Changes in anthelmintic resistance status of Haemonchus contortus and Trichostrongylus colubriformis exposed to different anthelmintic selection pressures in grazing sheep

CHANGES IN ANTHELMINTIC RESISTANCE STATUS OF HAE~ONCHU~ CONTORT~S AND TR~CHUSTRON~Y~~~ COLUBRIFORiWS EXPOSED TO DIFFERENT ANTHELMINTIC SELECTION PRESSURES IN GRAZING SHEEP P. J. WALLER,* A. D. DONALD,* R. J. DOBSON,* E. LACEY,* D. R. HENNESSY,” G. R. ALLERTON*

and R. K. PRICHARD~ “CSIRO Divisionof Animal Health,McMasterLaboratory,Glebe, NSW 2037, Australia thrstitute of Parasitology, McGill University, Macdonald College, Ste Anne-de-Bellevue, Quebec, Canada H9X 1CO (Received 1 fune 1988; accepted 1 September 1988)

Abstract-WALLERP. J., DONALD

A. D., DOESON R. J., LACEYE., HENNESSY D. R., ALLERTON G. R. and PRICHARDR. K. 1989. Changes in anthelmintic resistance status of Haemonchus contortus and Trichos~ro~~Ius ~olubriform~ exposed to different anthel~ntic seiection pressures in grazing sheep. internutionuf Jolourna! for Parasitology 19: 99-l 10. This experiment was designed to study, over a 5-year-period, the effect of different frequencies of treatment with three different anthelmintic groups, namely, benzimidazoles, levamisole and ivermectin, and different frequencies of ~ternation between them, on existing levels of anthehnintic resistance in the nematode parasites Haemonchus contortus and Trichostrong&s cofubriformis of grazing sheep. No evidence of ivermectin resistance emerged, even in suppressively treated groups. Likewise, H. contortus failed to develop resistance to levamisole under a similar selection regimen. Thiabend~ole was shown to seleot positively against ievamisole resistancein T. colzcbriform& resulting in significantly greater susceptibility to this drug than for the natural reversion which occurred in the untreated control. There was no evidence that an anthelmintic treatment combined with a movement of sheep to pastures of low infectivity selected more rapidly for resistance than where the same number of treatments were given to set-stocked sheep. Rotation between ~thel~ntic groups at yearly intervals appeared to be more beneficial in delaying resistance than rotation of drugs with each treatment.

INDEX KEY WORDS: Anthelmintic resistance; Haemonchus contorfus; Trichostron~l~ colubriformis; levamisole; benzimidazoies; ivermectin; reversion; counterselection; slow rotation; rapid rotation; treat and move.

INTRODUCTION

particular importance to the grazing industry in Australia, not only because of great breadth of spectrum and potency (Campbell & Benz, 1984), but also because they have been shown to be highly effective against both benzimidazole and levamisole/ morantel resistant sheep nematodes (Wailer & Donald, 1983). However, there is no reason to assume that resistance to these drugs could not develop, and therefore it is important to recognize those practices that are likely to facilitate and those that will delay the emergence of resistance to this class of compounds. At present, strategies recommended to minimize ~thel~ntic resistance include the use of one anthelmintic until it fails (Le Jambre, Southcott & Dash, 1977) followed by a change to another, and a slow rotation usually recommended at yearly intervals, between anthel~ntics with different modes of action (Prichard, Hall, Kelly, Martin & Donald, 1980). Also, it is generally considered that rapid alternation between anthel~ntic groups hastens the development of multiple resistance (for review see Donald &

ANTHELMINTIC resistance is the greatest threat to the continuing successful control of nematode parasites of sheep in Australia. Resistance to the benzimidazole and levamisole/morantel groups of broad spectrum anthelmjntics is widespread and at a high level in the three most important nematode genera of sheep, namely Haemonchus, Ostertagia and Trichostrongylus (for review, see Wailer, 1986). Sheep producers are becoming increasingly aware that their conventional anthelmintic treatment practices are not achieving the desired level of worm control and animal production benefits (Newman, 1984), and also that the various options for anthehnintic use are diminishing (Dash, Newman & Hall, 1985). Despite these problems, anthelmintics will remain the mainstay of nematode control because other non-che~ca~ methods such as helminth vaccines and breeding resistant animals are unlikely to be a commercial reality in the foreseeable future (Donald, 1985). Therefore, the avermectin class for compounds is of 99

P. J. WALLERrf al.

100

Wailer, 1982). These strategies have not been simultaneously evaluated and there has been very little experimental work on selection for anthelmintic

(‘Nilverm’, I.C.I. Australia Ltd) resistance, the return of benzimidazole (BZ) resistance and the development of IVM resistance. Two control treatments were included, namely, Treatment 1 which was the untreated control and Treatment 2 (b, iv) which maintained LEV selection pressure similar to that existing before the experiment. In addition, Treatment 2 (a, iii) was designed to test the hypothesis that anthelmintic treatment combined with a movement to ‘safe’ pasture selects strongly for resistance, and Treatment 2 (b, iii) was included Lodetermine whether the use of a newer generation and more effective BZ, namely, aibendazole (ABZ) (‘Valbazen’, Smith Kline Animal Health (Aust.) Ltd) would delay the return of BZ resistance. Each plot remained allotted to a treatment throughout the experiment but the sheep were replaced with spring-born weaners each year. Each plot contained its own water supply ‘and was isolated from adjacent plots by a buffer zone with a minimum width of 2 m, to prevent cross-contamination. This was also minimized by carrying out all sheep handling operations, e.g. faecal sampling, weighing and crutching within the plots using portable scales and disposable boot-covers worn by operators. Changes in anthelmintic resistance in each treatment group were monitored by faecal egg counts before and after anthelmintic treatment, accompanied by faecal culture and larval differentiation. In addition, samples of the parasite populations were obtained from each treatment in November 1984, 198.5 and 1986 and were tested for BZ, LEV and IVM resistance by in vitro methods. In vivo dose-response assays were also carried out on the initial parasite populations and on selected lines at the beginning and end of the experiment, respectively.

