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The development of anthelmintic resistance with best practice control of nematodes on commercial sheep farms in the UK
Veterinary Parasitology 229 (2016) 9–14
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Research paper
The development of anthelmintic resistance with best practice control of nematodes on commercial sheep farms in the UK Jane Learmount ∗ , Nathalie Stephens, Valerie Boughtflower, Alba Barrecheguren, Kayleigh Rickell Animal and Plant Health Agency, Sand Hutton, York YO41 1LZ, UK
a r t i c l e
i n f o
Article history: Received 30 April 2016 Received in revised form 7 September 2016 Accepted 8 September 2016 Keywords: Anthelmintic resistance Larval development test SCOPS Benzimidazole Imidazothiazole
a b s t r a c t Antimicrobial resistance threatens the effective prevention and treatment of an ever-increasing range of infections. The widespread development of anthelmintic resistance is a major global issue affecting the effective control of parasite diseases in grazing livestock production. Sustainable control strategies that reduce dependence on antimicrobials have the potential to slow the further development of resistance but there is little data on the effect of control strategies on resistance development in the field. This report documents a study undertaken to measure the temporal effect of the UK sustainable control of parasites in sheep (SCOPS) guidelines on the development of anthelmintic resistance. Farms carrying out SCOPS or traditional worm control (TRADITIONAL) were tested for resistance to the benzimidazole and imidazothiazole anthelmintics in vitro using a discriminating dose (dd) larval development test (LDT) in year 1 and then 7 years later. In years 5 and 7, resistance was also measured using a dose-response LDT assay. There was a significant increase in Teladorsagia survivors at the benzimidazole dd assay between year 1 and year 7 for both treatment groups, but the increase in survivors was greater for the farms carrying out their traditional worm control compared to the SCOPS farms. There was also a significant difference between benzimidazole dd results generated across years for Trichostrongylus, but the year and treatment interaction was not significant. Only one of the farm Teladorsagia populations had survivors in the imidazothiazole dd assay in years 1 and 7 and none of the Trichostrongylus populations survived in year 1 compared to isolates from three of the farms in year 7. Dose-response data showed a significant effect for time for both the benzimidazole and imidazothiazole anthelmintics and the increase was again significantly higher for the Teladorsagia populations in the TRADITIONAL group compared to the SCOPS group. This data suggests an increased sensitivity both to detect and to measure changes in response to anthelmintics with the dose-response assay compared to the dd and this is important particularly when allele frequencies are low as might be the case when novel compounds are released to the market. Anthelmintic use across years 5–7 was significantly lower for the farms in the SCOPS group compared to the TRADITIONAL group and farmers in the SCOPS group had selected products from the benzimidazole group less often than farmers in the TRADITIONAL group. Both groups had made minimal use of the imidazothiazole anthelmintic classes and the majority of ewe treatments were selected from the macrocyclic lactone class. Further research is required to determine the effect of these anthelmintic choices on the development of resistance to the macrocyclic lactones. Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
1. Introduction Antimicrobial resistance now ranks as one of the most important global health concerns of our age and threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi (WHO, 2014). Para-
∗ Corresponding author. E-mail address: [email protected] (J. Learmount). http://dx.doi.org/10.1016/j.vetpar.2016.09.006 0304-4017/Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
site diseases are widely acknowledged as a major economic threat to grazing livestock production worldwide and so the widespread development of anthelmintic resistance (dos Santos et al., 2014; Geurden et al., 2014; Karrow et al., 2014) is a major global issue severely affecting their control. This is of particular concern due to the need to produce more protein in a world with a rapidly expanding human population. Developing and monitoring the effectiveness of interventions is critical to the global effort to slow further resistance and will contribute to ensuring the sustainability of our livestock production. Developing and optimising sustain-
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able control strategies is particularly critical when promoting the use of much needed novel drug classes that may be discovered now and in the future. Research, particularly in the UK, Australia, South Africa and New Zealand, has resulted in the recognition of potential resistance-delaying strategies for anthelmintics that can be used on farms (Coles, 2002, 2003; Gaba et al., 2006; Michel, 1985; Van Wyk, 2001) and high and low risk practices have been identified. National guidelines for sustainable control of parasites in sheep (SCOPS) were produced in 2004, incorporating all available evidence for best practice control, with the aim of slowing the development of anthelmintic resistance on UK sheep farms (Abbott et al., 2012). Promotion and revision of the guidelines is ongoing and facilitated by the SCOPS committee, with broad membership representing industry and researchers in the field. This kind of coordinated national approach is critical to ensure maximum uptake and to minimise the potential for the spread of resistance alleles, for example through movement of sheep between farms via breeding stock or common grazing. However, the guidelines are based on data often derived from experimental infections and largely with single species in controlled environments. There is little data on the effect of the guidelines on resistance development in the field in the many commercial settings that exist in the UK. Such data is essential not only to better understand how resistance is selected for in the farm environment, which in turn should allow the optimisation of resistance delaying strategies, but also to convince farmers of the benefits and to guide policy on the safe and effective use of veterinary medicines. This report, therefore, documents a study undertaken to measure the temporal effect of the SCOPS guidelines on the development of anthelmintic resistance. Sixteen farms were engaged to the study with 14 of these being part of a previous study carried out between 2007 (year 1) and 2010 when resistance to the benzimidazole and imidazothiazole drugs were tested as part of an extension study to promote and monitor the uptake of the SCOPS guidelines on UK sheep farms. Half of the farms carried out SCOPS control and half carried out the traditional worm control that had been used on the farms for many years. In 2012 (year 5), the farms were engaged on a second 3-year study to evaluate the effect of the SCOPS guidelines in practice and the SCOPS guidance was intensified, while resistance to the benzimidazole and imidazothiazole anthelmintics was further investigated.
2. Materials and methods 2.1. Selection of study farms All study farms were those previously reported by Learmount et al. (2015). In year 1, data were from 14 of the farms, which were part of a wider cohort (n = 30) of farms engaged to a study that aimed to provide a qualitative evaluation of the practicality and effectiveness of the SCOPS guidelines in practice by deploying them across a network of representative farms. Farms were assigned to one of two experimental treatments: 1. SCOPS, for farms that were already using or were willing to implement the SCOPS guidelines; and 2. TRADITIONAL, for farms that wished to continue employing their traditional worm control without regard to SCOPS guidance. The farms were self-selecting to treatment group: farmers were given information about the trial and then, if they wished to participate, selected whether they did or did not wish to carry out worm control using SCOPS guidance. The SCOPS guidelines advocate a ‘toolbox’ of resistance delaying control methods, with their deployment dependent on individual farm requirements. Hence, evolving strategies were devised for each farm based on veterinary advice. A network of veterinarians was, therefore, established at the start of the project, with each vet visiting and monitoring their assigned
farms at least ten times across a three-year period. Results of this study demonstrated some reduction in anthelmintic use and no significant difference in infection levels in the lambs between the two groups. However, data clearly demonstrated that farmers had not used all of the potential resistance delaying strategies advocated by the SCOPS guidance. Fourteen of the farms (as well as an additional two) were then engaged to a second study (Learmount et al., 2015) in 2012, which aimed to intensify the intervention and collect robust evidence of outcomes relevant to policy makers and industry (years 5–7). The farms were selected based on treatment group, farm type and region to allow a balanced factorial design. Farmers in the SCOPS treatment group pro-actively adopted low-risk management practices while farmers in the TRADITIONAL treatment group were known to have adopted several high-risk management practices during the first study. Further detail is described by Learmount et al. (2015). As before, all study farms had a private veterinarian responsible for animal welfare, and sample and data collection who also developed a formalised farm plan for worm control and advised on diagnostic results for each of the SCOPS farms. As two other factors (Region and Farm Type) might have affected the epidemiology of gastrointestinal worms (Coyne et al., 1991; Crofton, 1965; Gibson et al., 1981), these were equally represented in SCOPS and TRADITIONAL treatment groups. Regional (South west or North east) grouping was carried out to account for the possible effects of climate on the measured effects and farms were divided for type (Lowland or Upland) using the criteria previously described (Learmount et al., 2015). 2.2. Evaluation of anthelmintic resistance In year 1, discriminating dose (dd) larval development tests (LDT) were conducted using 0.1 g/ml thiabenzidole or 1 g/ml levamisole as these doses are reported to be minimum inhibitory concentrations (MIC’s) for susceptible Teladorsagia and Trichostrongylus (Taylor et al., 2009). In years 5 and 7, dose response assays were carried out as evidence gathered during the study suggested that this may be a more sensitive method for determining smaller changes in drug sensitivity over time. In both cases, the LDT used a protocol based on the method originally described by Taylor (1990). Eggs used in the assays were harvested from faecal samples collected from ewes prior to treatment each season. Where possible, the same samples from each farm were used for the tests with levamisole and thiabenzidole at each of the time points. For each sample, larvae were exposed to thiabenzidole or levamisole, as well as left untreated (controls), using the following protocol. Stock solutions were prepared for the dd tests by dissolving drugs in methanol to give a final exposure concentration of 0.1 g/ml thiabenzidole or 1 g/ml levamisole. In years 5 and 7, a range of appropriate concentrations for each farm population, with the aim of killing between 5 and 10% at the lowest and 90–99% of the worms at the highest concentration, were prepared for the dose response assays. Doses ranged between 0.013 and 0.8 g/ml for thiabendazole and 0.003 and 0.4 g/ml for levamisole. The discriminating dose (dd) of 0.1 g/ml was incorporated into the dose ranges for thiabenzidole, and an additional dose of 1 g/ml used for levamisole for all respective dose response tests, to allow comparison of data over time. A 0.075% solution of lyophilized Escherichia coli (Sigma-Aldrich) was mixed with an equal volume of sieved sterile, worm free sheep faecal material in solution (25 g faeces: 85 ml water) and 1 ml of water containing the harvested eggs at the appropriate concentration to give 50–60 trichostrongyle eggs per assay. Aliquots of 190 l of the solution were added to each well of a 24-well plate, shaking the egg suspension well between each aliquot to ensure even dispersal of the eggs. A 10 l aliquot of each prepared drug solution was then added to each of the wells and methanol alone was added to the control wells. For each assay, 4 replicates were prepared for
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2.3. Evaluation of anthelmintic use
% survivors
100 90 80 70 60 50 40
Yr 1
30 20 10
Yr 7
0
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8 13 14 16
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Control
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Farm reference Fig. 1. Anthelmintic resistance to thiabenzidole in Teladorsagia farm isolates determined using discriminating dose test in Year 1 and Year 7.
