Comparison of two in vitro methods for the detection of ivermectin resistance in Haemonchus contortus in sheep

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Summary

Gastrointestinal parasitic nematodes in sheep cause severe economic losses. Anthelmintics are the most commonly used drugs for prophylaxis and therapy against parasitic helminths. The problem of drug resistance has developed for all commercially available anthelmintics in several genera and classes of helminths. In vitro and in vivo tests are used to detect anthelmintic resistance. Two in vitro methods (larval migration inhibition test and micromotility test) for the detection of ivermectin (IVM) resistance were compared using IVM-resistant and IVM-susceptible isolates of Haemonchus contortus. The degree of resistance for each test was expressed as a resistance factor (RF). The micromotility test was more sensitive for quantitatively measuring the degree of resistance between susceptible and resistant isolates. The RFs for this test for IVM and eprinomectin ranged from 1.00 to 108.05 and from 3.87 to 32.32, respectively.

Introduction

Resistance to anthelmintics has become a serious problem in countries with developed sheep and goat industries, especially Australia, New Zealand, South Africa, and South America (Várady et al., 2011). Macrocyclic lactones (MLs) are currently the most common group of broad-spectrum anthelmintics for the control of nematode parasites. Reports of resistance to MLs in nematodes of small ruminants, however, have increased over the last decade (Álvarez-Sanchez et al., 2006; Diez - Banos et al., 2008; Artho et al., 2007; Bartley et al., 2006; Čerňanská et al., 2006). A number of in vitro and in vivo tests have been developed for the detection of anthelmintic resistance (Taylor et al., 2002). Several in vitro tests have been described for testing the anthelmintic activity of MLs for the detection of resistance. These tests depend on an assessment of paralysis in larvae (Gill et al., 1991; Kotze et al., 2006) or of inhibited larval development (Coles et al., 1988; Hubert & Kerboeuf, 1992; Dolinská et al., 2013). A micromotility meter has also been developed to evaluate the motility of various larval and adult nematodes as a criterion of paralysis in the absence or presence of anthelmintics, based on a quantitative measurement of motility by photo-detectors (Folz et al., 1987a). In this method, infective larvae of Haemonchus contortus and Trichostrongylus colubriformis are exposed to an anthelmintic for 24 h and then transferred to the micromotility meter (Folz et al., 1987b,c; Coles et al., 1989). The present study was designed to evaluate the potential of two in vitro methods for the detection of ML resistance in the sheep nematode H. contortus. The results of the tests are compared, and the suitability of the methods for the field screening of ML resistance is discussed.

Material and Methods

Parasites isolates

Two susceptible isolates of H. contortus, McMaster and ISE (MHco3), and five resistant isolates, White River (WR), CAVR, MOX23, ISE-SL, and ISE-SLI were used in this study. The susceptible ISE isolate was obtained as an inbred isolate of MHCo3 (Roos et al., 2004). The McMaster isolate was isolated prior to the introduction of broad-spectrum anthelmintics and is routinely used as a reference susceptible isolate in similar studies (Gill et al., 1995). WR was isolated from the field in South Africa and demonstrated resistance to IVM (30 % efficacy at 0.2 mg/kg) and to the benzimidazoles, rafoxanide, and closantel (Van Wyk etal., 1988). CAVR is resistant to MLs and moderately resistant to the benzimidazoles (Le Jambre et al., 1995). MOX23 has been selected for 23 generations in the laboratory for moxidectin resistance (Ranjan et al., 2002). ISE-SL has been selected in the laboratory for IVM resistance (Coles et al., 2005). ISE-SLI, derived from ISE-SL, was selected further in our laboratory using 1.5× the recommended dose of IVM.

Trial design

All isolates had been routinely maintained by passage through individually housed, helminth-naive, 5-6 month-old lambs. The lambs were infected orally with 5000 third-stage (L3) larvae of each isolate. Faecal samples were collected 35-50 days after experimental infection, and coprocultures were subsequently prepared by the method described by Henriksen and Korsholm (1983). L3 larvae were isolated from the faecal cultures by standard Baermann filtration and stored at 10 °C in distilled water prior to use. The larvae were tested within one month after collection.

