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A comparison of in vitro tests and a faecal egg count reduction test in detecting anthelmintic resistance in horse strongyles
Veterinary Parasitology 85 (1999) 49–59
A comparison of in vitro tests and a faecal egg count reduction test in detecting anthelmintic resistance in horse strongyles J. Craven a,b,∗ , H. Bjørn a , E.H. Barnes b , S.A. Henriksen a , P. Nansen b b
a Danish Veterinary Laboratory, Bülowsvej 27, 1790 Copenhagen V, Denmark Danish Centre for Experimental Parasitology, Royal Veterinary and Agricultural University, Bülowsvej 13, 1870 Frederiksberg C, Denmark
Received 25 September 1998; accepted 25 April 1999
Abstract This study reports a comparison between faecal egg count reduction test (FECRT), egg hatch assay (EHA) and larval development assay (LDA) for detecting anthelmintic resistance in equine strongyles. Resistance to benzimidazoles was demonstrated in 33 of 42 (79%) farms tested by FECRT and in 32 (62%) of the 52 farms tested by EHA. As the reference strain used was not fully susceptible to benzimidazoles it was not possible to determine the level of resistance by LDA. Pyrantel resistance was indicated on three of 15 farms by faecal egg count reduction. Resistance was also indicated by LDA for one of these farms. In addition resistance was indicated by LDA on two more farms that were not tested by FECRT. Further testing is needed to confirm if these findings are truly indicative of resistance. Generally, correlations between the tests were poor and it was not possible to use the outcome of one test to predict the outcome of another. ©1999 Elsevier Science B.V. All rights reserved. Keywords: Horse; Larval development assay; Egg hatch assay; Faecal egg count reduction; Pyrantel; Benzimidazole
1. Introduction Resistance to one or more classes of anthelmintic compounds has been reported in gastrointestinal nematodes of each of the major farm animal species (Condor and Campbell, 1995). The situation is most severe in the trichostrongyle nematodes of sheep and goats ∗
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where resistance has been reported to all available anthelmintic classes. In horses, small strongyles resistant to benzimidazoles (BZs) have been found to be highly prevalent (Kelly et al., 1981; Bauer et al., 1986; Bjørn et al., 1991; Boersema et al., 1991; Craven et al., 1998) but only recently have studies reporting resistance to pyrantel been published (Chapman et al., 1996; Craven et al., 1998). As a result of the increasing prevalence of anthelmintic resistance (AR) a number of in vivo and in vitro techniques have been developed to detect this resistance. The faecal egg count reduction test (FECRT) is the most widely used in vivo method for detection of resistance in field surveys. In general, however, in vivo tests are time consuming, expensive and often characterised by poor data quality (low precision and reproducibility) due to interanimal variation and drug pharmacodynamics in the host (Lacey et al., 1990). Therefore, increasing interest is paid to in vitro tests, such as the egg hatch assay (EHA) and larval development assay (LDA). Despite being more technically demanding, the EHA for detection of BZ resistance (Le Jambre, 1976; Coles and Simpkin, 1977; Whitlock et al., 1980) is cheaper, more accurate and less time consuming than the FECRT (Hazelby et al., 1994). The use of anaerobic (Hunt and Taylor, 1989) or cold (4◦ C) (Smith-Buijs and Borgsteede, 1986) storage of faecal samples enables this test to be used for field surveys, but it is still considered as a technique best suited to research (Donald, 1985). LDA’s (Coles et al., 1988; Giordano et al., 1988; Lacey et al., 1990; Taylor, 1990; Hubert and Kerboeuf, 1992) have the additional advantage over the EHA of testing for resistance to a range of drugs. Despite the potential of this technique for field screening (Johansen, 1989), only a few surveys using this method appear in the literature. In horses the EHA, mostly based on the method of Whitlock et al. (1980), has been widely used for in vitro testing of AR (Kelly et al., 1981; Ullrich et al., 1988; Ihler, 1995) whereas the use of the LDA has only been reported twice (Preinsberger, 1992; Ihler, 1995). This study was designed to examine the relationship between results of the in vivo FECRT, and the in vitro EHA and LDA for detecting AR in horse strongyles.
2. Materials and methods 2.1. Experimental design Following a questionnaire study carried out in Denmark in 1995, 56 farms were selected for a practical examination of AR in horse strongyles. The criteria used were that 12 or more horses were available for the examination and these horses must not have been treated with anthelmintics in the eight weeks prior to testing. FECRT’s were undertaken with samples being collected on day 0 (day of treatment) and day 14. The drugs used were the commercially available oral paste formulations of Panacur® vet. (18.75% Fenbendazole (FBZ), Hoechst Veterinär, Frankfurt, Germany), Banminth® vet. (43.9% Pyrantel Embonate, Pfizer, London, Ontario, Canada) and Eqvalan® vet. (1.9% Ivermectin (IVM), MSD Agvet, Haarlem, Holland). An FECRT was also undertaken on the reference strain (six animals per group) using Panacur® and Banminth® . An untreated control group was also
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included for each farm. The samples were collected into plastic gloves, air was excluded and the glove was sealed by tying a knot close to the faecal material. The samples were then sent by overnight mail to the Danish Veterinary Laboratory for testing. 2.2. Techniques Details of the egg counts and FECR calculations are described elsewhere (Craven et al., 1998). The FECRT on the reference strain used a modified McMaster egg count technique with a lower detection limit of 25 epg. After faeces from the pre-treatment sampling had been allocated for egg counts and larval cultures, the faeces from animals from each farm with positive egg counts were mixed and the eggs separated using sieves and centrifugation in a sucrose gradient. An EHA and a LDA were then carried out. The EHA, used on 52 of the farms, was carried out according to the recommendations of the World Association for the Advancement of Veterinary Parasitology (WAAVP) (Coles et al., 1992), with one major change. The drug was added to the plates before the egg suspension, as this has been found to improve the reliability of the assay (Craven, unpublished data). Two replicates, each of 10 concentrations of thiabendazole (TBZ) dissolved in dimethylsulphoxide (DMSO), were used for each farm. The range of concentrations used was 0.10–5.0 M TBZ. On each occasion a reference parasite strain was included to give a measure of inter-assay variation. Eggs for the reference strain were obtained from a farm on which BZ anthelmintics had never been used. The LDA, used on 32 farms, was carried out according to Varady et al. (1996), using the drugs pyrantel citrate, levamisole hydrochloride, TBZ and mebendazole (MBZ) (all Sigma Chemical Company, St Louis, MO, USA). All drugs were dissolved in DMSO. TBZ and MBZ were tested over a concentration range of 0.0049–10 M, levamisole (LEV) 0.0325–66.67 M and pyrantel (PYR) 0.39–800 M. Final DMSO concentration was 1%. Two replicates were made for the drugs TBZ, MBZ and LEV and four replicates for PYR. Analysis of the ovicidal effect of the BZ’s (TBZ-O, MBZ-O) was also undertaken. The same reference strain used for the EHA was also used in the LDA testing. 2.3. Data analysis The average data from the two in vitro assays were analysed using the logit program (Dobson et al., 1987) to obtain estimates of the LD50 or ED50 data (the dose that prevents 50% of the population developing to L3 larvae or hatching, respectively). The data was further analysed with the aim of defining a delineating concentration for resistance in each of the assays. For the LDA resistance ratios (RR) were calculated as the LD50 of the test strain divided by the LD50 of the reference strain. Due to their low prevalence (<5–10%) (Craven et al., 1998), Strongylus and other non-cyathostome L3 larvae were not counted in the LDA, but eggs and early stage larvae will have contributed to the totals of these stages in both the LDA and the EHA. The relationships between the three tests were examined using Spearman rank correlation coefficients between the FECR, ED50 and LD50 values obtained in each test. In order to examine the feasibility of using the results of one of these tests to predict the results of another, regression lines were calculated using
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(a) FECR as explanatory variable and ED50 or LD50 as response variable, and (b) ED50 as explanatory variable and LD50 as response variable. 95% confidence intervals for the mean of the response variables for a given value of the explanatory variable were also calculated and the numbers of observations that fell outside these intervals were counted. An attempt was made to use the results of the FECRT to determine threshold or cutoff ED50s or LD50s that could be used to discriminate between resistant and susceptible parasites in the EHA or LDA. The threshold FECR percentage and ED50 or LD50 for declaring resistance were varied over a range of values and for each combination the number of observations where the results of the two tests agreed (i.e. both declared resistance or neither declared resistance) were counted. The values of FECR percentage tested were 90% and 95%, and values of ED50 or LD50 tested were the drug concentrations used in the assays.