100 90 80
% survivors
each drug concentration and control and larvae were exposed to a range of 5 concentrations of both thiabenzidole or levamisole. Replicate batches of worms were exposed to the drug at 27◦ C for 7 days. The total content of each well was then pipetted to a 40 m sieve in contact with water contained in a petri dish. This was left for 1 h to allow all surviving larvae to disperse below the sieve, at which point the sieve was removed. The number of surviving third stage larvae were then immediately counted under a microscope. Replicates from each drug dose and control were combined and the surviving third stage larvae, to a maximum of 50, identified to species. The number of third stage larvae surviving in each replicate well was then adjusted, using the appropriate species count (Van Wyk and Mayhew, 2013) for each anthelmintic concentration and control, to number of Teladorsagia and Trichostrongylus larvae surviving in each well. As Trichostrongylus is less prevalent than Teladorsagia on UK farms, particularly in spring when the samples were taken, numbers in control wells were occasionally low and so the results from two dd tests in year 1 and two in year 5 were combined for the Trichostrongylus results.
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70 60 50 40 30
Yr 1
20
Yr 7
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Full data sets for anthelmintic use on each of the farms were collected in the autumn each year after the lambs had been finished in year 5, 6 and 7. The total number of doses of anthelmintics per 1000 animals was calculated for the SCOPS and for the TRADITIONAL farms and the average for the study period was calculated for each group and for ewes and lambs. The percentage of treatments for each of the anthelmintic classes: benzimidazole; imidazothiazole; macrocyclic lactone; macrocyclic lactone + aminoacetyl derivate; spiroindole was calculated for years 5–7. In years 1 and 2, treatments were only recorded for a monitored group and so calculations of total doses were not made. However, the percentage of treatments used for each of the 3 anthelmintics available on the UK market at that time (benzimidazole; imidazothiazole and macrocyclic lactones) was calculated, as the treatments to the monitored group were representative of the wider flock. 2.4. Data and statistical analysis For the dd assays, the % of survivors in the treated wells compared to numbers counted in the control wells was calculated. Isolates were classified as benzimidazole or imidazothiazole resistant (according to exposure) using the Animal and Plant Health Agency (APHA) standard criteria for assessing resistance using this test method. This states that if more than 10% of control numbers survive at 0.1 g/ml for thiabenzidole and 1.0 g/ml for levamisole, the population is designated as benzimidazole or imidazothiazole resistant respectively, as these doses are reported to be minimum inhibitory concentrations (MIC’s) for susceptible Teladorsagia and Trichostrongylus. The totals for survivors in treated and untreated wells in year 1 and year 7 were analysed using a generalised linear mixed model (GLMM) fitted with a logit link function and binomial errors, with farm number as the random effect and treatment and years as the fixed effects. For the dose response assays, the number of third stage Teladorsagia or Trichostrongylus larvae surviving at each drug concentration after 7 days was then used to estimate the concentration required to prevent 50% of the population developing (LD50), using the probit analysis with logit transformation and correcting for Wadley’s problem of only estimating the number of eggs placed in each well. LD50 data were log10 transformed and repeated measures analysis of variance (ANOVA) used to examine the difference in log10 LD50 between treatments and years, with degrees of freedom
0
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7
8 13 14 16
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9 10 11 12 SCOPS
Control Farm Reference
Fig. 2. Anthelmintic resistance to thiabenzidole in Trichostrongylus farm isolates determined using discriminating dose test in Year 1 and Year 7.
corrected (using the Greenhouse-Geisser method) if the variance between years and in each treatment group was significant. All statistical analysis was carried out in Genestat v8 (VSN International, Hemel Hempstead, UK). 3. Results 3.1. Discriminating dose data Discriminating dose data generated for Teladorsagia exposed to thiabenzidole in year 1 and year 7 for the 14 farms engaged to both studies are plotted graphically in Fig. 1. Two of the farms in the latter study (farm refs 1 and 15) were new recruits and, therefore, had no year 1 data for comparison. Eight of the 14 Teladorsagia populations tested in year 1 had >10% of individuals surviving in the dd assay compared to the control survivors and were classified as benzimidazole resistant according to the Animal and Plant Health Agency (APHA) standard criteria for assessing resistance using this test method (Fig. 1). The % survivors ranged between 4 and 69% in year 1. All of the Teladorsagia spp. populations tested with thiabenzidole in Year 7 had >10% of individuals surviving (results ranged between 13 and 100%) at the dd and were therefore classified as benzimidazole resistant. Statistical analysis showed a significant increase in survivors at the dd between year 1 and year 7 (p < 0.001) and a significant effect for the treatment and year interaction (p = 0.046), i.e. the increase in survivors was greater for the TRADITIONAL farms compared to the SCOPS farms. However there was no significant effect of treatment (TRADITIONAL vs SCOPS) on survivors (p = 0.413). Discriminating dose data for the Trichostrongylus spp. population on each farm when tested with thiabenzidole were also calculated as % survivors and data are shown in Fig. 2. Five of the 14 farm populations tested were classed as benzimidazole resistant in year 1 and % survivors ranged between zero and 45. All of the farm Trichostrongylus populations tested in year 7 were classified as resistant according to the APHA criteria and results ranged between
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100 Spiroindole
Perecntage of total doses
90 80
Aminoacetyl derivate + abamecn
70 60
Macrocyclic lactone (milbemycin)
50
Macrocyclic lactone (avermecn)
40 30
Imidazothiazole
20 10
Benzimadazole
0 Ewes TR Ewes SCO Lambs TR Lambs SCO Fig. 3. Average number of anthelmintic doses per thousand animals per year (Years 5–7). SCO = Farms in the SCOPS treatment group. TR = Farms in the TRADITIONAL treatment group.
Fig. 4. Percentage of anthelmintic treatments allocated to each of the available classes during Years 5–7. SCO = Farms in the SCOPS treatment group. TR = Farms in the TRADITIONAL treatment group.
