Larval development assay for detection of anthelmintic resistance in cyathostomins of Swedish horses

Veterinary Parasitology 128 (2005) 261–269 www.elsevier.com/locate/vetpar

Larval development assay for detection of anthelmintic resistance in cyathostomins of Swedish horses Eva Osterman Lind*, Arvid Uggla, Peter Waller, Johan Ho¨glund Department of Parasitology (SWEPAR), Swedish University of Agricultural Sciences and National Veterinary Institute, SE-751 89 Uppsala, Sweden Received 10 June 2004; received in revised form 4 November 2004; accepted 23 November 2004

Abstract The aim of this study was to investigate the suitability of a larval development assay (LDA) for the determination of anthelmintic resistance in cyathostomin nematode populations of the horse. In addition, comparison of results between geographic regions, types of horse establishment, and the use of anthelmintics in Sweden, was established. Seventy horse herds from different parts of Sweden were sampled, and strongyle eggs from the faeces of 54 of those were investigated by an LDA TM (DrenchRite ). The following anthelmintics were tested: thiabendazole (TBZ), levamisole (LEV), ivermectin monosaccharide (IVM-MS), ivermectin aglycone (IVM-AG) and pyrantel (PYR). The LC50 values for TBZ and LEV were generally lower than those previously reported in other LDA studies on horse nematodes. This could be related to the infrequent use of these compounds for the past 20 years in Sweden. In this study, there was a great variation within and between assay plates that could not be explained. Still the LC50 values differed significantly between the regions for all anthelmintics, except for pyrantel. The highest LC50s were observed in parasite populations from the south of Sweden. There were no significant differences between riding schools and studs. Limitations of this technique exist, namely the lack of established cut-off values for susceptible and resistant populations and interpretation problems related to multi-species infections. Although there are advantages with LDA such as the possibility of testing several compounds simultaneously without interference with the deworming programmes on the farms, we conclude that LDA currently is not a reliable alternative to the faecal egg count reduction test (FECRT). # 2004 Elsevier B.V. All rights reserved. Keywords: Horse; Cyathostomins; Anthelmintic Resistance; Larval development assay; Sweden

1. Introduction Anthelmintic resistance in populations of cyathostomin nematodes of the horse is a well-known and * Corresponding author. Tel.: +46 18 674037; fax: +46 18 309162. E-mail address: [email protected] (E.O. Lind).

widespread problem (Kelly et al., 1981; Uhlinger and Johnstone, 1985; Bauer et al., 1986; Nilsson et al., 1989; MacG. King et al., 1990; Bjørn et al., 1991; Boersema et al., 1991; Fisher et al., 1992; Ihler and Bjørn, 1996; Borgsteede et al., 1997; Craven et al., 1998). Until the end of the 1990s, resistance in horse parasites was restricted to benzimidazole anthelmintic compounds. However, reports from the USA and

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Denmark have indicated the existence also of pyrantel resistance (Chapman et al., 1996; Craven et al., 1998; Kaplan et al., 2004). The traditional method for detection of anthelmintic resistance under field conditions is to perform a faecal egg count reduction test (FECRT). However, there are also several in vitro methods, such as egg hatch assays (EHA), larval development assays (LDA) and larval motility assays, which can be used to demonstrate anthelmintic resistance in nematode parasites of livestock. These methods, which are technically more demanding, have not been evaluated in Sweden. The LDA, in particular, has been frequently used in Australia on nematode parasites of sheep (Johansen and Waller, 1989; Lacey et al., 1990; Gill et al., 1995). A study performed on Haemonchus contortus and Teladorsagia circumcincta of sheep showed that EHA and LDA appear to be the most suitable in vitro methods to be used with sheep nematodes under field conditions (Va´ rady and Corba, 1999). Whether this is the case also for horse nematodes has not been established; there are only a few studies published on in vitro methods for detection of anthelmintic resistance in cyathostomins and the results from these vary. The aims of this study were to: (1) evaluate LDA as a method for monitoring anthelmintic resistance in Swedish horse herds; (2) investigate the occurrence and levels of resistance in cyathostomins by use of the LDA.