3. Results Table 1 shows the results of the FECRT on the reference strain. Five of the six horses in the BZ group had positive egg counts after treatment, as did one horse from the pyrantel group. The reference strain was shown to be suspected resistant to BZ’s (96% reduction, lower 95% confidence limit 88%) but sensitive to PYR and IVM. FECR data for the other farms is summarised in Table 2. FBZ FECR values (Table 3) obtained from the 42 farms examined varied from 0 to 100%, with 11 farms having less than 60% reduction and nine having 95% or greater. PYR FECR values (Table 4) varied from 87 to 100% with only the three farms where resistance was detected having values lower than 99%. Table 1 Faecal egg count reduction test results for the reference strain showing group egg counts pre- and post-treatment, the egg count reduction percentage (FECR) and lower and upper 95% confidence limits (LCL and UCL) Drug
n
Pre epg
Post epg
FECR
LCL
UCL
Control BZ Pyrantel
6 6 6
567 500 454
508 18 4
– 96 99
– 88 93
– 99 100
Table 2 Summary of the results from the faecal egg count reduction testsa,b,c (Craven et al., 1998) Drug
Farms Tested
Benzimidazole Pyrantel Ivermectin
42 15 16
Suspected Sensitive 5 11 16
Resistant 4 1 0
Resistant: <95% FECR and <90% lower 95% confidence limit. Suspected resistant: either >95% FECR or >90% lower 95% confidence limit. c Susceptible: >95% FECR and >90% lower 95% confidence limit. a
b
Resistant 33 3 0
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Table 3 Faecal egg count reduction (FECR), egg hatch assay (EHA) and larval development assay (LDA) data for the two benzimidazoles used Farm ID 1351 1352 1362 1363 1364 1482 1498 1507 1520 1523 1524 1525 1586 1602 1609 1610 1611 1716 1717 1723 1724 1725 1729 1730 1741 1742 1752 1756 1761 1762 1763 1767 1768 1853 1854 1859 1862 1863 1870 1871 1880 1881 1882 1887 1888 1891 1892 1895 1896 1993 2000 2517 a
BZ FECR 91 93 96 – 34 – 87 – 59 0 29 78 – 100 59 84 100 – 88 – 99 81 88 71 61 81 76 90 98 81 98 90 66 46 – 100 – 2 57 72 18 0 – 46 100 – 87 85 75 69 95 0
EHA (uM)
MBZ (uM)
MBZ-Oa (uM)
TBZ (uM)
TBZ-Oa (uM)
0.50 5.00 0.28 0.60 0.67 0.91 0.78 0.38 0.86 0.81 0.67 0.52 0.90 0.47 0.95 0.71 0.45 0.65 0.38 0.66 0.51 0.41 0.42 0.43 1.09 0.27 0.56 0.33 0.42 0.51 0.38 0.43 0.56 0.73 1.42 1.60 0.70 2.14 0.81 0.62 0.48 0.40 0.20 1.37 0.63 1.76 0.31 0.70 0.37 5.00 0.36 2.16
0.13 0.047 0.13 0.13 0.24 0.51 0.43 0.40 0.13 0.35 0.28 0.16 – 0.19 0.29 0.22 0.084 0.12 0.11 0.35 0.15 0.19 0.05 0.26 0.11 – – – – – – – – – – – – – – – 0.20 0.12 0.12 0.23 0.26 – 0.088 0.18 0.16 – – –
3.00 – – – – – – – – – – – – 0.45 0.44 0.38 0.62 1.20 0.55 2.00 1.70 1.52 1.90 1.57 1.54 0.39 0.40 0.50 0.96 1.09 0.64 1.04 1.03 – – – – – – – 0.31 0.54 0.29 1.44 0.36 – 0.35 0.63 0.33 – – –
0.098 0.077 0.11 0.13 0.19 0.30 0.077 0.086 0.10 0.056 0.21 0.10 – 0.075 0.16 0.084 0.09 0.086 0.088 0.073 0.057 0.066 0.046 0.05 0.034 – – – – – – – – – – – – – – – 0.26 0.14 0.081 0.23 0.22 – 0.079 0.11 0.097 – – –
1.01 – 0.21 0.30 0.55 0.57 0.76 0.23 0.35 0.40 0.40 0.25 – 0.12 0.17 0.09 0.23 0.36 0.27 0.55 0.47 0.34 0.45 0.27 0.59 0.17 0.54 0.24 0.24 0.34 0.19 0.29 0.36 – – – – – – – 0.19 0.23 0.16 0.70 0.46 – 0.19 0.21 0.16 – – –
The suffix ’O’ refers to the ovicidal effect of the drug.