15 and 100%. The statistical analysis showed a significant difference between results generated in year 1 and year 7 (p < 0.001), but the year and treatment interaction was not significant (p = 0.270) and there was no effect of treatment (p = 0.703). Only one of the farm Teladorsagia populations was classified as imidazothiazole resistant using the dd assay and this was true for this farm in year 1 (55% surviving) and year 7 (11% surviving). None of the Trichostrongylus populations were classified as resistant according to the APHA criteria in year 1, but three were classified as resistant in year 7 (11–14%), and one was the same farm as that which had the levamisole resistant Teladorsagia population.
submitted). Between years 5 and 7 there were, on average, 596 treatments carried out per 1000 ewes on the farms in the SCOPS group and 2421 treatments per thousand ewes on farms in the TRADITIONAL group. The majority of anthelmintic doses to ewes in the SCOPS group were products from the macrocyclic lactones class (84%), with 7% of the treatments with benzimidazole products and 9% from the imidazothiazole anthelmintic class. Farmers in the TRADITIONAL group also predominantly treated with macrocyclic lactones products (70%), 21% of treatments with benzimidazole products and 9% being imidazothiazole anthelmintics. For lambs, 2477 treatments were carried out on average per 1000 animals on the farms in the SCOPS group and 4076 per 1000 lambs on farms in the TRADITIONAL group. Of these treatments, the majority of treatments were with benzimidazole anthelmintics for both SCOPS and TRADITIONAL farms (47 and 64% respectively), followed by macrocyclic lactones anthelmintic products (29 and 24% respectively). The least used anthelmintic class was the imidazothiazoles, with these making up 13 and 12% of the total treatments for the SCOPS and TRADITIONAL groups respectively. Farmers in the SCOPS group had also made some use of the two novel drug classes to treat lambs, with 5% of treatments with Startect TM (abamectin [macrocyclic lactone] and derquantel [spiroindole], Zoetis, UK) and 6% with ZolvixTM (monepantel [aminoacetyl derivate], Novartis Animal Health, UK).
3.2. Dose response data For the Teladorsagia field isolates exposed to thiabenzidole in LDT dose response assays across both SCOPS and TRADITIONAL groups, LD50s ranged between 0.008 and 0.205 g/ml in Year 5 and between 0.014 and 0.443 g/ml in Year 7. Results of the ANOVA show that there was no difference in log10 LD50 between SCOPS and TRADITIONAL farms (p-value = 0.095). There was, however, an effect of year and a treatment-by-year interaction (p-value = 0.007, p-value = 0.039, respectively). Overall, the mean LD50 was higher in year 7 (0.17) than year 5 (0.07) and the increase in LD50 was greater in TRADITIONAL farms (mean values 0.06 in Year 5 and 0.24 in Year 7) than SCOPS farms (mean values 0.08 in Year 5 and 0.09 in Year 7). TRADITIONAL farms therefore had a higher mean log10 LD50 in year 7 than SCOPS farms. For the Teladorsagia field isolates exposed to levamisole in the LDT dose response assays, LD50s ranged between 0.002 and 0.223 g/ml in year 1 and between 0.017 and 0.197 g/ml in Year 3. Results of the ANOVA show that there was no difference in log10 LD50 between SCOPS and TRADITIONAL farms or treatmentby-year effect (p-value = 0.948, p-value = 0.848, respectively). There was however a year effect (p-value = 0.002). 3.3. Anthelmintic treatments In the first study (year 1 and year 2) the benzimidazole and macrocyclic lactones compounds were used more often than imidazothiazole anthelmintics when comparing percentages for all the treatments carried out on all farms in the study regardless of experimental group (44 and 37% compared to 19% respectively).The average annual number of treatments per 1000 ewes or lambs between years 5–7 is shown in Fig. 3 and the percentage of treatments in each anthelmintic class within treatment groups was calculated and is shown in Fig. 4. The mean number of treatments carried out on the SCOPS farms was significantly lower than were carried out by the TRADITIONAL farmers (Learmount et al.,
4. Discussion Although there is compelling evidence from controlled experiments for the ability of low risk management to delay the development of anthelmintic resistance (AR) (Kenyon and Jackson, 2012; Leathwick et al., 2006, 2008), there are no data currently available for the effect of best practice worm control on the development of anthelmintic resistance on commercial UK farms. Furthermore, a systematic review and meta-analysis of factors associated with anthelmintic resistance in sheep carried out by Falzon et al. (2014) suggested that unclear risk of selection bias was present in all of the available published trials. Their analysis of ten independent studies found only high frequency of treatment was a significant risk factor for AR. Furthermore, as there is currently no consensus on the preferred method for anthelmintic resistance diagnosis, the use of different diagnostic tools (e.g. in vivo FECRT and in vitro tests) means that it is difficult to compare data from different studies and draw robust conclusions on the selection of anthelmintic resistance in the field and temporal shifts in resistance. Most of the evidence for potential effects on resistance alleles comes from computer modelling exercises (Learmount et al., 2012; Leathwick, 2012) or artificial infections (reviewed by Falzon et al., 2014).
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Our study aimed to produce robust evidence of resistance over time on well characterised farms to allow the evaluation of the effectiveness of proposed resistance delaying strategies and to optimise proposed guidelines as well as to gain insight to field selection of resistance mechanisms. Data from our study showed that benzimidazole resistance was present on a majority of the study farms and this is perhaps not suprising as this is the drug class that has been on the market for the longest time compared to the other four, and reports of benzimidazole are widespread, not only in the UK but globally. However, the farm study data clearly demonstrated that benzimidazole resistance prevalence and levels are increasing in both the Teladorsagia and the Trichostrongylus populations despite the fact that benzimidazole resistance is well documented and has been for a number of years (e.g. Coop et al., 1993). There was also clear evidence that resistance had developed less rapidly on the SCOPS study farms compared to those carrying out more traditional control methods, with a significant effect demonstrated in the case of benzimidazole resistance in the Teladorsagia populations using the dd method over 7 years and the dose reponse assay over 2 years. Teladorsagia are the most prevalent species on UK farms and, as the benzimidazole anthelmintics were the first of the five classes examined to be released on to the UK market, it is likely that resistance changes will be most apparent for this species and anthelmintic class. In year 1, dd data suggested that benzimidazole resistance in Trichostrongylus was absent or at low levels on our study farms and the data suggest a clear increase in prevalence on this cohort of farms. Although Haemonchus was rare on the study farms, where it did occur, there was evidence of benzimidazole resistance in this species also (unpublished data) and so the evidence from our study suggests that multi species resistance to benzimidazole anthelmintics may be more likely at the end compared to the start of the study. Data from dose response lines generated for this study suggest that this may be a more sensitive method for measuring changes in response to anthelmintics over time than the dd dose method. There was only a two year gap between results generated in dose response assays in year 5 and year 7 compared to the five year interval for dd data generated in year 1 and year 5. Despite this shorter time period, a significant effect was shown with a reduced increase in resistance levels for isolates exposed to thiabenzidole from the SCOPS farms compared to isolates from the TRADITIONAL group. The dd method was not sensitive enough to measure changes over this shorter time period (year 5–7, unpublished). In addition, the levamisole dd assays only detected resistance on one study farm in Year 1 and three of the study farms in Year 3 and the increase in resistance levels was minimal. However, using the dose response method, a significant increase in dose required to kill 50% of the population was shown for the farms from both of the study groups, although this effect was not significantly different for the 2 treatment groups. The data suggest an increased sensitivity both to detect and to measure change with this method and this is important, particularly when allele frequencies are low, as might be the case when novel compounds are released to the market. Diagnosing resistance in parasitic worms is not easy, particularly as their host dependence makes laboratory experimentation difficult and this severely constrains early detection and anthelmintic resistance surveillance in the field, as well as inhibiting the development and implementation of control strategies. There is a general acceptance based on experimental evidence, that resistance allele frequencies may need to be significant (>25%) before a reduction in field efficacy is observed and most reported field resistance is from on farm Faecal Egg Count Reduction Testing (FECRT). Improving the sensitivity of laboratory methods is, therefore, of great value to the research effort. The development of more sensitive methods may also aid research into the mechanisms of AR, which are poorly understood and research in this area has also
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been severely constrained by a lack of effective tools with which to investigate the problem and deliver practical solutions. Anthelmintic use across years 5–7 was significantly lower for the farms in the SCOPS group compared to the TRADITIONAL group (Learmount et al., submitted). Farmers in the SCOPS group had selected products from the benzimidazole group less often than farmers in the TRADITIONAL group to treat their lambs, but both groups had made minimal use of the imidazothiazole anthelmintic class for ewe treatments, with the majority of treatments selected from the macrocyclic lactones class. Although farmers in the SCOPS group had relied less on the benzimidazole class for ewe treatments, they had replaced these with persistant macrocyclic lactoness. Much the same pattern was observed for lamb treatments, but here, the SCOPS farmers made more use of the novel products Startect and Zolvix. Selection of the macrocyclic lactones anthelmintic class had increased proportionally at the end compared to the start of the study. It will be important, therefore, to measure the effect of this product shift on the development of resistance to the other anthelmintic classes and particularly the macrocyclic lactones, which have been available in the UK for more than 10 years. Data for the anthelmintic resistance results and drug use on the farms suggest that farmers in the SCOPS group could have made greater use of products from the imidazothiazole anthelminitic group as well as the two novel products. Early integration of the new products is of particular benefit while older products are still effective in the field, so that their use is not relied on and so the development of effective strategies for their use forms an important agenda. Long term use of single products presents a high risk for resistance development and there are already reports of anthelmintic resistance to Zolvix from New Zealand, Uruguay and Holland (Mederos et al., 2014; Scott et al., 2013; Van den Brom et al., 2015) and single product use has been identified as a common risk factor. In order to realise the full benefit of investment in initiatives such as SCOPS, it is critical that the guidelines are adopted by as many farmers as possible, particularly where there is the potential for gene flow between farms and so convincing farmers of the value of resistance delaying strategies is critical to maximise the impact of research in this field. Furthermore, farmers are often reluctant to acknowledge the effect that resistance development may have on production on their farm and routine measurement of the efficacy of products on UK farms may be rare. Thus early detection of resistance and development of robust practical advice to mitigate negative impact remains an important research goal.