2. Materials and methods 2.1. Animals A total of 70 horse farms, of which 26 were located in the south, 23 in the central and 21 in the north of Sweden, were selected for the survey, which was conducted in late spring 1998. Of the farms tested, 36 (51%) were studs and/or trotting stables and 34 (49%) were riding schools. The criteria for a farm to be included in the survey were that there had to be at least 10 horses on the farm and that no anthelmintic treatment had been undertaken for at least 8 weeks prior to the testing. The horse owners/managers were instructed to collect fresh faecal samples from 10 to 20 of the youngest horses on their farm, place them individually in plastic bags, evacuate the air and

promptly send these samples to the laboratory. In connection with the sampling procedure, six questions on anthelmintic usage were completed. 2.2. Processing of samples Samples were processed within 3 days after arrival to the parasitology Laboratory of the National Veterinary Institute in Sweden. Faecal egg counts were carried out using a modified McMaster technique based on 3 g of faeces (Anon., 1986) with a minimum detection level of 50 eggs per gram of faeces (EPG). Faeces from egg positive horses were used for the LDA. In order to estimate the proportion of cyathostomin eggs, pooled samples from each farm were incubated at 25 8C for at least 10 days. Third stage larvae were then collected by means of the Baermann procedure, preserved in Lugol’s solution and identified either as Strongylus spp. or Cyathostominae spp. by morphological criteria according to Thienpont et al. (1979). At least 100 larvae per farm were examined. The LDA used in this study was originally developed and commercialised for detection of anthelmintic resistance in nematodes of sheep (DrenchRiteTM, Horizon Technology, Australia). The original assay plate contains duplicate wells of thiabendazole (TBZ), levamisole (LEV), TBZ/LEV combination, and single wells of ivermectin monosaccharide (IVM-MS) and ivermectin aglycone (IVMAG). In the present study, however, we used a modified DrenchRite plate where the TBZ/LEV wells were substituted for pyrantel (PYR) by the manufacturer; other wells contained the same drugs and concentrations as the original. However, because of technical problems with the pyrantel formulation, the delivery of the specially designed plates was delayed and the first 19 farms were therefore tested by use of original plates without pyrantel. The assay was performed as described in the DrenchRiteTM user manual (Anon., 1996), with some minor changes:  Before the eggs were dispensed into the wells, the plate was inspected and if necessary, rehydrated with distilled water.  The sugar gradient was prepared with 10 ml of each sugar solution instead of 20 ml.

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Approximately 200 g of faeces and 0.5 l of water were stirred to form a slurry, which was washed over copper sieves of 180 and 75 mm, and a nylon sieve of 25 mm. The material collected on the latter sieve, contained microscopic debris together with nematode eggs and this was gently flushed with deionised water into a beaker from which 10 ml was applied to a sucrose gradient (10%, 25% and 40%) and centrifuged for 8 min at 3000 rpm. The eggs were pipetted from the upper interface, flushed over a 25 mm nylon sieve, and then re-suspended 2–4 times with distilled water in a test tube. Between 70 and 100 eggs were added to each of the 96 wells on the DrenchRite plate. The assay plate was incubated for 24 h at 25 8C, before the addition of a bacterial broth to enable the first and second larval stages to feed, and then further incubated for another 6 days. On day 7, the larvae were killed with Lugol’s iodine and the numbers of L1/L2 and L3 in each well were counted, either directly or after storage at 4 8C for a maximum of 3 days. One assay plate was analysed for each farm. 2.3. Data analysis The data were analysed using the LOGIT program and an LC50 value (the concentration that prevents 50% of the eggs to develop to the L3 stage) was obtained for each anthelmintic (Dobson et al., 1987). Two-way analysis of variance (ANOVA) was used to compare the mean LC50 values between different regions and types of herds. The LC50 values were reciprocal root transformed owing to a non-normal distribution. Correlations between LC50 values for IVM-MS and IVM-AG and between LEV and PYR were calculated by a linear regression. Two years after the present study was completed, 15 of the farms were included in a FECRT with fenbendazole where the calculations were performed on geometric means of EPG according to Bjørn et al. (1991) (details not shown). The correlation between the LC50TBZ values

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and FECRT results was analysed by a Spearman rank correlation test. The statistical analyses were performed using Stata1 (Stata corporation, College Station, USA), and the significance level was set to 0.05. 2.4. Anthelmintic sales Data on the horse anthelmintic sales in Sweden 1985–2003 was obtained from the Apoteksbolaget (Swedish Pharmacy Chain), which is a state owned company and the sole retailer for medical products in Sweden. The calculations were performed on various paste formulations that have been registered for horses during this period of time.