Larval migration inhibition test (LMIT)

The LMIT was performed as described by Kotze et al. (2006) in 96-well microtiter plates (Millipore, Australia). Stock drug solutions of IVM (10 mg/ml in DMSO) were serially diluted 2-fold, 0.5 μL of each dilution were added to the wells of drug plates, followed by 20 μl of distilled water and 30 μl of a solution containing infective L3 larvae (55 – 60 larvae mixed with amphotericin B – 250 mg/ml). The plates were placed into plastic bags and incubated for 24 h at 27 °C. Rinse plates were prepared at the same time and in the same format as their corresponding drug plates. The culture medium comprised 4.75 μl of each drug dilution and 400 μl of distilled water. These plates were kept at room temperature. Agar/filter plates were also prepared on the same day: 75 μl of agar (0.125 %) were added to the 20-μm filter of each well, and the plates were stored at room temperature. The following day, 325 μl of the solution from the rinse plate were added to the corresponding well of a plate, and the agar/ filter plate was lowered into this plate and was incubated several hours at 27 °C. Twenty-four hours after the establishment of the drug plates, the worms from the drug plates were transferred to the agar/filter plates at the corresponding positions. The remaining worms in the drug plates were collected by adding 50 μl of the corresponding solution from the rinse plates to the drug plates, mixed, and then added to the agar/filter plates. The total content of each well consisted of 325 μl of solution from the rinse plates, 75 μl of 0.125 % agar, 50 μl of L3 larvae from the drug plates, and 50 μl of the solution from the rinse plates. The prepared plates were placed into clear plastic bags and incubated under a light at 27 °C for 48 h. The filters were then removed, and all worms were counted.

Micromotility test

The micromotility test was performed as described by Folz et al. (1987). Movement of the larvae caused a variation in light rays refracting from the meniscus, and consequently a variation in the electrical signal produced by a photo-detector located at the level of the meniscus. The numerical representation of the modulated signal is termed the motility index. H. contortus L3 larvae were mixed with water (500 L3/ml). A stock solution was prepared by dissolving 1 mg of anthelmintic (IVM or eprinomectin) in a mixture of 200 μl of acetone and 50 μl of Tween 20 and then adding 750 μl of distilled water. An identical blank solution but with distilled water replacing the anthelmintic was also prepared. L3 larvae were exposed to anthelmintic in four concentrations: 0.1,1.0,10.0, and 100.0 μg/ml. Eprinomectin at the tested concentrations was less effective than IVM against all isolates, so we increased its concentration to 300 μg/ml to obtain reductions in motility. Two susceptible and four resistant isolates of H. contortus were tested. For each drug to be tested, 450 μl of the suspension containing H. contortus larvae and 50 μl of the acetone/drug solutions at the four concentrations or the blank solution were added to culture tubes (10 × 75 mm). The culture tubes were covered with parafilm to prevent evaporation. The tubes were incubated at 8 – 12 °C for 24 h, and the worms were then processed with the micromotility meter.

Table 1

Origin and status of anthelmintic susceptibility (S) or resistance (R) of ISE, McMaster, ISE-SL, ISE-SLI, MOX-23, WR and CAVR isolates of Haemonchus contortus

IsolateR/SReferenceOrigin
ISEsusceptibleRoos et al. (2004)Kenya
McMastersusceptibleCSIRO, ArmidaleAustralia
ISE-SLIVMColes (2005)Great Britain
ISE-SLIIVMColes (2005)Great Britain
MOX-23IVMPrichard et al. (2002)Canada
WRBZ, CLO, IVM, RAFVan Wyk and Malan (1988)South Africa
CAVRIVM, BZLeJambre et al. (1995)Australia

BZ, benzimidazole; CLO, closantel; IVM, ivermectin; RAF, rafoxanide

Data analysis

Motility indexes for the larval micromotility test were transformed to percent reductions of motility by the formula: [(vehicle control index - treatment index)/(vehicle control index - background index)] ×100. The results are presented as a resistance factor: the ratio of the LD50 or LD99 for the resistant strain to the LD50 or LD99 for the susceptible strain. The LD50 and LD99 values were determined by a logistic regression model (Dobson et al., 1987).