5. Conclusions In summary, the study data suggests that implementation of SCOPS worm control principles on English and Welsh farms leads to a significant reduction in anthelmintic use and has the potential to delay the development of anthelmintic resistance. This effect was significant for the benzimidazole anthelmintics against Teladorsagia, the most prevalent species on UK sheep farms. Further research is required to measure the full effect for other anthelmintic classes.
Acknowledgements We are grateful to the UK Veterinary Medicines Directorate for funding this work. Also to the private veterinarians: Mike Glover, Paul Roger, Kate Hovers, Erica Moks, Tim Bebbington, John Hughes, Helen Fielding and Debby Brown, without whom the study could not have been carried out. Thanks are also due to Rebecca Callaby for carrying out the statistical analysis.
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References Abbott, K., Taylor, M., Stubbings, L., 2012. Sustainable worm control strategies for sheep. In: A Technical Manual for Veterinary Surgeons and Advisers. Sustainable Control of Parasites in Sheep (SCOPS). Context Publications. Coles, G., 2002. Sustainable use of anthelmintics in grazing animals. Vet. Rec. 151, 165–169. Coles, G., 2003. Strategies to minimise anthelmintic resistance in large animal practice. Practice 25, 191–199. Coop, R., Jackson, F., Coles, G., Hong, C., 1993. Anthelmintic resistant nematodes in farm animals in the United Kingdom. In: Anthelmintic Resistance in Nematodes of Farm Animals. Report of a Seminar Organised for the European Commission, Brussels, from 8 to 9 November, pp. 79–89. Coyne, M., Smith, G., Johnstone, C., 1991. Fecundity of gastrointestinal trichostrongylid nematodes of sheep in the field. Am. J. Vet. Res. 52, 1182–1188. Crofton, H., 1965. Ecology and biological plasticity of sheep nematodes. I. The effect of temperature on the hatching of eggs of some nematode parasites of sheep. Cornell Vet. 55, 242–250. Falzon, L., O’Neill, T., Menzies, P., Peregrine, A., Jones-Bitton, A., Mederos, A., 2014. A systematic review and meta-analysis of factors associated with anthelmintic resistance in sheep. Prev. Vet. Med. 117, 388–402. Gaba, S., Cabaret, J., Ginot, V., Silvestre, A., 2006. The early drug selection of nematodes to anthelmintics: stochastic transmission and population in refuge. Parasitology 133, 345. Geurden, T., Hoste, H., Jacquiet, P., Traversa, D., Sotiraki, S., di Regalbono, A.F., Tzanidakis, N., Kostopoulou, D., Gaillac, C., Privat, S., 2014. Anthelmintic resistance and multidrug resistance in sheep gastro-intestinal nematodes in France, Greece and Italy. Vet. Parasitol. 201, 59–66. Gibson, T., Everett, G., Whitehead, J., 1981. The survival of the free living stages of Ostertagia circumcincta during drought. Int. J. Biometeorol. 25, 223–225. Karrow, N.A., Goliboski, K., Stonos, N., Schenkel, F., Peregrine, A., 2014. Review: genetics of helminth resistance in sheep. Can. J. Anim. Sci. 94, 1–9. Kenyon, F., Jackson, F., 2012. Targeted flock/herd and individual ruminant treatment approaches. Vet. Parasitol. 186, 10–17. Learmount, J., Taylor, M., Bartram, D., 2012. A computer simulation study to evaluate resistance development with a derquantel-abamectin combination on UK sheep farms. Vet. Parasitol. 187, 244–253. Learmount, J., Gettinby, G., Boughtflower, V., Stephens, N., Hartley, K., Allanson, P., Gutierrez Barrecheguren, A., Perez, D., Taylor, M., 2015. Evaluation of ‘best practice’(SCOPS) guidelines for nematode control on commercial sheep farms in England and Wales. Vet. Parasitol. 207, 259–265.
Learmount, J., Stephens, N., Boughtflower, V., Gutierrez Barrecheguren, A., Massei, G., Taylor, M., submitted. Three-year evaluation of best practice guidelines for nematode control on commercial sheep farms in the UK. Vet. Parasitol. Leathwick, D., Miller, C., Atkinson, D., Haack, N., Alexander, R., Oliver, A., Waghorn, T., Potter, J., Sutherland, I., 2006. Drenching adult ewes: implications of anthelmintic treatments pre-and post-lambing on the development of anthelmintic resistance. N. Z. Vet. J. 54, 297–304. Leathwick, D., Miller, C., Atkinson, D., Haack, N., Waghorn, T., Oliver, A., 2008. Managing anthelmintic resistance: untreated adult ewes as a source of unselected parasites, and their role in reducing parasite populations. N. Z. Vet. J. 56, 184–195. Leathwick, D., 2012. Modelling the benefits of a new class of anthelmintic in combination. Vet. Parasitol. 186, 93–100. Mederos, A.E., Ramos, Z., Banchero, G.E., 2014. First report of monepantel Haemonchus contortus resistance on sheep farms in Uruguay. Parasites Vectors 7, 1–4. Michel, J., 1985. Strategies for the use of anthelmintics in livestock and their implications for the development of drug resistance. Parasitology 90, 621–628. dos Santos, J.M.L., Monteiro, J.P., Ribeiro, W.L.C., Macedo, I.T.F., Camurc¸a-Vasconcelos, A.L.F., da Silva Vieira, L., Bevilaqua, C.M.L., 2014. Identification and quantification of benzimidazole resistance polymorphisms in Haemonchus contortus isolated in Northeastern Brazil. Vet. Parasitol. 199, 160–164. Scott, I., Pomroy, W., Kenyon, P., Smith, G., Adlington, B., Moss, A., 2013. Lack of efficacy of monepantel against Teladorsagia circumcincta and Trichostrongylus colubriformis. Vet. Parasitol. 198, 166–171. Taylor, M.A., Learmount, J., Lunn, E., Morgan, C., Craig, B.H., 2009. Multiple resistance to anthelmintics in sheep nematodes and comparison of methods used for their detection. Small Rumin. Res. 86, 67–70. Taylor, M., 1990. A larval development test for the detection of anthelmintic resistance in nematodes of sheep. Res. Vet. Sci. 49, 198–202. Van Wyk, J.A., Mayhew, E., 2013. Morphological identification of parasitic nematode infective larvae of small ruminants and cattle: a practical lab guide. Onderstepoort J. Vet. Res. 8 (1), 14, Art. 539. Van Wyk, J.A., 2001. Refugia—overlooked as perhaps the most potent factor concerning the development of anthelmintic resistance. Onderstepoort J. Vet. Res. 68, 55. Van den Brom, R., Moll, L., Kappert, C., Vellema, P., 2015. Haemonchus contortus resistance to monepantel in sheep. Vet. Parasitol. 209, 278–280. WHO, 2014. Antimicrobial resistance: global report on surveillance.