resistance in the field, where the effects of other selective agencies and the overall fitness of resistant phenotypes can be expressed. This experiment, which was designed to extend over 5 years, studied the effect of different frequencies of anthelmintic treatment and of ~temation between different anthel~ntic groups on existing levels of resistance in nematode parasites of sheep. EXPERIMENTAL DESIGN At the CSIRO McMaster Farm, Badgery’s Creek, New South Wales, thiabendazole resistance was first detected in the late 1960s in Haernonchus contortus and Trichostrongylus colubriformis parasites of sheep. Since 1973 only levamisole has been used routinely. The experiment reported here was performed on the farm from January 1982 to December 1986. At the commencement, theexperimentai area carried a reasonably uniform pasture composed predominantly of perennial ryegrass (Lnlium perenne), Phalaris aquutica and white clover (Trifoliurn repens). The pastures had been contaminated with the local parasite fauna by sheep grazing during the previous spring and early summer. The design of the experiment is summarized in Table I. The primary experimental unit was a 0.4 ha plot for each of the two replications of 12 treatments. Excluding Treatments 1,2 (a, iii), 2 (b, iii) and 2 (b, iv), the experiment was a balanced factorial design testing the effect of drenching frequency with, and the frequency of alternation between, ivermectin (IVM) (‘Ivomec’, Merck, Sharp & Dohme (Aust.) Ltd) and thiabendazole (TBZ) (‘Thibenzolc’, Merck, Sharp & Dohme (Aust.) Ltd) on the decline in levamisole (LEV) TABLE

~-DESIGN

MATERIALS AND METHODS Management. Each of the 24 plots was stocked in January

of each year with six spring-born Merino weaners, reducing to five weaners when pasture growth declined with the onset OF THE EXPERIMENI

Treatment 1. No treatment 2. Continuous use of the same drug (set stocked) (a) 3;;e;y;*ts/year (strategic) (il) TBZt (iii) TBZ-moved to ‘safe’ pasture with each treatment (b) 8~~~~ts/year (suppressive)

1

(ii) TBZ (iii) ABZS (iv) LEV§ 3. Drugs alternated at each treatment (rapid alternation) (a) 3 trea;E;yy (strategic) (b) 8 tre;rGstsar

Plots

Replicates

2

2

2

2

4

2

2 2 2 2

2 2 2 2

2

2

2

2

2

2

2

2

(suppressive)

4. Drugs alternated each year (slow alternation) (a) 3 tre~~~~~ (strategic) (b) 8 tre~~~~~ *IVM, Ivermectin; levamisole.

(suppressive) ITBZ, thiabendazole;

SABZ, albendazole;

$LEV,

Worm control and anthelmintic resistance

of winter each year. Supplement~y feeding with grain and hay was necessary from June 1982 until April 1983 because of drought, and also during the winters of 1984 and 1986. Warm, moist conditions during the autumn months of each year, except 1983, resulted in clinical haemonchosis in many treatments. The sheep were inspected daily and any that were unable to travel more than 200 m at walking pace were classed as ‘dead’. They were removed from the experiment and given appropriate anthehnintic treatment. In an attempt to prevent ‘deaths’, one-two doses of nap~~ophos (‘Rametin H’, Bayer Australia Ltd) at 30 mg kg-‘, or rafoxanide (‘Ranide’, Merck Sharp & Dohme (Amt.) Ltd) at 20 mg kg-‘, were administered to sheep in all treatments during late summer/autumn in all years except 1983. On plots where ‘deaths’ occurred, grazing pressure was maintained by the addition of worm-free sheep of similar age and class as the experimental sheep. From these replacement sheep, faecal samples were taken and body weights were recorded only after they had been on the plots for at least 2 months. In the suppressive groups, ~thelmintic was administered approximately every 6 weeks, with seven-eight treatments given each year except during the drought year of 1983 when only three treatments were given. A single anthelmintic treatment was given in autumn, winter and spring in each of the strategic treatments. Sheep in the strategic dose-and-move treatment 2(a, iii) were swapped between two plots following each anthelmintic dose, with pasture growth on the unstacked plots kept to a comparable level as the stocked plots by mowing when necessary. All antheimintics were used according to the manufacturer’s dose rate, namely thjabend~ole at 44 mg kg-‘, levamisole at 7.5 mg kg-‘, albendazole at 3.8 mg kg-’ and ivermectin at 0.2 mg kg-‘. Replacement sheep from a spare mob were given both levamisole (15 mg kg-‘) and albendazole (7.6 mg kg-‘) within 24 h of being placed on the plots; subsequently they received the same anthelmintic treatments as the experimental sheep on their plot. All lambs were weighed at the time of the suppressive drenching treatment. Monitoring anthelmintic resistance status. In December 198 1, before the experiment began, a sample of the parasite populations was obtained by bulk culturing faeces collected from 10 infected sheep. Third stage larvae from this source were used to infect two worm-free lambs. After 4 weeks both lambs were slaughtered and H. contortus and T. colubriformis, which were by far the most prevalent species, were separately recovered and each species surgically implanted into a worm-free lamb. Faeces from these lambs were cultured to yield larvae for an in vivo dose-response assay for TBZ, LEV and IVM. Fifty-four worm-free lambs were each given 5000 H. contortus and 6000 T. colubriform~ infective larvae. The lambs were randomly allocated to treatment groups with four lambs at each of four dose rates for each anthelmintic and six lambs serving as untreated controls. Anthelmintic doses according to individual liveweight were given 4 weeks after infection by intraruminal injection. with controls receiving 5 ml of water. Lambs were slaughtered 4-5 days later for worm recovery and estimation of total numbers present by the methods of Donald, Morley, Wailer, Axelsen & Donnelly (1978). Throughout the experiment, the effectiveness of anthehnintic treatments in each group was monitored by differential faecal egg counts (Donald et nl., 1978) on five sheep per plot, the same sheep being sampled on each occasion. Faecal samples were taken on the day treatment was given, and again lo-14 days later for all groups. In addition, the strategic groups were sampled in late winter and early summer of each year. Control groups were sampled at the same time as the strate-