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Table 4 Comparison of faecal egg count reduction (FECR) and larval development assay (LDA) LD50 (M) and resistance ratio (RR) data for pyrantel and levamisole Farm ID
PYR FECR
PYR−LD50
PYR−RR
LEV−LD50
LEV−RR
1351 1352 1362 1363 1364 1482 1498 1507 1520 1523 1524 1525 1602 1609 1610 1611 1716 1717 1723 1724 1725 1729 1730 1761 1762 1871 1880 1881 1882 1887 1888 1892 1895 1996
100 100 100 – 100 – – – – – – – 100 – 100 87 – 100 0 0 0 0 92 99 100 92 0 100 0 0 0 0 99 100
5.3 2.5 10.9 6.9 6.1 4.6 1.6 4.9 5.3 0.92 4.8 5.5 9.8 6.9 7.8 7.1 10.0 4.1 7.5 6.4 4.6 2.4 10.4 – – – 12.0 5.5 7.4 8.1 7.6 6.3 5.6 6.6
0.94 0.58 2.7 1.7 1.5 0.75 0.78 2.4 0.86 0.15 0.77 0.89 0.91 0.65 0.73 0.66 2.14 0.87 1.59 1.36 0.97 0.52 2.21 – – – 2.0 0.90 1.2 1.3 1.3 1.0 0.93 1.1
0.24 0.19 1.1 0.85 0.58 0.81 0.14 0.41 0.30 0.18 0.25 1.3 1.1 0.5 0.56 0.33 0.8 0.43 0.33 0.38 0.8 0.25 0.35 – – – 1.7 0.68 1.1 0.55 0.52 0.46 0.77 1.1
1.2 0.92 2.3 1.7 1.2 0.92 1.0 3.1 0.34 0.20 0.28 1.5 0.72 0.33 0.37 0.22 1.76 0.95 0.73 0.84 1.8 0.55 0.76 – – – 1.6 0.60 1.0 0.49 0.46 0.40 0.69 1.0
The EHA results are shown in Table 3. For two farms the maximum inhibition of hatching was less than 50%, so for the data analysis these farms were assigned the highest tested concentration (5 M) as their ED50. The reference strain was tested on 30 separate occasions with a mean ED50 of 0.39 M (Table 5). In the LDA the control isolate was tested on eight separate occasions (Table 5). For all isolates the development of eggs to L3 larvae was high (>80%) in control wells but there was a large variation in development within each assay. The LDA data for the two BZ’s is shown in Table 3. Due to the suspected FBZ resistance of the reference strain, RR values for the BZ’s were not calculated. The LDA data for LEV and PYR is presented in Table 4. For both the LDA and EHA it was not possible to determine a delineating dose for resistance to BZ’s.
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Table 5 Egg hatch assay (EHA) results (M TBZ) and larval development assay (LDA) results (M) for the reference strain
Number Mean Std Dev
EHA ED50a
PYR LD50b
LEV LD50b
MBZ LD50b
MBZ-Oc ED50a
TBZ LD50b
TBZ-Oc ED50a
30 0.39 0.093
8 5.98 2.74
7 0.69 0.47
8 0.12 0.049
5 0.80 0.35
8 0.068 0.024
8 0.26 0.088
a
ED50 is the point at which 50% of the eggs fail to hatch. LD50 is the point at which 50% of the eggs develop into L3 larvae. c The suffix ’O’ refers to the ovicidal effect of the drug. b
Table 6 Spearman rank correlation coefficients and p-values for comparison of faecal egg count reduction test (FECRT), egg hatch assay (EHA) and larval development assay (LDA) Comparison
n
EHAa
42
0.48
0.001
26 26 26 33 12
−0.39 0.1 −0.28 −0.13 −0.37
0.048 0.6 0.2 0.5 0.2
32 29 32 39
0.49 0.33 0.23 0.56
vs.
FECRTb
Spearman correlation coefficient
p-value
LDAc
vs. FECRT MBZ MBZ-Od TBZ TBZ-Od PYR
LDA vs. EHA MBZ MBZ-Od TBZ TBZ-Od
0.004 0.08 0.21 0.0002
ED50 (M TBZ). Faecal egg count reduction percentage. c LD50 (M). d The suffix ’O’ refers to the ovicidal effect of the drug. a
b
The Spearman rank correlations between the tests are shown in Table 6. The correlation coefficients were generally fairly low and only statistically significant (p < 0.05) in four cases out of 10. The regressions to examine the feasibility of using the results of one test to predict another were significant (p < 0.05) in four cases out of 10. On average about half of all observations fell outside the 95% confidence intervals, and in many cases the ED50/LD50 of the actual observations were two to three times higher than the predicted values (farm no. 1993 was five times higher). For the LDA using PYR there were only 12 observations, of which 10 had 100% FECR so it was not feasible to determine a threshold concentration for PYR in the LDA. In all other cases, maximal agreement between each pair of tests (percentage of observations where both tests made the same declaration) was approximately 80% and this occurred for FECR of 95%. The highest threshold concentrations (M) that yielded maximum agreement are shown in Table 7. However, in all cases, the concentrations listed in Table 7 result in all or almost all the observations obtained in this study being declared resistant by the EHA/LDA.
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Table 7 Range of benzimidazole concentrations (M) yielding maximal agreement of FECRT results (for 95% reduction) with either egg hatch assay (EHA) or larval development assay (LDA) EHA LDA (MBZ) LDA (MBZ-O) LDA (TBZ) LDA (TBZ-O)
0.4 ≤ ED50 < 0.65 0.039 ≤ LD50 < 0.078 0.156 ≤ LD50 < 0.3125 0.0195 ≤ LD50 < 0.039 0.156 ≤ LD50 < 0.3125
4. Discussion Whereas Coles et al. (1992) added the drug to the eggs, in this study the eggs were added to the drug. This has been seen to improve the reproducibility of the data obtained from the EHA, most likely as a result of a more thorough mixing of the two solutions. From the results observed in this study an ED50 of between 0.4 M and 0.65 M TBZ was indicated as the threshold for declaring BZ resistance (Table 7). This is in agreement with the recommendation of the WAAVP (Coles et al., 1992) that an ED50 of 0.5 M (0.1 g/ml) TBZ is the threshold for declaring resistance in horses. Using this threshold, resistance is declared on 32 (62%) of the 52 farms examined in this study. This compares well with the prevalence of resistance detected by FECRT in this study (78%). The assay would become easier to interpret if hatching at or above a specific dose could be used to indicate resistance. This principle was used in two reports from Germany (Ullrich, 1987; Ullrich et al., 1988) that used a less than 96% inhibition of egg hatch at 0.75 M (0.15 g/ml) TBZ as the threshold for declaring resistance. Using this approach all farms examined in the present study would be declared resistant. The average ED50 of the reference strain was higher than expected and a subsequent FECR test indicated that the worm population was suspected resistant to FBZ, despite the fact that BZ’s have never been used on the property. We attribute this finding to the importation of resistant worms when buying replacement horses. Despite the fact that most of the testing was carried out during the atypically hot 1995 summer, the eggs had not visibly commenced development 24 h after the samples were taken. This would indicate that eliminating most of the air from the glove was sufficient to create anaerobic conditions, thus halting/slowing the development of the eggs. Anaerobic storage for up to 7 days has been shown to halt egg development without impact on their subsequent development in sheep trichostrongyles (Hunt and Taylor, 1989). Faecal material for the reference strain was collected only once per week and stored at 4◦ C until required. However, EHAs were sometimes conducted four times per week. Despite storage for 0 to 80 h before testing, variation in the ED50 of the control isolate was acceptable (Table 4). This is in agreement with the findings for Haemonchus contortus by Smith-Buijs and Borgsteede (1986). Although the development of eggs to third stage larvae in the absence of drugs was quite high (80–85%) in the LDA used in this study, it was not as high as reported by others (Lacey et al., 1990; Hubert and Kerboeuf, 1992). This may be the result of the higher temperature used (26◦ C) which has been shown to reduce the development of the sheep trichostrongyles (Hubert and Kerboeuf, 1992). Additionally, there was greater variability in the development