3. Results 3.1. Faecal egg counts, larval culture and questionnaire Faecal samples and completed questionnaires were obtained from 34 riding schools and 36 studs located in three different regions of Sweden (Table 1). The EPG values varied considerably within and between farms. Faecal egg counts and larval cultures showed that 95–100% of the population were cyathostomin nematodes. Eggs/larvae from large strongyles, Parascaris equorum and Triodontophorus spp. were also found. Eight farms were excluded from the LDA because the faecal samples contained too few eggs, i.e. the mean EPG was <200 for the egg positive horses. Twelve percent of the farms in the south stated that anthelmintic treatment was performed 6 times per year or more, whereas in contrast, no horse farms in the north of Sweden treated as frequently (Fig. 1). For 5 years prior to the study, 4 (6%) of the farms had been using ivermectin exclusively, 49 (71%) ivermectin and pyrantel and 17 (23%) ivermectin, pyrantel and benzimidazoles. Two-third of the farms had used

Table 1 Data on 70 horse farms in Sweden sampled for LDA test Region

No. of riding schools

No. of studs

No. of horses per farm (S.D.)

Horse age (S.D.)

Arithmetic mean of EPG (range)

Geometric mean of EPG (range)

South Central North

12 11 11

15 11 10

50 (42) 63 (58) 37 (20)

6 (3) 7 (3) 6 (3)

388 (1–1186) 321 (4–1683) 378 (0–1165)

155 (1–757) 132 (1–1317) 141 (1–712)

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Fig. 1. Drenching frequencies of horses in different regions of Sweden.

ivermectin at the latest treatment occasion prior to sampling; the remaining third had used pyrantel. 3.2. LDA The average development of eggs to L3s in absence of anthelmintics was 87%  4.1 (S.D.). Overall, we observed a decreased level of development with increased drug concentrations, where the data generally showed a sigmoidal relationship between the logarithm of the drug concentration and the proportion of larvae affected by the drug. However, 14 (26%) of the plates contained at least two wells where the development of L1/L2 had stopped for some inexplicable reason. Samples from seven farms also had to be re-tested owing to a poor development in all the wells in the first assay. Still, eight farms were

excluded from the data analysis: three because of poor development of the eggs and larvae in the majority of the wells, including the control wells, and five because of bacterial overgrowth on the assay plates. For two farms the LC50LEV values obtained from the statistical analysis were lower than the lowest LEV concentration in the plate; those values were excluded. The mean LC50 values obtained for different drugs in different regions of the country are presented in Table 2. There was a significant positive correlation between the LC50 values for the ivermectin analogues (r = 0.61) but not between LEV and PYR (r = 0.06). The LC50 values for TBZ, IVM-AG, IVM-MS and LEV differed significantly between the regions. The highest LC50s were observed in parasite populations from the south of Sweden. There were no significant differences in the LC50s between riding schools and studs. In 7 (13%) of the plates, 1–4 third stage larvae were found also in the highest concentrations of the ivermectin analogues. These larvae were 50–100 mm longer and 10 mm wider than other third stage larvae, found in the lower concentrations. 3.3. Anthelmintic sales The total sale of anthelmintics for equines in Sweden, expressed as the number of doses per 500 kg body weight, has increased from 321,433 doses in 1985 till 525,432 doses in 2003 (Fig. 2). The use of benzimidazoles decreased dramatically in 1987, and since 1994 these compounds have comprised less than

Table 2 Summary of mean LC50 values, ranges and standard deviations (S.D.) from three regions in Sweden Region

TBZ (mM)

LEV (mM)

IVM-AG (nM)

IVM-MS (nM)

PYR (mM)

South (n = 20) Mean LC50 Range S.D.

0.069 0.027–0.178 0.035

0.60 0.23–1.79 0.38

7.67 2.75–23.89 4.38

6.48 1.25–10.94 2.51

2.43a 1.08–3.27 0.80

Central (n = 19) Mean LC50 Range S.D.

0.051 0.030–0.104 0.021

0.34 0.20–0.64 0.11

5.23 3.06–10.76 1.62

4.30 2.29–8.67 1.45

2.17b 1.30–3.81 0.75

North (n = 15) Mean LC50 Range S.D.

0.048 0.031–0.073 0.013

0.31 0.18–0.42 0.07

5.92 3.20–8.30 1.43

4.96 2.52–9.44 1.91

2.54 1.54–4.47 0.86

a b

n = 7. n = 14.