101

pie groups. In November 1984,1985 and 1986, H. contortus and T. colubriformis populations were isolated from each treatment by grazing plots with lambs that had been reared ureviouslv in pens for a minimum of 4 weeks after being tieated with double the manufacturer’s dose rate of both TBZ and LEV. Lambs grazed for 2 weeks on each replicate plot for each treatme& and were then held in pens for an additional 2 weeks before slaughtering. H. contortus and T. ~o~ubr~fo~~ were separately recovered from each lamb and surgically implanted into worm-free lambs. Faeces from these 24 recipient lambs, representing H. contortus and T. colubriformis pure infections from each of the 12 treatments, were collected to obtain stocks of both eggs and infective larvae for in vitro anthelmintic assays. These were the tubulin binding assay, described by Lacey & Snowdon (1988), carried out on infective larvae to determine BZ resistance, the egg-hatch assay of Dobson, Donald, Waller & Snowdon (1986) to estimate LEV resistance and the larval culture assay (Wailer & Lacey, 1986) which was performed on the 1986 isolates only, to detect the possible emergence of IVM resistance. In vivo dose-response assays were carried out on H. contortus and T. coiubriform~ populations derived from various treatment groups in November 1986. Two hundred and twenty-five worm-free lambs were randomly allocated to assays of these isolates and were each infected with 3500 H. contortus and 4000 or 10,000 T. colubriformis infective larvae of the appropriate line. Procedures described for the in vivo assay on the initial parasite isolates were used, except that five sheep were used both for the untreated control and the treatment groups. Stutist~~al methods. The results of egg hatch assays were fitted by a mixed logit model, describedby Waller, Dobson, Donald. Griffiths &Smith c19851. and estimates of nercentage resistance in a mixed population and EC,,, of tesistant and susceptible sub-populations, together with standard errors for each of these parameters, were obtained from this fit. Data from the tubulin binding assays were presented as susceptibility factors. These were calculated as the ratio of the [W’] mebendazole (MBZ) binding (in p moles per mg crude protein) of the treatment isolates to that of the known BZ susceptible isolates (McMaster strains, Lacey & Snowdon. 19881. Data were derived from a burns of four binding determinations. For the egg hatch, tubulin binding and ivermectin in vitro assays, there was no significant difference between variance estimates for individual determinations of resistant sub-populations, susceptibility factors and EC+ Thus a pooled variance estimate was calculated which gave a standard error of 4.83%, 0.045 and 0.046 for resistant sub-population, susceptibility factor determinations and EC,,+, respectively. (Least significant differences (lsd) for P = 0.05 were determined from the pooled variance estimates.) It should be noted that estimates of error associated with the assay techniques do not incorporate between-replicate (within-treatment) variability as parasite strains used in the assays were derived from treatments in which isolates from each replicate were pooled. RESULTS resistance at the commence-

of anthelrnintic ment of the experiment

Levels

Results of in vivo dose and slaughter assays of IVM, TBZ and LEV against the parent H. ~~ntortMs and

T. colubriformis populations are shown in Tables 2 and 3, respectively. Resistance to thiabendazole was recorded in H. contortus, with a 72% reduction in worm numbers at the recommended dose rate

102

P. J. TABLE

2-h

vivo ANTHELMINTIC

WALLER

el al.

ASSAYS* AGAINST

H. contortus POPULATIONS

ISOLATED AT

THE COMMENCEMENT OF THE EXPERIMENT AND FOR CERTAIN TREATMENTS IN DECEMBER

Mean worm numbers

Control 2650

Initial isolate 198 1 Treatment isolates Untreated 3TBZ 3TBZ and move BTBZ BIVM BLEV

Initial isolate Treatment Untreated BTBZ

reduction

TBZ 5.5 concentra$x 11 8 46 57

‘) (rn;cg 72

88 -

91 42 5 0 61 61

96 73 77 79 81

1986 970 1200 525 2000 9.50 1210

Control 2650

198 1

isolates

Percentage

1986

-

-

-

-

-

-

LEV concentration 0.28 0.83 2.5 54 96 100

(mg kg-‘) 7.5t 22.5 100 100

1986

Initial isolate 198 1 Treatment isolates 1986 Untreated 31VM 81VM SIVM/TBZ/treatment BIVM/TBZ/year

1570 2000

Control 2650 1570 850 560 1360 1587

*Sheep numbers: 6/control, 4/treatment, TManufacturers’ recommended dose.