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of larvae within each plate than has been observed for Oesophagostomum spp. in pigs either in earlier (unpublished) work using the method of Lacey et al. (1990) or using this method (Varady et al., 1996). The LD50 values obtained for the reference strain in this study for LEV are similar to values that have been reported for horse strongyles (Ihler, 1995), sheep trichostrongyles (Lacey et al., 1990; Hubert and Kerboeuf, 1992) and Oesophagostomum spp. in pigs (Varady et al., 1996). Interestingly, the average LD50 value obtained for PYR is less than half that obtained in sheep strongyles (Hubert and Kerboeuf, 1992) and approximately 10 times lower than that seen in Oesophagostomum spp. in pigs (Varady et al., 1996). Four (13%) of the 31 farms tested with PYR had an RR greater than two. Two of these were not tested by FECRT, the third tested resistant and the fourth susceptible. Two of the four farms also had LEV RRs of greater than two, suggesting side resistance to LEV has developed after treatment with PYR. Further testing will be undertaken to confirm the presence of PYR resistance on these farms. For the BZ’s there is no data published for susceptible cyathostome isolates. Ihler (1995) observed a range of LD50 values of 0.15–0.36 M TBZ for seven isolates of varying degrees of resistance, while Preinsberger (1992) recorded an average value of 0.2 M TBZ for a single resistant isolate. In this study the reference strain had an average LD50 against TBZ and MBZ of 0.068 M and 0.12 M, respectively, which is similar to the values obtained by Varady et al. (1996) for Oesophagostomum spp. and by Lacey et al. (1990) for sheep trichostrongyles. However, it must be remembered that the reference strain in this study may have had elevated LD50s since it was suspected to be FBZ resistant. Due to this suspected resistance it was not possible to calculate RRs, nor therefore the number of farms on which resistance was present, for TBZ or MBZ. When comparing the in vitro assays it can be seen that the EHA ED50 is higher than that for the LDA (TBZ-O). TBZ has the effect of delaying, not preventing, hatching so this is expected given the shorter run time of the EHA. Analysis of the data to find a delineating dose for BZ resistance for both the LDA and EHA was unsuccessful. While the LD50 values obtained for the reference strain were grouped closely there was a wide variation in values when LD99 was considered. This variation may have been due to the suspected resistant population of the reference strain. The results of the correlations obtained here were, with a few exceptions, poor. From this we might conclude that one of the tests cannot measure AR status, however it is more likely that the different tests are measuring different attributes of the parasites’ response to anthelmintics, especially since the horses are most likely carrying a mixture of parasite species. At present it is not possible to identify the mixture of species present in the horse, nor obtain in vitro data for mono-specific populations, although samples have been preserved from this study to enable such identification to be made when the techniques become available. The only way to decide on the optimal test would be to compare its performance to some agreed standard of AR. For parasites of smaller animals this has often been worm burden reduction (compared with non-treated controls) in a treat-and-slaughter assay but this is virtually impossible for parasites in horses. Furthermore, the best test to use in any given situation is likely to also depend on the drug against which we need to measure resistance, the purpose for which we are testing for resistance and the resources available.
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Acknowledgements The study was supported by the Danish National Research Foundation, the Danish Veterinary Laboratory, MSD Agvet (Copenhagen, Denmark) and Pfizer A/S (Copenhagen, Denmark). The participating equine veterinarians are cordially thanked for their efforts in treatment and sampling of the horses. References Bauer, C., Merkt, J.C., Janke-Grimm, G., 1986. Prevalence and control of BZ resistant small strongyles on German thoroughbred studs. Vet. Parasitol. 21, 189–203. Bjørn, H., Sommer, C., Schougård, H., Nansen, P., 1991. Resistance to benzimidazole anthelmintics in small strongyles (Cyathostominae) of horses in Denmark. Acta Vet. Scan. 32, 253–260. Boersema, J.H., Borgsteede, F.H.M., Eysker, M., Elena, T.E., Gaasenbeek, C.P.H., van den Burg, W.P.J., 1991. The prevalence of AR of horse strongyles in the Netherlands. Vet. Quarterly 13 (4), 209–217. Chapman, M.R., French, D.D., Monahan, C.M., Klei, T.R., 1996. Identification and characterization of a pyrantel pamoate resistant cyathostome population. Vet. Parasitol. 66, 205–212. Coles, G.C., Simpkin, K.G., 1977. Resistance of nematode eggs to the ovicidal activity of benzimidazoles. Res. Vet. Sci. 22, 386–387. Coles, G.C., Tritschler, J.P., Giordano, D.J., Laste, N.J., Schmidt, A.L., 1988. Larval development test for detection of anthelmintic resistant nematodes. Res. Vet. Sci. 45, 50–53. Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World association for the advancement of veterinary parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 44, 35–44. Condor, G.A., Campbell, W.C., 1995. Chemotherapy of nematode infections of veterinary importance, with special reference to drug resistance. Adv. Parasitol. 35, 1–84. Craven, J., Bjørn, H., Henriksen, S.A., Nansen, P., Larsen, M., Lendal, S., 1998. A survey of anthelmintic resistance on Danish horse farms, using 5 different methods of calculating faecal egg count reduction. Equine Vet. J. 30, 289–293. Dobson, R.J., Griffiths, D.A., Donald, A.D., Waller, P.J., 1987. A genetic model describing the evolution of levamisole resistance in T. colubriformis, a nematode parasite of sheep. IMA Journal of Mathematics Applied in Medicine and Biology 4, 279–293. Donald A.D., 1985. Research priorities in anthelmintic resistance. In: Anderson, N., Waller, P.J. (Eds.), Resistance of Nematodes to Anthelmintic Drugs, CSIRO Division of Animal Health, Glebe, NSW, pp. 171–181. Giordano, D.J., Tritschler, J.P., Coles, G.C., 1988. Selection of ivermectin-resistant Trichostrongylus colubriformis in lambs . J. Vet. Pharmacol. Therap. 30, 139–148. Hazelby, C.A., Probert, A.J., 1994. Anthelmintic resistance in nematodes causing parasitic gastroenteritis of sheep in the UK. J. Vet. Pharmacol. Therap. 17, 245–252. Hubert, J., Kerboeuf, D., 1992. A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes. Vet. Rec. 130, 442–446. Hunt, K.R., Taylor, M.A., 1989. Use of the egg hatch assay on sheep faecal samples for the detection of benzimidazole resistant nematodes. Vet. Rec. 125, 153–154. Ihler, C.F., 1995. Use of two in vitro methods in the detection of benzimidazole resistance in equine small strongyles (Cyathostoma spp.). Vet. Parasitol. 65, 117–125. Johansen, M.V., 1989. An evaluation of techniques used for the detection of anthelmintic resistance in nematode parasites of domestic livestock. Vet. Res. Comm. 13, 455–466. Kelly, J.D., Webster, J.H., Griffin, D.L., Whitlock, H.V., Martin, I.C.A., Gunawan, M., 1981. Resistance to benzimidazole anthelmintics in equine strongyles. Frequency, geographical distribution and relationship between occurrence, animal husbandry procedures and anthelmintic usage. Aust. Vet. J. 7, 163–171. Lacey, E., Redwin, J.M., Gill, J.H., Demargheriti, V.M., Waller, P.J., 1990. A larval development assay for the simultaneous detection of broad spectrum anthelmintic resistance. In: Boray, J.C., Martin, P.J., Roush, R.T. (Eds.), Resistance of Parasites to Antiparasitic Drugs, MSD Agvet, Rahway, NJ, pp. 177–184.
