of reduced ML efficacy against other equine nematodes. There are numerous reports of Parascaris equorum resistance to MLs (Schougaard and Nielsen, 2007; Lyons et al., 2008a; Veronesi et al., 2009; Armstrong et al., 2014; Bishop et al., 2014; Beasley et al., 2015), and growing concerns about ML efficacy against Oxyuris equi (Scháňková et al., 2013; Wolf et al., 2014; Felippelli et al., 2015). Combined, these events highlight the danger of suppressive treatment regimes, still widely practiced on Australian horse properties, and underline the importance of routine 129

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from the UK (Dudeney et al., 2008; Relf et al., 2014; Hallowell-Evans et al., 2016), Europe (van Doorn et al., 2014; von SamsonHimmelstjerna et al., 2007), the USA (Little et al., 2003; Lyons et al., 2008b), and Sweden (Lind et al., 2007). In some of these studies, ERPs were as short as 4 weeks (Dudeney et al., 2008; Lyons et al., 2008b; Lyons et al., 2011) although there is a tendency for these shorter ERPs to reflect the time when strongylid eggs first reappeared in faeces following treatment, rather than when FECR (%) passed the 90% threshold as described earlier. In one UK study (Relf et al., 2014), the first positive FEC was observed just 2 weeks after treatment of horses with IVM but the FECR had not decreased to ≤ 90% until week 6. Similarly, both Woods et al. (1998) and Tarigo-Martinie et al. (2001) reported a ≤ 90% FECR at 6 weeks post IVM-treatment on US farms. Although only two properties were examined in this study, our results mirror this trend, with the ERP following IVM treatment being 6 weeks. MOX is generally considered to have an ERP of at least 14–16 weeks, and initial ERPs reported in Australia did exceed 14 weeks (Rolfe et al., 1998). ERPs exceeding 19 weeks were initially reported from Belgium and Canada (Slocombe and Lake, 1996; Demeulenaere et al., 1997), but, there have been a number of more recent reports indicating a dramatically shortened ERP. Rossano et al. (2010) reported only a 67% FECR at 6 weeks post MOX-treatment in a group of yearling horses in Kentucky. Similarly, Relf et al. (2014) reported ERPs following MOX administration of 6, 8 and 9 weeks respectively in 3 groups of horses in the UK. The ERP following MOX treatment reported here for property C was 11 weeks, shorter than the 14+ weeks originally reported. This indicates a shift toward decreasing ERP following MOX treatment on this Australian property, consistent with international observations. There is a well-documented relationship between age and FEC in horses, with young animals having higher counts than mature horses (Klei and Chapman, 1999; Kornaś et al., 2010). There is also an assumption that ERPs are shorter in younger horses (Herd, 1986; Herd and Gabel, 1990), but the relationship between age and ERP has not been thoroughly investigated and the evidence presented thus far is conflicting. Reasons proposed for a shorter ERP in young horses include the resumption of development of a higher number of encysted larvae, and higher rates of re-infection as a result of a less developed immune response. Hallowell-Evans et al. (2016) carried out resistance testing (FECRTs) and ERP measurements on Thoroughbreds in the UK and asserted that young stock showed reduced FECRs following treatment with PYR compared to adults on the same farm. The ERPs for IVM and MOX were 6 and 8 weeks, respectively, however data from mature horses was not collected for comparison. Boersema et al. (1996) reported no significant difference in the ERPs of foals, yearlings and adult horses even though the FECs of the younger horses were consistently higher. Further investigations measuring ERP in both young and mature horses on the same farm are warranted to specifically test the hypothesis. Properties A and B in the present study differed markedly in the mean age of horses (mature/aged vs weanlings < 1 year of age, respectively) but the younger cohort did not have significantly higher pre-treatment FECs than their mature counterparts, nor did they have a shorter ERP following treatment with IVM. Factors other than age and immunity are also known to influence FECs, such as the magnitude of larval challenge that horses are exposed to which is very difficult to quantify in the field. ERP measurement was carried out in the Spring for all 3 groups, a time of year in southeast QLD that provides favourable conditions for L3 s on pasture, however no assumptions can be made about differences in larval challenge between the 3 properties. Although strongylid eggs were first detected one week earlier in the younger group (week 4 for property A versus week 5 for property B), the FECR threshold of 90% was reached in week 6 for both IVM-treated groups. In this case, it seems that age alone was not a useful predictor of ERP, however, it would be dangerous to extrapolate from such a limited dataset. Clearly, more work involving a larger number of Australian properties would be useful to further define the

Table 2 Arithmetic mean faecal egg count reduction following treatment with ivermectin (0.2 mg/kg) (properties A and B) or moxidectin (0.4 mg/kg) (property C), and 95% confidence limits. Week posttreatment

2 3 4 5 6 7 8 9 10 11 12

Faecal egg count reduction (%)a A (IVM)

B (IVM)

C (MOX)

100 100 100 99.6 80.4 66.1

100 100 94.8 93.8 76.8 69.3

100 100 100 100 100 100 100 98.4 98.8 90.4 80.9

(98.8–100) (61.0–99.9) (29.3–100)

(89.6–99.9) (88.3–99.3) (69.3–84.3) (49.0–83.6)

(95.5–100) (96.7–100) (77.53–100) (57.3–100)

a Bold indicates the week in which the egg reappearance period (defined as < 90% FECR) was reached.