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Research paper
The development of anthelmintic resistance with best practice control of nematodes on commercial sheep farms in the UK Jane Learmount ∗ , Nathalie Stephens, Valerie Boughtflower, Alba Barrecheguren, Kayleigh Rickell Animal and Plant Health Agency, Sand Hutton, York YO41 1LZ, UK
a r t i c l e
i n f o
Article history: Received 30 April 2016 Received in revised form 7 September 2016 Accepted 8 September 2016 Keywords: Anthelmintic resistance Larval development test SCOPS Benzimidazole Imidazothiazole
a b s t r a c t Antimicrobial resistance threatens the effective prevention and treatment of an ever-increasing range of infections. The widespread development of anthelmintic resistance is a major global issue affecting the effective control of parasite diseases in grazing livestock production. Sustainable control strategies that reduce dependence on antimicrobials have the potential to slow the further development of resistance but there is little data on the effect of control strategies on resistance development in the field. This report documents a study undertaken to measure the temporal effect of the UK sustainable control of parasites in sheep (SCOPS) guidelines on the development of anthelmintic resistance. Farms carrying out SCOPS or traditional worm control (TRADITIONAL) were tested for resistance to the benzimidazole and imidazothiazole anthelmintics in vitro using a discriminating dose (dd) larval development test (LDT) in year 1 and then 7 years later. In years 5 and 7, resistance was also measured using a dose-response LDT assay. There was a significant increase in Teladorsagia survivors at the benzimidazole dd assay between year 1 and year 7 for both treatment groups, but the increase in survivors was greater for the farms carrying out their traditional worm control compared to the SCOPS farms. There was also a significant difference between benzimidazole dd results generated across years for Trichostrongylus, but the year and treatment interaction was not significant. Only one of the farm Teladorsagia populations had survivors in the imidazothiazole dd assay in years 1 and 7 and none of the Trichostrongylus populations survived in year 1 compared to isolates from three of the farms in year 7. Dose-response data showed a significant effect for time for both the benzimidazole and imidazothiazole anthelmintics and the increase was again significantly higher for the Teladorsagia populations in the TRADITIONAL group compared to the SCOPS group. This data suggests an increased sensitivity both to detect and to measure changes in response to anthelmintics with the dose-response assay compared to the dd and this is important particularly when allele frequencies are low as might be the case when novel compounds are released to the market. Anthelmintic use across years 5–7 was significantly lower for the farms in the SCOPS group compared to the TRADITIONAL group and farmers in the SCOPS group had selected products from the benzimidazole group less often than farmers in the TRADITIONAL group. Both groups had made minimal use of the imidazothiazole anthelmintic classes and the majority of ewe treatments were selected from the macrocyclic lactone class. Further research is required to determine the effect of these anthelmintic choices on the development of resistance to the macrocyclic lactones. Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
1. Introduction Antimicrobial resistance now ranks as one of the most important global health concerns of our age and threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi (WHO, 2014). Para-
∗ Corresponding author. E-mail address: [email protected] (J. Learmount). http://dx.doi.org/10.1016/j.vetpar.2016.09.006 0304-4017/Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
site diseases are widely acknowledged as a major economic threat to grazing livestock production worldwide and so the widespread development of anthelmintic resistance (dos Santos et al., 2014; Geurden et al., 2014; Karrow et al., 2014) is a major global issue severely affecting their control. This is of particular concern due to the need to produce more protein in a world with a rapidly expanding human population. Developing and monitoring the effectiveness of interventions is critical to the global effort to slow further resistance and will contribute to ensuring the sustainability of our livestock production. Developing and optimising sustain-
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able control strategies is particularly critical when promoting the use of much needed novel drug classes that may be discovered now and in the future. Research, particularly in the UK, Australia, South Africa and New Zealand, has resulted in the recognition of potential resistance-delaying strategies for anthelmintics that can be used on farms (Coles, 2002, 2003; Gaba et al., 2006; Michel, 1985; Van Wyk, 2001) and high and low risk practices have been identified. National guidelines for sustainable control of parasites in sheep (SCOPS) were produced in 2004, incorporating all available evidence for best practice control, with the aim of slowing the development of anthelmintic resistance on UK sheep farms (Abbott et al., 2012). Promotion and revision of the guidelines is ongoing and facilitated by the SCOPS committee, with broad membership representing industry and researchers in the field. This kind of coordinated national approach is critical to ensure maximum uptake and to minimise the potential for the spread of resistance alleles, for example through movement of sheep between farms via breeding stock or common grazing. However, the guidelines are based on data often derived from experimental infections and largely with single species in controlled environments. There is little data on the effect of the guidelines on resistance development in the field in the many commercial settings that exist in the UK. Such data is essential not only to better understand how resistance is selected for in the farm environment, which in turn should allow the optimisation of resistance delaying strategies, but also to convince farmers of the benefits and to guide policy on the safe and effective use of veterinary medicines. This report, therefore, documents a study undertaken to measure the temporal effect of the SCOPS guidelines on the development of anthelmintic resistance. Sixteen farms were engaged to the study with 14 of these being part of a previous study carried out between 2007 (year 1) and 2010 when resistance to the benzimidazole and imidazothiazole drugs were tested as part of an extension study to promote and monitor the uptake of the SCOPS guidelines on UK sheep farms. Half of the farms carried out SCOPS control and half carried out the traditional worm control that had been used on the farms for many years. In 2012 (year 5), the farms were engaged on a second 3-year study to evaluate the effect of the SCOPS guidelines in practice and the SCOPS guidance was intensified, while resistance to the benzimidazole and imidazothiazole anthelmintics was further investigated.
2. Materials and methods 2.1. Selection of study farms All study farms were those previously reported by Learmount et al. (2015). In year 1, data were from 14 of the farms, which were part of a wider cohort (n = 30) of farms engaged to a study that aimed to provide a qualitative evaluation of the practicality and effectiveness of the SCOPS guidelines in practice by deploying them across a network of representative farms. Farms were assigned to one of two experimental treatments: 1. SCOPS, for farms that were already using or were willing to implement the SCOPS guidelines; and 2. TRADITIONAL, for farms that wished to continue employing their traditional worm control without regard to SCOPS guidance. The farms were self-selecting to treatment group: farmers were given information about the trial and then, if they wished to participate, selected whether they did or did not wish to carry out worm control using SCOPS guidance. The SCOPS guidelines advocate a ‘toolbox’ of resistance delaying control methods, with their deployment dependent on individual farm requirements. Hence, evolving strategies were devised for each farm based on veterinary advice. A network of veterinarians was, therefore, established at the start of the project, with each vet visiting and monitoring their assigned
farms at least ten times across a three-year period. Results of this study demonstrated some reduction in anthelmintic use and no significant difference in infection levels in the lambs between the two groups. However, data clearly demonstrated that farmers had not used all of the potential resistance delaying strategies advocated by the SCOPS guidance. Fourteen of the farms (as well as an additional two) were then engaged to a second study (Learmount et al., 2015) in 2012, which aimed to intensify the intervention and collect robust evidence of outcomes relevant to policy makers and industry (years 5–7). The farms were selected based on treatment group, farm type and region to allow a balanced factorial design. Farmers in the SCOPS treatment group pro-actively adopted low-risk management practices while farmers in the TRADITIONAL treatment group were known to have adopted several high-risk management practices during the first study. Further detail is described by Learmount et al. (2015). As before, all study farms had a private veterinarian responsible for animal welfare, and sample and data collection who also developed a formalised farm plan for worm control and advised on diagnostic results for each of the SCOPS farms. As two other factors (Region and Farm Type) might have affected the epidemiology of gastrointestinal worms (Coyne et al., 1991; Crofton, 1965; Gibson et al., 1981), these were equally represented in SCOPS and TRADITIONAL treatment groups. Regional (South west or North east) grouping was carried out to account for the possible effects of climate on the measured effects and farms were divided for type (Lowland or Upland) using the criteria previously described (Learmount et al., 2015). 2.2. Evaluation of anthelmintic resistance In year 1, discriminating dose (dd) larval development tests (LDT) were conducted using 0.1 g/ml thiabenzidole or 1 g/ml levamisole as these doses are reported to be minimum inhibitory concentrations (MIC’s) for susceptible Teladorsagia and Trichostrongylus (Taylor et al., 2009). In years 5 and 7, dose response assays were carried out as evidence gathered during the study suggested that this may be a more sensitive method for determining smaller changes in drug sensitivity over time. In both cases, the LDT used a protocol based on the method originally described by Taylor (1990). Eggs used in the assays were harvested from faecal samples collected from ewes prior to treatment each season. Where possible, the same samples from each farm were used for the tests with levamisole and thiabenzidole at each of the time points. For each sample, larvae were exposed to thiabenzidole or levamisole, as well as left untreated (controls), using the following protocol. Stock solutions were prepared for the dd tests by dissolving drugs in methanol to give a final exposure concentration of 0.1 g/ml thiabenzidole or 1 g/ml levamisole. In years 5 and 7, a range of appropriate concentrations for each farm population, with the aim of killing between 5 and 10% at the lowest and 90–99% of the worms at the highest concentration, were prepared for the dose response assays. Doses ranged between 0.013 and 0.8 g/ml for thiabendazole and 0.003 and 0.4 g/ml for levamisole. The discriminating dose (dd) of 0.1 g/ml was incorporated into the dose ranges for thiabenzidole, and an additional dose of 1 g/ml used for levamisole for all respective dose response tests, to allow comparison of data over time. A 0.075% solution of lyophilized Escherichia coli (Sigma-Aldrich) was mixed with an equal volume of sieved sterile, worm free sheep faecal material in solution (25 g faeces: 85 ml water) and 1 ml of water containing the harvested eggs at the appropriate concentration to give 50–60 trichostrongyle eggs per assay. Aliquots of 190 l of the solution were added to each well of a 24-well plate, shaking the egg suspension well between each aliquot to ensure even dispersal of the eggs. A 10 l aliquot of each prepared drug solution was then added to each of the wells and methanol alone was added to the control wells. For each assay, 4 replicates were prepared for
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2.3. Evaluation of anthelmintic use
% survivors
100 90 80 70 60 50 40
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30 20 10
Yr 7
0
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6
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8 13 14 16
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Farm reference Fig. 1. Anthelmintic resistance to thiabenzidole in Teladorsagia farm isolates determined using discriminating dose test in Year 1 and Year 7.