Results

Larval migration inhibition test

The results of the LMIT for IVM resistance are shown in Table 2. The LD50 values of the resistant strains differed from those of the susceptible strains by an RF from 1.7 to 4.9, and the LD99 values differed by an RF from 2.9 to 17.2 (Table 3). The LD99 values were more variable, producing a higher coefficient of variation. The mean RFs for the LMIA are shown in Table 3. The LD99 data were better able to distinguish between the susceptible and resistant isolates.

Table 2

Arithmetic mean ± SD of LD50 and LD99 for susceptible and resistant isolates obtained in the larval migration inhibition test with ivermectin

IsolateLD50(μg/ml) ± SDLD99(μg/ml) ± SD
McMaster1.03 ±0.722.80 ±0.61
ISE1.25 ±0.483.00 ±1.24
MOX232.13 ±1.7734.36 ±25.14
CAVR2.80 ± 0.7220.10 ±12.12
WR3.80 ± 0.358.77 ± 4.92
ISE-SL3.65 ± 0.4848.18 ±57.59
ISE-SLI5.05 ±1.9822.87 ± 7.45
Table 3

Resistance factors for ivermectin (IVM) in the larval migration inhibition test

StrainsIVM RF50IVM RF99
WR/McM3.683.13
CAVR/McM2.717.17
MOX23/McM2.0612.27
ISE-SL/McM3.5417.20
ISE-SLI/McM4.908.16
WR/ISE3.042.92
CAVR/ISE2.246.70
MOX23/ISE1.7011.45
ISE-SL/ISE2.9216.06
ISE-SLI/ISE4.047.62

Micromotility test

The mean reduction in motility and the LD50 values for the susceptible and resistant isolates of H. contortus after treatment with IVM and eprinomectin are presented in Tables 4 and 5. An IVM concentration of 10 μg/ml (Table 4) significantly reduced the larval motility of the susceptible H. contortus isolates, but only the treatment with 100 μg/ml IVM substantially reduced motility in the resistant isolates. The sensitive isolates displayed higher susceptibilities to the drugs tested, as indicated by the LD50 values.

Table 4

Mean reduction (%) in motility of L3 larvae of resistant and susceptible strains of H. contortus after incubation in different concentrations of ivermectin (IVM)

Concentration of IVM (μg/ml)
Isolate0.1110100LD50
WR0026.249.790.76
CAVR055.252.876.14.2
McMaster039.788.980.91.57
ISE059.397.891.40.84
MOX2313.741.278.996.41.57
ISE SL11.2326.780.578.33
Table 5

Mean reduction (%) in motility of L3 larvae of resistant and susceptible strains of H. contortus after incubation in different concentrations of eprinomectin (EPM)

Concentration of EPM (μg/ml)
Isolate0.1110100200300LD50
WR43.44353.372.271.741.04
CAVR3549.4829.365.364.749.77
McM037.176.463.91001003.98
ISE34.653.2365.269.31001001.54
MOX2315.827.740.370.710010015.4
ISE SL14.2611.6568.550.1645.166.947.27

Discussion

The values of RFs in LMIT in our study were as high as 12.2 for MOX-23 and 7.17 for CAVR. Kotze et al. (2006) with identical test obtained RFs for CAVR and MOX-23 of 1.7 and 5.7, respectively. LMIT utilize the ability of larvae to migrate through a filter mesh.

A layer of 0.125 % agar is applied to the filter mesh to create an additional barrier for the migrating larvae. The test was able to detect a level of resistance of 10 % in a population. Kotze et al. (2006) detected resistance to MLs in H. contortus but not in T. colubriformis and Ostertagia circumcincta. Despite the potential of LMITs, the tests present some difficulties. Several factors may contribute to poor sensitivity. In mixed field parasitic populations, IVM may have different potencies against different species of gastro-intestinal parasites. The LMIT is suitable only for H. contortus, which seriously limits the utility of this test for monitoring resistance in field surveys. The RFs obtained by LMITs are significantly lower than those obtained by larval development tests (Dolinská et al., 2012, 2013), which may indicate a lower sensitivity of the LMIT for the detection of IVM-resistant parasites. Additionally the cost of the microtiter plates (Millipore), in which the LMIT is performed, is considerable. From a practical point of view, the test cannot thus be used for the detection of IVM resistance in mixed populations of trichostrongylids.

