InrerMrionalJournalfor ParrifologyVol.21, No.7,PP.771-776, 1991 Printed in Greet Britain 0
DETECTION
OF RESISTANCE TO IVERMECTIN CONTORTUS
JENNIFER H. GILL,*?
JUDITH M. REDWIN,* JAN
002(t7519/91 $3.00 + 0.00 Pergmon Press plc 1991 Aurrralim Societyfor Parasitology
IN HAEMONCHUS
A. VAN WYK$ and ERNEST LACEY*
*C.S.I.R.O. Division of Animal Health, McMaster Laboratory, Private Bag No. 1, P.O. Glebe, New South Wales 2037, Australia .$Veterinary Research Institute, Onderstepoort 0110, Republic of South Africa (Received 12 February 1991; accepted20 June 1991)
Ahstract-GtLr_ J. H., REDWINJ. M., WYKJ. A. VAN and LACEY E. 1991. Detection of resistance to ivermectin in Haemonchus contorts. International Journal for Parasitology 21: 771-776. Infective, third-stage (L3) larvae of Haemonchus conrortus isolates resistant to ivermectin (IVM) show a decreased sensitivity to IVMinduced paralysis in vitro. The inhibition of larval motility by IVM can be detected in L3 larvae incubated in the dark on an agar matrix containing IVM, by the failure of affected larvae to move when stimulated by exposure to light. Optimally, avermectin (AVM) potency is quantified after three cycles, each involving storage in the dark for 24 h followed by a brief exposure to light. For IVM-susceptible isolates, a 50% inhibition of motility (LP,) was achieved with IVM concentrations between 0.30 and 0.49 PM, while LP,, values in IVM-resistant isolates ranged from 0.8 to 2.6 PM depending on the in vivo resistance status of the isolate. A limited study of structure-activity relationships within the AVM class indicated that in vitro inhibition of L3 motility was consistent with the known in vivo efficacy of each analogue. Resistance factors for IVM-resistant isolates were dependent on AVM structure with the more polar AVM B, analogue being a particularly sensitive probe of IVM-resistance status. INDEX KEY WORDS: Anthelmintic resistance; Haemonchus contortus; ivermectin; motility; nematode.
INTRODUCTION THE
avermectins (AVMs, Fig. 1) are a family of 16membered macrocyclic lactones which show potent, broad spectrum nematocidal and insecticidal activity (Campbell, Fisher, Stapley, Albers-Schiinberg & Jacob, 1983; Campbell & Benz, 1984). Two analogues, avermectin B, (AVM B,) and ivermectin (IVM, 22,23dihydro-AVM B,), are commercially available for the control of nematode parasites in domestic livestock.
Although the AVMs were only introduced in the mid198Os, resistance to these drugs has already been observed in the field. In South Africa (Anonymous, 1986; Carmichael, Visser, Schneider & Soll, 1987; van Wyk & Malan, 1988; van Wyk, Malan, Gerber & Alves, 1989), Brazil (Echevarria & Trindade, 1989) and the U.S.A. (Craig & Miller, 1990) IVM resistance has been observed in Haemonchus contortus populations, while IVM-resistant populations of Ostertagia spp. have been isolated in New Zealand (Watson & Hosking, 1990). In addition, IVM-resistant lines of H. contortus and Trichostrongylus colubriformis have been produced in laboratory selection studies (Egerton, Suhayda & Eary, 1988; Giordano, Tritschler & Coles, 1988; Shoop, Egerton, Eary & Suhayda, 1990). The development of drug resistance in parasitic nematode populations has placed limitations on the
t To whom all correspondence should be addressed.
continued use of many anthelmintics. However, responsible use of new drugs should minimize the development of resistance and prolong their useful life (Waller, 1986). Techniques for the detection of resistance are necessary in order to monitor the extent and development of the problem in parasite populations and thus provide a database from which rational control programmes can be developed. In vivo techniques for the diagnosis of anthelmintic resistance are time consuming and expensive; in vitro techniques have the advantages of greater sensitivity and precision, speed and cheapness, although they are often limited to specific drug classes. At present there is no in vitro assay available for the routine diagnosis of resistance to IVM in the important trichostrongylid parasites of sheep. Such an assay would be valuable not only for the detection of field resistance but also as an aid to research investigating the mode of action of the AVMs in nematodes and mechanisms of resistance to this class of drugs. Although the mechanism of action of the AVMs in target organisms is yet to be elucidated, the physiological response to this group of compounds is known to involve an increase in membrane permeability to chloride ions which results, it is thought, from some interaction of AVMs with chloride ion channels (Turner & Schaeffer, 1989). The activity of AVMs against nematodes has been assayed in vitro by measuring inhibition of the motility of Cuenorhabditis 771
712
J. H. GILL, J.
Anologue AVM 81 AVM 82 IVM
M. REDWIN, J. A. VANWYKand E. LACEY
x-_-y CH=CH CH2-CH(OH)
CH3
OH
CH2-CH2
FIG. 1. Chemical structure of the avennectins. elegans, a free-living nematode (Pong, Wang & Fritz,
1980; Kass, Wang, Walrond & Stretton, 1980; Schaeffer & Haines, 1989). A direct correlation has been found between the observed potency of AVMs as inhibitors of C. efegans motility and their ability to displace [‘HI-IVM from a specific, high-affinity binding site in C. cfegans preparations (Schaeffer & Haines, 1989); the functional significance of this binding site is unknown. Exsheathed L3 larvae of the parasitic nematodes, T. colubriformis and H. contortus, also show impaired motility in response to the presence of AVMs (Folz, Pax, Thomas, Bennett, Lee & Conder, 1987; Boisvenue, Brand& Galloway & Hendrix, 1983). In addition, Ibarra & Jenkins (1984) demonstrated that IVM is a potent inhibitor of larval development in faecal slurries for a number of trichostrongylid nematodes, including H. contortus and T. colubriformis. Giordano et al. (1988) used an assay which monitored inhibition of larval development (Coles, Tritschler, Giordano, Laste & Schmidt, 1988) in the only reported attempt to detect IVM resistance in vitro. Although these workers failed to achieve a normal dose response for IVM in this assay, they did observe a shift in response which indicated some decrease in sensitivity to IVM in a laboratory selected, IVM-resistant T. colubrformis isolate. In this paper we report an in vitro larval motility assay for the detection of IVM resistance in isolates of H. contortus. In this technique, infective third-stage (L3) larvae are incubated in the dark for 72 h on an agar matrix containing the test compound, after which time they are exposed to light to stimulate movement in non-paralysed larvae. For IVM-resistant isolates a clear shift in the dose response is observed, reflecting the reduced potency of IVM against these nematodes. MATERIALS AND METHODS AVM B,, avermectin B, (AVM B2),IVM, IVMmonosaccharide and IVM-aglycone were gifts from Dr Chemicals.