100 90 80
% survivors
each drug concentration and control and larvae were exposed to a range of 5 concentrations of both thiabenzidole or levamisole. Replicate batches of worms were exposed to the drug at 27◦ C for 7 days. The total content of each well was then pipetted to a 40 m sieve in contact with water contained in a petri dish. This was left for 1 h to allow all surviving larvae to disperse below the sieve, at which point the sieve was removed. The number of surviving third stage larvae were then immediately counted under a microscope. Replicates from each drug dose and control were combined and the surviving third stage larvae, to a maximum of 50, identified to species. The number of third stage larvae surviving in each replicate well was then adjusted, using the appropriate species count (Van Wyk and Mayhew, 2013) for each anthelmintic concentration and control, to number of Teladorsagia and Trichostrongylus larvae surviving in each well. As Trichostrongylus is less prevalent than Teladorsagia on UK farms, particularly in spring when the samples were taken, numbers in control wells were occasionally low and so the results from two dd tests in year 1 and two in year 5 were combined for the Trichostrongylus results.
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70 60 50 40 30
Yr 1
20
Yr 7
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Full data sets for anthelmintic use on each of the farms were collected in the autumn each year after the lambs had been finished in year 5, 6 and 7. The total number of doses of anthelmintics per 1000 animals was calculated for the SCOPS and for the TRADITIONAL farms and the average for the study period was calculated for each group and for ewes and lambs. The percentage of treatments for each of the anthelmintic classes: benzimidazole; imidazothiazole; macrocyclic lactone; macrocyclic lactone + aminoacetyl derivate; spiroindole was calculated for years 5–7. In years 1 and 2, treatments were only recorded for a monitored group and so calculations of total doses were not made. However, the percentage of treatments used for each of the 3 anthelmintics available on the UK market at that time (benzimidazole; imidazothiazole and macrocyclic lactones) was calculated, as the treatments to the monitored group were representative of the wider flock. 2.4. Data and statistical analysis For the dd assays, the % of survivors in the treated wells compared to numbers counted in the control wells was calculated. Isolates were classified as benzimidazole or imidazothiazole resistant (according to exposure) using the Animal and Plant Health Agency (APHA) standard criteria for assessing resistance using this test method. This states that if more than 10% of control numbers survive at 0.1 g/ml for thiabenzidole and 1.0 g/ml for levamisole, the population is designated as benzimidazole or imidazothiazole resistant respectively, as these doses are reported to be minimum inhibitory concentrations (MIC’s) for susceptible Teladorsagia and Trichostrongylus. The totals for survivors in treated and untreated wells in year 1 and year 7 were analysed using a generalised linear mixed model (GLMM) fitted with a logit link function and binomial errors, with farm number as the random effect and treatment and years as the fixed effects. For the dose response assays, the number of third stage Teladorsagia or Trichostrongylus larvae surviving at each drug concentration after 7 days was then used to estimate the concentration required to prevent 50% of the population developing (LD50), using the probit analysis with logit transformation and correcting for Wadley’s problem of only estimating the number of eggs placed in each well. LD50 data were log10 transformed and repeated measures analysis of variance (ANOVA) used to examine the difference in log10 LD50 between treatments and years, with degrees of freedom
0
5
6
7
8 13 14 16
2
3
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9 10 11 12 SCOPS
Control Farm Reference
Fig. 2. Anthelmintic resistance to thiabenzidole in Trichostrongylus farm isolates determined using discriminating dose test in Year 1 and Year 7.
corrected (using the Greenhouse-Geisser method) if the variance between years and in each treatment group was significant. All statistical analysis was carried out in Genestat v8 (VSN International, Hemel Hempstead, UK). 3. Results 3.1. Discriminating dose data Discriminating dose data generated for Teladorsagia exposed to thiabenzidole in year 1 and year 7 for the 14 farms engaged to both studies are plotted graphically in Fig. 1. Two of the farms in the latter study (farm refs 1 and 15) were new recruits and, therefore, had no year 1 data for comparison. Eight of the 14 Teladorsagia populations tested in year 1 had >10% of individuals surviving in the dd assay compared to the control survivors and were classified as benzimidazole resistant according to the Animal and Plant Health Agency (APHA) standard criteria for assessing resistance using this test method (Fig. 1). The % survivors ranged between 4 and 69% in year 1. All of the Teladorsagia spp. populations tested with thiabenzidole in Year 7 had >10% of individuals surviving (results ranged between 13 and 100%) at the dd and were therefore classified as benzimidazole resistant. Statistical analysis showed a significant increase in survivors at the dd between year 1 and year 7 (p < 0.001) and a significant effect for the treatment and year interaction (p = 0.046), i.e. the increase in survivors was greater for the TRADITIONAL farms compared to the SCOPS farms. However there was no significant effect of treatment (TRADITIONAL vs SCOPS) on survivors (p = 0.413). Discriminating dose data for the Trichostrongylus spp. population on each farm when tested with thiabenzidole were also calculated as % survivors and data are shown in Fig. 2. Five of the 14 farm populations tested were classed as benzimidazole resistant in year 1 and % survivors ranged between zero and 45. All of the farm Trichostrongylus populations tested in year 7 were classified as resistant according to the APHA criteria and results ranged between
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100 Spiroindole
Perecntage of total doses
90 80
Aminoacetyl derivate + abamecn
70 60
Macrocyclic lactone (milbemycin)
50
Macrocyclic lactone (avermecn)
40 30
Imidazothiazole
20 10
Benzimadazole
0 Ewes TR Ewes SCO Lambs TR Lambs SCO Fig. 3. Average number of anthelmintic doses per thousand animals per year (Years 5–7). SCO = Farms in the SCOPS treatment group. TR = Farms in the TRADITIONAL treatment group.
Fig. 4. Percentage of anthelmintic treatments allocated to each of the available classes during Years 5–7. SCO = Farms in the SCOPS treatment group. TR = Farms in the TRADITIONAL treatment group.