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Le Jambre, L.F., 1976. Egg hatch as an in vitro assay of thiabendazole resistance in nematodes. Vet. Parasitol. 2, 385–391. Preinsberger, H., 1992. Zur Brauchbarkeit und Standardisierung des Larvenentwicklungstest bei equinen Strongyliden. Inaugural Dissertation zur Erlangung der Würde eines Doctor Medicinae Veterinariae, Wien. Smith-Buijs, C.M.C., Borgsteede, F.H.M., 1986. Effect of cool storage of faecal samples containing Haemonchus contortus eggs on the results of an in vitro egg development assay to test anthelmintic resistance. Res. Vet. Sci. 40, 4–7. Taylor, M.A., 1990. A larval development test for the detection of anthelmintic resistance in nematodes of sheep. Res. Vet. Sci. 49, 198–202. Ullrich, D., 1987. Verbreitung benzimidazol-resistenter Strongyliden in Nordrhein-Westfalen. Inaugural Dissertation zur Erlangung des Grades eines Doctor Medicinae Veterinariae, Tierärztliche Hochschule, Hannover. Ullrich, D., Bauer, C., 1988. Benzimidazolresistenz bei kleinen Strongyliden (Cyathostominae): Verbreitung in Pferdebeständen Nordrhein-Westfalens. Berl. Münch. Tierärztl. Wschr. 101, 406–408. Varady, M., Bjørn, H., Nansen, P., 1996. In vitro characterization of anthelmintic susceptibility of field isolates of the pig nodular worm Oesophagostomum spp. susceptible or resistant to various anthelmintics. Int. J. Parasitol. 26, 733–740. Whitlock, H.V., Kelly, J.D., Porter, C.J., Griffin, D.L., Martin, I.C.A., 1980. In vitro field screening for anthelmintic resistance in strongyles of sheep and horses. Vet. Parasitol. 7, 215–232.
A comparison of in vitro tests and a faecal egg count reduction test in detecting anthelmintic resistance in horse strongyles J. Craven a,b,∗ , H. Bjørn a , E.H. Barnes b , S.A. Henriksen a , P. Nansen b b
a Danish Veterinary Laboratory, Bülowsvej 27, 1790 Copenhagen V, Denmark Danish Centre for Experimental Parasitology, Royal Veterinary and Agricultural University, Bülowsvej 13, 1870 Frederiksberg C, Denmark
Received 25 September 1998; accepted 25 April 1999
Abstract This study reports a comparison between faecal egg count reduction test (FECRT), egg hatch assay (EHA) and larval development assay (LDA) for detecting anthelmintic resistance in equine strongyles. Resistance to benzimidazoles was demonstrated in 33 of 42 (79%) farms tested by FECRT and in 32 (62%) of the 52 farms tested by EHA. As the reference strain used was not fully susceptible to benzimidazoles it was not possible to determine the level of resistance by LDA. Pyrantel resistance was indicated on three of 15 farms by faecal egg count reduction. Resistance was also indicated by LDA for one of these farms. In addition resistance was indicated by LDA on two more farms that were not tested by FECRT. Further testing is needed to confirm if these findings are truly indicative of resistance. Generally, correlations between the tests were poor and it was not possible to use the outcome of one test to predict the outcome of another. ©1999 Elsevier Science B.V. All rights reserved. Keywords: Horse; Larval development assay; Egg hatch assay; Faecal egg count reduction; Pyrantel; Benzimidazole
1. Introduction Resistance to one or more classes of anthelmintic compounds has been reported in gastrointestinal nematodes of each of the major farm animal species (Condor and Campbell, 1995). The situation is most severe in the trichostrongyle nematodes of sheep and goats ∗
Corresponding author. Tel.: +45-35282791; fax: +45-35282774; e-mail: [email protected]
0304-4017/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 9 ) 0 0 1 1 3 - 2
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where resistance has been reported to all available anthelmintic classes. In horses, small strongyles resistant to benzimidazoles (BZs) have been found to be highly prevalent (Kelly et al., 1981; Bauer et al., 1986; Bjørn et al., 1991; Boersema et al., 1991; Craven et al., 1998) but only recently have studies reporting resistance to pyrantel been published (Chapman et al., 1996; Craven et al., 1998). As a result of the increasing prevalence of anthelmintic resistance (AR) a number of in vivo and in vitro techniques have been developed to detect this resistance. The faecal egg count reduction test (FECRT) is the most widely used in vivo method for detection of resistance in field surveys. In general, however, in vivo tests are time consuming, expensive and often characterised by poor data quality (low precision and reproducibility) due to interanimal variation and drug pharmacodynamics in the host (Lacey et al., 1990). Therefore, increasing interest is paid to in vitro tests, such as the egg hatch assay (EHA) and larval development assay (LDA). Despite being more technically demanding, the EHA for detection of BZ resistance (Le Jambre, 1976; Coles and Simpkin, 1977; Whitlock et al., 1980) is cheaper, more accurate and less time consuming than the FECRT (Hazelby et al., 1994). The use of anaerobic (Hunt and Taylor, 1989) or cold (4◦ C) (Smith-Buijs and Borgsteede, 1986) storage of faecal samples enables this test to be used for field surveys, but it is still considered as a technique best suited to research (Donald, 1985). LDA’s (Coles et al., 1988; Giordano et al., 1988; Lacey et al., 1990; Taylor, 1990; Hubert and Kerboeuf, 1992) have the additional advantage over the EHA of testing for resistance to a range of drugs. Despite the potential of this technique for field screening (Johansen, 1989), only a few surveys using this method appear in the literature. In horses the EHA, mostly based on the method of Whitlock et al. (1980), has been widely used for in vitro testing of AR (Kelly et al., 1981; Ullrich et al., 1988; Ihler, 1995) whereas the use of the LDA has only been reported twice (Preinsberger, 1992; Ihler, 1995). This study was designed to examine the relationship between results of the in vivo FECRT, and the in vitro EHA and LDA for detecting AR in horse strongyles.