Wesley Shoop, Merck, Sharpe & Dohme, U.S.A. Bacto-agar (Difco) was purchased from Bacto Laboratories (Sydney, Australia). Dimethyl sulphoxide (DMSO, analytical grade)
was obtained from BDH Chemicals (Sydney, Australia). All other reagents used were of the highest grade commercially available. Larvae. Table 1 contains particulars of the larvae used in these investigations including their anthelmintic resistance profiles and details of their in vivo susceptibility to IVM. The IVM-resistant H. conformswere derived from field isolations in South Africa. The McMaster and Onderstepoort H. contortus isolates, which have had little or no exposure to any anthelmintic, are reference susceptible strains, routinely maintained at the McMaster Laboratory, Sydney, Australia and the Veterinary Research Institute, Onderstepoort, South Africa, respectively. Neither the benzimidazole (BZ)resistant VRSG (Hall, Kelly, Campbell, Whitlock & Martin, 1978) nor the BZ/levamisole (LVS)-resistant Lawes (Green, Forsyth, Rowan & Payne, 1981) Australian H. contortus isolates have been exposed to IVM. All isolates were routinely maintained by passage in 46-month-old wormfree Merino wethers. L3 larvae were isolated from faecal cultures by standard Baermann filtration and stored at 10°C in tap-water prior to use. Mo+r assay. Stock drug solutions were serially diluted 1 in 2 with DMSO and aliquots dispensed in duplicate into the wells of 96-well microtitre plates. Agar (2%, 200 ~1) was added to give final drug concentrations of 5w.024 PM in 1% (v/v) DMSO. Control wells contained 1% (v/v) DMSO alone. Plates were chilled to 4°C prior to the addition of L3 larvae (20-30 per well) in water (20 $,4”C) as the L3 larvae of H. contortus adopt a coiled stationary position at this temperature, which facilitated the counting of the total number of larvae in each well (Nr). Plates were incubated at 25’C in the dark for 24 h before being illuminated for 20 min either on a light box (60 W) or during counting (see below). Unless otherwise indicated, this procedure was repeated for two further 24 h cycles. The numbers of motile and nonmotile larvae in each well were assessed under a light microscope after an initial illumination sufficient to activate > 90% of larvae in control wells (l-2 mm). Motile larvae were defined as those moving with a normal sinusoidal thrashing motion; still larvae and those moving in a restricted manner were counted as non-motile. To accelerate counting, only motile larvae (N,.,) were counted in wells containing predominantly non-motile larvae and only non-motile larvae (NN) were counted in wells containing predominantly motile larvae. In the intervening wells both motile and non-motile larvae were counted.
Ivermectin resistance in H. contortus TABLE I-ANMELMINTICRESISTANCESTATIJSOF H. contortus
773 ISOLATES
In vivo
No. Isolate IVM-susceptible 1 McMaster 2 VRSG 3 Law& 4 Onderstepoort* IVM-resistant 5 White River I* 6 White River II* 7 Swellendam* 8 Stellenbosch* 9 Tulbach* 10 Cullinan/DAS*
IVM efficacy at 0.2 mg kg-’
Other known resistance
100% 100% 100% 100%
nil BZ BZ,LVS nil
49.4%5 33.0% 51.2% 63.6% n.a. 72.5%
BZ, RAF BZ. CST. RAF n.a.S nil n.a. n.a.
Reference?
1 2 394 5 3.4 3;4 3,4,6,7 3,4
* South African isolates. t 1. Hallet al., 1978; 2. Green et al., 1981; 3. van Wyk & Malan, 1988; 4. van Wyk et al., 1989; 5. van Wyk, Malan, Gerber & Alves, 1987; 6. Anon, 1986; 7. Carmichael ef al., 1987. $ Not available. 4 J. A. Van Wyk, unpublished data. Data treatment. Numbers of non-motile larvae (NN or NT- Nhl) as a proportion of the total larvae present (NT) at each drug concentration were calculated and a logconcentration-logit model (Wailer, Dobson, Donald, Griffiths & Smith, 1985) was fitted to the data to estimate the concentration of drug required to paralyse 50% of the larvae present (LP,). Data were corrected for PO,the mean number of non-motile larvae in six control wells (POwas generally < 5%). The susceptibility of the H. contortus isolates to a given AVM was expressed as a resistance factor (RF): LP, for the test isolate RF= LP, for McMaster isolate RESULTS
After incubation in the dark on agar at 25”C, the L3 larvae of H. contortus can be stimulated to move in a rapid sinusoidal motion by exposure to light; they then remain active for at least 10 min before resuming resting positions which range from coiled to straight at room temperature (Fig. 2). The effect of the number of motility cycles (each consisting of 24 h incubation in the dark, followed by exposure to light) on the length of the period of maximum activity is shown in Fig. 2. Maximum activity was reached after a short lag time of l-2 min and continued for up to 15 min. Although a progressive reduction in the vigour and duration of movement was observed as the number of motility cycles increased, there was an increase in the uniformity of the response of the larvae to the light stimulus. A typical dose response for the inhibition of the motility of McMaster L3 larvae after incubation for 72 h (three motility cycles) on agar containing IVM (500.024 PM) is shown in Fig. 3. The effect of the number of motility cycles on IVM LP,, values against the McMaster isolate was monitored over 7 days (Fig. 4).
Time
(min)
FIG. 2. Effect of duration of exposure to light on the motility of H. contortus L3 larvae. Motility was monitored at 24 (-), 48 (---) and 72 h (-.-). Larvae were otherwise incubated in the dark on 2% agar.