(44 mg kg-‘) and an estimated ED,, of 129.7 mg kg-‘. This isolate was shown to have a high level of susceptibility to both ivermectin and levamisole. The T. cofubriformis isolate was found to be highly resistant to levamisole, showing no reduction in worm numbers at three times recommended dose rate the (22.5 mg kg-‘). It was highly susceptible to both ivermectin and thiabendazole. Faecal egg counts

Although anthelmintic treatment was carried out approximately every 6 weeks in the suppressively treated groups, both H. contortus and T. colubriformis were able to complete development from the infective larva to egg-laying adult within this interval. This was shown by the persistent positive egg counts for both species in lambs from the IVM suppressive, set-stocked treatment, despite the 100% efficacy in egg count reduction produced by this drug. However, as expected, the overall level of egg counts was much lower in the IVM intensivelv-treated grouts than in all other treatments. Egg counfs were hi&es;in the newly allocated lambs in autumn, due principally to H. contortus, tending to stabilize in winter before falling in

-

-

-

IVM concentration 0.0075 0.015 0.03 46 93 97

198 1 assay; S/control,

77 69 0 37 29

98 95 60 >99 91

S/treatment,

100 100

-

(mg kg-‘) 0.06 0.2t 100 >99 98 >99 >99

100 100 100 100 >99

1986 assay.

spring when the sheep exceeded 12 months of age. The percentage reduction in post-treatment faecal egg count is shown in Figs. la-f to illustrate changes in resistance status of both H. contortus and T. colubriformis over the course of the experiment. Strategic treatment groups. The percentage reductions in faecal egg counts of the three groups treated continuously with IVM, TBZ or TBZ accompanied by a move to safe pasture are shown in Figs. la and b. IVM remained highly efficacious against both species for the duration of the experiment. There was no indication of any consistent differences between the TBZ set-stocked and the TBZ dose-and-move treatments for either species. Suppressive treatment groups. Faecal egg count reductions of the four set-stocked suppressivelytreated groups are shown in Figs. lc and d for H. contortus and T. colubriformis, respectively. Thirty-one anthelmintic doses were given to sheep in each of these groups over the 5 years of study. IVM was highly effective against both species, with virtually 100% reduction in faecal egg counts in lambs on all occasions. Likewise, LEV remained highly efficacious against H. contortus throughout, whereas in most

103

Worm control and anthelmintic resistance TABLE 3-h vivo ANTHELMINTIC ASSAYS*AGAINSTT. colubrijiormisPOPULATIONS ISOLATED AT THECOMMENCEMENT OF THEEXPERIMENT AND FORCERTAINTREATMENTS IN DECEMBER 1986 Mean

Percentage reduction

worm numbers

Initial isolate 198 1 Treatment isolates 1986 Untreated 3TBZ 3TBZ and move BTBZ BIVM BLEV

Initial isolate 198 1 Treatment isolates 1986 Untreated BTBZ

Initial isolate 198 1 Treatment isolates 1986 Untreated 31VM BIVM BIVM/TBZ/treatment BIVM/TBZ/year

Control 3320 2650 2750 1588 7670 2490 4760

Control 3320 4960 7670 Control 3320 4960 2450 2290 2380 1380

TBZ concentration (mg kg-‘) 5.5 11 22 44t 88 8 83 88 96 -

-

-

-

-

88 51 35 0 88 88

100 100 100 100 100

LEV concentration (mg kg-‘) 0.28 0.85 2.5 7.5t 22.5 1 3 11 14 2 -

-

60 84

-

IVM concentration (mg kg-‘) 0.0075 0.015 0.03 0.06 0.2t 2 25 78 100 -

14 10 0 0 57

52 95 49 20 97

97 97 98 99

100 100 >lOO >99 100

*Sheep numbers: 6/control, 4/treatment, 1981 assay; S/control, S/treatment, 1986 assay. ‘FManufacturers’ recommended dose. instances it failed to reduce egg counts of T. colubriforma. Efficacy of TBZ against T. colubrtformis exceeded 90% in 1982, declining to a plateau which fluctuated between 70 and 80% reduction in egg counts for the following 18 months. During this time the majority of TBZ treatments failed to reduce the faecal egg counts of H. contortus by more than 50%. From the latter half of 1984, faecal egg count reduction of both species was similar, showing an irregular pattern which reflected an overall poor level of efficacy of this drug. ABZ was highly effective against both species during 1982 and 1983 but showed a decline over the following 2 years to a poor level of efficacy in 1986. Drug alternation groups. Faecal egg count reductions of the rapid and slow alternation, strategic and suppressively treated groups are shown in Figs. le and f for H. contortus and T. colubriformis, respectively. Slow alternation between IVM and TBZ appeared to be more effective than rapid alternation in the reduction by TBZ of H. contortus egg counts of lambs. This was most evident in the suppressive treatment groups where in the 2 years that TBZ was used in the yearly rotation with IVM, faecal egg count reduction was

mainly greater than 50%. However, when TBZ was alternated with IVM at each treatment it usually had no effect, resulting in a saw-tooth pattern in faecal egg count reduction between 100% and zero efficacy when IVM and TBZ were used respectively (see Fig. le). This difference between rapid and slow alternation was not as apparent for T. colubriformis (see Fig. lf). Faecal egg count reduction following TBZ treatment exceeded 75% until the latter half of 1985 in the rapid alternation suppressively-treated groups. It was during this year that a marked reduction in efficacy of TBZ in the slow rotation suppressive groups was also recorded. In vitro assaysfor anthelmintic resistance Benzimidazole resistance. Results of the tubulin binding assay to determine levels of benzimidazole resistance in both H. contortus and T. colubriformis isolates from all treatments are shown in Table 4. Tubulin extracts from resistant strains bind substantially less drug than those from susceptible strains. As expected, the highest rate of return of BZ resistance and greatest absolute levels of benzimidazole resistance occurred in both the H. contortus and