15 and 100%. The statistical analysis showed a significant difference between results generated in year 1 and year 7 (p < 0.001), but the year and treatment interaction was not significant (p = 0.270) and there was no effect of treatment (p = 0.703). Only one of the farm Teladorsagia populations was classified as imidazothiazole resistant using the dd assay and this was true for this farm in year 1 (55% surviving) and year 7 (11% surviving). None of the Trichostrongylus populations were classified as resistant according to the APHA criteria in year 1, but three were classified as resistant in year 7 (11–14%), and one was the same farm as that which had the levamisole resistant Teladorsagia population.
submitted). Between years 5 and 7 there were, on average, 596 treatments carried out per 1000 ewes on the farms in the SCOPS group and 2421 treatments per thousand ewes on farms in the TRADITIONAL group. The majority of anthelmintic doses to ewes in the SCOPS group were products from the macrocyclic lactones class (84%), with 7% of the treatments with benzimidazole products and 9% from the imidazothiazole anthelmintic class. Farmers in the TRADITIONAL group also predominantly treated with macrocyclic lactones products (70%), 21% of treatments with benzimidazole products and 9% being imidazothiazole anthelmintics. For lambs, 2477 treatments were carried out on average per 1000 animals on the farms in the SCOPS group and 4076 per 1000 lambs on farms in the TRADITIONAL group. Of these treatments, the majority of treatments were with benzimidazole anthelmintics for both SCOPS and TRADITIONAL farms (47 and 64% respectively), followed by macrocyclic lactones anthelmintic products (29 and 24% respectively). The least used anthelmintic class was the imidazothiazoles, with these making up 13 and 12% of the total treatments for the SCOPS and TRADITIONAL groups respectively. Farmers in the SCOPS group had also made some use of the two novel drug classes to treat lambs, with 5% of treatments with Startect TM (abamectin [macrocyclic lactone] and derquantel [spiroindole], Zoetis, UK) and 6% with ZolvixTM (monepantel [aminoacetyl derivate], Novartis Animal Health, UK).
3.2. Dose response data For the Teladorsagia field isolates exposed to thiabenzidole in LDT dose response assays across both SCOPS and TRADITIONAL groups, LD50s ranged between 0.008 and 0.205 g/ml in Year 5 and between 0.014 and 0.443 g/ml in Year 7. Results of the ANOVA show that there was no difference in log10 LD50 between SCOPS and TRADITIONAL farms (p-value = 0.095). There was, however, an effect of year and a treatment-by-year interaction (p-value = 0.007, p-value = 0.039, respectively). Overall, the mean LD50 was higher in year 7 (0.17) than year 5 (0.07) and the increase in LD50 was greater in TRADITIONAL farms (mean values 0.06 in Year 5 and 0.24 in Year 7) than SCOPS farms (mean values 0.08 in Year 5 and 0.09 in Year 7). TRADITIONAL farms therefore had a higher mean log10 LD50 in year 7 than SCOPS farms. For the Teladorsagia field isolates exposed to levamisole in the LDT dose response assays, LD50s ranged between 0.002 and 0.223 g/ml in year 1 and between 0.017 and 0.197 g/ml in Year 3. Results of the ANOVA show that there was no difference in log10 LD50 between SCOPS and TRADITIONAL farms or treatmentby-year effect (p-value = 0.948, p-value = 0.848, respectively). There was however a year effect (p-value = 0.002). 3.3. Anthelmintic treatments In the first study (year 1 and year 2) the benzimidazole and macrocyclic lactones compounds were used more often than imidazothiazole anthelmintics when comparing percentages for all the treatments carried out on all farms in the study regardless of experimental group (44 and 37% compared to 19% respectively).The average annual number of treatments per 1000 ewes or lambs between years 5–7 is shown in Fig. 3 and the percentage of treatments in each anthelmintic class within treatment groups was calculated and is shown in Fig. 4. The mean number of treatments carried out on the SCOPS farms was significantly lower than were carried out by the TRADITIONAL farmers (Learmount et al.,
4. Discussion Although there is compelling evidence from controlled experiments for the ability of low risk management to delay the development of anthelmintic resistance (AR) (Kenyon and Jackson, 2012; Leathwick et al., 2006, 2008), there are no data currently available for the effect of best practice worm control on the development of anthelmintic resistance on commercial UK farms. Furthermore, a systematic review and meta-analysis of factors associated with anthelmintic resistance in sheep carried out by Falzon et al. (2014) suggested that unclear risk of selection bias was present in all of the available published trials. Their analysis of ten independent studies found only high frequency of treatment was a significant risk factor for AR. Furthermore, as there is currently no consensus on the preferred method for anthelmintic resistance diagnosis, the use of different diagnostic tools (e.g. in vivo FECRT and in vitro tests) means that it is difficult to compare data from different studies and draw robust conclusions on the selection of anthelmintic resistance in the field and temporal shifts in resistance. Most of the evidence for potential effects on resistance alleles comes from computer modelling exercises (Learmount et al., 2012; Leathwick, 2012) or artificial infections (reviewed by Falzon et al., 2014).
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Our study aimed to produce robust evidence of resistance over time on well characterised farms to allow the evaluation of the effectiveness of proposed resistance delaying strategies and to optimise proposed guidelines as well as to gain insight to field selection of resistance mechanisms. Data from our study showed that benzimidazole resistance was present on a majority of the study farms and this is perhaps not suprising as this is the drug class that has been on the market for the longest time compared to the other four, and reports of benzimidazole are widespread, not only in the UK but globally. However, the farm study data clearly demonstrated that benzimidazole resistance prevalence and levels are increasing in both the Teladorsagia and the Trichostrongylus populations despite the fact that benzimidazole resistance is well documented and has been for a number of years (e.g. Coop et al., 1993). There was also clear evidence that resistance had developed less rapidly on the SCOPS study farms compared to those carrying out more traditional control methods, with a significant effect demonstrated in the case of benzimidazole resistance in the Teladorsagia populations using the dd method over 7 years and the dose reponse assay over 2 years. Teladorsagia are the most prevalent species on UK farms and, as the benzimidazole anthelmintics were the first of the five classes examined to be released on to the UK market, it is likely that resistance changes will be most apparent for this species and anthelmintic class. In year 1, dd data suggested that benzimidazole resistance in Trichostrongylus was absent or at low levels on our study farms and the data suggest a clear increase in prevalence on this cohort of farms. Although Haemonchus was rare on the study farms, where it did occur, there was evidence of benzimidazole resistance in this species also (unpublished data) and so the evidence from our study suggests that multi species resistance to benzimidazole anthelmintics may be more likely at the end compared to the start of the study. Data from dose response lines generated for this study suggest that this may be a more sensitive method for measuring changes in response to anthelmintics over time than the dd dose method. There was only a two year gap between results generated in dose response assays in year 5 and year 7 compared to the five year interval for dd data generated in year 1 and year 5. Despite this shorter time period, a significant effect was shown with a reduced increase in resistance levels for isolates exposed to thiabenzidole from the SCOPS farms compared to isolates from the TRADITIONAL group. The dd method was not sensitive enough to measure changes over this shorter time period (year 5–7, unpublished). In addition, the levamisole dd assays only detected resistance on one study farm in Year 1 and three of the study farms in Year 3 and the increase in resistance levels was minimal. However, using the dose response method, a significant increase in dose required to kill 50% of the population was shown for the farms from both of the study groups, although this effect was not significantly different for the 2 treatment groups. The data suggest an increased sensitivity both to detect and to measure change with this method and this is important, particularly when allele frequencies are low, as might be the case when novel compounds are released to the market. Diagnosing resistance in parasitic worms is not easy, particularly as their host dependence makes laboratory experimentation difficult and this severely constrains early detection and anthelmintic resistance surveillance in the field, as well as inhibiting the development and implementation of control strategies. There is a general acceptance based on experimental evidence, that resistance allele frequencies may need to be significant (>25%) before a reduction in field efficacy is observed and most reported field resistance is from on farm Faecal Egg Count Reduction Testing (FECRT). Improving the sensitivity of laboratory methods is, therefore, of great value to the research effort. The development of more sensitive methods may also aid research into the mechanisms of AR, which are poorly understood and research in this area has also
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been severely constrained by a lack of effective tools with which to investigate the problem and deliver practical solutions. Anthelmintic use across years 5–7 was significantly lower for the farms in the SCOPS group compared to the TRADITIONAL group (Learmount et al., submitted). Farmers in the SCOPS group had selected products from the benzimidazole group less often than farmers in the TRADITIONAL group to treat their lambs, but both groups had made minimal use of the imidazothiazole anthelmintic class for ewe treatments, with the majority of treatments selected from the macrocyclic lactones class. Although farmers in the SCOPS group had relied less on the benzimidazole class for ewe treatments, they had replaced these with persistant macrocyclic lactoness. Much the same pattern was observed for lamb treatments, but here, the SCOPS farmers made more use of the novel products Startect and Zolvix. Selection of the macrocyclic lactones anthelmintic class had increased proportionally at the end compared to the start of the study. It will be important, therefore, to measure the effect of this product shift on the development of resistance to the other anthelmintic classes and particularly the macrocyclic lactones, which have been available in the UK for more than 10 years. Data for the anthelmintic resistance results and drug use on the farms suggest that farmers in the SCOPS group could have made greater use of products from the imidazothiazole anthelminitic group as well as the two novel products. Early integration of the new products is of particular benefit while older products are still effective in the field, so that their use is not relied on and so the development of effective strategies for their use forms an important agenda. Long term use of single products presents a high risk for resistance development and there are already reports of anthelmintic resistance to Zolvix from New Zealand, Uruguay and Holland (Mederos et al., 2014; Scott et al., 2013; Van den Brom et al., 2015) and single product use has been identified as a common risk factor. In order to realise the full benefit of investment in initiatives such as SCOPS, it is critical that the guidelines are adopted by as many farmers as possible, particularly where there is the potential for gene flow between farms and so convincing farmers of the value of resistance delaying strategies is critical to maximise the impact of research in this field. Furthermore, farmers are often reluctant to acknowledge the effect that resistance development may have on production on their farm and routine measurement of the efficacy of products on UK farms may be rare. Thus early detection of resistance and development of robust practical advice to mitigate negative impact remains an important research goal.