LP, values were found to be dependent on the number of motility cycles, decreasing to a constant value by 72 h (three cycles). As the number of motility cycles increased the dose response data became less scattered and the LP,, values obtained were more consistent between experiments. In order to determine whether it was the length of exposure to IVM or the number of motility cycles which was responsible for the observed reduction in LP, values over 72 h, the LP, values for IVM against McMaster L3 larvae at 24, 48 and 72 h were compared to the LP, value obtained at 72 h for larvae that had been maintained in the dark. The LP,, values of the cycled larvae were 0.78,0.56 and 0.42 ,UM after 24,48 and 72 h, respectively, while for the larvae kept continuously in the dark, an LP, value of 0.84 ,UM was obtained at 72 h. This suggests that activation of the larvae at 24 hourly intervals by brief exposure to light is an important determinant of the LP,, value
174
J. H. GILL, J. M. REDWIN,J. A. VANWYK and E. LACEY
TABLE~-ACTIVITYOFSOME AVM
ANALOGUES
OFTHE MOTILITY OF THE L3 RESISTANT H. COniOIWS ISOLATES
AS INHIBITORS
Rfl
LPXl*
OF NM-SUSCEPTIBLE
AVM B,
IVM Isolate
LARVAE
LP,,
AND
IVM-
AVM B, RF
RF
LPm
McMaster
0.30f0.11(14)
0.22 f 0.05(S)
0.84*0.31(5)
VRSG
0.44* 0.08(2)
1.5
0.24f 0.05(2)
1.1
0.57 f 0.20(2)
0.7
Lawes
0.47+0.13(3)
1.6
0.32 f 0.06(2)
1.5
1.7 f 0.5(2)
2.0
Ondersterpoort
0.27
0.9
0.41
1.9
0.90
1.1
White River I
0.80
2.1
0.74
3.4
2.1
2.5
White River II
1.5+0.5(2)
5.0
2.22 f 0.04(2)
10
8.90&0.14(2)
11
Swellendam
1.Of 0.6(2)
3.3
1.3*0.6(2)
5.9
7.19+0.12(2)
8.6
Stellenbosch
1.5 f 0.9(7)
5.0
1.34 f 0.08(2)
6.1
6.7 f 1.9(2)
8.0
Tulbach
2.6 f 0.7(3)
8.7
4.9 f 1.4(2)
22
16.9 f 0.7(2)
20
DAS
1.1 l0.6(3)
3.7
1.3 f 0.6(2)
5.9
7.7 f 3.0(2)
9.2
* LP,,_”(PM) . , values are auoted as means f S.D.(n) \ where n is the number of independent determinations. t Resistance factor. I
100 -
0 _
*___.___D.e 0.1
1.0
Drug concentration
10
24
46
72 Time
96
120
144
(h)
(j&4)
FIG. 3. Effect of IVM on the motility of H. contortus L3 larvae after 72 h. Each data point is the mean of two determinations. Lines are the fitted logistic curves (see Methods). Isolates are
McMaster (O-O) and White River II (O-O). obtained at 72 h. Whether the length of the incubation time in the dark between periods of light is important was not ascertained. For routine use an incubation period of 72 h, which included 20 min periods in the light at 24 and 48 h, was employed. In a series of independent determinations a 72 h (three cycle) LP,, value of 0.30 f 0.11 pM (s.D., n = 14) was obtained for IVM against the McMaster isolate indicating good between-assay reproducibility (Table 2). The effect of the age of the larvae used on LP,, values was also examined. No differences were found in LP, values for larvae up to 2 months old, although there was a decrease in the vigour of movement as the larvae
FIG. 4. Effect of incubation time on LP,, values for IVMsusceptible and IVM-resistant H. contortus isolates. L3 larvae were incubated in the dark except for 15 min periods of exposure to light each day during counting. LP, values are means f S.D. for two or more experiments. The isolates are McMaster (O-O), Tulbach (A-A) and White River II (O-O).
aged, making assessment of motility more difficult. Larvae less than 1 month old are preferred. IVM-naive isolates from Australia and South Africa were compared to assess the effect of genetic background and pre-existing resistance to other anthelmintic classes on IVM LP, values (Table 2). The BZ-resistant VRSG and BZ/LVS-resistant Lawes isolates were both found to be as sensitive to IVM as the McMaster isolate (Table 2), indicating no cross resistance between existing broad spectrum anthelmintics and the AVMs. The IVM LP, value obtained against the susceptible South African Onderstepoort
Ivertnectin resistance in H. contortus reference strain was also within the range of values obtained against the McMaster isolate. For IVM-resistant H. contortus isolates (e.g. White River II) a clear shift was observed in the IVM dose response profile (Fig. 3), indicating a reduced sensitivity compared to the McMaster isolate. IVM LP, values of 0.34.5 ,UM(Table 2) were observed for the IVM-susceptible isolates of H. contortus tested, while LP,, values from 0.8 to 2.6 PM, corresponding to resistance factors of 2.7-8.8, were recorded for the six IVM-resistant isolates examined. The effect of the number of motility cycles on the IVM LP,, values against IVM-resistant isolates was examined. As was the case with the McMaster isolate, a stabilization in IVM LP,, values was observed with all isolates after three motility cycles (Fig. 4). LP, values against all except the Tulbach isolate decreased from a maximum at 24 h to a constant value (e.g. White River II, Fig. 4). In contrast, the LP,, values for the Tulbach isolate increased to a peak at 48 h, which suggests that the mechanism responsible for the observed IVM resistance of this isolate may differ from that causing IVM resistance in the other South African isolates. The potency of a series of related AVMs as inhibitors of larval motility was examined (Table 2). Against the IVM-susceptible McMaster isolate the order of potency was: AVM B, = IVM > AVM B, > > IVM-monosaccharide, IVM-aglycone. For the latter two compounds less than 50% inhibition of motility was observed for susceptible isolates at 50 PM, the highest concentration tested. LP,, values recorded for AVM B, and AVM B, against the other IVMsusceptible isolates tested were within the range of values obtained for these compounds against the McMaster isolate (Table 2) with the exception that the Australian Lawes isolate appears slightly less sensitive to AVM B,. The rank order of potency of these compounds against the IVM-resistant isolates was the same as observed against the IVM-susceptible isolates, except that AVM B, was slightly less potent than IVM against the most resistant isolates, White River II and Tulbach. The largest resistance factors were obtained with the more polar AVM B,, which makes this the compound of choice for the detection of IVM resistance using this assay. These data suggest that resistance to AVMs in H. contortus is dependent on the structure of the analogue. Inhibition of L3 motility in vitro ranks these isolates in order of AVM resistance as: White River I < Swellendam, DAS, Stellenbosch, White River II < Tulbach. DISCUSSION
Under constant environmental conditions most nematodes undergo low rate, short duration periods of movement. If an environmental change occurs such as an increase in light intensity or a change in the temperature, a period of activity results (Croll & Matthews, 1977). When L3 larvae of H. contortus
775
which have been incubated in the dark are exposed to light a response can be elicited which can be made more uniform by conditioning the larvae through several light/dark cycles. This response to changes in light intensity has been exploited to allow the detection of AVM-induced paralysis in a larval motility assay. The assay is capable of detecting the resistance of a number of H. contortus isolates to IVM. L3 larvae are easily obtained in large numbers from faecal cultures and are normally quite hardy, making this a convenient stage in the parasite life-cycle with which to work. It is not fully understood why AVM LP,, values are dependent on the number of motility cycles. The increasing potency of the drug over the first three motility cycles (72 h) is more likely a reflection of a progressive impairment of the motor functions controlled by the site concerned, rather than a slower phase of accumulation of drug at receptor sites, since LP, values were dependent on the number of motility cycles and not the length of incubation. The reduction in potency of IVM against the Tulbach isolate at 48 h, which was not observed for other isolates, may reflect subtle differences in the manner in which the site of action regulates motor function. Australian and South