P. J. WALLER et al.

104

T.colubriformis

Hxontortus use - 3 treatments

Continuous (al loo-

/ year

(strategic) Ibl

0

=

L

0

-z 3 IVM

'0 505 0 *

6

i’“l

i

r---w

loo-

3

TBZ

+

so-

Continuous

use - 8 treatments

(cl

/ year

(suppressive)

p

loo-o---a-.=

a

50-

IVM

6 . = loo2 p 50L ?k m ‘ooE 0 50e 0

oloo- ---50-

Alternation

=

---P 8 LEV

treatments [fl

(el 100

;roo G 2 50

- strategic

11

- s.uPPrassiva

Rapid

: 2100 : E

r- Rapid

50 c .o

alternation

alternation

50

am 100 50

Slow alternation ,982

:983

1984

- suppressive 1986

1986

FIG. 1. Percentage reduction in faecal egg count of H. contorfus and T. colubriformis quencies of treatment and alternation of anthehnintics from 1984 to 1986 inclusive. thiabendazole; w, levamisole.

in sheep receiving different freIvermectin; A, albendazole; l,

0,

Worm control TABLE

r

~-VARIATION

colubriformis

(NOTE-THE

IN

ISOLATES

in-vitro

and anthelmintic

resistance

BZ ~USCEPT~~L~TYFACTORS

FROM EACH EXPERIMENTAL

LOWER THE SUSCEPTEIIL~TY

105 OF

TREATMENT

H. contortus AND

FROM

1984 TO 1986

FACTOR THE GREATER THE LEVEL OF

H.

BZ

RESISTANCE)

T. colubriformis

contortus

Treatment

1984

1985

1986

1984

1985

1986

1. Nil 2. Continuous use of same drug (a) 3 treatments/year (i) IVM (ii) TBZ (iii) TBZ and move (b) 8 treatments/year (i) IVM (ii) TBZ (iii) ABZ (iv) LEV 3. Drugs alternated each treatment (a) 3/year (IVM/TBZ) @) 8/year (IVMRBZ) 4. Drugs alternated each yeart (a) 3/year (IVM/TBZ) @) S/year (IVM/TBZ)

0.71

0.74

0.82

0.69

0.52

0.56

0.80 0.54 0.44

0.69 0.41 0.41

0.74 0.36 0.33

0.67 0.48 0.30

0.62 0.32 0.23

0.66 0.22 0.45

0.65 0.35 0.59 0.80

0.78 0.29 0.50 0.70

0.74 0.43 0.37 0.68

0.55 0.47 0.67 0.66

0.79 0.20 0.41 0.55

0.66 0.24 0.33 0.58

0.66 0.30

0.60 0.46

0.41 0.33

0.72 0.37

0.47 0.35

0.64 0.58

0.66 0.71

0.65 0.41

0.68 0.45

0.62 0.65

0.42 0.49

0.56 0.51

1.0

1.0

1.0

1.0

1.0

1.0 -

McMaster

Susc.

Data are presented as the mean of susceptibility ratio. Data that differ by more than 0.13 (the Isd) are significantly different at P< 0.05. All data are derived from a minimum of four observations. iIVM was used in even numbered years.

T. colubriformis lines isolated

from

the suppressively

treated TBZ group. In contrast, resistance developed more slowly with ABZ given at the same frequency of treatment. The dose-and-move treatment appeared to select rapidly for resistance judged by the results in 1984. However, resistance in both species isolated from sheep treated the same number of times, but setstocked (TBZ three treatments/year), subsequently increased rapidly to a comparable level to the doseand-move treatment. Although rapid alternation hastened the selection for resistance, this only occurred in the suppressively treated groups. As expected, the level of benzimidazole resistance remained low in the intensively treated, slow rotation group during 1984 when IVM was used. However, resistance increased rapidly during 1985 when TBZ was used, resulting in little difference in BZ resistance in both H. contortus and T. colubriformis populations isolated from either of the alternation frequencies. A marked increase in resistance occurred in H. contortus isolated from the rapid alternation strategic treatment in 1986, but apart from this, levels of resistance of the rapid and slow alternation strategic groups remained comparable to each other for both species. Levamisole resistance. The EC,,, estimates for H. contortus isolates from all treatments were very

low, with no evidence of resistance emerging in the LEV suppressive group isolated in either 1985 or

1986 (Table 5). The T. colubriformis population at the commencement of the experiment was shown to consist of two distinct sub-populations: one susceptible (30%) and the other highly resistant to LEV (70%). Estimates of the highly resistant portion of the population for each treatment from 1984 to 1986 inclusive are shown in Table 5. The highly resistant fraction of the suppressively treated LEV line remained consistent with the level of the initial isolate, fluctuating about a mean of approximately 75%. This fraction showed a significant and rapid reduction to 30% of the T. colubriformis population isolated from the untreated control groups in 1984, thereafter remaining at this level. A further significant reduction in the percentage of the population highly resistant to LEV was recorded in the suppressively treated TBZ group in 1985, and also in the following year for both the suppressive and strategically treated TBZ groups. Ivermectin resistance. Tests for IVM resistance were carried out on standard reference strains and populations isolated in 1986 from untreated controls, all the IVM treatments and the suppressively treated LEV isolate for both H. contortus and T. colubriformis (Table 6). There was no evidence of resistance to IVM emerging in any treatment for either species. Levels of anthelmintic resistance at the end of the experiment Results of the in vivo anthelmintic assays carried out on the H. contortus and T. colubriformis 1986

P. J. WALLER et al.

106

TABLE ~-VARIATION IN in-vitro LEVAMISOLERESLSTANCELEVELSIN H. contortus AND T cofubriformis ISOLATESFROMEACH EXPER~MENTALTREATMENTFROM 1984 TO 1986

H. contortus” 1986

1984

1985

1986

0.18

(30)

(30)

(36)

0.50 0.20 0.17

0.08 0.07 0.04

(30) (45) (50)

(21) (31) (30)

(;:I

0.11 0.15 0.21 0.19

0.09 0.14 0.21 0.23

(40) (35) (45) (75)

(47) (14) (23) (66)

(“,$

0.20 0.22

0.09 0.16

(55) (60)

(32) (39)

(12) (29)

0.18 0.25

0.13 0.12

(35) (65)

(22) (44)

(19) (38)

0.14

0.19

(0)

(0)

(0)

Treatment

1985

1. Nil 2. Continuous use of same drug (a) 3 treatments/year (i) IVM (ii) TBZ (iii) TBZ and move (b) 8 treatments/year (i) IVM (ii) TBZ (iii)ABZ (iv) LEV 3. Drugs alternated each treatment (a) 3/year (IVM/TBZ) (b) 8/year (IVM/TBZ) 4. Drugs alternated each year+ (a) 3/year (IVM/TBZ) (b) S/year (IVM/TBZ)

0.1

McMaster

Susc.