5. Conclusions In summary, the study data suggests that implementation of SCOPS worm control principles on English and Welsh farms leads to a significant reduction in anthelmintic use and has the potential to delay the development of anthelmintic resistance. This effect was significant for the benzimidazole anthelmintics against Teladorsagia, the most prevalent species on UK sheep farms. Further research is required to measure the full effect for other anthelmintic classes.
Acknowledgements We are grateful to the UK Veterinary Medicines Directorate for funding this work. Also to the private veterinarians: Mike Glover, Paul Roger, Kate Hovers, Erica Moks, Tim Bebbington, John Hughes, Helen Fielding and Debby Brown, without whom the study could not have been carried out. Thanks are also due to Rebecca Callaby for carrying out the statistical analysis.
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References Abbott, K., Taylor, M., Stubbings, L., 2012. Sustainable worm control strategies for sheep. In: A Technical Manual for Veterinary Surgeons and Advisers. Sustainable Control of Parasites in Sheep (SCOPS). Context Publications. Coles, G., 2002. Sustainable use of anthelmintics in grazing animals. Vet. Rec. 151, 165–169. Coles, G., 2003. Strategies to minimise anthelmintic resistance in large animal practice. Practice 25, 191–199. Coop, R., Jackson, F., Coles, G., Hong, C., 1993. Anthelmintic resistant nematodes in farm animals in the United Kingdom. In: Anthelmintic Resistance in Nematodes of Farm Animals. Report of a Seminar Organised for the European Commission, Brussels, from 8 to 9 November, pp. 79–89. Coyne, M., Smith, G., Johnstone, C., 1991. Fecundity of gastrointestinal trichostrongylid nematodes of sheep in the field. Am. J. Vet. Res. 52, 1182–1188. Crofton, H., 1965. Ecology and biological plasticity of sheep nematodes. I. The effect of temperature on the hatching of eggs of some nematode parasites of sheep. Cornell Vet. 55, 242–250. Falzon, L., O’Neill, T., Menzies, P., Peregrine, A., Jones-Bitton, A., Mederos, A., 2014. A systematic review and meta-analysis of factors associated with anthelmintic resistance in sheep. Prev. Vet. Med. 117, 388–402. Gaba, S., Cabaret, J., Ginot, V., Silvestre, A., 2006. The early drug selection of nematodes to anthelmintics: stochastic transmission and population in refuge. Parasitology 133, 345. Geurden, T., Hoste, H., Jacquiet, P., Traversa, D., Sotiraki, S., di Regalbono, A.F., Tzanidakis, N., Kostopoulou, D., Gaillac, C., Privat, S., 2014. Anthelmintic resistance and multidrug resistance in sheep gastro-intestinal nematodes in France, Greece and Italy. Vet. Parasitol. 201, 59–66. Gibson, T., Everett, G., Whitehead, J., 1981. The survival of the free living stages of Ostertagia circumcincta during drought. Int. J. Biometeorol. 25, 223–225. Karrow, N.A., Goliboski, K., Stonos, N., Schenkel, F., Peregrine, A., 2014. Review: genetics of helminth resistance in sheep. Can. J. Anim. Sci. 94, 1–9. Kenyon, F., Jackson, F., 2012. Targeted flock/herd and individual ruminant treatment approaches. Vet. Parasitol. 186, 10–17. Learmount, J., Taylor, M., Bartram, D., 2012. A computer simulation study to evaluate resistance development with a derquantel-abamectin combination on UK sheep farms. Vet. Parasitol. 187, 244–253. Learmount, J., Gettinby, G., Boughtflower, V., Stephens, N., Hartley, K., Allanson, P., Gutierrez Barrecheguren, A., Perez, D., Taylor, M., 2015. Evaluation of ‘best practice’(SCOPS) guidelines for nematode control on commercial sheep farms in England and Wales. Vet. Parasitol. 207, 259–265.
Learmount, J., Stephens, N., Boughtflower, V., Gutierrez Barrecheguren, A., Massei, G., Taylor, M., submitted. Three-year evaluation of best practice guidelines for nematode control on commercial sheep farms in the UK. Vet. Parasitol. Leathwick, D., Miller, C., Atkinson, D., Haack, N., Alexander, R., Oliver, A., Waghorn, T., Potter, J., Sutherland, I., 2006. Drenching adult ewes: implications of anthelmintic treatments pre-and post-lambing on the development of anthelmintic resistance. N. Z. Vet. J. 54, 297–304. Leathwick, D., Miller, C., Atkinson, D., Haack, N., Waghorn, T., Oliver, A., 2008. Managing anthelmintic resistance: untreated adult ewes as a source of unselected parasites, and their role in reducing parasite populations. N. Z. Vet. J. 56, 184–195. Leathwick, D., 2012. Modelling the benefits of a new class of anthelmintic in combination. Vet. Parasitol. 186, 93–100. Mederos, A.E., Ramos, Z., Banchero, G.E., 2014. First report of monepantel Haemonchus contortus resistance on sheep farms in Uruguay. Parasites Vectors 7, 1–4. Michel, J., 1985. Strategies for the use of anthelmintics in livestock and their implications for the development of drug resistance. Parasitology 90, 621–628. dos Santos, J.M.L., Monteiro, J.P., Ribeiro, W.L.C., Macedo, I.T.F., Camurc¸a-Vasconcelos, A.L.F., da Silva Vieira, L., Bevilaqua, C.M.L., 2014. Identification and quantification of benzimidazole resistance polymorphisms in Haemonchus contortus isolated in Northeastern Brazil. Vet. Parasitol. 199, 160–164. Scott, I., Pomroy, W., Kenyon, P., Smith, G., Adlington, B., Moss, A., 2013. Lack of efficacy of monepantel against Teladorsagia circumcincta and Trichostrongylus colubriformis. Vet. Parasitol. 198, 166–171. Taylor, M.A., Learmount, J., Lunn, E., Morgan, C., Craig, B.H., 2009. Multiple resistance to anthelmintics in sheep nematodes and comparison of methods used for their detection. Small Rumin. Res. 86, 67–70. Taylor, M., 1990. A larval development test for the detection of anthelmintic resistance in nematodes of sheep. Res. Vet. Sci. 49, 198–202. Van Wyk, J.A., Mayhew, E., 2013. Morphological identification of parasitic nematode infective larvae of small ruminants and cattle: a practical lab guide. Onderstepoort J. Vet. Res. 8 (1), 14, Art. 539. Van Wyk, J.A., 2001. Refugia—overlooked as perhaps the most potent factor concerning the development of anthelmintic resistance. Onderstepoort J. Vet. Res. 68, 55. Van den Brom, R., Moll, L., Kappert, C., Vellema, P., 2015. Haemonchus contortus resistance to monepantel in sheep. Vet. Parasitol. 209, 278–280. WHO, 2014. Antimicrobial resistance: global report on surveillance.