African IVM-naive H. contortus isolates were equally sensitive to the paralytic effects of IVM. No cross resistance was detected between the AVMs and two other major classes of broad spectrum anthelmintics, LVS/morantel and the BZs. This is consistent with the observations of Kass et al. (1980), who found no difference in the response of LVS-susceptible and LVS-resistant C. eleguns lines to theparalyticeffects ofAVM B,, and with in vivaefficacy data (Wailer & Donald, 1983). These results support the hypothesis that the mode of action of the AVMs is independent of that of LVS, a known acetylcholine agonist (Lewis, Wu, Levine & Berg, 1980) and that of the BZs, which act by inhibiting the polymerization of tubulin (Lacey, 1988). Limited data were obtained on structure-activity relationships within the AVM class. The observed potency of the AVM analogues tested reflected in vivo efficacy against a number of nematode parasites in sheep (Chabala, Mrozik, Tolman, Eskola, Lusi, Peterson, Woods, Fisher, Campbell, Egerton & Ostlind, 1980). Hydration of the 22,23-double bond of AVM B, to produce AVM B, caused a two-fold loss in activity against the IVM-susceptible McMaster isolate; reduction of this bond to give IVM, although resulting in a similar change in the geometry of the spiroketal region, caused little change in activity. Loss of one or both of the oleandrose sugar moieties from IVM caused a greater than lOO-fold loss in potency against the McMaster isolate. Comparison between in vitro LP,, values for IVM (Table 2) and in vivo efficacy data (Table 1) gave a reasonable correlation for the H. contortus isolates tested. Provided there are parallels in the mechanisms of resistance it should be possible to extend the use of
116
J. H. GILL, J. M. REDWIN, J. A. VAN WYK and E. LACEY
this larval motility assay to the detection of IVM resistance in other important trichostrongylid parasites such as T. colubriformis and 0. circumcincta. While the site(s) of action of the AVMs remain(s) unknown, the results of this study of IVM-resistant isolates add further support to the hypothesis that the mechanism of AVM action in nematodes is associated with motor function. Acknowledgements-The authors would like to thank Dr Nicholas Sangster for the supply of larvae from the Lawes H. contortus isolate. The work reported here was supported by a grant from the Australian Wool Corporation (CSO7P). REFERENCES ANONYMOUS1986. Directorate of Veterinary Services, South Africa: Laboratory Services Monthly Reports, January, p. 4. BOISVENUER. J., BRANDTM. C., GALLOWAYR. B. & HENDRIX J. C. 1983. In vitro activity of various anthelmintic compounds against Haemonchus contortus larvae. Veterinary Parasitology 13: 341-347. CAMPBELL W. C., FISHER M. H., STAPLEY E. O., ALBERSSCHONBERGG. & JACOB T. A. 1983. Ivermectin: a potent new antiparasitic agent. Science 221: 823-828. CAMPBELLW. C. & BENZ G. W. 1984. Ivermectin: a review of efficacy and safety. Journal of Veterinary Pharmacology and Therapeutics 7: 1-16. CARMICHAELI., VISSER R., SCHNEIDERD. & SOLL M. 1987. Haemonchus contortus resistance to ivermectin. Journal of the South African Veterinary Association 58: 93. CHABALAJ. C., MROZIK H., TOLMANR. L., ESKOLAP., Lust A., PETERSONL. H., WOODS M. F., FISHER M. H., CAMPBELLW. C., EGERTONJ. R. & OSTLINDD. A. 1980. Ivermectin, a new broad spectrum antiparasitic agent. Journal of Medicinal Chemistry 23: 11341136. COLES G. C., TRITSCHLERJ. P. II, GIORDANOD. J., LASTEN. J. & SCHMIDT A. L. 1988. Larval development test for detection of anthelmintic resistant nematodes. Research in Veterinary Science 45: 50-53. CRAIG T. M. & MILLER D. K. 1990. Resistance bv Haemonchus contortus to ivermectin in angora goats. Veterinary Record 126: 580. CROLL N. A. & MATTHEWSB. E. 1977. Biology of Nematodes. Blackie, Glasgow. ECHEVARRIA F. A. M. & TRINDADE G. N. P. 1989. Anthelmintic resistance by Haemonchus contortus to ivermectin in Brazil: a preliminary report. Veterinary Record 124: 147-148. EGERTONJ. R., SUHAYDAD. & EARY C. H. 1988. Laboratory selection of Haemonchus contortus for resistance to ivermectin. Journal of Parasitology 74: 614617. FOLZ S. D., PAX R. A., THOMASE. M., BENNETTJ. L., LEE B. L. & CONDER G. A. 1987. Development and validation of an in vitro Trichostrongylus colubriformis motility assay. International Journalfor-Parasitology 17: 1441-1444. _ GIORDANO D. J., TRITSCHLERJ. P. II & COLES G. C. 1988. Selection of ivermectin-resistant Trichostrongylus colubriformis in lambs. Veterinary Parasitology 30: 139-148. GREENP. E., FORSYTHB. A., ROWAN K. J. & PAYNE G. 1981. The isolation of a field strain of Haemonchus contortus in Queensland showing multiple anthelmintic resistance. Australian Veterinary Journal 57: 79-84. HALL C. A., KELLY J. D., CAMPBELLN. J., WHITLOCKH. V. & MARTIN I. C. A. 1978. The dose response of several
benzimidazole anthelmintics against resistant strains of Haemonchus contortus and Trichostrongylus colubrtformis selected with thiabendazole. Research in Veterinary Science 25: 364-367. IBARRA0. F. & JENKINSD. C. 1984. The relevance of in vitro anthelmintic screening tests employing the free-living stages of trichostrongylid nematodes. Journal of Helminthology 58: 107-l 12. KASS I. S., WANG C. C., WALRONDJ. P. & STRET~ONA. 0. W. 1980. Avermectin B,,, a paralysing anthelmintic that affects interneurons and inhibitory motoneurons in Ascaris. Proceedings of the National Academy of Sciences of the United States of America 77: 6211-6215. LACEY E. 1988. The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles. International Journal for Parasitology 18: 885-936. LEWIS J. A., Wu C. -H., LEVINE J. H. & BERG H. 1980. Levamisole-resistant mutants of the nematode Caenorhabditis elegans appear to lack pharmacological acetylcholine receptors. Neuroscience 5: 967-989. PONG S. -S., WANG C. C. & FRITZ L. C. 1980. Studies on the mechanism of action of avermectin B,a: stimulation of release of y-aminobutyric acid from brain synaptosomes. Journal of Neurochemistry 34: 351-358. SCHAEFFERJ. M. & HAINES H. W. 1989. Avermectin binding in Caenorhabditis elegans: a two-state model for the avermectin binding site. Biochemical Pharmacology 38: 2329-2338. SHEEP W. L., EGERTONJ. R., EARYC. H. & SUHAYDAD. 1990. Laboratory selection of a benzimidazole-resistant isolate of Trichostrongylus colubrtformis for ivermectin resistance. Journal of Parasitology 76: 186-189. TURNER M. J. & SCHAEFFERJ. M. 1989. Mode of action of ivermectin. In: Zvermectin and Abamectin (Edited by CAMPBELLW. C.), pp. 73-88. Springer, New York. WALLER P. J. & DONALD A. D. 1983. New perspectives in helminth control. In: Recent Developments in the Controlof Animal Parasites: Proceedings, MSD AGVET Symposium in Association with the XXII World Veterinary Congress, Perth, 25 and 26 August 1983 (Edited by LEANING W. H. D., SIEGMUND0. H. & FRASER C. M.), pp. __ 215-230. MSD AGVET, Rahway, NJ. WALLER P. J.. DOBSON R. J.. DONALDA. D.. GRIFFITHS D. A. & SMITHE. F. 1985. Selection studies’on anthelmintic resistant and susceptible populations of Trichostrongylus colubrtformis of sheep. International Journal for Parasitology 15: 669-616. WALLER P. J. 1986. Anthelmintic resistance in Australia. Parasitology Today 2: S16-S18. WAGON T. G. & HOSKING B. C. 1990. Evidence for multiple anthelmintic resistance in two nematode parasite genera on a Saanen goat dairy. New Zealand Veterinary Journal 38: 5&53. WYKJ. A. VAN,MALANF. S., GERBERH. M. & ALVESR. M. R. 1987. Two field strains of Haemonchus contortus resistant to rafoxanide. Onderstepoort Journal of Veterinary Research 54: 143-146. WYK J. A. VAN& MALAN F. S. 1988. Resistance of field strains of Haemonchus conforms to ivermectin, closantel, rafoxanide and the benzimidazoles in South Africa. Veterinary Record 123: 22&228. WYK J. A. VAN,MALANF. S., GERBERH. M. & ALVESR. M. R. 1989. The problem of escalating resistance of Haemonchus contortus to the modem anthelmintics in South Africa. Onderstepoort Journal of Veterinary Research 56: 4149.