T colubriformist

1

(11)

(15) (89)

*Data are presented on the interpolated EC,,, @g ml-’ value derived according to the method of Wailer et al., 1985). H. contortus not examined in 1984. tThe proportion of highly LEV resistant larvae are estimated according to the model proposed by Wailer et al. (1985). Percentages that differ by more than 14% (the Isd) are significantly different (P< 0.05). $IVM was used in even numbered years.

TABLE ~-VARIATION IN in-vitro IVERMECTFN RESISTANCE LEVELS IN H contortus AND i? colubriformis ~SOLATEDFROMEXPER~MENTAL TREATMENTS IN 1986

H. contortus*

T. colubriformis+

1. Nil 2. Continuous use of same drug (a) 3 treatments/year (i) IVM (ii) TBZ (iii) TBZ and move (b) 8 treatments/year (i) IVM (ii) TBZ (iii) ABZ (iv) LEV 3. Drugs alternated each treatment (a) 3/year (IVM/TBZ) (b) g/year (IVM/TBZ) 4. Drugs alternated each year+ (a) 3/year (IVM/TBZ) (b) 8/year (IVM/TBZ)

0.020

0.066

0.022 -t

0.036

0.038 -

0.061

0.023

0.048

0.036 0.022

0.012 0.059

0.042 0.025

0.055 0.060

McMaster

0.017

0.050

Treatment

Susc.

-

*Data are presented as EC,,, values (,~g ml-‘). Data that differ by more than 0.13 (the Isd) are significantly different at P< 0.05. tNot tested. $IVM was used in even numbered years.

Worm control and anthelmintic resistance isolates from the control and the TBZ and IVM suppressive, set-stocked treatment are shown in Tables 2 and 3, respectively. IVM at the recommended dose rate was 100% effective against both species derived from the IVM suppressive treatment. Selection with TBZ resulted in a return to LEV susceptibility in T. colubriformis, with an 84% removal following administration of LEV at the recommended dose rate compared to 14% removal in the parent strain in 1982. However, suppressive TBZ selection produced a high level of resistance to this drug in both H. contortus and T. colubriformis. Levels of TBZ resistance in the strategic set-stocked and dose-and-move treatments also reached unacceptable levels from the control standpoint. Production data

Because of the small number of original sheep on some plots, largely as a result of clinical haemonchosis in autumn of all years, except 1983, comparison between treatments overestimated the performance of those treatments in which many ‘deaths’ occurred. Mean sheep live weights at the end of each year, and over the entire experiment, together with a ranking on performance, are shown in Table 7. Overall, the IVM suppressively treated sheep and the ‘dose-and-move’ sheep performed the best. Likewise, sheep ‘deaths’ largely reflected live weight gain (Table S), with the suppressively treated sheep and dose-and-move treatments having the lowest mortalities. DISCUSSION The levels of anthehnintic resistance of H. contortus and T. colubriformis populations at the commencement of the experiment were consistent with the expectations of Le Jambre et al. (1977) who suggested using one anthelmintic for as long as it remained effective. By implication, a change is then made to another drug (strictly drug group) until that, in turn, fails. In effect, this process had been completed, firstly with the BZ group and then the LEV/ MT group of anthelmintics at the McMaster Farm. Isolates made at the commencement of the study showed that a high level of resistance to LEV had developed in T. colubriformis. However, this species had reverted to BZ susceptibility. H. contortus, on the other hand, had retained a marked resistance to BZ despite the drug having been withdrawn from use for 8 years, but had remained fully susceptible to LEV. Although multiple resistance did not exist for either parasite species, effective control presented a difficulty as both species were abundant and each showed a high level of resistance to one or other of the commercially available broad spectrum groups. Similar situations are commonly arising on commercial properties in Australia (Wailer, 1986). Ivermectin represented an entirely new group of anthelmintics, against which resistance had not been specifically

107

selected. Thus, there is an urgent need to determine the best, or worst, strategies for the use of this drug. As expected, the highest rate of return to BZ resistance and highest levels of BZ resistance occurred in both the H. contortus and T. colubriformis lines isolated from the suppressively-treated TBZ group. It is of interest to note that ABZ given at the same frequency delayed the development and magnitude of resistance, which may be attributed to the higher efficacy of this drug (see Figs. lc, d), resulting in fewer survivors following treatment to pass on their resistance genes to subsequent generations. Such findings have not been previously recorded for nematode parasites, but they are in general accord with the model, described by Muggleton (1986), who investigated various strategies to control the stored product insect pest, Oryzaephilus surinamensis. Despite the failure of TBZ soon after its reintroduction, this drug was shown to counterselect against LEV resistance in T. colubriformis. Resistance was significantly lower in the three and eight TBZ/year groups than in the population isolated from the untreated control group by the end of the experiment (Table 5). These findings confirm the laboratory selection studies on this strain isolated at the commencement of the study and reported in detail by Waller et al. (1985). Although LEV resistance was at an extremely high level at the start, shown by the lack of effect of LEV even when administered at three times the recommended dose rate, susceptibility alleles were present in the population. This conclusion was supported by in vitro estimates of resistance which showed that the strain consisted of two distinct sub-populations: one susceptible and the other highly resistant to LEV, and also that in the absence of LEV selection, susceptible genotypes had a survival advantage over individuals carrying resistant genes. This finding is of considerable practical importance, because it suggests that alternating different drug groups may prevent very high levels of resistance from developing, thus prolonging the practical usefulness of such drugs. Throughout the experiment NM was highly effective in controlling both H. contortus and T. colubriformis, even in the suppressively treated groups which were included as the treatment most likely to give not only the greatest measure of parasite control, but also the greatest opportunity to select for resistance to this drug. Faecal egg count monitoring of sheep showed no evidence of resistance following IVM treatment which was also supported by in vitro assays. Comparison between in vivo assays of IVM against isolates of both species taken at the beginning and end of the experiment showed 100% efficacy of IVM at 0.2 mg kg-‘, the recommended dose rate, on both occasions. There was, however, a greater survival of both H. contortus and T. colubriformis at 0.03 mg kg-’ in the 1986 isolates, but it must be recognized that this represents only 15% of the recommended dose rate and it is reasonable to expect fluctuations in efficacy when the

