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
Two susceptible isolates of
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.
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.
The micromotility test was performed as described by Folz et
Origin and status of anthelmintic susceptibility (S) or resistance (R) of ISE, McMaster, ISE-SL, ISE-SLI, MOX-23, WR and CAVR isolates of
Isolate | R/S | Reference | Origin |
---|---|---|---|
ISE | susceptible | Roos et al. (2004) | Kenya |
McMaster | susceptible | CSIRO, Armidale | Australia |
ISE-SL | IVM | Coles (2005) | Great Britain |
ISE-SLI | IVM | Coles (2005) | Great Britain |
MOX-23 | IVM | Prichard et al. (2002) | Canada |
WR | BZ, CLO, IVM, RAF | Van Wyk and Malan (1988) | South Africa |
CAVR | IVM, BZ | LeJambre et al. (1995) | Australia |
BZ, benzimidazole; CLO, closantel; IVM, ivermectin; RAF, rafoxanide
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
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.
Arithmetic mean ± SD of LD50 and LD99 for susceptible and resistant isolates obtained in the larval migration inhibition test with ivermectin
Isolate | LD50(μg/ml) ± SD | LD99(μg/ml) ± SD |
---|---|---|
McMaster | 1.03 ±0.72 | 2.80 ±0.61 |
ISE | 1.25 ±0.48 | 3.00 ±1.24 |
MOX23 | 2.13 ±1.77 | 34.36 ±25.14 |
CAVR | 2.80 ± 0.72 | 20.10 ±12.12 |
WR | 3.80 ± 0.35 | 8.77 ± 4.92 |
ISE-SL | 3.65 ± 0.48 | 48.18 ±57.59 |
ISE-SLI | 5.05 ±1.98 | 22.87 ± 7.45 |
Resistance factors for ivermectin (IVM) in the larval migration inhibition test
Strains | IVM RF50 | IVM RF99 |
---|---|---|
WR/McM | 3.68 | 3.13 |
CAVR/McM | 2.71 | 7.17 |
MOX23/McM | 2.06 | 12.27 |
ISE-SL/McM | 3.54 | 17.20 |
ISE-SLI/McM | 4.90 | 8.16 |
WR/ISE | 3.04 | 2.92 |
CAVR/ISE | 2.24 | 6.70 |
MOX23/ISE | 1.70 | 11.45 |
ISE-SL/ISE | 2.92 | 16.06 |
ISE-SLI/ISE | 4.04 | 7.62 |
The mean reduction in motility and the LD50 values for the susceptible and resistant isolates of
Mean reduction (%) in motility of L3 larvae of resistant and susceptible strains of
Concentration of IVM (μg/ml) | |||||
---|---|---|---|---|---|
Isolate | 0.1 | 1 | 10 | 100 | LD50 |
WR | 0 | 0 | 26.2 | 49.7 | 90.76 |
CAVR | 0 | 55.2 | 52.8 | 76.1 | 4.2 |
McMaster | 0 | 39.7 | 88.9 | 80.9 | 1.57 |
ISE | 0 | 59.3 | 97.8 | 91.4 | 0.84 |
MOX23 | 13.7 | 41.2 | 78.9 | 96.4 | 1.57 |
ISE SL | 11.2 | 32 | 6.7 | 80.5 | 78.33 |
Mean reduction (%) in motility of L3 larvae of resistant and susceptible strains of
Concentration of EPM (μg/ml) | |||||||
---|---|---|---|---|---|---|---|
Isolate | 0.1 | 1 | 10 | 100 | 200 | 300 | LD50 |
WR | 43.4 | 43 | 53.3 | 72.2 | 71.7 | 41.04 | |
CAVR | 35 | 49.48 | 29.3 | 65.3 | 64.7 | 49.77 | |
McM | 0 | 37.1 | 76.4 | 63.9 | 100 | 100 | 3.98 |
ISE | 34.6 | 53.23 | 65.2 | 69.3 | 100 | 100 | 1.54 |
MOX23 | 15.8 | 27.7 | 40.3 | 70.7 | 100 | 100 | 15.4 |
ISE SL | 14.26 | 11.65 | 68.5 | 50.16 | 45.1 | 66.9 | 47.27 |
The values of RFs in LMIT in our study were as high as 12.2 for MOX-23 and 7.17 for CAVR. Kotze
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
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
In our study the micromotility meter was used to evaluate the effect of two ML anthelmintics on the motility of
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
Resistance factors for ivermectin (IVM) and eprinomectin (EPM) in the micromotility test
Strains | IVM RF50 | EPM RF50 |
---|---|---|
WR/McM | 57.81 | 10.31 |
CAVR/McM | 2.67 | 12.50 |
MOX23/McM | 1.00 | 3.87 |
ISE-SL/McM | 49.89 | 11.88 |
WR/ISE | 108.05 | 26.65 |
CAVR/ISE | 5.00 | 32.32 |
MOX23/ISE | 1.87 | 10.00 |
ISE-SL/ISE | 93.25 | 30.69 |
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
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
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.