DETECTION
OF RESISTANCE TO IVERMECTIN CONTORTUS
JENNIFER H. GILL,*?
JUDITH M. REDWIN,* JAN
002(t7519/91 $3.00 + 0.00 Pergmon Press plc 1991 Aurrralim Societyfor Parasitology
IN HAEMONCHUS
A. VAN WYK$ and ERNEST LACEY*
*C.S.I.R.O. Division of Animal Health, McMaster Laboratory, Private Bag No. 1, P.O. Glebe, New South Wales 2037, Australia .$Veterinary Research Institute, Onderstepoort 0110, Republic of South Africa (Received 12 February 1991; accepted20 June 1991)
Ahstract-GtLr_ J. H., REDWINJ. M., WYKJ. A. VAN and LACEY E. 1991. Detection of resistance to ivermectin in Haemonchus contorts. International Journal for Parasitology 21: 771-776. Infective, third-stage (L3) larvae of Haemonchus conrortus isolates resistant to ivermectin (IVM) show a decreased sensitivity to IVMinduced paralysis in vitro. The inhibition of larval motility by IVM can be detected in L3 larvae incubated in the dark on an agar matrix containing IVM, by the failure of affected larvae to move when stimulated by exposure to light. Optimally, avermectin (AVM) potency is quantified after three cycles, each involving storage in the dark for 24 h followed by a brief exposure to light. For IVM-susceptible isolates, a 50% inhibition of motility (LP,) was achieved with IVM concentrations between 0.30 and 0.49 PM, while LP,, values in IVM-resistant isolates ranged from 0.8 to 2.6 PM depending on the in vivo resistance status of the isolate. A limited study of structure-activity relationships within the AVM class indicated that in vitro inhibition of L3 motility was consistent with the known in vivo efficacy of each analogue. Resistance factors for IVM-resistant isolates were dependent on AVM structure with the more polar AVM B, analogue being a particularly sensitive probe of IVM-resistance status. INDEX KEY WORDS: Anthelmintic resistance; Haemonchus contortus; ivermectin; motility; nematode.
INTRODUCTION THE
avermectins (AVMs, Fig. 1) are a family of 16membered macrocyclic lactones which show potent, broad spectrum nematocidal and insecticidal activity (Campbell, Fisher, Stapley, Albers-Schiinberg & Jacob, 1983; Campbell & Benz, 1984). Two analogues, avermectin B, (AVM B,) and ivermectin (IVM, 22,23dihydro-AVM B,), are commercially available for the control of nematode parasites in domestic livestock.
Although the AVMs were only introduced in the mid198Os, resistance to these drugs has already been observed in the field. In South Africa (Anonymous, 1986; Carmichael, Visser, Schneider & Soll, 1987; van Wyk & Malan, 1988; van Wyk, Malan, Gerber & Alves, 1989), Brazil (Echevarria & Trindade, 1989) and the U.S.A. (Craig & Miller, 1990) IVM resistance has been observed in Haemonchus contortus populations, while IVM-resistant populations of Ostertagia spp. have been isolated in New Zealand (Watson & Hosking, 1990). In addition, IVM-resistant lines of H. contortus and Trichostrongylus colubriformis have been produced in laboratory selection studies (Egerton, Suhayda & Eary, 1988; Giordano, Tritschler & Coles, 1988; Shoop, Egerton, Eary & Suhayda, 1990). The development of drug resistance in parasitic nematode populations has placed limitations on the
t To whom all correspondence should be addressed.
continued use of many anthelmintics. However, responsible use of new drugs should minimize the development of resistance and prolong their useful life (Waller, 1986). Techniques for the detection of resistance are necessary in order to monitor the extent and development of the problem in parasite populations and thus provide a database from which rational control programmes can be developed. In vivo techniques for the diagnosis of anthelmintic resistance are time consuming and expensive; in vitro techniques have the advantages of greater sensitivity and precision, speed and cheapness, although they are often limited to specific drug classes. At present there is no in vitro assay available for the routine diagnosis of resistance to IVM in the important trichostrongylid parasites of sheep. Such an assay would be valuable not only for the detection of field resistance but also as an aid to research investigating the mode of action of the AVMs in nematodes and mechanisms of resistance to this class of drugs. Although the mechanism of action of the AVMs in target organisms is yet to be elucidated, the physiological response to this group of compounds is known to involve an increase in membrane permeability to chloride ions which results, it is thought, from some interaction of AVMs with chloride ion channels (Turner & Schaeffer, 1989). The activity of AVMs against nematodes has been assayed in vitro by measuring inhibition of the motility of Cuenorhabditis 771