P. J. WALLERn ni.

108

TABLE7---MEAN

SHEEP

BODY

WEIGHTS

(KG)

AT DIFFERENT MEANS

TIMES AND

DURING

986

1%2-l

INCLUSIVE

TOGETHER

WITH

OVERALL

RANKING

Treatment

1982

1983

1984

1985

1986

Overall mean

Rank

1. Control 2. Continuous use of same drug 3 lVM/year 3 TBUyear 3 TBZ/year (move) 8 IVMiyear 8 TBZ/year 8 ABZ/year 8 LEV/year 3. Drugs alternated each treatment 3 IVM-TBZ/treatment 8 IVM-TBZ/treatment 4. Drugs alternated each year 3 IVM-TBZiyear 8 IVM-TBZ/year Means calculated from final weighing date Means include the sheep in trial from this date

25.6

42.4

26.9’

31.5

23.7

30.0

IO

26.8 26.8 30.3 28.5 26.2 28.5 25.8

43.0 40.8 41.3 45.5 41.3 41.2 42.2

27.1 29.7 35.5 35.4 28.9 32.6 29.7

27.0 32.1

22.7 24.4

29.3 30.7

12 6

33.2 33.6 31.6 31.1 31.7

27.5 22.8 24.4 23.3 22.0

33.2 33.5 30.5 31.3 30.3

: 8 5 9

27.9 29.7 IVM 28.3 27.8

44.7 42.5 TBZ 39.4 41.6

36.4 32.2 IVM 30.4 29.5

31.2 31.2 TBZ 27.8 31.3

22.3 21.5 IVM 22.9 22.8

32.5 31.5

3 4

29.8 30.6

I1 7

29.9.82

22.11.83

9.10.84

24.10.85

4.9.86

22.7.82

t

15.584

257.85

10.7.86

*One plot only; tNo deaths this year.

drug is administered at such a low dose rate. Although 5 years of suppressive IVM treatment was an extreme test in selection for resistance, its failure to emerge in this study does not mean that it will not occur in other environments involving other parasite ecotypes. Unfortunately this appears to be the case in South Africa where field reports of IVM resistance in H. contortus following intensive use of this drug have emerged (van Wyk, Malan, Gerber & Alves, 1987). It is likely that the parasite strains in our study lacked the genetic diversity to develop fVM resistance. This is also apparent for H. con~o~~s which failed to develop LEV resistance despite being exposed exclusively to this drug for 9 years prior to the commencement of TABLE

8-SHEEP

‘DEATHS’

PROM

1982 to 1986

the study and subsequently for 5 years in the LEV suppressive treatment. Similar findings elsewhere contrast with the prevalence of BZ resistance in this species, which comprises the majority of field reports of resistance throughout the world (Wailer, 1986). The dose-and-move treatment was included to specifically investigate the misgivings expressed initially by Le Jambre (1978), that anthehnintic treatment combined with movement to pastures of low infectivity would select more potently for resistance than would occasional treatments given to sheep on contaminated pastures. This dose-and-move principle forms the basis of many integrated parasite control schemes and provides the foundation for efficient, INCLUSIVE,

TOGETHER

WITH

OVERALL

TOTAL

AND

RANKING

Treatment 1. Controls

1982

1983

1984

1985

25

0

10

12

8 15 2 0 1

0 0 0 0 0

11 8 2 3 8

:

12 11 3 0 4 2 3

0 0 TBZ 0 0

11 8 IVM 21 4

5 3 TBZ 5 6

Overall total

Rank

7

54

12

16 5 2 3 3 5 2

47 39 9 6 16 9 9

10 9 2

7 11 IVM 12 8

28 23

8 7

51 18

11 6

1986

2. Continuous use of same drug 3 IVM/year 3 TBZ/year 3 TBZ/year (move) 8 IVM/year 8 TBZ/year 8 ABZ/year 8 LEV/year 3. Drugs ~ternated each treatment 3 I~-TBZ/treatment 8 IVM-TBZ/treatment 4. Drugs alternated each year 3 IVM-TBZ/year 8 IVM-TBZ/year

1 2 15 1 IVM 13 1

:

1 5 2 2

109

Worm control and anthelmintic resistance

slow rotation with IVM, and that simulation studies suggest that this is the preferred control option (Dobson, Griffiths, Donald & Waller, 1987), there is no reason to depart from the recommendation made initially by Prichard et al. (1980) and currently being widely promoted, of using different classes of anthelmimics in rotation at approximately yearly intervals. Taken together, these results suggest that to maintain the effectiveness of be~i~d~ole ~thelmintics in a parasite control program, where high levels of resistance to this drug group previously existed, their infrequent use in slow alternation with a highly effective drug (e.g. IVM) is the best option. However, productivity of young sheep is unlikely to be satisfactory unless treatment is followed by movement of the sheep to pastures of relatively low infectivity.