monitoring of drug efficacy across farms. It has been estimated that by the time a FECRT can detect a reduction in drug efficacy, > 25% of the nematode population are resistant (Martin et al., 1989), so that appreciable levels of resistance may develop undetected at the farm level. Delaying changes to parasite control strategies until resistance is demonstrated via FECRT may be too little too late, since very little can be done to reverse the resistance status of a nematode population once established (Borgsteede and Duyn, 1989). There has been debate in the literature over the most appropriate method for statistical analysis of equine FECRT data. There are inherent difficulties in investigating anthelmintic efficacy of horses including; acquiring adequate group sizes with sufficiently high pre-treatment FECs; lack of untreated control groups; poor sensitivity and high variability of the FEC methodologies used; and the overdispersed nature of FEC data (Lester et al., 2013). Statistical analyses applied to FECR data have varied from calculating basic arithmetic group means (Coles et al., 1992) to the use of parametric bootstrap and likelihoodbased analyses, or Bayesian parametric methods such as Markov chain Monte Carlo algorithms (Vidyashankar et al., 2012). The variation in reported statistical approaches highlights the need for consensus so that meaningful comparisons between papers can be made. In the absence of a clear consensus, the most widely used approach in recent literature, and outlined in the AAEP guidelines, has been adopted for use in the current study. A similar complication exists for comparing ERP results across multiple studies. Until the AAEP guidelines were published (Nielsen et al., 2016), a consensus on the precise definition of the ERP had not been reached. Earlier studies generally defined ERP as either the time taken for the mean FEC to exceed a predetermined threshold, usually in the range of 100–200 epg (Jacobs et al., 1995; Boersema et al., 1996; Mercier et al., 2001) or the time between anthelmintic treatment and the first positive FEC (Little et al., 2003; Molento et al., 2008, Fischer et al., 2015). Both methods are biased by individual differences in susceptibility to worm infection which are inherent in pre-treatment FECs. The AAEP recommendations, followed in the current study, require horses be sampled weekly from 2 weeks post-treatment and define the ERP as the time when the mean FECR (%) first declines below 90% of the mean pre-treatment FEC. Shortly after the MLs became available on the Australian market, Rolfe et al. (1998) reported an 11-week ERP following IVM treatment of horses on a NSW property, and ERPs of similar length, (9–13 weeks), were also reported in Belgium and the Netherlands (Borgsteede et al., 1993; Boersema et al., 1996; Demeulenaere et al., 1997). More recently, however, there have been a number of reports of reduced ERPs following IVM treatment and ERPs of < 8 weeks have been reported

130

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ERP following treatment with different ML products. Industry practice still largely involves suppressive treatment programs; apart from the inherent problems in such programs selecting for anthelmintic resistance, there is a significant risk that they are also based on incorrect assumptions about ERPs. The history of anthelmintic treatment frequency on a property may influence drug sensitivity of the resident cyathostomin population. Treatment frequency dictates the level of drug exposure to nematode populations and higher treatment frequencies select more strongly for resistance (Barton, 1983). Under this scenario, as the level of resistance increases, ERPs would potentially shorten. Properties A and B in the present study were grazed on properties with reported differences in historic ML treatment frequency. Property A had received infrequent ML treatments (1–2 times per year) for at least 10 years prior to the study, while management of horses on property B traditionally consisted of at least 4 ML treatments per year. Despite this, the properties' ERPs were similar, suggesting comparable reductions in drug sensitivity on both properties. Frequent movement of horses between properties, both locally, nationally and internationally for breeding and competition, is more common in the equine industry compared with production animal industries and has implications for the dispersal of resistance genes. In terms of anthelmintic resistance among cyathostomin populations, it could be speculated that the geographic dissemination of resistance genes between horse properties may be more significant than historic treatment frequency on a specific property. This would have implications for biosecurity protocols at equine events and more broadly for the general movement of horses on and off properties. Genetic studies would be required to examine this hypothesis further. Despite the encouraging FECRT results presented here, which provide evidence of continued high efficacy of the MLs against cyathostomins, there should be no complacency regarding the need for more strategic and targeted approaches to worm control. The shortening of ERPs reported here is a warning of impending resistance. There is a need for the development and adoption of more modern Australian parasite control guidelines, such as those provided by the AAEP (Nielsen et al., 2016). Surveillance of drug efficacy, along with routine monitoring of FECs, should form the foundation of such guidelines. In terms of identifying resistance at the earliest possible stage on properties where ML drugs have been shown to be 100% effective via FECRT, and in the absence of viable molecular or in vitro assays to assist, gathering ERP data is highly beneficial. The laborious and costly nature of weekly ERP sampling may be overcome by advising one additional sample at 4–6 weeks post-treatment. This would require further work on defining ERP thresholds for determining drug resistance status of cyathostomin populations, but is certainly a topic worthy of debate among equine parasitologists. Acknowledgements The authors would like to acknowledge the valuable contributions of the participating properties, Ms. Lyn Knott for her technical contributions, and the Rural Industries Research and Development Corporation (RIRDC, PRJ-008135) for generously funding this work. References Armstrong, S., Woodgate, R., Gough, S., Heller, J., Sangster, N., Hughes, K., 2014. The efficacy of ivermectin, pyrantel and fenbendazole against Parascaris equorum infection in foals on farms in Australia. Vet. Parasitol. 205, 575–580. Australian Pesticides and Veterinary Medicines Authority, 2014. Public Chemicals Registration Information System. Australian Government. Database accessed 15/4/ 2016 https://portal.apvma.gov.au/pubcris. Bairden, K., Davies, H., Gibson, N., Hood, A., Parker, L., 2006. Efficacy of moxidectin 2 per cent oral gel against cyathostomins, particularly third-stage inhibited larvae, in horses. Vet. Rec. 158, 766–768. Barton, N., 1983. Development of anthelmintic resistance in nematodes from sheep in Australia subjected to different treatment frequencies. Int. J. Parasitol. 13, 125–132. Beasley, A., Coleman, G., Kotze, A., 2015. Suspected ivermectin resistance in a south-east

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