Calculation of LD99 values in the in vitro tests can significantly increase test sensitivity and identify resistance when only a small proportion of the worm population is resistant (Várady et al., 2007). Based on our data from LMIT the RF99 values obtained from CAVR, MOX23, ISE-SL and ISE-SLI isolates were 1.6 – 6.7 times higher compare to RF50. Only LD99 values from WR isolate were almost identical with LD50 values. The possible reason for this could be the different proportion of resistant individuals in the isolates. While CAVR, MOX23, ISE-SL and ISE-SLI isolates showed high resistance in vivo, WR isolated from the field in South Africa demonstrated moderate resistance to IVM.

In our study the micromotility meter was used to evaluate the effect of two ML anthelmintics on the motility of H. contortus L3 larvae. Paralysis tests described previously by Martin and Le Jambre (1979), Barton (1983), and Geerts et al. (1989) were based on visual evaluations of larval paralysis, which is considered to be subjective and not sufficiently reproducible. By using a micromotility meter, the degree of subjectivity (assessing whether a larva is in motion) can be minimized. The micromotility meter has been described as a sensitive tool for measuring the in vitro motility of larval and adult H. contortus and T. colubriformis (Bennett and Pax, 1986; Folz et al., 1987b, c). Some studies, however, have demonstrated a lower ability to distinguish between susceptible and resistant isolates of H. contortus (Coles et al., 1989; Várady & Čorba, 1998).

In the current study, the RFs ranged from 1.00 to 108.05 for IVM and from 3.87 to 32.32 for eprinomectin. Similarly high RFs (maximum 88.51-345.6) were obtained by Demeler (2005) in a comparison of the motilities of susceptible and resistant adult T. colubriformis and O. circumcincta. The use of adult parasites, however, requires the sacrifice of animals and so is not financially practical for field surveys. Our micromotility testing provided relatively high RFs, indicating a good ability to differentiate between IVM-resistant and -susceptible strains of H. contortus. The ability to measure the motility of larvae in mixed infections of gastrointestinal parasites, however, is questionable, because different species of gastrointestinal nematodes have different motilities, e.g. the motility of H. contortus is much higher initially but is markedly lower after about 10 minutes (Gill et al., 1995). The present version of the micromotility test is thus unsuitable for measuring levels of resistance under field conditions.

Table 6

Resistance factors for ivermectin (IVM) and eprinomectin (EPM) in the micromotility test

StrainsIVM RF50EPM RF50
WR/McM57.8110.31
CAVR/McM2.6712.50
MOX23/McM1.003.87
ISE-SL/McM49.8911.88
WR/ISE108.0526.65
CAVR/ISE5.0032.32
MOX23/ISE1.8710.00
ISE-SL/ISE93.2530.69

As it was demonstrated in the previous studies, the use of avermectin analogs (eprinomectin, ivermectin aglycone) significantly increased the ability of the in vitro tests to differentiate between IVM-resistant and -susceptible isolates (Dolinská et al., 2013). These analogues produce 2 – 3 times higher RR compared to IVM, which was not a case of our study in MMT. The reason for this is unclear and could be related to the motility cycles of the larvae. After incubation in the dark at 25 °C, the L3 larvae of H. contortus were stimulated to move in a rapid sinusoidal motion by exposure to light. They then remain active for at least 10 minutes and maximum activity was reached after a short lag time of l-2 min. Thus LD50 values were more dependent on exposure of L3 larvae to the light and not the length of incubation.

The lowest RR obtained for MOX23 isolate in both tests suggested different or additional genetic mechanism in MOX resistance compared to IVM resistance. IVM is >130-fold more potent than moxidectin at inhibiting pharyngeal pumping (and thus blocking feeding) in Caenorhabditis elegans, and IVM initially stimulates motility before paralyzing this nematode. Moxidectin appears to only cause paralysis (Ardelli et al., 2009). Additionally slightly higher RR (especially for LD99) obtained in LMIT suggest that LMIT may be a superior tool to monitor resistance to MOX.