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Fig. 2. Number of anthelmintic doses sold per 500 kg bodyweight of horse 1985–2003 in Sweden.

5% of the market. The benzimidazoles have been replaced by tetrahydropyromidines and macrocyclic lactones, with macrocyclic lactones currently comprising 60% of the market (2003). 3.4. Comparison of LDA and FECRT results A Spearman rank correlation test showed a significant correlation between the LC50TBZ values and FECR 14 days after fenbendazole treatment, although there were two outliers (Fig. 3). Five farms with FECR >90% had a mean LC50TBZ of 0.04  0.01 (95% confidence interval) and 5 farms with FECR <80% had a mean LC50TBZ of 0.09  0.04.

In the present study, the average development of eggs in the control wells was 87%, which is comparable with results from a Danish study on horse nematodes (Craven et al., 1999). The LC50 values for TBZ and LEV were generally lower than what has been reported from LDA studies performed in Norway (Ihler and Bjørn, 1996), Denmark (Craven et al., 1999), Australia (Pook et al., 2002) and the USA (Tandon and Kaplan, 2004), but slightly higher than those obtained for a cyathostomin population of feral

4. Discussion Data from 54 farms were analysed after the LDAs had been performed. When comparing these results with those performed elsewhere it must be stressed that there are often considerable variations in results from different laboratories. Furthermore, only a few papers have been published on LDA as a tool for measuring anthelmintic resistance in cyathostomin populations in horses and there are no suggested cutoff values for susceptible and resistant populations.

Fig. 3. Faecal egg count reduction (FECR) following fenbendazole treatment versus LC50TBZ for 15 farms that participated in the present LDA study as well as in a faecal egg count reduction test (FECRT) 2 years later. A Spearman rank correlation test showed significant (p = 0.0027) correlation between the two tests.

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Table 3 Overview of means and ranges of LC50 values from six LDA studies on horse strongyles Mean LC50

USA Young et al.

Norway Ihler and Bjørn

Denmark Craven et al.

Australia Pook et al.

USA Tandon and Kaplan

Sweden current

No. of herds

1a

7

32

9

15

54

b

TBZ Range

0.031

0.29 0.15–0.36

0.114 0.034–0.30

0.137 0.062–0.19

0.185 0.042–0.32

0.057 0.027–0.18

LEV Range

0.677

0.99 0.20–1.87

0.61 0.14–1.7

0.54b 0.11–1.0

1.57 0.52–4.42

0.43 0.18–1.8

IVM-AG Range

11

n.p.c

n.p.c

3.95b 1.9–7.07

88 0.03–835

6.3 2.7–23.9

IVM-MS Range

5.5

15.8 4.3–27.8

n.p.c

1.86b 0.71–3.63

7.57 0.18–54.6

5.28 1.25–10.94

Pyr Range

n.p.c

n.p.c

6.3 0.92–12.0

n.p.c

n.p.c

2.37 1.08–4.47

a b c

Feral herd. Pre-treatment. n.p.: not performed.

horses in the USA (Young et al., 1999). The average LC50 values and ranges from these studies are summarised in Table 3. Possibly, low LC50TBZ values could be explained by a low frequency of treatment with benzimidazole anthelmintics on Swedish horse farms for the past 20 years; the Swedish market for horse anthelmintics has been dominated by macrocyclic lactones and pyrantel (Fig. 2). Interestingly, five out of seven farms with LC50TBZ values 0.10 had used benzimidazole compounds at least once during 5 years prior to this study. Three of the seven farms were also tested by FECRT, which showed egg reductions of 74%, 77% and 82%, respectively, 14 days after fenbendazole treatment. Broad-spectrum anthelmintics are usually classified into three groups according to their modes of action. It is generally considered that resistance to one drug automatically implies resistance to other drugs within the same group. This is certainly the case for the benzimidazoles (Hall et al., 1978; Webb and McCully, 1979), and resistance to levamisole also results in resistance to morantel (Le Jambre and Martin, 1979; Sangster et al., 1979). However, selection with morantel does not automatically select for resistance to levamisole in sheep parasites (McKenna, 1985; Waller et al., 1986), and parasites of pigs (Bjørn et al., 1990). In the current LDA, the efficacy of both pyrantel and levamisole was tested on