cost-effective programs being promoted in many countries at the present time (Wailer, 1986). This warning by Le Jambre (1978), if substantiated, would depreciate the value of such schemes. Studies by Martin, Anderson & Jarrett (1985) showed a higher level of BZ resistance in Ostertagia spp. in sheep given TBZ prior to introduction to a property that previously carried no sheep, than in parasites of sheep from the supply farms. They considered that this result provided evidence that ‘dose-and-move’ parasite control strategy is a strong selector for resistance. However in their study, 3 years had elapsed between transfer of sheep and tests for resistance, samples for parasite isolation were taken on only one occasion and no information was given as to whether the supply farms had acquired sheep, and thus parasites with possibly a lower level of resistance, during this period. In this investigation, comparison of the ‘dose-andmove’ and the set-stocked treatments where TBZ was given three times each year, showed the former appeared to select rapidly for BZ resistance in both H. contortus and T. colubriformis, judged by the results in 1984 (Table 4). However, this difference for both species was not observed in subsequent years, suggesting that while an initial faster rate of selection may occur, the long term effect of such strategies on development of resistance is ~~rn~, This result, together with the substantial production benefits of the ‘dose-and-move’, which were equivaient to suppressive IVM treatment (Tables 7 & 8) indicates that warnings against the adoption of integrated parasite control schemes needed to be tempered to prevent their being unjustifiably discredited. Although frequent alternation of anthehnintic groups hastened the selection for BZ resistance, this only occurred in H. contortus isolated from the intensively treated group. Prior to 1985, TBZ was used on only three occasions during the drought year of 1983, in the slow rotation treatment. As expected, BZ resistance was low in H. contortus isolated from this treatment in December 1984, the year in which IVM was used for the second time. Although TBZ appeared to be more efficacious in the slow compared

would also like to thank D. Bircliiin. K. Parsons, M. Peonies. J. Redwin and P. Stein for techhidal assistance. We are grateful to Drs P. Scott and J. Eagleson and staff of Merck Sharp & Dohme (Australia) Pty Ltd, Ingleburn for conducting and analysing the ivermectin in vivo assays. This trial was partially funded in 1982 by the Rural Credits Development Fund of the Reserve Bank of Australia, and from 1983 to 1986 by Merck Sharp & Dohme (Australia) Pty Ltd.

to the rapid rotation group in 1985, as judged by faecal egg count depression (Fig. le), the level of BZ resistance in fi. contorts isolated at the end of this

DONALDA. D., MORLEYF. H. W., WALLER P. J., AXELSEN

year had increased to be similar in both treatment strategies. Howewever, over the last year of study, rapid alternation increased the level of BZ resistance whereas it remained static in the slow rotation treatment (Table 4). It is also relevant to note that reversion towards BZ susceptibility was recorded in the slow rotation, strategic treatment in 1986 when TBZ was withdrawn. There was no evidence, either from faecal egg count depression (Fig. lf) or in vitro assays (Table 4), of any difference in the level of BZ resistance in T. colubriformis isolates from either the rapid or slow rotation groups. However, given that TBZ would remain more effective in the control of H. contortus when used in

Ac~nuwledgemen~-We

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A. & DONNELLY J. R. 1978. Av~lability to grazing sheep of gastrointestinal nematode infection arising from summer contamination of pastures. Australian Journal of Agricultural Research 29:*189-204.

DONAI.D A. D. & WALLER P. J. 1982. Problems and prospects in the control of helminthiasis in sheep. In: kol&y and Control of Endoparasites (Edited by SVMONS L. E. A.. DONALDA. D. & DINEENJ. K.). DO. 171-186. Academic Press, Sydney. DONALDA. D. 1985. Research priorities in anthehnintic resistance. In: Resistance in Nematodes fo Anthetmint~c I)rugs (Edited by ANDERSON N. & WALLER P. J.), DO. 17 I- 186. CSIRO, Melbourne. LA&Y E. & SNOWDON’K. L. 1988. A routine diagnostic assay for the detection of benzimidazole resistance in parasitic nematodes using tritiated benzimidazole carbamates. Veterinary Parasitology 27: 309-324. I

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mintic resistance in nematodes. Australian Veterinary Journal 56: 239-251. WALLER P. J. & DONALD A. D. 1983. New perspectives in helminth control. In: Recent Developments in the Control of Animal Parasites: Proceedings, MSD AGVET Symposium in association with the XXII World Veterinary Congress. Perth, 25 and 26 August 1983 (Edited by LEANING h. G. D., SIEGMUND 0. H’. & FRASER C. M.); pp. 215230. MSD AGVET. Rahwav. NJ. WALLER P. J., DOB&N R. J.: ‘DONALLI A. D., GRIFFITHS D. A. & SMITH E. F. 1985. Selection studies on anthelmintic resistant and susceptible populations of Trichostrongylus colubriformis of sheep. International Journal for Parasitology 15: 669-676. WALLER P. J. 1986. Anthehnintic resistance in nematode parasites of sheep. Agricultural Zoology Reviews 1: 333373. WALLER P. J. & LACEY E. 1986. The effect of triflumuron (SIR8514), on the free-living stages of sheep nematodes. i/eterina$ Parasitology 2 1: i 194 26. WYK J. A. VAN. MALAN F. S., GERBER H. M. & ALVES R. M. R. 198;. Two field strains of Haemonchus contortus resistant to rafoxanide. Onderstepoort Journal of Veterinary Research 54: 143-146.