The potential of the two tests for use in monitoring IVM resistance is questionable and additional experimental work is require optimizing the test in field condition.

Acknowledgements

This study was supported by Slovak Research and Development Agency project No. APVV 0539-10 and by the project “INFEKTZOON – Centre of Excellence for Animal Infections and Zoonoses (ITMS-26220120002)”.

References

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  • Ardelli B.F. Stitt L.E. Tompkins J.B. Prichard R.K. (2009): A comparison of the effects of ivermectin and moxidectin on the nematode Caenorhabditis elegans. Vet. Parasitol. 165(1-2): 96 – 108. DOI: 10.1016/j.vetpar.2009.06.043

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  • Artho R. Schnyder M. Kohler L. Torgerson P.R. Hertzberg H. (2007): Avermectin – resistance in gastrointestinal nematodes of Boer goats and Dorper sheep in Switzerland. Vet. Parasitol. 144(1-2): 68 – 73. DOI: 10.1016/j.vetpar.2006.09.032

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  • Bartley D.J. Donnan A.A. Jackson E. Sargison N. Mitchell G.B.B. Jackson F. (2006): A small scale survey of ivermectin resistance in sheep nematodes using the faecal egg count reduction test on samples collected from Scottish sheep. Vet. Parasitol. 137(1-2): 112 – 118. DOI: 10.1016/j.vetpar.2005.12.014

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  • Álvarez-Sánchez M.A. Pérez – García J. Cruz – Rojo M.A. Rojo Vázquez F.A. (2006): Anthelmintic resistance in trichostrongylid nematodes of sheep farms in Northwest Spain. Parasitol. Res. 99(1): 78 – 83. DOI: 10.1007/s00436-006-0130-2

    • Crossref
    • Export Citation
  • Ardelli B.F. Stitt L.E. Tompkins J.B. Prichard R.K. (2009): A comparison of the effects of ivermectin and moxidectin on the nematode Caenorhabditis elegans. Vet. Parasitol. 165(1-2): 96 – 108. DOI: 10.1016/j.vetpar.2009.06.043

    • Crossref
    • Export Citation
  • Artho R. Schnyder M. Kohler L. Torgerson P.R. Hertzberg H. (2007): Avermectin – resistance in gastrointestinal nematodes of Boer goats and Dorper sheep in Switzerland. Vet. Parasitol. 144(1-2): 68 – 73. DOI: 10.1016/j.vetpar.2006.09.032

    • Crossref
    • Export Citation
  • Bartley D.J. Donnan A.A. Jackson E. Sargison N. Mitchell G.B.B. Jackson F. (2006): A small scale survey of ivermectin resistance in sheep nematodes using the faecal egg count reduction test on samples collected from Scottish sheep. Vet. Parasitol. 137(1-2): 112 – 118. DOI: 10.1016/j.vetpar.2005.12.014

    • Crossref
    • Export Citation
  • Barton N. J. (1983): Development of anthelmintic resistance in nematodes from sheep in Australia subjected to different treatment frequencies. Int. J. Parasitol. 13(2): 125 – 132. DOI:10.1016/0020-7519(83)90002-4

    • Crossref
    • Export Citation
  • Bennett J.L. Pax R.A. (1986): Micromotility meter: an instrument designed to evaluate the action of drugs on motility of larval and adult nematodes. Parasitology 93(Pt 2): 341 – 346

    • Crossref
    • Export Citation
  • Coles G.C. Tritschler Ii J.P. Laste N.J. Schmidt A.L. (1988): Larval development test for detection of anthelmintic resistant nematodes. Res. Vet. Sci. 45(1): 50 – 53

  • Coles G.C. Folz D. Tritschler I.J.P. (1989): Motility response of levamisole/benzimidazole-resistant Haemonchus contortus larvae. Vet. Parasitol. 31(3-4): 253 – 257

    • Crossref
    • Export Citation
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