2. Materials and methods 2.1. Experimental design Following a questionnaire study carried out in Denmark in 1995, 56 farms were selected for a practical examination of AR in horse strongyles. The criteria used were that 12 or more horses were available for the examination and these horses must not have been treated with anthelmintics in the eight weeks prior to testing. FECRT’s were undertaken with samples being collected on day 0 (day of treatment) and day 14. The drugs used were the commercially available oral paste formulations of Panacur® vet. (18.75% Fenbendazole (FBZ), Hoechst Veterinär, Frankfurt, Germany), Banminth® vet. (43.9% Pyrantel Embonate, Pfizer, London, Ontario, Canada) and Eqvalan® vet. (1.9% Ivermectin (IVM), MSD Agvet, Haarlem, Holland). An FECRT was also undertaken on the reference strain (six animals per group) using Panacur® and Banminth® . An untreated control group was also
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included for each farm. The samples were collected into plastic gloves, air was excluded and the glove was sealed by tying a knot close to the faecal material. The samples were then sent by overnight mail to the Danish Veterinary Laboratory for testing. 2.2. Techniques Details of the egg counts and FECR calculations are described elsewhere (Craven et al., 1998). The FECRT on the reference strain used a modified McMaster egg count technique with a lower detection limit of 25 epg. After faeces from the pre-treatment sampling had been allocated for egg counts and larval cultures, the faeces from animals from each farm with positive egg counts were mixed and the eggs separated using sieves and centrifugation in a sucrose gradient. An EHA and a LDA were then carried out. The EHA, used on 52 of the farms, was carried out according to the recommendations of the World Association for the Advancement of Veterinary Parasitology (WAAVP) (Coles et al., 1992), with one major change. The drug was added to the plates before the egg suspension, as this has been found to improve the reliability of the assay (Craven, unpublished data). Two replicates, each of 10 concentrations of thiabendazole (TBZ) dissolved in dimethylsulphoxide (DMSO), were used for each farm. The range of concentrations used was 0.10–5.0 M TBZ. On each occasion a reference parasite strain was included to give a measure of inter-assay variation. Eggs for the reference strain were obtained from a farm on which BZ anthelmintics had never been used. The LDA, used on 32 farms, was carried out according to Varady et al. (1996), using the drugs pyrantel citrate, levamisole hydrochloride, TBZ and mebendazole (MBZ) (all Sigma Chemical Company, St Louis, MO, USA). All drugs were dissolved in DMSO. TBZ and MBZ were tested over a concentration range of 0.0049–10 M, levamisole (LEV) 0.0325–66.67 M and pyrantel (PYR) 0.39–800 M. Final DMSO concentration was 1%. Two replicates were made for the drugs TBZ, MBZ and LEV and four replicates for PYR. Analysis of the ovicidal effect of the BZ’s (TBZ-O, MBZ-O) was also undertaken. The same reference strain used for the EHA was also used in the LDA testing. 2.3. Data analysis The average data from the two in vitro assays were analysed using the logit program (Dobson et al., 1987) to obtain estimates of the LD50 or ED50 data (the dose that prevents 50% of the population developing to L3 larvae or hatching, respectively). The data was further analysed with the aim of defining a delineating concentration for resistance in each of the assays. For the LDA resistance ratios (RR) were calculated as the LD50 of the test strain divided by the LD50 of the reference strain. Due to their low prevalence (<5–10%) (Craven et al., 1998), Strongylus and other non-cyathostome L3 larvae were not counted in the LDA, but eggs and early stage larvae will have contributed to the totals of these stages in both the LDA and the EHA. The relationships between the three tests were examined using Spearman rank correlation coefficients between the FECR, ED50 and LD50 values obtained in each test. In order to examine the feasibility of using the results of one of these tests to predict the results of another, regression lines were calculated using
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(a) FECR as explanatory variable and ED50 or LD50 as response variable, and (b) ED50 as explanatory variable and LD50 as response variable. 95% confidence intervals for the mean of the response variables for a given value of the explanatory variable were also calculated and the numbers of observations that fell outside these intervals were counted. An attempt was made to use the results of the FECRT to determine threshold or cutoff ED50s or LD50s that could be used to discriminate between resistant and susceptible parasites in the EHA or LDA. The threshold FECR percentage and ED50 or LD50 for declaring resistance were varied over a range of values and for each combination the number of observations where the results of the two tests agreed (i.e. both declared resistance or neither declared resistance) were counted. The values of FECR percentage tested were 90% and 95%, and values of ED50 or LD50 tested were the drug concentrations used in the assays.