712
J. H. GILL, J.
Anologue AVM 81 AVM 82 IVM
M. REDWIN, J. A. VANWYKand E. LACEY
x-_-y CH=CH CH2-CH(OH)
CH3
OH
CH2-CH2
FIG. 1. Chemical structure of the avennectins. elegans, a free-living nematode (Pong, Wang & Fritz,
1980; Kass, Wang, Walrond & Stretton, 1980; Schaeffer & Haines, 1989). A direct correlation has been found between the observed potency of AVMs as inhibitors of C. efegans motility and their ability to displace [‘HI-IVM from a specific, high-affinity binding site in C. cfegans preparations (Schaeffer & Haines, 1989); the functional significance of this binding site is unknown. Exsheathed L3 larvae of the parasitic nematodes, T. colubriformis and H. contortus, also show impaired motility in response to the presence of AVMs (Folz, Pax, Thomas, Bennett, Lee & Conder, 1987; Boisvenue, Brand& Galloway & Hendrix, 1983). In addition, Ibarra & Jenkins (1984) demonstrated that IVM is a potent inhibitor of larval development in faecal slurries for a number of trichostrongylid nematodes, including H. contortus and T. colubriformis. Giordano et al. (1988) used an assay which monitored inhibition of larval development (Coles, Tritschler, Giordano, Laste & Schmidt, 1988) in the only reported attempt to detect IVM resistance in vitro. Although these workers failed to achieve a normal dose response for IVM in this assay, they did observe a shift in response which indicated some decrease in sensitivity to IVM in a laboratory selected, IVM-resistant T. colubrformis isolate. In this paper we report an in vitro larval motility assay for the detection of IVM resistance in isolates of H. contortus. In this technique, infective third-stage (L3) larvae are incubated in the dark for 72 h on an agar matrix containing the test compound, after which time they are exposed to light to stimulate movement in non-paralysed larvae. For IVM-resistant isolates a clear shift in the dose response is observed, reflecting the reduced potency of IVM against these nematodes. MATERIALS AND METHODS AVM B,, avermectin B, (AVM B2),IVM, IVMmonosaccharide and IVM-aglycone were gifts from Dr Chemicals.
Wesley Shoop, Merck, Sharpe & Dohme, U.S.A. Bacto-agar (Difco) was purchased from Bacto Laboratories (Sydney, Australia). Dimethyl sulphoxide (DMSO, analytical grade)
was obtained from BDH Chemicals (Sydney, Australia). All other reagents used were of the highest grade commercially available. Larvae. Table 1 contains particulars of the larvae used in these investigations including their anthelmintic resistance profiles and details of their in vivo susceptibility to IVM. The IVM-resistant H. conformswere derived from field isolations in South Africa. The McMaster and Onderstepoort H. contortus isolates, which have had little or no exposure to any anthelmintic, are reference susceptible strains, routinely maintained at the McMaster Laboratory, Sydney, Australia and the Veterinary Research Institute, Onderstepoort, South Africa, respectively. Neither the benzimidazole (BZ)resistant VRSG (Hall, Kelly, Campbell, Whitlock & Martin, 1978) nor the BZ/levamisole (LVS)-resistant Lawes (Green, Forsyth, Rowan & Payne, 1981) Australian H. contortus isolates have been exposed to IVM. All isolates were routinely maintained by passage in 46-month-old wormfree Merino wethers. L3 larvae were isolated from faecal cultures by standard Baermann filtration and stored at 10°C in tap-water prior to use. Mo+r assay. Stock drug solutions were serially diluted 1 in 2 with DMSO and aliquots dispensed in duplicate into the wells of 96-well microtitre plates. Agar (2%, 200 ~1) was added to give final drug concentrations of 5w.024 PM in 1% (v/v) DMSO. Control wells contained 1% (v/v) DMSO alone. Plates were chilled to 4°C prior to the addition of L3 larvae (20-30 per well) in water (20 $,4”C) as the L3 larvae of H. contortus adopt a coiled stationary position at this temperature, which facilitated the counting of the total number of larvae in each well (Nr). Plates were incubated at 25’C in the dark for 24 h before being illuminated for 20 min either on a light box (60 W) or during counting (see below). Unless otherwise indicated, this procedure was repeated for two further 24 h cycles. The numbers of motile and nonmotile larvae in each well were assessed under a light microscope after an initial illumination sufficient to activate > 90% of larvae in control wells (l-2 mm). Motile larvae were defined as those moving with a normal sinusoidal thrashing motion; still larvae and those moving in a restricted manner were counted as non-motile. To accelerate counting, only motile larvae (N,.,) were counted in wells containing predominantly non-motile larvae and only non-motile larvae (NN) were counted in wells containing predominantly motile larvae. In the intervening wells both motile and non-motile larvae were counted.
Ivermectin resistance in H. contortus TABLE I-ANMELMINTICRESISTANCESTATIJSOF H. contortus
773 ISOLATES
In vivo
No. Isolate IVM-susceptible 1 McMaster 2 VRSG 3 Law& 4 Onderstepoort* IVM-resistant 5 White River I* 6 White River II* 7 Swellendam* 8 Stellenbosch* 9 Tulbach* 10 Cullinan/DAS*
IVM efficacy at 0.2 mg kg-’
Other known resistance
100% 100% 100% 100%
nil BZ BZ,LVS nil
49.4%5 33.0% 51.2% 63.6% n.a. 72.5%
BZ, RAF BZ. CST. RAF n.a.S nil n.a. n.a.
Reference?
1 2 394 5 3.4 3;4 3,4,6,7 3,4
* South African isolates. t 1. Hallet al., 1978; 2. Green et al., 1981; 3. van Wyk & Malan, 1988; 4. van Wyk et al., 1989; 5. van Wyk, Malan, Gerber & Alves, 1987; 6. Anon, 1986; 7. Carmichael ef al., 1987. $ Not available. 4 J. A. Van Wyk, unpublished data. Data treatment. Numbers of non-motile larvae (NN or NT- Nhl) as a proportion of the total larvae present (NT) at each drug concentration were calculated and a logconcentration-logit model (Wailer, Dobson, Donald, Griffiths & Smith, 1985) was fitted to the data to estimate the concentration of drug required to paralyse 50% of the larvae present (LP,). Data were corrected for PO,the mean number of non-motile larvae in six control wells (POwas generally < 5%). The susceptibility of the H. contortus isolates to a given AVM was expressed as a resistance factor (RF): LP, for the test isolate RF= LP, for McMaster isolate RESULTS
After incubation in the dark on agar at 25”C, the L3 larvae of H. contortus can be stimulated to move in a rapid sinusoidal motion by exposure to light; they then remain active for at least 10 min before resuming resting positions which range from coiled to straight at room temperature (Fig. 2). The effect of the number of motility cycles (each consisting of 24 h incubation in the dark, followed by exposure to light) on the length of the period of maximum activity is shown in Fig. 2. Maximum activity was reached after a short lag time of l-2 min and continued for up to 15 min. Although a progressive reduction in the vigour and duration of movement was observed as the number of motility cycles increased, there was an increase in the uniformity of the response of the larvae to the light stimulus. A typical dose response for the inhibition of the motility of McMaster L3 larvae after incubation for 72 h (three motility cycles) on agar containing IVM (500.024 PM) is shown in Fig. 3. The effect of the number of motility cycles on IVM LP,, values against the McMaster isolate was monitored over 7 days (Fig. 4).