32 horse farms. Both anthelmintics exert their anthelmintic effect by binding to nicotinic acetylcholine receptors and, therefore, drug selected populations could be expected to show side resistance. Contrary to Va´ rady et al. (1997), in our study the LC50 values for the two compounds were not significantly correlated. Unfortunately, no LC50 values were obtained for pyrantel from the herds that had the highest values for levamisole, and it was therefore difficult to validate the possibility of extrapolating LEV LDA results to PYR efficacy. Tandon and Kaplan (2004) reported that the LC50 and LC95 values obtained for LEV were not consistent with results from a FECRT of pyrantel, in which a high prevalence of resistance was found. The LC50IVM-MS values in this study were lower than those reported by Ihler and Bjørn (1996), but higher than those found for IVM-MS and IVM-AG by Pook et al. (2002). Although ivermectin resistance have not yet been reported in cyathostomins, huge variations in LC50 values were observed in one LDA study of horse cyathostomins (Tandon and Kaplan, 2004). For studies on nematode parasites in sheep, a good correlation between the in vivo susceptibility of isolates and LDA has been shown for H. contortus (Gill et al., 1995). However, Palmer et al. (1998) showed a poor sensitivity for the detection of ivermectin resistance in Ostertagia spp. by the

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LDA, when compared with the definitive in vivo estimation (anthelmintic dose-and-slaughter trials) for resistance to this drug. In the light of these findings, the interpretation regarding the ivermectin susceptibility of cyathostomins in the present study may be uncertain. Interestingly, there were plates where third stage larvae were found also in the highest concentrations of the ivermectin analogues. Unfortunately, based on the appearance in the wells we were not able to identify the species of these larvae, but the contemporary finding of Triodontophorus eggs in the highest concentrations of TBZ in six of the plates possibly suggests the identity. The presence of a small subpopulation of cyathostomins resistant to both IVM-MS and IVM-AG has been reported by Young et al. (1999). From the questionnaire responses we can conclude that the most intensive drenching programmes were used in the south and central of Sweden, where 12% and 9% of the respondents stated that they deworm their horses 6 times per year or more. Although there are no cut-off values for resistant and susceptible cyathostomin populations, we observed significant differences in mean LD50 values for all anthelmintic classes (thiabendazole, ivermectin analogues and levamisole) between the geographical regions. These differences may reflect the general use of anthelmintics in the different regions. By the use of mono-specific reference strains, a correlation between estimated level of resistance and the actual in vivo anthelmintic efficacy has been demonstrated for sheep nematodes (Lacey et al., 1990; Taylor, 1990; Hubert and Kerboeuf, 1992). Also for Oesophagostomum spp. in pigs, a good correlation between LDA and in vivo results has been observed (Va´ rady et al., 1996). There are only a few studies where attempts have been made to compare FECRT and LDA for horse nematodes. A Danish study including 56 herds showed overall low correlation coefficients for the relationship between in vitro tests and FECRT (Craven et al., 1999). The maximal agreement of FECRT and LDA for TBZ was seen at concentrations ranging from 0.0195  LC50 < 0.039 mM, i.e. in the low concentrations (wells 3– 4). In a recent study of oxibendazole resistant farms, low LC50TBZ values were obtained for some farms and there were great variations in results between the farms, suggesting low reliability of the test for

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assessment of benzimidazole resistance on individual farms (Tandon and Kaplan, 2004). Totally 15 of the farms in the present study were also examined for benzimidazole resistance by FECRT. In accordance with data published by Ko¨ nigova´ et al. (2003), a significant correlation was found between the LC50TBZ values and FECR 14 days after fenbendazole treatment. Still, there were two outliers with low FECR and unexpectedly low LC50TBZ values. In conclusion, the great advantage with LDA is that anthelmintics with different modes of action can be tested simultaneously. Furthermore, there is no interference with the deworming program used on a farm. The interpretation of the data is somewhat complicated by the presence of mixed infections involving many cyathostomin species (Osterman Lind et al., 2003). The lack of cut-off values for resistant cyathostomin populations and the variations in the larval development within and between assay plates show the need of further evaluation and verification, preferably including various reference strains. For Swedish horse owners, a standardised test for the detection of resistance to pyrantel and ivermectin – the two main drugs used – would be valuable. Unfortunately, at this stage we do not consider LDA being a reliable alternative to FECRT in equine practice.

Acknowledgements Financial support was obtained from the Swedish Horse Race Totalisator Board (ATG) and Agria Insurance Co., Stockholm, Sweden. We are very grateful to Drs Jeffrey Craven and Robert Dobson for advise on technical and statistical procedures.

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