3. Results Table 1 shows the results of the FECRT on the reference strain. Five of the six horses in the BZ group had positive egg counts after treatment, as did one horse from the pyrantel group. The reference strain was shown to be suspected resistant to BZ’s (96% reduction, lower 95% confidence limit 88%) but sensitive to PYR and IVM. FECR data for the other farms is summarised in Table 2. FBZ FECR values (Table 3) obtained from the 42 farms examined varied from 0 to 100%, with 11 farms having less than 60% reduction and nine having 95% or greater. PYR FECR values (Table 4) varied from 87 to 100% with only the three farms where resistance was detected having values lower than 99%. Table 1 Faecal egg count reduction test results for the reference strain showing group egg counts pre- and post-treatment, the egg count reduction percentage (FECR) and lower and upper 95% confidence limits (LCL and UCL) Drug
n
Pre epg
Post epg
FECR
LCL
UCL
Control BZ Pyrantel
6 6 6
567 500 454
508 18 4
– 96 99
– 88 93
– 99 100
Table 2 Summary of the results from the faecal egg count reduction testsa,b,c (Craven et al., 1998) Drug
Farms Tested
Benzimidazole Pyrantel Ivermectin
42 15 16
Suspected Sensitive 5 11 16
Resistant 4 1 0
Resistant: <95% FECR and <90% lower 95% confidence limit. Suspected resistant: either >95% FECR or >90% lower 95% confidence limit. c Susceptible: >95% FECR and >90% lower 95% confidence limit. a
b
Resistant 33 3 0
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Table 3 Faecal egg count reduction (FECR), egg hatch assay (EHA) and larval development assay (LDA) data for the two benzimidazoles used Farm ID 1351 1352 1362 1363 1364 1482 1498 1507 1520 1523 1524 1525 1586 1602 1609 1610 1611 1716 1717 1723 1724 1725 1729 1730 1741 1742 1752 1756 1761 1762 1763 1767 1768 1853 1854 1859 1862 1863 1870 1871 1880 1881 1882 1887 1888 1891 1892 1895 1896 1993 2000 2517 a
BZ FECR 91 93 96 – 34 – 87 – 59 0 29 78 – 100 59 84 100 – 88 – 99 81 88 71 61 81 76 90 98 81 98 90 66 46 – 100 – 2 57 72 18 0 – 46 100 – 87 85 75 69 95 0
EHA (uM)
MBZ (uM)
MBZ-Oa (uM)
TBZ (uM)
TBZ-Oa (uM)
0.50 5.00 0.28 0.60 0.67 0.91 0.78 0.38 0.86 0.81 0.67 0.52 0.90 0.47 0.95 0.71 0.45 0.65 0.38 0.66 0.51 0.41 0.42 0.43 1.09 0.27 0.56 0.33 0.42 0.51 0.38 0.43 0.56 0.73 1.42 1.60 0.70 2.14 0.81 0.62 0.48 0.40 0.20 1.37 0.63 1.76 0.31 0.70 0.37 5.00 0.36 2.16
0.13 0.047 0.13 0.13 0.24 0.51 0.43 0.40 0.13 0.35 0.28 0.16 – 0.19 0.29 0.22 0.084 0.12 0.11 0.35 0.15 0.19 0.05 0.26 0.11 – – – – – – – – – – – – – – – 0.20 0.12 0.12 0.23 0.26 – 0.088 0.18 0.16 – – –
3.00 – – – – – – – – – – – – 0.45 0.44 0.38 0.62 1.20 0.55 2.00 1.70 1.52 1.90 1.57 1.54 0.39 0.40 0.50 0.96 1.09 0.64 1.04 1.03 – – – – – – – 0.31 0.54 0.29 1.44 0.36 – 0.35 0.63 0.33 – – –
0.098 0.077 0.11 0.13 0.19 0.30 0.077 0.086 0.10 0.056 0.21 0.10 – 0.075 0.16 0.084 0.09 0.086 0.088 0.073 0.057 0.066 0.046 0.05 0.034 – – – – – – – – – – – – – – – 0.26 0.14 0.081 0.23 0.22 – 0.079 0.11 0.097 – – –
1.01 – 0.21 0.30 0.55 0.57 0.76 0.23 0.35 0.40 0.40 0.25 – 0.12 0.17 0.09 0.23 0.36 0.27 0.55 0.47 0.34 0.45 0.27 0.59 0.17 0.54 0.24 0.24 0.34 0.19 0.29 0.36 – – – – – – – 0.19 0.23 0.16 0.70 0.46 – 0.19 0.21 0.16 – – –
The suffix ’O’ refers to the ovicidal effect of the drug.
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Table 4 Comparison of faecal egg count reduction (FECR) and larval development assay (LDA) LD50 (M) and resistance ratio (RR) data for pyrantel and levamisole Farm ID
PYR FECR
PYR−LD50
PYR−RR
LEV−LD50
LEV−RR
1351 1352 1362 1363 1364 1482 1498 1507 1520 1523 1524 1525 1602 1609 1610 1611 1716 1717 1723 1724 1725 1729 1730 1761 1762 1871 1880 1881 1882 1887 1888 1892 1895 1996
100 100 100 – 100 – – – – – – – 100 – 100 87 – 100 0 0 0 0 92 99 100 92 0 100 0 0 0 0 99 100
5.3 2.5 10.9 6.9 6.1 4.6 1.6 4.9 5.3 0.92 4.8 5.5 9.8 6.9 7.8 7.1 10.0 4.1 7.5 6.4 4.6 2.4 10.4 – – – 12.0 5.5 7.4 8.1 7.6 6.3 5.6 6.6
0.94 0.58 2.7 1.7 1.5 0.75 0.78 2.4 0.86 0.15 0.77 0.89 0.91 0.65 0.73 0.66 2.14 0.87 1.59 1.36 0.97 0.52 2.21 – – – 2.0 0.90 1.2 1.3 1.3 1.0 0.93 1.1
0.24 0.19 1.1 0.85 0.58 0.81 0.14 0.41 0.30 0.18 0.25 1.3 1.1 0.5 0.56 0.33 0.8 0.43 0.33 0.38 0.8 0.25 0.35 – – – 1.7 0.68 1.1 0.55 0.52 0.46 0.77 1.1
1.2 0.92 2.3 1.7 1.2 0.92 1.0 3.1 0.34 0.20 0.28 1.5 0.72 0.33 0.37 0.22 1.76 0.95 0.73 0.84 1.8 0.55 0.76 – – – 1.6 0.60 1.0 0.49 0.46 0.40 0.69 1.0
The EHA results are shown in Table 3. For two farms the maximum inhibition of hatching was less than 50%, so for the data analysis these farms were assigned the highest tested concentration (5 M) as their ED50. The reference strain was tested on 30 separate occasions with a mean ED50 of 0.39 M (Table 5). In the LDA the control isolate was tested on eight separate occasions (Table 5). For all isolates the development of eggs to L3 larvae was high (>80%) in control wells but there was a large variation in development within each assay. The LDA data for the two BZ’s is shown in Table 3. Due to the suspected FBZ resistance of the reference strain, RR values for the BZ’s were not calculated. The LDA data for LEV and PYR is presented in Table 4. For both the LDA and EHA it was not possible to determine a delineating dose for resistance to BZ’s.
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Table 5 Egg hatch assay (EHA) results (M TBZ) and larval development assay (LDA) results (M) for the reference strain
Number Mean Std Dev
EHA ED50a
PYR LD50b
LEV LD50b
MBZ LD50b
MBZ-Oc ED50a
TBZ LD50b
TBZ-Oc ED50a
30 0.39 0.093
8 5.98 2.74
7 0.69 0.47
8 0.12 0.049
5 0.80 0.35
8 0.068 0.024
8 0.26 0.088
a
ED50 is the point at which 50% of the eggs fail to hatch. LD50 is the point at which 50% of the eggs develop into L3 larvae. c The suffix ’O’ refers to the ovicidal effect of the drug. b
Table 6 Spearman rank correlation coefficients and p-values for comparison of faecal egg count reduction test (FECRT), egg hatch assay (EHA) and larval development assay (LDA) Comparison
n
EHAa
42
0.48
0.001
26 26 26 33 12
−0.39 0.1 −0.28 −0.13 −0.37
0.048 0.6 0.2 0.5 0.2
32 29 32 39
0.49 0.33 0.23 0.56
vs.