Time
(min)
FIG. 2. Effect of duration of exposure to light on the motility of H. contortus L3 larvae. Motility was monitored at 24 (-), 48 (---) and 72 h (-.-). Larvae were otherwise incubated in the dark on 2% agar.
LP, values were found to be dependent on the number of motility cycles, decreasing to a constant value by 72 h (three cycles). As the number of motility cycles increased the dose response data became less scattered and the LP,, values obtained were more consistent between experiments. In order to determine whether it was the length of exposure to IVM or the number of motility cycles which was responsible for the observed reduction in LP, values over 72 h, the LP, values for IVM against McMaster L3 larvae at 24, 48 and 72 h were compared to the LP, value obtained at 72 h for larvae that had been maintained in the dark. The LP,, values of the cycled larvae were 0.78,0.56 and 0.42 ,UM after 24,48 and 72 h, respectively, while for the larvae kept continuously in the dark, an LP, value of 0.84 ,UM was obtained at 72 h. This suggests that activation of the larvae at 24 hourly intervals by brief exposure to light is an important determinant of the LP,, value
174
J. H. GILL, J. M. REDWIN,J. A. VANWYK and E. LACEY
TABLE~-ACTIVITYOFSOME AVM
ANALOGUES
OFTHE MOTILITY OF THE L3 RESISTANT H. COniOIWS ISOLATES
AS INHIBITORS
Rfl
LPXl*
OF NM-SUSCEPTIBLE
AVM B,
IVM Isolate
LARVAE
LP,,
AND
IVM-
AVM B, RF
RF
LPm
McMaster
0.30f0.11(14)
0.22 f 0.05(S)
0.84*0.31(5)
VRSG
0.44* 0.08(2)
1.5
0.24f 0.05(2)
1.1
0.57 f 0.20(2)
0.7
Lawes
0.47+0.13(3)
1.6
0.32 f 0.06(2)
1.5
1.7 f 0.5(2)
2.0
Ondersterpoort
0.27
0.9
0.41
1.9
0.90
1.1
White River I
0.80
2.1
0.74
3.4
2.1
2.5
White River II
1.5+0.5(2)
5.0
2.22 f 0.04(2)
10
8.90&0.14(2)
11
Swellendam
1.Of 0.6(2)
3.3
1.3*0.6(2)
5.9
7.19+0.12(2)
8.6
Stellenbosch
1.5 f 0.9(7)
5.0
1.34 f 0.08(2)
6.1
6.7 f 1.9(2)
8.0
Tulbach
2.6 f 0.7(3)
8.7
4.9 f 1.4(2)
22
16.9 f 0.7(2)
20
DAS
1.1 l0.6(3)
3.7
1.3 f 0.6(2)
5.9
7.7 f 3.0(2)
9.2
* LP,,_”(PM) . , values are auoted as means f S.D.(n) \ where n is the number of independent determinations. t Resistance factor. I
100 -
0 _
*___.___D.e 0.1
1.0
Drug concentration
10
24
46
72 Time
96
120
144
(h)
(j&4)
FIG. 3. Effect of IVM on the motility of H. contortus L3 larvae after 72 h. Each data point is the mean of two determinations. Lines are the fitted logistic curves (see Methods). Isolates are
McMaster (O-O) and White River II (O-O). obtained at 72 h. Whether the length of the incubation time in the dark between periods of light is important was not ascertained. For routine use an incubation period of 72 h, which included 20 min periods in the light at 24 and 48 h, was employed. In a series of independent determinations a 72 h (three cycle) LP,, value of 0.30 f 0.11 pM (s.D., n = 14) was obtained for IVM against the McMaster isolate indicating good between-assay reproducibility (Table 2). The effect of the age of the larvae used on LP,, values was also examined. No differences were found in LP, values for larvae up to 2 months old, although there was a decrease in the vigour of movement as the larvae
FIG. 4. Effect of incubation time on LP,, values for IVMsusceptible and IVM-resistant H. contortus isolates. L3 larvae were incubated in the dark except for 15 min periods of exposure to light each day during counting. LP, values are means f S.D. for two or more experiments. The isolates are McMaster (O-O), Tulbach (A-A) and White River II (O-O).
aged, making assessment of motility more difficult. Larvae less than 1 month old are preferred. IVM-naive isolates from Australia and South Africa were compared to assess the effect of genetic background and pre-existing resistance to other anthelmintic classes on IVM LP, values (Table 2). The BZ-resistant VRSG and BZ/LVS-resistant Lawes isolates were both found to be as sensitive to IVM as the McMaster isolate (Table 2), indicating no cross resistance between existing broad spectrum anthelmintics and the AVMs. The IVM LP, value obtained against the susceptible South African Onderstepoort
Ivertnectin resistance in H. contortus reference strain was also within the range of values obtained against the McMaster isolate. For IVM-resistant H. contortus isolates (e.g. White River II) a clear shift was observed in the IVM dose response profile (Fig. 3), indicating a reduced sensitivity compared to the McMaster isolate. IVM LP, values of 0.34.5 ,UM(Table 2) were observed for the IVM-susceptible isolates of H. contortus tested, while LP,, values from 0.8 to 2.6 PM, corresponding to resistance factors of 2.7-8.8, were recorded for the six IVM-resistant isolates examined. The effect of the number of motility cycles on the IVM LP,, values against IVM-resistant isolates was examined. As was the case with the McMaster isolate, a stabilization in IVM LP,, values was observed with all isolates after three motility cycles (Fig. 4). LP, values against all except the Tulbach isolate decreased from a maximum at 24 h to a constant value (e.g. White River II, Fig. 4). In contrast, the LP,, values for the Tulbach isolate increased to a peak at 48 h, which suggests that the mechanism responsible for the observed IVM resistance of this isolate may differ from that causing IVM resistance in the other South African isolates. The potency of a series of related AVMs as inhibitors of larval motility was examined (Table 2). Against the IVM-susceptible McMaster isolate the order of potency was: AVM B, = IVM > AVM B, > > IVM-monosaccharide, IVM-aglycone. For the latter two compounds less than 50% inhibition of motility was observed for susceptible isolates at 50 PM, the highest concentration tested. LP,, values recorded for AVM B, and AVM B, against the other IVMsusceptible isolates tested were within the range of values obtained for these compounds against the McMaster isolate (Table 2) with the exception that the Australian Lawes isolate appears slightly less sensitive to AVM B,. The rank order of potency of these compounds against the IVM-resistant isolates was the same as observed against the IVM-susceptible isolates, except that AVM B, was slightly less potent than IVM against the most resistant isolates, White River II and Tulbach. The largest resistance factors were obtained with the more polar AVM B,, which makes this the compound of choice for the detection of IVM resistance using this assay. These data suggest that resistance to AVMs in H. contortus is dependent on the structure of the analogue. Inhibition of L3 motility in vitro ranks these isolates in order of AVM resistance as: White River I < Swellendam, DAS, Stellenbosch, White River II < Tulbach. DISCUSSION
Under constant environmental conditions most nematodes undergo low rate, short duration periods of movement. If an environmental change occurs such as an increase in light intensity or a change in the temperature, a period of activity results (Croll & Matthews, 1977). When L3 larvae of H. contortus
775
which have been incubated in the dark are exposed to light a response can be elicited which can be made more uniform by conditioning the larvae through several light/dark cycles. This response to changes in light intensity has been exploited to allow the detection of AVM-induced paralysis in a larval motility assay. The assay is capable of detecting the resistance of a number of H. contortus isolates to IVM. L3 larvae are easily obtained in large numbers from faecal cultures and are normally quite hardy, making this a convenient stage in the parasite life-cycle with which to work. It is not fully understood why AVM LP,, values are dependent on the number of motility cycles. The increasing potency of the drug over the first three motility cycles (72 h) is more likely a reflection of a progressive impairment of the motor functions controlled by the site concerned, rather than a slower phase of accumulation of drug at receptor sites, since LP, values were dependent on the number of motility cycles and not the length of incubation. The reduction in potency of IVM against the Tulbach isolate at 48 h, which was not observed for other isolates, may reflect subtle differences in the manner in which the site of action regulates motor function. Australian and South African IVM-naive H. contortus isolates were equally sensitive to the paralytic effects of IVM. No cross resistance was detected between the AVMs and two other major classes of broad spectrum anthelmintics, LVS/morantel and the BZs. This is consistent with the observations of Kass et al. (1980), who found no difference in the response of LVS-susceptible and LVS-resistant C. eleguns lines to theparalyticeffects ofAVM B,, and with in vivaefficacy data (Wailer & Donald, 1983). These results support the hypothesis that the mode of action of the AVMs is independent of that of LVS, a known acetylcholine agonist (Lewis, Wu, Levine & Berg, 1980) and that of the BZs, which act by inhibiting the polymerization of tubulin (Lacey, 1988). Limited data were obtained on structure-activity relationships within the AVM class. The observed potency of the AVM analogues tested reflected in vivo efficacy against a number of nematode parasites in sheep (Chabala, Mrozik, Tolman, Eskola, Lusi, Peterson, Woods, Fisher, Campbell, Egerton & Ostlind, 1980). Hydration of the 22,23-double bond of AVM B, to produce AVM B, caused a two-fold loss in activity against the IVM-susceptible McMaster isolate; reduction of this bond to give IVM, although resulting in a similar change in the geometry of the spiroketal region, caused little change in activity. Loss of one or both of the oleandrose sugar moieties from IVM caused a greater than lOO-fold loss in potency against the McMaster isolate. Comparison between in vitro LP,, values for IVM (Table 2) and in vivo efficacy data (Table 1) gave a reasonable correlation for the H. contortus isolates tested. Provided there are parallels in the mechanisms of resistance it should be possible to extend the use of
116
J. H. GILL, J. M. REDWIN, J. A. VAN WYK and E. LACEY
this larval motility assay to the detection of IVM resistance in other important trichostrongylid parasites such as T. colubriformis and 0. circumcincta. While the site(s) of action of the AVMs remain(s) unknown, the results of this study of IVM-resistant isolates add further support to the hypothesis that the mechanism of AVM action in nematodes is associated with motor function. Acknowledgements-The authors would like to thank Dr Nicholas Sangster for the supply of larvae from the Lawes H. contortus isolate. The work reported here was supported by a grant from the Australian Wool Corporation (CSO7P). REFERENCES ANONYMOUS1986. Directorate of Veterinary Services, South Africa: Laboratory Services Monthly Reports, January, p. 4. BOISVENUER. J., BRANDTM. C., GALLOWAYR. B. & HENDRIX J. C. 1983. In vitro activity of various anthelmintic compounds against Haemonchus contortus larvae. Veterinary Parasitology 13: 341-347. CAMPBELL W. C., FISHER M. H., STAPLEY E. O., ALBERSSCHONBERGG. & JACOB T. A. 1983. Ivermectin: a potent new antiparasitic agent. Science 221: 823-828. CAMPBELLW. C. & BENZ G. W. 1984. Ivermectin: a review of efficacy and safety. Journal of Veterinary Pharmacology and Therapeutics 7: 1-16. CARMICHAELI., VISSER R., SCHNEIDERD. & SOLL M. 1987. Haemonchus contortus resistance to ivermectin. Journal of the South African Veterinary Association 58: 93. CHABALAJ. C., MROZIK H., TOLMANR. L., ESKOLAP., Lust A., PETERSONL. H., WOODS M. F., FISHER M. H., CAMPBELLW. C., EGERTONJ. R. & OSTLINDD. A. 1980. Ivermectin, a new broad spectrum antiparasitic agent. Journal of Medicinal Chemistry 23: 11341136. COLES G. C., TRITSCHLERJ. P. II, GIORDANOD. J., LASTEN. J. & SCHMIDT A. L. 1988. Larval development test for detection of anthelmintic resistant nematodes. Research in Veterinary Science 45: 50-53. CRAIG T. M. & MILLER D. K. 1990. Resistance bv Haemonchus contortus to ivermectin in angora goats. Veterinary Record 126: 580. CROLL N. A. & MATTHEWSB. E. 1977. Biology of Nematodes. Blackie, Glasgow. ECHEVARRIA F. A. M. & TRINDADE G. N. P. 1989. Anthelmintic resistance by Haemonchus contortus to ivermectin in Brazil: a preliminary report. Veterinary Record 124: 147-148. EGERTONJ. R., SUHAYDAD. & EARY C. H. 1988. Laboratory selection of Haemonchus contortus for resistance to ivermectin. Journal of Parasitology 74: 614617. FOLZ S. D., PAX R. A., THOMASE. M., BENNETTJ. L., LEE B. L. & CONDER G. A. 1987. Development and validation of an in vitro Trichostrongylus colubriformis motility assay. International Journalfor-Parasitology 17: 1441-1444. _ GIORDANO D. J., TRITSCHLERJ. P. II & COLES G. C. 1988. Selection of ivermectin-resistant Trichostrongylus colubriformis in lambs. Veterinary Parasitology 30: 139-148. GREENP. E., FORSYTHB. A., ROWAN K. J. & PAYNE G. 1981. The isolation of a field strain of Haemonchus contortus in Queensland showing multiple anthelmintic resistance. Australian Veterinary Journal 57: 79-84. HALL C. A., KELLY J. D., CAMPBELLN. J., WHITLOCKH. V. & MARTIN I. C. A. 1978. The dose response of several
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