FECRTb
Spearman correlation coefficient
p-value
LDAc
vs. FECRT MBZ MBZ-Od TBZ TBZ-Od PYR
LDA vs. EHA MBZ MBZ-Od TBZ TBZ-Od
0.004 0.08 0.21 0.0002
ED50 (M TBZ). Faecal egg count reduction percentage. c LD50 (M). d The suffix ’O’ refers to the ovicidal effect of the drug. a
b
The Spearman rank correlations between the tests are shown in Table 6. The correlation coefficients were generally fairly low and only statistically significant (p < 0.05) in four cases out of 10. The regressions to examine the feasibility of using the results of one test to predict another were significant (p < 0.05) in four cases out of 10. On average about half of all observations fell outside the 95% confidence intervals, and in many cases the ED50/LD50 of the actual observations were two to three times higher than the predicted values (farm no. 1993 was five times higher). For the LDA using PYR there were only 12 observations, of which 10 had 100% FECR so it was not feasible to determine a threshold concentration for PYR in the LDA. In all other cases, maximal agreement between each pair of tests (percentage of observations where both tests made the same declaration) was approximately 80% and this occurred for FECR of 95%. The highest threshold concentrations (M) that yielded maximum agreement are shown in Table 7. However, in all cases, the concentrations listed in Table 7 result in all or almost all the observations obtained in this study being declared resistant by the EHA/LDA.
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Table 7 Range of benzimidazole concentrations (M) yielding maximal agreement of FECRT results (for 95% reduction) with either egg hatch assay (EHA) or larval development assay (LDA) EHA LDA (MBZ) LDA (MBZ-O) LDA (TBZ) LDA (TBZ-O)
0.4 ≤ ED50 < 0.65 0.039 ≤ LD50 < 0.078 0.156 ≤ LD50 < 0.3125 0.0195 ≤ LD50 < 0.039 0.156 ≤ LD50 < 0.3125
4. Discussion Whereas Coles et al. (1992) added the drug to the eggs, in this study the eggs were added to the drug. This has been seen to improve the reproducibility of the data obtained from the EHA, most likely as a result of a more thorough mixing of the two solutions. From the results observed in this study an ED50 of between 0.4 M and 0.65 M TBZ was indicated as the threshold for declaring BZ resistance (Table 7). This is in agreement with the recommendation of the WAAVP (Coles et al., 1992) that an ED50 of 0.5 M (0.1 g/ml) TBZ is the threshold for declaring resistance in horses. Using this threshold, resistance is declared on 32 (62%) of the 52 farms examined in this study. This compares well with the prevalence of resistance detected by FECRT in this study (78%). The assay would become easier to interpret if hatching at or above a specific dose could be used to indicate resistance. This principle was used in two reports from Germany (Ullrich, 1987; Ullrich et al., 1988) that used a less than 96% inhibition of egg hatch at 0.75 M (0.15 g/ml) TBZ as the threshold for declaring resistance. Using this approach all farms examined in the present study would be declared resistant. The average ED50 of the reference strain was higher than expected and a subsequent FECR test indicated that the worm population was suspected resistant to FBZ, despite the fact that BZ’s have never been used on the property. We attribute this finding to the importation of resistant worms when buying replacement horses. Despite the fact that most of the testing was carried out during the atypically hot 1995 summer, the eggs had not visibly commenced development 24 h after the samples were taken. This would indicate that eliminating most of the air from the glove was sufficient to create anaerobic conditions, thus halting/slowing the development of the eggs. Anaerobic storage for up to 7 days has been shown to halt egg development without impact on their subsequent development in sheep trichostrongyles (Hunt and Taylor, 1989). Faecal material for the reference strain was collected only once per week and stored at 4◦ C until required. However, EHAs were sometimes conducted four times per week. Despite storage for 0 to 80 h before testing, variation in the ED50 of the control isolate was acceptable (Table 4). This is in agreement with the findings for Haemonchus contortus by Smith-Buijs and Borgsteede (1986). Although the development of eggs to third stage larvae in the absence of drugs was quite high (80–85%) in the LDA used in this study, it was not as high as reported by others (Lacey et al., 1990; Hubert and Kerboeuf, 1992). This may be the result of the higher temperature used (26◦ C) which has been shown to reduce the development of the sheep trichostrongyles (Hubert and Kerboeuf, 1992). Additionally, there was greater variability in the development
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of larvae within each plate than has been observed for Oesophagostomum spp. in pigs either in earlier (unpublished) work using the method of Lacey et al. (1990) or using this method (Varady et al., 1996). The LD50 values obtained for the reference strain in this study for LEV are similar to values that have been reported for horse strongyles (Ihler, 1995), sheep trichostrongyles (Lacey et al., 1990; Hubert and Kerboeuf, 1992) and Oesophagostomum spp. in pigs (Varady et al., 1996). Interestingly, the average LD50 value obtained for PYR is less than half that obtained in sheep strongyles (Hubert and Kerboeuf, 1992) and approximately 10 times lower than that seen in Oesophagostomum spp. in pigs (Varady et al., 1996). Four (13%) of the 31 farms tested with PYR had an RR greater than two. Two of these were not tested by FECRT, the third tested resistant and the fourth susceptible. Two of the four farms also had LEV RRs of greater than two, suggesting side resistance to LEV has developed after treatment with PYR. Further testing will be undertaken to confirm the presence of PYR resistance on these farms. For the BZ’s there is no data published for susceptible cyathostome isolates. Ihler (1995) observed a range of LD50 values of 0.15–0.36 M TBZ for seven isolates of varying degrees of resistance, while Preinsberger (1992) recorded an average value of 0.2 M TBZ for a single resistant isolate. In this study the reference strain had an average LD50 against TBZ and MBZ of 0.068 M and 0.12 M, respectively, which is similar to the values obtained by Varady et al. (1996) for Oesophagostomum spp. and by Lacey et al. (1990) for sheep trichostrongyles. However, it must be remembered that the reference strain in this study may have had elevated LD50s since it was suspected to be FBZ resistant. Due to this suspected resistance it was not possible to calculate RRs, nor therefore the number of farms on which resistance was present, for TBZ or MBZ. When comparing the in vitro assays it can be seen that the EHA ED50 is higher than that for the LDA (TBZ-O). TBZ has the effect of delaying, not preventing, hatching so this is expected given the shorter run time of the EHA. Analysis of the data to find a delineating dose for BZ resistance for both the LDA and EHA was unsuccessful. While the LD50 values obtained for the reference strain were grouped closely there was a wide variation in values when LD99 was considered. This variation may have been due to the suspected resistant population of the reference strain. The results of the correlations obtained here were, with a few exceptions, poor. From this we might conclude that one of the tests cannot measure AR status, however it is more likely that the different tests are measuring different attributes of the parasites’ response to anthelmintics, especially since the horses are most likely carrying a mixture of parasite species. At present it is not possible to identify the mixture of species present in the horse, nor obtain in vitro data for mono-specific populations, although samples have been preserved from this study to enable such identification to be made when the techniques become available. The only way to decide on the optimal test would be to compare its performance to some agreed standard of AR. For parasites of smaller animals this has often been worm burden reduction (compared with non-treated controls) in a treat-and-slaughter assay but this is virtually impossible for parasites in horses. Furthermore, the best test to use in any given situation is likely to also depend on the drug against which we need to measure resistance, the purpose for which we are testing for resistance and the resources available.
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